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The MECP2 Duplication Database (MDBase): Deep Phenotyping to Understand a Rare Developmental Epileptic Encephalopathy

Daniel Ta¹, Jenny Downs^1,2, Gareth Baynam^1,3, Andrew Wilson^1,4, Peter Richmond^1,4 and Helen Leonard¹

¹Telethon Kids Institute, University of Western Australia, Perth, WA, Australia, ²Curtin School of Allied Health, Curtin University, Perth, WA, Australia, ³Genetic Services of Western Australia, WA Department of Health, Perth, WA, Australia and ⁴Perth Children’s Hospital, Perth, WA, Australia

Background: MECP2 duplication syndrome (MDS) remains a poorly characterized, X-linked neurodevelopmental disorder caused by duplication of the methyl CPG-binding protein 2 (MECP2) gene, with an incidence of ∼1/150,000 live births. Much better characterization of this disease is needed. Aim: A) ascertain a large population of patients with MDS to address the scarcity of phenotypic information and expand upon ill-defined features; B) compare acquisition of sitting, walking, and onset across other developmental epileptic encephalopathies (DEEs): Rett syndrome (RTT) and CDKL5 deficiency disorder (CDD). Methods: The international MECP2 Duplication Database (MDBase) was established in 2020 to collect in-depth health-related data. Further data were extracted from the InterRett database (n = 619), AussieRett (n = 289), and International CDKL5 Disorder Database (n = 333). Descriptive statistics were used to report prevalence of comorbidities; time-to-event survival analysis was used to estimate median age of motor skill development onset. Results: Data were ascertained on 148 males (88%; med = 9.80 years) and 21 females (12%; med = 11.05 years) with MDS. Common comorbidities such as respiratory infections (119/143 [83%]) and epilepsy (90/146 [61%]) were highly prevalent. Previously poorly characterized features included fractures (46/138 [33%]), urinary retention (38/138 [28%]), and autonomic problems such as cold hands/feet (100/144 [69%]) and temperature regulation difficulties (82/145 [57%]). Developmental milestones delay in MDS was of intermediate severity compared with RTT and CDD. Conclusion: This is the largest case series on patients with MDS to date. Further natural history data is important for understanding how MDS develops with age, between males and females and for a better comparison with other DEEs.

Insights from Five Years of Genomics Workforce and Education Research

Amy Nisselle^1,2,3, Belinda McClaren^1,2,3, Chriselle Hickerton^1,2, Fiona Lynch^1,2,3, Erin Crellin^1,2,3, Brigitte Cusack^1,4, Bronwyn Terrill^1,5,6, Debra Graves^1,7, Kate Dunlop^1,8, Sylvia Metcalfe^1,2,3 and Clara Gaff^1,2,3

¹Australian Genomics Health Alliance, ²Murdoch Children’s Research Institute, ³University of Melbourne, VIC, Australia , ⁴Centre for Genetics Education, NSW Health, Sydney, NSW, Australia, ⁵Kinghorn Centre for Clinical Genetics, Garvan Institute of Medical Research, Sydney, NSW, Australia, ⁶University of New South Wales, St Vincent’s Clinical School, Sydney, NSW, Australia, ⁷Royal College of Pathologists of Australasia and ⁸The Daffodil Centre, The University of Sydney, a joint venture with Cancer Council NSW, Sydney, NSW, Australia

Aim/Methods: The Australian Genomics Workforce & Education program was tasked with: mapping the Australian landscape of genomics practice and education; assessing education needs across genomic workforce sectors; and developing tools to support effective genomics education and evaluation. Here we report results and deliverables from the five-year program. From 2016 to 2020, mixed-methods projects used desk-top audits, interviews, focus groups, surveys, and/or Delphi consensus process. Results:Data were collected from 1067 participants across 20 projects, including genetic and nongenetic clinicians (36 disciplines), education providers and evaluators (spanning 11 countries, 16 genomics alliances/consortia), relevant organizations, and system influencers. 54% of nongenetic specialists practice genomic medicine but only 25% feel prepared; 69.7% want support from Genetics services. The impact of different genomics topics and ways of learning suggests ‘not all education is equal’. GPs access genomics education but genomics is yet to impact practice beyond NIPT. Other nonmedical health practitioners practice consumer-driven genomic medicine with limited access to impartial genomics education. Multidisciplinary workplace learning engenders increased confidence in acute care clinicians. In 2016 only 13% of Australian genomics education providers had education qualifications. We therefore developed tools: survey assessing genomic practice, attitudes and educational needs; program logic model to plan, develop, and deliver education; evaluation framework; and reporting standards to build an evidence base for effective genomics education. Conclusion: Our research provides evidence of adoption of genomic medicine, informing education as part of national implementation strategies. International collaborators are adapting our tools for local use. Factors facilitating workplace learning in genomics are now being explored.

Population Genomic Screening of All Young Adults in Australia to Detect Familial Hypercholesterolemia: A Cost-Effectiveness Analysis

Paul Lacaze1,*, Clara Marquina1,*, Jane Tiller¹, Moeen Riaz¹, Amy C Sturm², Mark R Nelson^1,6, Brian A. Ference³, Jing Pang⁴, Gerald F Watts^4,5, Stephen J Nicholls¹, Sophia Zoungas¹, Danny Liew¹, John McNeil¹ and Zanfina Ademi¹

¹School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC, Australia, ²Genomic Medicine Institute, Geisinger, PA, USA, ³University of Cambridge, Centre for Naturally Randomised Trials, Cambridge, UK, ⁴School of Medicine, Faculty of Health and Medical Sciences, University of Western Australia, Perth, WA, Australia, ⁵Lipid Disorders Clinic, Cardiometabolic Service, Departments of Cardiology and Internal Medicine, Royal Perth Hospital, Perth, WA, Australia and ⁶Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia

Background: Familial hypercholesterolemia (FH) is a life-threatening inherited condition that causes high cholesterol and early coronary heart disease (CHD), affecting 1 in 250 Australians. FH is significantly underdiagnosed in Australia, with an estimated >90% of carriers remaining unidentified by current criteria-based genetic testing. Aim: To assess the impact and cost-effectiveness of offering population genomic screening to all young adults in Australia to detect heterozygous FH. Methods: We designed a decision analysis model to compare the current standard-of-care for heterozygous FH diagnosis (opportunistic cholesterol screening and genetic cascade testing) with population genomic screening of adults aged 18–40 years to detect pathogenic variants in the LDLR/APOB/PCSK9 genes. The model captured morbidity/mortality due to CHD over a lifetime horizon, from a healthcare perspective. CHD risk, treatment effects, prevalence, and healthcare costs were estimated from published studies. Outcomes included quality adjusted life years (QALYs), costs, and incremental cost-effectiveness ratio (ICER), discounted 5% annually. Sensitivity analyses explored the impact of key input parameters on model robustness. Results: Over the population’s lifetime (4,167,768 men; 4,129,961 women), the model estimated 62,722 years gained and 73,959 QALYs due to CHD prevention. Population genomic screening for FH would be cost-effective from a healthcare perspective if the cost per test was ≤AU$300, yielding an ICER <AU$28,000 per QALY gained. From a societal perspective, population genomic screening would be cost-saving. Conclusion: Based on our model, offering population genomic screening to all young adults to detect FH could be cost-effective in the Australian healthcare system, at testing costs that are currently feasible.

Enabling Mitochondrial Donation in Australia – The Path to Legislative Change

David R. Thorburn^1,2,3,4, John Christodoulou^1,2,3,4 and Sean Murray⁴

¹Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia, ²Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia, ³Victorian Clinical Genetics Services, Royal Children’s Hospital, Melbourne, VIC, Australia and ⁴The Mito Foundation, Sydney, NSW, Australia

Background: Mitochondrial DNA (mtDNA) mutations cause severe health problems in ∼60 individuals born in Australia each year. Mitochondrial donation (MD) can potentially allow couples to have a healthy child related to both parents who has healthy donor mtDNA. Aim: To describe the processes that led to legislation enabling MD to proceed to a conscience vote in Australia and how it is expected to roll out. Methods: The authors are mitochondrial researchers (DRT, JC) and CEO of the Mito Foundation (SM); all played lead roles in MD advocacy and will describe how the pending legislative change has been reached. Results: Following a series of scientific and ethical reviews plus community engagement, the UK legalized MD in 2015. In 2014, the Mito Foundation began lobbying for MD to be legalized in Australia, via encouraging families to speak with their political representatives plus direct lobbying of key position holders across the political spectrum. This led to a Senate Inquiry in 2018, followed by an NHMRC Expert Working Committee review and multiple forms of public engagement. In March 2021, the Federal Government introduced MD legislation that is expected to go to a conscience vote in mid-2021. In May 2021, the Federal Budget allocated $10M over 10 years to introduce MD into research settings in Australia and to facilitate a clinical trial. Conclusion: If MD proceeds in Australia, it will be a boon to affected families and the result of over 7 years of co-ordinated effort from families, scientists, clinicians, ethicists, politicians, and professional organizations.

Reproductive Options for mtDNA Disease in Light of Current Australian Mitochondrial Donation Law Reform (Maeve’s Law) Bill

Suzanne C.E.H. Sallevelt¹, Christine E.M. de Die-Smulders², Joseph C.F.M. Dreesen², Irenaeus F.M. de Coo³, Patrick Lindsey³, Debby M.E.I. Hellebrekers² and Hubert J.M. Smeets³

¹Women’s and Children’s Hospital, Adelaide, SA, Australia (note: work presented originates from previous affiliation: Maastricht University Medical Centre+, the Netherlands), ²Maastricht University Medical Centre+, Maastricht, the Netherlands and ³Maastricht University, Maastricht, the Netherlands

Background: Mitochondrial DNA (mtDNA) mutations can be maternally inherited or de novo and are often heteroplasmic. Given the clinical severity and lack of treatment, couples with an affected child may request preventing transmission to subsequent offspring. Due to limitations in phenotype prediction, prenatal diagnosis (PND) is only suitable if a high likelihood exists of offspring without mutation or mutation load below the expression threshold. This applies to mtDNA deletions and skewing mtDNA mutations. Preimplantation Genetic Testing (PGT) can be offered to heteroplasmic mtDNA mutation carriers with a higher (or unpredictable) recurrence risk, transferring embryos with mutant load below a mutation-specific or general expression threshold. For homoplasmic carriers, PGT is by definition not feasible and the same may apply to carriers with high mtDNA mutation loads. For those, mitochondrial donation, replacing the carrier’s mitochondria with healthy donor mitochondria, can offer a solution. Aim: To assess, optimize and maximize reproductive options for mtDNA mutations. A summary of currently available techniques apart from egg donation will be presented from European experience. Methods: Systematic study of PND and PGT analyses for mtDNA mutations. Results: PND can also be offered to mothers of patients with most likely de novo mtDNA point mutations, which account for at least 25% of pediatric patients. In PGT, blastomere mutation load generally represents the whole embryo. Conclusion: Reproductive options are available for most mtDNA mutation carriers. Mitochondrial donation is currently in the process of (hopefully) being legislated in Australia and completes the portfolio of options to prevent mtDNA disease transmission.

Genetic Landscape of Infantile Spasms With Focal Brain Malformations

Matthew Coleman^1,2, Min Wang¹, Cas Simons¹, Colleen D’Arcy³, Simon Harvey³, Sarah EM Stephenson^1,2, Wei Shern Lee^1,2, Richard J Leventer^1,2,3, Katherine B Howell^1,2,3 and Paul J Lockhart^1,2

¹Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²University of Melbourne, Melbourne, VIC, Australia and ³Royal Children’s Hospital, Melbourne, VIC, Australia

Background: Infantile spasms (IS) are a relatively common developmental and epileptic encephalopathy, typically arising between 4 and 8 months of age. IS are often associated with malformations of cortical development (MCDs). Aim: To investigate the genetic landscape of IS in individuals that underwent epilepsy surgery. Methods: We performed histopathologic review and genomic testing in 52 individuals with IS who underwent resective neurosurgery for seizure control at the Royal Children’s Hospital, Melbourne. Genomic testing was performed on brain tissue using deep exome sequencing (n = 28), or targeted sequencing with a gene panel (n = 24). Results: Histopathologic analysis of brain tissue demonstrated tuberous sclerosis (TSC) (n = 22), focal cortical dysplasia (FCD) type I (16) and II (9) and nonspecific findings (5). The genetic basis of IS was identified in 35/52 (67%) individuals. Germline putative pathogenic variants were identified in 22 individuals, predominantly in genes associated with the mTOR pathway, such as TSC2, DEPDC5, and PIK3CA. Putative pathogenic somatic variants in brain (allele frequency ∼1% to 41%) were identified in 13 individuals in SLC35A2, AKT3, DEPDC5, and MTOR. Brain somatic variants in SLC35A2 were identified in most individuals with FCD I, whereas mTOR pathway variants were identified in most individuals with TSC and FCD II. Conclusion: The genetic landscape of IS with focal brain malformations comprises germline and brain somatic variants. Given the under-diagnosis of focal brain malformations, brain somatic variants are likely an under-recognized cause of IS. Our data highlights somatic variants in SLC35A2 as a major cause of IS with focal malformations.

Males with FMR1 Premutation Alleles of Less Than 71 CGG Repeats Have Low Risk of Being Affected with Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS)

Ellenore M. Martin¹, Zhu Ying^2,3, Claudine M. Kraan^4,5, Kishore R. Kumar^6,7, David E. Godler^4,5 and Michael J. Field¹

¹Genetics of Learning Disability Service (GOLD Service), Hunter Genetics, Newcastle, NSW, Australia, ²Randwick Genomics Laboratory, NSW Health Pathology, Prince of Wales Hospital, Sydney, NSW, Australia, ³Kolling Institute of Medical Research, Sydney, NSW, Australia, ⁴Diagnosis and Development, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia, ⁵Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, VIC, Australia, ⁶Molecular Medicine Laboratory and Neurology Department, Concord Repatriation General Hospital, Concord Clinical School, The University of Sydney, Sydney, NSW, Australia and ⁷Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia

Background: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset condition characterized by cerebellar ataxia and intention tremor, that can affect individuals with FMR1 premutation (PM) CGG expansion (PM alleles — 55-199 repeats). Current penetrance estimates suggest 45% of males with a PM will develop FXTAS by age 80. Aim: To delineate FXTAS penetrance estimates for males based on PM CGG repeat size. Methods: Using pooled data detailing the incidence of PM alleles and corresponding CGG repeat size in nine adult onset ataxia cohorts (17/1,320 males), and PM rates in the community (61/41,847), we applied a Bayesian approach to calculate the probability of being identified with FXTAS as a function of allele size, assuming a lifetime risk of adult onset ataxia of 1 in 2000. Results: Our penetrance estimates suggest the risk of developing FXTAS for males with ≤70 repeats is less than 1% (0.031%; CI [0.007, 0.141]) which is over 40-fold lower than previously reported. Ataxia penetrance increases with allele size; 71–80 repeats (0.630%; CI [0.130, 2.992]), 81-90 repeats (3.074%; CI [0.806, 11.020]) and is greatest for alleles >90 repeats (4.542%; CI [1.749- 11.270]) with a broad confidence range reflecting a small sample size. Conclusion: In the absence of a family history of FXS, the risk of developing FXTAS for males with a PM of ≤70 CGG repeats is significantly lower than existing estimates. This data is critical to accurately counsel individuals with PM alleles, who are increasingly being identified through community carrier screening programs.

The Role of AGG Interrupt Testing in Preconception Screening for Fragile X

Katrina Fisk¹, Bruce Bennetts^1,2, Tiffany Lai¹, Lauren Patterson McDonald¹ and Gladys Ho^1,2

¹Sydney Genome Diagnostics; Western Sydney Genetics Program, The Children’s Hospital at Westmead, Sydney, NSW, Australia and ²Disciplines of Child & Adolescent Health and Genomic Medicine, University of Sydney, Sydney, NSW, Australia

Background: Fragile X syndrome (FXS) is primarily due to the expansion of an unstable CGG repeat in the 5’ UTR of FMR1. Premutation alleles (PM, 55-200 repeats) can expand to a full mutation (>200 repeats) upon a single maternal transmission; however, the risk is mitigated by the presence of AGG interrupts within the repeat region. Aim: To offer Australia-wide AGG interrupt testing with a turnaround time of <2 weeks to improve risk stratification for women with a low-range PM. Methods: The polymorphic (CGG)n triplet repeat was amplified by a modified Amplidex™ (Asuragen) PCR, in conjunction with an AGG-specific primer assay to provide unambiguous genotypes for PM alleles. Results: Since Jan 2020, 63 women were tested. The majority of these (57%) had a PM between 55-59 repeats, all of which carried at least 1 AGG interrupt and are not at risk of expansion to a FM. Overall 54 of the 63 women tested (86%) were considered to be very low-risk of having an expansion to a FM after AGG interrupt testing. Conclusion: AGG interrupt testing can better inform the risks for PM carriers, reducing unnecessary parental anxiety, counseling time and the need for invasive prenatal testing. We recommend that AGG interrupt testing be incorporated as a second tier test for low range premutations (55-69 repeats) in preconception screening, and only those <70 without AGG interrupts be reported as high risk. We are able to offer second tier testing for Fragile X screening for preconception programs.

Clinical Impact of Whole Genome Sequencing in Early Onset Dementia Patients

Aamira J. Huq^1,2,3, Bryony A. Thompson^1,4, Mark F. Bennett⁵, Adam Bournazos⁶, Shobhana Bommireddipalli⁶, Alexandra Gorelik^2,7,8, Joshua Schultz¹, Adrienne Sexton^1,2, Rebecca Purvis¹, Kirsty West¹, Megan Cotter³, Giulia Valente³, Andrew Hughes⁹, Moeen Riaz⁷, Sarah Farrand¹⁰, Samantha Loi^2,10, Trevor Kilpatrick^2,11, Amy Brodtman^11,12, David Darby¹², Dhamidu Eratne^2,10, Mark Walterfang^2,10, Martin Delatycki^3,13, Elsdon Storey^1,7, Michael Fahey¹, Sandra Cooper⁶, Paul Lacaze⁷, Colin L. Masters¹⁴, Dennis Velakoulis¹⁰, Melanie Bahlo⁵, Paul A. James¹ and Ingrid M. Winship^1,2

¹Department of Genomic Medicine, Royal Melbourne Hospital, Melbourne, VIC, Australia, ²Department of Medicine, University of Melbourne, Melbourne, VIC, Australia, ³Department of Clinical Genetics, Austin Health, Melbourne, VIC, Australia, ⁴Department of Pathology, Royal Melbourne Hospital, Melbourne, VIC, Australia, ⁵Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia, ⁶Kids Neuroscience Centre, The Children’s Hospital at Westmead, Sydney, NSW, Australia, ⁷Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, VIC, Australia, ⁸Monash Department of Clinical Epidemiology, Cabrini Institute, Melbourne, VIC, Australia, ⁹Department of Neurology, Austin Health, Melbourne, VIC, Australia, ¹⁰Department of Psychiatry, Royal Melbourne Hospital, Melbourne, VIC, Australia, ¹¹Department of Neurology, Royal Melbourne Hospital, Melbourne, VIC, Australia, ¹²Department of Neurology, Box Hill Hospital, Melbourne, VIC, Australia, ¹³Victorian Clinical Genetics Service Melbourne, VIC, Australia and ¹⁴The Florey Institute, University of Melbourne, Melbourne, VIC, Australia

Background: Clinical genetic testing in early onset dementia (EOD) patients is challenging due to multiple types of diagnostic tests, undertaken through different laboratories, in order to arrive at a precise diagnosis. Whole genome sequencing (WGS) has the potential to serve as a single diagnostic platform for EOD genetics, due to its ability to detect common, rare, and structural genetic variations. Methods: WGS analysis was performed in 50 patients with EOD. Investigation of a targeted panel of 117 genes, followed by a ‘Mendeliome’ approach was undertaken. Subsequently, structural variant (SV) and short tandem repeat (STR) analysis were performed. An Alzheimer’s disease (AD) age of onset specific polygenic hazard score (PHS) was calculated in patients with AD. Results: Clinically diagnostic pathogenic variants were identified in 7/50 (14%) of the patients. A further eight patients (16%) were found to have established risk factor alleles. All relevant variants were in the targeted set of genes with no additional findings through Mendeliome analysis. Two of the clinically diagnostic variants were identified through SV analysis. No expanded STRs were found in this study cohort, but a blinded analysis with a positive control identified a C9orf72 expansion accurately. Nine of the 19 AD patients (∼47%) had a PHS equivalent to >90th percentile risk. Conclusion: WGS acts as a single genetic test to identify different types of clinically relevant genetic variations in patients with EOD. WGS, if used as a first-line clinical diagnostic test, has the potential to increase the diagnostic yield and reduce time to diagnosis for EOD.

Lessons Learned From the First 170 Trios from the Genomic Autopsy Study

T. T. Ha^3,5, A. B. Byrne^3,4,5, P. Arts^3,5, J. Feng^6,5, P. S. Wang⁷, A. W. Schreiber^6,5,8, P. Brautigan³, M. Babic³, W. Waters⁹, L. Pais⁴, S. Yu¹⁰, J. Lipsett¹⁰, L. Moore¹⁰, N. Manton¹⁰, Y. Khong¹⁰, E. Thompson1Genomic Autopsy Study Research Network, J. Liebelt¹, L. McGregor¹, S. Sallevelt¹, M. Dinger¹¹, D. G. MacArthur⁴, S. L. King-Smith^12,13, C. N. Hahn^12,14,5, K. S. Kassahn^3,8, M. R. Jackson^12,13, H. S. Scott^3,15,16,14 and C. P. Barnett^1,2

¹Paediatric and Reproductive Genetics Unit, Women’s and Children’s Hospital, Adelaide, SA, Australia, ²University of Adelaide, Adelaide, SA, Australia, ³Genetics and Molecular Pathology Research Laboratory, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ⁴Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, MA, United States, ⁵School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia, ⁶ACRF Cancer Genomics Facility, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ⁷3ACRF Cancer Genomics Facility, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ⁸School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia, ⁹Department of Genetics and Molecular Pathology, SA Pathology, North Adelaide, SA, Australia, ¹⁰Department of Anatomical Pathology, SA Pathology at Women’s and Children’s Hospital, Adelaide, SA, Australia, ¹¹Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia, ¹²Genetics and Molecular Pathology Research Laboratory, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ¹³Australian Genomic Health Alliance, Melbourne, VIC, Australia, ¹⁴School of Medicine, University of Adelaide, Adelaide, SA, Australia, ¹⁵ACRF Cancer Genomics Facility, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia and ¹⁶Department of Genetics and Molecular Pathology, SA Pathology, Adelaide, SA, Australia

Background: The cause of pregnancy loss and perinatal death remains unexplained in at least 25% of cases, despite a high perinatal autopsy rate in Australia. The Genomic autopsy study is a national collaborative study aimed at determining the cause. Aim: To apply WES/WGS to identify genetic causes of fetal/newborn abnormalities that result in termination of pregnancy, death in utero or in the newborn period, in view to providing families with answers regarding cause and likelihood of recurrence. Methods: WES/WGS is being performed on parent-fetus trios/quads following standard autopsy and noninformative microarray. High priority cases are consanguineous families, fetuses with multiple malformations, and unexplained fetal/newborn death. Statistical, bioinformatic, and experimental laboratory techniques are used to confirm causality of variants. Results: 170 prospective trios (150) or quads (20) have been recruited and sequenced. 89/170 (52%) are either solved or have a strong candidate gene identified. 42/170 (25%) of cases have been clearly solved by identification of a pathogenic mutation in a known gene. An additional 27/170 (16%) have a candidate variant in a known gene that expands phenotype. In 20/170 (12%) of cases, a mutation was identified in a gene not yet linked to human disease. Discussion: Our results provide justification for genomic investigation of pregnancy loss and perinatal death, particularly when congenital abnormalities are present. Establishing a clear diagnosis on clinical grounds alone is often challenging. Late stillbirth remains largely unexplained by genomics. Novel techniques for genomic investigation of unexplained stillbirth need further consideration, including genomic investigation of the placenta.

USP9X Associated Neurodevelopmental Disorders

Lachlan A. Jolly¹, Euan Parnell², Sehyoun Yoon², Maria A Kasherman³, Laura Currey^3,4, Stephen A. Wood⁴, Thomas H. J. Burne³, Michael Piper³, Peter Penzes² and Jozef Gecz^1,5

¹University of Adelaide and Robinson Research Institute, SA, Australia, ²Northwestern University Feinberg School of Medicine, Il, USA., ³University of Queensland, Brisbane, QLD, Australia, ⁴Griffith University, Brisbane, QLD, Australia and ⁵South Australian Health and Medical Research Institute, Adelaide, SA, Australia

Variants in USP9X, an atypical X-chromosome gene that escapes X-inactivation, cause neurodevelopmental disorders (NDDs) in males and females, but by different mechanisms. Males are impacted by de-novo or maternally inherited hemizygous partial loss-of-function missense variants, while females are impacted by de-novo heterozygous complete loss-of-function variants. We now provide evidence of the contribution of USP9X missense variants in female NDDs also by reporting 14 likely pathogenic missense variants, arising de novo or inherited from mosaic mothers. Aggregate phenotypic assessment of 35 currently known females with pathogenic USP9X variation revealed a clinically recognisable USP9X-female syndrome. Comparison of this syndrome against all known male USP9X associated phenotypes highlighted shared neurological features, and discordant female-only congenital features. We thus used a brain-specific USP9X knockout mouse to investigate neuropathology. Consistent with human phenotypes, knockout mice displayed abnormal learning, memory, and communication. At a cellular level, advanced diffusion tensor MRI of knockout brains revealed deficits in major forebrain commissures, and identified long-range hypo-connectivity between cortical and subcortical regions. At a molecular level, USP9X encodes a deubiquitinating enzyme which promotes the stability and abundance of other target proteins. Using knockout mice, patient cells, and interrogation of all known USP9X target proteins, we show USP9X preferentially functions to stabilize proteins encoded by other NDD genes. Collectively, this new data expand, collate, and compare the genetic and phenotypic landscape of male and female USP9X associated NDDs, and reveal mechanisms of shared neuropathology involving disrupted neuronal connectivity and destabilization of proteins known to cause NDDs through loss of dosage.

Cancer Diagnosis Following Abnormal Noninvasive Prenatal Testing: A Case Series and Proposed Management Model

Eryn Dow^1,2, Alison Freimund^1,3, Kortnye Smith¹, Rodney J. Hicks^1,3, Peter Jurcevic⁴, Mark Shackleton⁵, Paul A. James^2,3, Andrew Fellowes¹, Martin B. Delatycki⁶, Susan Fawcett⁴, Nicola Flowers⁶, Mark D. Pertile⁶, George McGillivray^4,6 and Linda Mileshkin^1,3

¹Peter MacCallum Cancer Centre, Melbourne, VIC, Australia, ²Parkville Familial Cancer Centre, Melbourne, VIC, Australia, ³University of Melbourne, Melbourne, VIC, Australia, ⁴Royal Women’s Hospital, Melbourne, VIC, Australia, ⁵Alfred Health, Melbourne, VIC, Australia and ⁶Victorian Clinical Genetics Service, Melbourne, VIC, Australia

Background: Noninvasive prenatal testing (NIPT) is a screening test for fetal chromosomal aneuploidy utilizing cell-free DNA (cfDNA) derived from maternal blood. It has been rapidly accepted into obstetric practice due to its application from 10-weeks’ gestation, and its high sensitivity and specificity. NIPT results can be influenced by several factors including placental or maternal mosaicism and co-twin demise; cfDNA from a maternal origin can also complicate interpretation, with evidence that NIPT can detect asymptomatic maternal malignancies. Aim: This study aimed to develop management guidelines for women with NIPT results suspicious of maternal malignancy. Methods: The Peter MacCallum Cancer Centre’s experience of seven cases where abnormal NIPT results led to investigation for maternal malignancy between 2016 and 2019 were reviewed, along with the published literature. Results: Six of the seven women (86%) were diagnosed with advanced malignancies, including colorectal cancer, breast cancer, melanoma, and Hodgkin’s lymphoma. Reviewing the diagnostic yield of investigations preformed contributed to the development of the proposed guidelines, including the utilization low dose FDG PET-CT, which had a high concordance with other investigations and diagnoses. Conclusion: Based on our experiences, as well as a review of the literature, guidelines for the investigation and management of women with NIPT results suspicious of malignancy are proposed. These guidelines include maternal and fetal investigations as well as consideration of the complex medical, psychological, social, and ethical needs of these patients and their families.

Assessing the Contribution of Mosaic Genetic Variation to Cerebral Palsy Etiology

Nandini Sandran^1,3, Clare van Eyk^1,3, Dani Webber^1,3, Mark Corbett^1,3, André Minoche⁴, Jeffrey Craig², Kelly Harper³, Jesia Berry³, Alastair MacLennan³ and Jozef Gécz^1,3

¹Neurogenetics Research Program, Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia, ²Centre for Molecular and Medical Research, School of Medicine, Deakin University, Geelong, VIC, Australia, ³Australian Collaborative Cerebral Palsy Research Group, Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia and ⁴Kinghorn Centre for Clinical Genomics, Garvin Institute of Medical Research, Sydney, NSW, Australia

Cerebral palsy (CP) results from nonprogressive damage to the developing brain and is the most common cause of childhood physical disability. At least 1/4 of individuals have an underlying genetic cause, with most variants arising de novo. This suggests that variants either: (1) arise during embryonic development (postzygotic mosaicism) or (2) are inherited from a germline mosaic parent. These mechanisms have different implications for recurrence risk and therefore, differentiating between them is important. We use genomic data from the Australian CP Biobank to assess the contribution of each mechanism to CP. We develop a pipeline using GATK, Mutect², and MosaicHunter to identify somatic variants in parent-child trios, enabling identification of both germline and post-zygotic events. We also examine the causes of CP discordance in identical (monozygotic) twins, hypothesising that post-zygotic genetic variation could explain clinical discordance. We assessed both germline (i.e., shared between identical twins) and postzygotic (i.e., discordant in identical twins) genetic variation. For 5/13 twin pairs, we identified de novo shared variants of interest. In 1/13, we identified a novel de novo discordant missense variant in MYT1L, which we also validated by digital droplet PCR (alternate allele frequency:0.04 in CP-affected twin, 0.00 in unaffected twin). MYT1L drives neuronal differentiation by repressing the expression of nonneuronal genes and has been associated with intellectual disability. We are now performing functional analysis to assess pathogenicity of this variant. Together, these data demonstrate that genetic mosaicism is likely to be a rare but important cause of CP, with implications for estimating recurrence risk within families.

Specifications of the Acmg/Amp Variant Curation Guidelines for Myoc: Recommendations from the Clingen Glaucoma Expert Panel

Emmanuelle Souzeau¹, Kathryn Burdon², Jamie Craig¹, Patricia Graham^2,3, Johanna Hadler^1,4, Alex Hewitt², John Hulleman⁵, David Mackey^2,3, Emi Nakahara⁵, Francesca Pasutto⁶, Owen Siggs¹, Terri Young⁷ and Andrew Dubowsky⁴

¹Flinders University, Flinders Medical Centre, Adelaide, SA, Australia, ²University of Tasmania, Hobart, TAS, Australia, ³University of Western Australia, Perth, WA, Australia, ⁴SA Pathology, Adelaide, SA, Australia, ⁵University of Texas Southwestern Medical Center, Dallas, TX, USA, ⁶University Erlangen-Nuremberg, Erlangen, Germany and ⁷University of Wisconsin-Madison, Madison, WI, USA

Background: Standardized variant curation criteria are essential for accurate interpretation of genetic results and clinical care of patients. The variant curation guidelines developed by ACMG/AMP in 2015 are widely used by the genetic community but are not gene-specific. The Clinical Genome Resource (ClinGen) Variant Curation Expert Panels (VCEP) are tasked with developing gene-specific variant curation guidelines. Aim & Methods: The Glaucoma VCEP was created in 2019 and assembled clinicians, researchers, and laboratory scientists that aimed to develop and pilot rule specifications of the ACMG/AMP variant curation guidelines for MYOC variants, the most common cause of Mendelian glaucoma. Results: Among the 28 ACMG/AMP criteria, the Glaucoma VCEP adapted 15 rules to MYOC (including 10 strength specifications), while 13 rules were deemed not applicable. Key specifications included calculations of minor allele frequency thresholds, developing an approach to counting multiple independent probands and segregations, and reviewed functional assays that reported on the solubility and secretion of mutant proteins. The specified rules were piloted on 81 variants and led to a change in classification in 14/37 (38%) of those that were classified in ClinVar. Functional evidence led to the reclassification of 20 (45%) of VUS (including 13 from VUS to likely pathogenic and 7 from VUS to likely benign). Conclusion: The standardized variant curation guidelines for MYOC from the Glaucoma VCEP improved variant classification. MYOC variant curation and classifications will be submitted to ClinVar with expert-level status.

An Approach to Genetic Counseling and Diagnostic Genetic Testing for Mnd and/or Ftd: Results of a Modified Delphi Consensus Survey

Ashley Crook^1,3, Chris Jacobs¹, Toby Newton-John¹ and Alison McEwen¹

¹University of Technology Sydney, Graduate School of Health, Sydney, NSW, Australia, ²Centre for MND research, Department of Biomedical Science, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia and ³Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney. NSW, Australia

Background: Genetic counseling and diagnostic genetic testing is part of the multidisciplinary care of people with motor neuron disease (MND) and/or frontotemporal dementia (FTD). There are no consistent, evidence-based genetic counseling approaches/guidelines for MND/FTD. Aim: Identify areas of consensus and conflict on the ideal components of genetic counseling for diagnostic genetic testing among various stakeholders, and propose a best practice statement. Methods: Experts in genetic counseling/testing for MND/FTD were invited, including health professionals (e.g. genetic health professionals, neurologists) and consumer experts (e.g., patients, relatives, MND/FTD support organization staff). An online, modified, multiround Delphi consensus survey was conducted using REDCap. Items in the first round were informed by two systematic literature reviews and qualitative interviews with consumers who had experienced diagnostic genetic testing. Descriptive and content analysis informed the development of the subsequent round and final results. Results: Forty-six experts participated: 100% completed round 1, 95.65% completed round 2. After round 1, items were updated based on comments regarding content and wording and presented for consensus in round 2. At survey completion, consensus was reached (>75% agreement) on all items, but there were conflicts regarding the timing of various discussions. Sixteen consensus items will be presented. Conclusion: The high level of consensus among health professionals and consumers indicates that the level of information, counseling, and support provided throughout the genetic counseling process should be adapted to the client’s needs. The findings are critical as genetic testing becomes more routinely part of adult-onset neurodegenerative disease management due to emerging genotype-driven therapies.

Understanding the Needs of Adolescent and Young Adult Women Undergoing Predictive Genetic Counseling for Hereditary Breast and/or Ovarian Cancer

Anastasia Lewis^1,2, Rowan Forbes Shepherd^1,3, Lucy Holland⁴ and Alexandra Lewis¹

¹Parkville Familial Cancer Centre, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, VIC, Australia, ²Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia, ³Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Australia and ⁴Queensland University of Technology, Brisbane, QLD, Australia

Background: Adolescents and young adults (aged 15–25 years; AYAs) experience formative biopsychosocial development which has implications for the genetic counseling process. At the Parkville Familial Cancer Centre, Melbourne, AYA women are increasingly seeking predictive genetic testing for hereditary breast and/or ovarian cancer (HBOC), prior to the age of recommended risk management strategies (25 years). The specific genetic counseling needs of AYA women at this life stage have not yet been explored. Aim: This study explored the information and psychosocial support needs of AYA women who underwent predictive genetic testing for HBOC. Method: We conducted semistructured interviews with AYA women who underwent predictive genetic testing for HBOC and received results after January 2019. Reflexive thematic analysis was used to develop findings. Results: Nineteen AYAs (12 BRCA1/2+ve, 7 BRCA1/2 or ATM -ve) participated with a mean age at genetic testing of 21 years (range 17–24 years). Participants often found genetic counseling to be overwhelming, felt uncertain of their place in the health system and deprioritized their own needs to that of their loved ones, impacting their ability to process information and seek support. Test positive participants grappled with integrating the reproductive implications of their results into their view of the future, causing some participants discomfort and uncertainty. Conclusion: This study adds to growing evidence that AYAs require age-appropriate discussions and longitudinal specialized AYA care. Genetic health professionals should ascertain a young person’s developmental stage, family dynamics and their future outlook to provide tailored developmentally appropriate genetic counseling that addresses their needs.

Evaluating Psychological Resources Codeveloped With Caregivers of Children With Genetic Epilepsy

Suzanne Nevin^1,2, Claire E. Wakefield^1,2, Fleur Le Marne^1,3, Erin Beavis^1,3, Rebecca Macintosh^1,4, Rani Sachdev^1,4, Ann Bye^1,3, Elizabeth E. Palmer^1,4 and Kenneth Nun¹

¹School of Women’s and Children’s Health, UNSW Medicine, UNSW Sydney, NSW, Australia, ²Behavioural Sciences Unit, Kids Cancer Centre, Sydney Children’s Hospital, Sydney, NSW, Australia, ³Department of Neurology, Sydney Children’s Hospital, Sydney, NSW, Australia, ⁴Centre for Clinical Genetics, Sydney Children’s Hospital, Sydney, NSW, Australia and ⁵Department of Psychological Medicine, Children’s Hospital at Westmead, Sydney, NSW, Australia

Background: Developmental and epileptic encephalopathies (DEE) are chronic and often life-threatening genetic conditions. Caring for a child with a genetic DEE is associated with a high risk of depression, anxiety, and chronic-traumatic stress. Caregivers report pervasive challenges accessing psychological support tailored to the realities and contexts of their child’s diagnostic trajectory. However, acceptable methods of supporting caregiver psychological needs and adaptation to their child’s diagnosis are unknown. Aim: We aimed to codesign and evaluate an empirically informed suite of audio-visual psychological resources tailored to DEE caregivers needs. Methods: We collaborated with caregivers using in-depth interviews (N = 25) and interactive focus group workshops (N = 8) to iteratively codesign the content of the psychological resources. Informed by this data, we co-produced six audio-visual resources, including caregiver quotes and practical psychoeducational summaries. We administered a web-based survey to assess the acceptability, relevance, and emotional impact of the resources, using validated and purpose-designed questionnaires. Results: Sixty-six caregivers, recruited from 10 international rare disease organizations and 4 tertiary children’s hospitals reviewed and evaluated the resources. Caregivers rated the resources as highly acceptable and reported that they accurately represented their caregiving experiences and psychological support needs. Frequently described emotional responses included feeling ‘comforted’, ‘hopeful’, ‘connected’, and ‘reassured’. Caregivers considered that providing the resources at diagnosis would address their need for reassurance and strengthen their coping skills. Conclusion: Our codesigned psychological resources appear to be acceptable and relevant to caregivers. Results suggest that these psychological resources offer valuable emotional support and can be delivered to DEE caregivers in healthcare settings.

Recording Our Genes: Stakeholder Views on Genetic Test Results in Networked Electronic Medical Records

Megan Prictor¹, Maria Rychkova¹, Mark J. Taylor¹, Jane Kaye^1,2, Wendy Chapman³, Tiffany Boughtwood⁴, Paul Fennessy⁵, Dianne Nicol⁶, Lynda O’Brien⁷ and Danya Vears⁸

¹Melbourne Law School, University of Melbourne, Melbourne, VIC, Australia, ²Faculty of Law, University of Oxford, Oxford, UK, ³Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, VIC, Australia, ⁴Australian Genomics Health Alliance, Melbourne, VIC, Australia, ⁵Department of Health and Human Services, Melbourne, VIC, Australia, ⁶Faculty of Law, University of Tasmania, Hobart, TAS, Australia, ⁷Murdoch Children’s Research Institute, Melbourne, VIC, Australia and ⁸Murdoch Children’s Research Institute, Melbourne, VIC, Australia

Background: Australia has seen rapid growth in clinical genetic testing and networked electronic medical records (EMRs): digital records that can be shared across different settings and may be patient-accessible. Incorporating genetic information into EMRs is a national policy priority to benefit care coordination and clinician decision making. Yet little is known about stakeholder views on its desirability and implementation. For instance, would patients prefer a more or less restrictive approach to genetic information accessibility? When should consent be sought for information access? What legal and information governance risks do clinicians perceive in networked EMRs? Aim: Within a project on legal and governance issues in the use of networked EMRs for genetic information, we aimed to examine the needs and concerns of patients, family members, patient advocates, and clinicians. Methods: We conducted semistructured qualitative interviews (mid 2021) and analyzed them using an inductive thematic approachin NVivo. Results: Preliminary data suggest: (1) Complex record-keeping systems have evolved to suit local clinical needs. (2) Stakeholders recognize the significance of the family context in the collection and disclosure of genetic information — both in determining individual risk and in familial health surveillance, prevention, diagnosis, and treatment. EMR design may evolve to better reflect this. (3) Laws, policies, and professional ethics protect individual health information but may fail to reflect family interests. Australian states and territories should pursue alignment with Commonwealth law on disclosing genetic information without patient consent (Privacy Act 1988 (Cth) s 16B(4)). Conclusion: The interview findings will inform recommendations to address barriers to this national policy priority.

Metaphors and Why These are Important in Genetic Counseling

Adrienne Sexton^1,2 and Paul James^1,3

¹Genomic Medicine, The Royal Melbourne Hospital, Melbourne, VIC, Australia, ²Department of Medicine, The University of Melbourne, Melbourne, VIC, Australia and ³Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia

Background: Metaphors appear simple but are fundamental schemata allowing expression and processing of complex emotions and information. Often so embedded in language and thinking that we are unaware of their impact, metaphor is recognized as a valuable tool in related fields of psychotherapy, education, linguistics, cancer care, palliative care, pain management, and counseling but has received little attention in genetic counseling aside from explanations for genes. Hypothesis: A deeper understanding of how to recognize and work with client-generated and counselor-generated metaphors has great potential as an addition to the genetic counseling ‘tool-box’. Methods: This is a discussion paper, with reference to metaphor theory and studies from related health and psychotherapy fields, to present how working purposefully with metaphors may offer a powerful way to enhance communication within a reciprocally engaged client-counselor relationship. Results: Metaphors present ways to explain complex genetic concepts in a personally meaningful form, to gain a deeper understanding of client’s experiences and emotions, to assist processing of experiences, emotions, and concepts, and to assist client and counselor to access and reflect on subconscious emotions, self-concept and motivations. In addition, working with metaphors has been shown to facilitate coping and action. Conclusion: This paper sets the scene for why and how genetic health professionals can utilize client-generated and counselor-generated metaphors purposefully to enhance the therapeutic interaction with clients.

My Research Results: A Genetic Counselor-Led Program to Facilitate Return of Clinically Actionable Genetic Research Findings

Amanda Willis^1,2, Bronwyn Terrill^1,2, Angela Pearce¹, Alison McEwen³, Mandy L Ballinger^2,4, Roger Milne^5,6,7, Fiona Bruinsma⁵, Jane Tiller⁸, Paul Lacaze⁸ and Mary-Anne Young^1,2

¹Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia, ²St Vincent’s Clinical School, UNSW Sydney, Sydney, NSW, Australia, ³Genetic Counselling, Graduate School of Health, University of Technology Sydney, Sydney, NSW, Australia, ⁴Cancer Theme, Garvan Institute of Medical Research, Sydney, NSW, Australia, ⁵Cancer Epidemiology Division, Cancer Council Victoria, Melbourne, VIC, Australia, ⁶Centre for Epidemiology and Biostatistics, School of Population and Global Health, The University of Melbourne, Melbourne, VIC, Australia, ⁷Precision Medicine, School of Clinical Sciences at Monash Health, Monash University, Melbourne, VIC, Australia and ⁸Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC, Australia

Background: Australian researchers increasingly support returning clinically actionable genetic research findings to participants, but may lack the skills and resources to do so. Aim: Develop a program to support researchers and facilitate the return of clinically actionable research findings to participants. Methods: The My Research Results (MyRR) program has been developed by a steering committee of clinicians, researchers, genetic educators, and consumers. MyRR supports researchers to return clinically actionable research findings to participants. MyRR is staffed by genetic counselors and available to researchers Australia-wide. Participants are notified of findings by letter, with a follow-up phone call from a genetic counselor. The MyRR experience of returning findings from the Melbourne Collaborative Cohort Study and the ASPREE Study is reported. Results: 23 individuals across the two studies were notified of clinically actionable findings from February to May 2021. Notification letters were sent to probands (n = 21) or, if deceased, the nominated next-of-kin (n = 2). MyRR genetic counselors successfully contacted 21 individuals (12 women, 9 men) regarding pathogenic variants in BRCA1 (n = 6), BRCA2 (n = 13), MSH6 (n = 1) and PMS2 (n = 1). The average age of notified probands was 81 years. Findings were disclosed to 20 individuals, one declined to receive the findings. Thirteen probands expressed an intention to attend a clinical genetics service for confirmatory testing and risk management advice. Five individuals were already aware of the findings. Conclusion: MyRR is a translational program promoting and facilitating access to clinically actionable genetic research findings, filling an important gap for Australian research studies and delivering health benefits to research participants.

Making Sense of Severity in the Context of Genetic Screenings

Lisa Dive^1,2 and Ainsley Newson^1,2

¹Sydney Health Ethics, The University of Sydney, Sydney, NSW, Australia and ²Australian Reproductive Carrier Screening Programme (Mackenzie’s Mission)

Background: For interventions such as reproductive genetic carrier screening (RCS), severity is a key issue. This concept influences both the inclusion of a genetic condition on a screening panel and couples’ subsequent decisions. Yet despite its crucial role, arriving at a specific definition of what severity means and how it can be applied to a range of genetic conditions remains elusive. Aim: We analyse severity as an ethically contested concept whose meaning is dependent on context and stakeholder role, and which can change over time. It is important for the design and implementation of programs like RCS to acknowledge and respond to this inherent complexity and uncertainty. Our conceptualization of severity achieves this. Methods: Our bioethical analysis of severity brings together both normative/theoretical and empirical investigations into this concept as it arises in healthcare decision making. We also critically evaluate attempts to develop objective approaches to quantifying severity in the context of genetic conditions. Results: Attending to different dimensions of severity helps anchor RCS policy and program design in a notion of severity that acknowledges the variability and uncertainty inherent in the concept, while still allowing it to play a crucial role in decision-making. Conclusion: While severity has an inherent uncertainty that depends on context and perspective, it is such a pivotal concept in RCS that we must find a way to explain and apply it clearly and consistently. Analysing its different dimensions is a way of bringing rigour to how severity is conceptualized.

Modelling Patient and Healthcare Services Outcomes From Applying Polygenic Risk Scores for Breast and Ovarian Cancer in Pathogenic Variant Carriers

Lara Petelin^1,2,3, Lucinda Salmon⁴, Gillian Mitchell¹, Danny Liew⁵, Alison H. Trainer^1,6 and Paul A. James^1,6

¹Parkville Familial Cancer Centre, Peter MacCallum Cancer Centre & the Royal Melbourne Hospital, Melbourne, VIC, Australia, ²The Daffodil Centre, University of Sydney, a joint venture with Cancer Council New South Wales, Sydney, NSW, Australia, ³Melbourne School of Population & Global Health, University of Melbourne, Melbourne, VIC, Australia, ⁴Clinical Genetics, Austin Hospital, Melbourne, VIC, Australia, ⁵School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC, Australia and ⁶Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia

Background: Polygenic risk modifies breast and ovarian cancer risk in carriers of moderate and high-risk rare pathogenic variants (PV). The addition of a polygenic risk score (PRS) to genomic risk assessments would facilitate more personalized risk estimates and risk management advice, but the effectiveness of this approach is unknown. Aim: Estimate the long-term clinical and cost-effectiveness outcomes of a genomic risk assessment including a PRS for breast and ovarian cancer for carriers of PV in high/moderate-risk genes using microsimulation. Methods: A validated simulation model (miBRovaCAre) was adapted to include a PRS. The target population was cancer-unaffected women aged 20–39 years, with a PV (genes included BRCA1, BRCA2, PALB2, ATM, BRIP1, CHEK2, RAD51C, RAD51D). Using accepted 10-year and lifetime risk management thresholds, the intervention was incorporation of a PRS to determine uptake and timing of risk management strategies, compared to current practice (based on the PV alone). Results: Introducing a PRS resulted in 0.16 quality-adjusted life-years saved per carrier at an average additional cost of $AUD1454, likely representing a cost-effective addition to current care. In general, the greatest benefit was seen in women in the highest quintile for PRS (breast and/or ovarian). An important contribution arose from improved personalization of the recommended age for risk-reducing salpingo-oophorectomy (RRSO). In women in the highest quintile of the PRS, the estimated average age of RRSO was reduced from 42.05 under current practice to 37.16 years by addition of the PRS. Conclusion: Tailoring cancer risk management through personalized germline risk assessments with a PRS could greatly benefit moderate- and high-risk PV carriers.

Clinical and Laboratory Reporting Impact of ACMG and Modified ClinGen Variant Classification Frameworks in MYH7-Related Cardiomyopathy

Christopher M. Richmond^1,2,3, Paul A. James^4,5, Sarah-Jane Pantaleo¹, Belinda¹, Sebastian Lunke^1,5, Tiong Y. Tan⁵ and Ivan Macciocca¹

¹Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²Genetic Health Queensland, Royal Brisbane & Women’s Hospital, Brisbane, QLD, Australia, ³School of Medicine, Griffith University, Gold Coast, QLD, Australia, ⁴Genomic Medicine Department, Royal Melbourne Hospital, QLD, Australia and ⁵University of Melbourne, Melbourne, VIC, Australia

Background: ClinGen’s expert panel adaptation of the ACMG guidelines provides gene-specific recommendations for interpretation of MYH7 sequence variants in patients with inherited cardiomyopathy. Aim: We assessed laboratory and clinical impact of reclassification by ACMG and ClinGen in 43 MYH7 variants reported by a diagnostic laboratory between 2013 and 2017. Methods: 52 proband reports containing MYH7 variants were reinterpreted by original ACMG and ClinGen guidelines. Evidence items were compared across schemes and reasons for classification differences recorded. Laboratory impact was assessed by number of recommended report reissues, and reclassifications coded as clinically ‘actionable’ or ‘equivalent’. Available pedigrees were reviewed to describe projected cascade impact. Results: ClinGen produced a higher proportion of diagnostic classifications (65% of variants) compared with ACMG (54%) and fewer variants of uncertain significance (30% versus 42%). ClinGen classification resulted in actionable changes in 18% of variants with equal upgrades and downgrades from original report. ClinGen’s revisions to PM1 and PS4 contributed to classification differences in 21% and 19% of variants, respectively. Each classification change per proband report impacted, on average, 3.1 cascade reports with a further 6.3 first- and second-degree relatives potentially available for genotyping per family. Conclusion: ClinGen’s gene-specific criteria provide expert-informed guidance for interpretation of MYH7 sequence variants. Periodic re-evaluation improves diagnostic confidence and should be considered by clinical and laboratory teams.

Identification of an Oncogene Duplication By Genomewide NonInvasive Prenatal Screening

Samantha Sundercombe¹, Tristan Hardy², Michael Friedlander³, Edwin Kirk⁴ and Fergus Scott⁵

¹NSW Health Pathology Randwick Genomics, Sydney, Australia, ²Repromed, Adelaide, SA, Australia, SA Pathology, Adelaide, SA, Australia, Monash IVF Group, ³Prince of Wales Clinical School, UNSW and Department of Medical Oncology, The Prince of Wales Hospital, Sydney, NSW, Australia, ⁴NSW Health Pathology Randwick Genomics, Sydney, NSW, Australia; School of Women’s and Children’s Health UNSW, Sydney Children’s Hospital, Sydney, NSW, Australia and ⁵Sydney Ultrasound for Women, Sydney, NSW, Australia

Background: Genomewide noninvasive prenatal screening (NIPS) assesses for aneuploidy of all chromosomes, including segmental aneuploidy ≥7Mb. This can generate incidental findings that would not be identified by more focused NIPS platforms. Identification of multiple chromosome aneuploidies in cell-free DNA can suggest maternal malignancy. Aim: To provide an example of NIPS leading to the diagnosis of maternal malignancy and illustrate limitations of NIPS in the context of maternal malignancy. Methods: A 40-year-old woman presenting for routine prenatal screening had genomewide NIPS (Illumina VeriSeq NIPT Solution v2). The NIPS result was unhelpful for pregnancy assessment so combined first trimester screening was performed leading to chorionic villus sample testing. She had a standard clinical workup for malignancy of unknown origin. Results: NIPS revealed a fetal fraction of 13% with multiple genomic aberrations including monosomies on chromosomes 1, 2, 3, 4, 5, 10, 11, 17, and 18, and trisomies on chromosomes 6, 7, 9, 13, 16, 20, 21, and X. A duplication of the entire long arm of chromosome 8 was reported, including the MYC proto-oncogene region at 8q24.21, the first oncogene duplication found by NIPS. The patient was diagnosed with colorectal adenocarcinoma. SNP microarray analysis of the CVS sample revealed trisomy 21. Microarray performed on the tumor DNA found comparable results to the cfDNA; however, showed the tumor was disomic for chromosome 21. Conclusion: This case highlights the potential of genomewide NIPS to identify oncogene duplications and asymptomatic maternal malignancy as well as the potential masking of fetal aneuploidy by acquired maternal aneuploidies.

Integration of Genomic Testing in Mainstream Healthcare: Lessons from Oncology

Rosie O’Shea¹, Nicole M. Rankin¹, Maira Kentwell^2,3, Margaret Gleeson⁴, Katherine M Tucker⁵, Heather Hampel⁶, Natalie Taylor^1,7 and Sarah Lewis¹

¹Faculty of Medicine and Health, University of Sydney, NSW, Australia, ²Parkville Familial Cancer Centre, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, VIC, Australia, ³Department of Oncology, Royal Women’s Hospital, Melbourne, VIC, Australia, ⁴Hunter Family Cancer Service, Newcastle, NSW, Australia, ⁵Hereditary Cancer Clinic, Prince of Wales Hospital, Sydney, NSW, Australia, ⁶Division of Human Genetics, Department of Internal Medicine, The Ohio State University Comprehensive Cancer Center, Columbus, OH, U.S., and ⁷Daffodil Centre, The University of Sydney, a joint venture with Cancer Council NSW

Background: ‘Mainstreaming’ is a proposed strategy to integrate genetic testing into oncology services, enhancing the identification of hereditary cancer and overcoming the barriers of genetics referral and clinician awareness. Aims: To identify health system interventions and implementation factors for mainstreaming genetic testing in oncology; to draft a mainstreaming oncology genomics model. Methods: Qualitative interviews and a quantitative survey with health professionals were conducted using the Consolidated Framework for Implementation Research. An exploratory cyclical design of two phases allowed the qualitative implementation data to inform the quantitative survey design. Theory-informed implementation data was collectively mapped to the Genomic Medicine Integrative Research framework. Results: The qualitative phase had 22 participants from 12 health organizations. The quantitative survey had 198 responses: 26% were genetic health professionals, 66% oncology health professionals, and 8% pathologists. The relative advantage and clinical utility of mainstreaming to improve genetic test access and to streamline care were identified. Optimization of current process was recognized for results delivery and follow-up. The barriers identified focused on funding, infrastructure and resources, and the need for process and role delineation for mainstreaming programs. Recommended interventions included improved communication and collaboration between specialties, embedded mainstream genetic counselors, electronic medical record genetic test ordering, with centralized results tracking systems, and development of dedicated resources. Conclusions: The Australian health system implementation factors identified informed the development of a preliminary mainstreaming oncology genomics model. Implementation and evaluation of this model are required to inform future mainstreaming initiatives and can translate to other disease contexts.

Polygenic Score Modifies Risk for Alzheimer’s Disease in Apoe E4 Homozygotes at Phenotypic Extremes

Aamira J. Huq^1,2,†, Brian Fulton-Howard^3,4,†, Moeen Riaz¹, Simon M. Laws^5,6,7, Robert Sebra⁴, Joanne Ryan1Alzheimer’s Disease Genetics Consortium, Alan E. Renton^3,4, Alison M. Goate^3,4, Colin L. Masters⁸, Elsdon Storey¹, Raj C. Shah⁹, Anne Murray¹⁰, John McNeil¹, Ingrid Winship², Paul James² and Paul Lacaze¹

¹Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC, Australia, ²Department of Genomic Medicine, Royal Melbourne Hospital; Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Australia, ³Nash Family Department of Neuroscience and Ronald Loeb Center for Alzheimer’s Disease, Icahn School of Medicine at Mount Sinai, New York, NY, USA, ⁴Departments of Neurology and Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, ⁵Collaborative Genomics and Translation Group, School of Medical and Health Sciences, Edith Cowan University, Perth, WA, Australia, ⁶Centre for Precision Health, Edith Cowan University, Perth, WA, Australia, ⁷School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Perth, Western Australia, Australia, ⁸The Florey Institute, University of Melbourne, Melbourne, VIC, Australia, ⁹Department of Family Medicine and Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago Illinois, USA and ¹⁰Berman Center for Outcomes and Clinical Research, Hennepin Healthcare Research Institute, Hennepin Healthcare, and University of Minnesota, Minneapolis, MN, USA;

Background: Phenotypic diversity in cognition among APOE e4 homozygotes can range from early-onset Alzheimer’s disease (AD) to a lifetime with no symptoms of cognitive impairment. In this study, we investigated the role of polygenic risk between APOE e4/e4 phenotypic extremes as one of the explanations for this diversity. Methods: Using the clumping and thresholding method, based on a published genomewide association study summary statistics, we evaluated a polygenic risk score (PRS) for AD, between cognitively healthy APOE e4 homozygotes aged ≥75 years (n = 213) and early-onset APOE □4 homozygote AD cases aged ≤65 years (n = 223) of European ancestry. Odds ratios (OR) for AD were calculated between the lowest 20% and highest 20% of PRS. Results: The PRS for AD was significantly higher in APOE e4 homozygote AD cases compared with older cognitively healthy APOE e4/e4 controls (OR 8.39; CI 2.0 to 35.2; p = .003). The difference in the same PRS between APOE e3/e3 extremes was not as significant (OR 3.13; CI 0.98 to 9.92; p = .053) despite similar numbers and power. There was no statistical difference in a PRS for educational attainment between the APOE e4/e4 or the APOE e3/e3 extremes, indicating that genetically determined education attainment did not act as a confounding factor. Conclusion: This APOE e4/e4 risk modifying PRS could contribute to improved risk stratification for those with the APOE e4/e4 genotype both in the research and clinical setting. It will also become more relevant as effective therapies for AD are developed.

Clinicians and Policy Reform: Towards the Best Outcomes for Patients With a Rare Genetic Disease

Nicole Millis

Rare Voices Australia

Background: Rare genetic diseases pose a significant collective burden in Australia. Diagnostic delays, together with limited access to treatments and health technologies, translate to a high level of unmet need in this patient group. The only way to see systemic changes for better care in this space is through effective policy reform. But how is this achieved? What is the role of clinicians in this work? Aims: A review of strengths and learnings from recent rare disease advocacy and policy activity, and the important role clinicians play in this. Methods: Case studies of recent national rare disease advocacy and policy activity in key policy areas, including the Life Saving Drugs Program, Newborn Bloodspot Screening, and the Parliamentary Inquiry into the approval processes for new drugs and novel medical technologies. Analysis of the role of clinicians and other stakeholders: Analysis of what is most and least effective in policy reform. Results: Clinicians lend real-world evidence and practical demonstrations of healthcare and clinical research to all stages of policy development. Their unique position enables a coordinated national approach to policy reform for integrated and innovative models of rare disease care. Conclusion: Effective policy reform cannot happen without strong advocacy and active collaboration. Clinicians and other stakeholders must work together at all stages in policy development to ensure nationwide systemic changes for rare genetic disease patients. Ongoing review of advocacy and policy reform activities would help ensure that this work remains strategic and effective in facilitating the best outcomes for patients.

A Model of Care for Clinical Implementation of Pharmacogenomics: Stage 1 of the Enact Study

Alison K. McLean¹, Sophie Devery¹, Renee Smyth¹, Michael Millard², Zhixin Liu⁴, Jonathan Brett³ and Kathy H. C. Wu^1,5,6,7,8

¹Clinical Genomics, St Vincent’s Hospital Sydney, Sydney, NSW, Australia, ²Department of Psychiatry St Vincent’s Hospital Sydney, Sydney, NSW, Australia, ³Clinical Pharmacology, St Vincent’s Hospital Sydney, Sydney, NSW, Australia, ⁴Stats Central, UNSW, Sydney, NSW, Australia, ⁵Disciplines of Medicine and Genomic Medicine, University of Sydney, Sydney, NSW, Australia, ⁶School of Medicine, University of New South Wales, Sydney, NSW, Australia, ⁷School of Medicine, University of Notre Dame Australia, Sydney, NSW, Australia and ⁸Garvan Institute of Medical Research, Sydney, NSW, Australia

Background: Genetic factors contribute to inter-individual variabilities in response to pharmacological agents. Pharmacogenomics (PG) has been shown to significantly enhance clinical outcomes in mental illness treatment. Despite growing evidence, Australia has been slow in adopting PG testing to guide therapy. Aims: To understand the current knowledge and experience of PG testing in clinicians working in mental health. To propose a model of care based on the findings. Methods: A survey was created in RedCap to capture the knowledge and acceptability of PG testing, as well as the experience with PG, perceived barriers and resource needs for PG implementation, among clinicians working in the Psychiatry and Pharmacy departments in a single institution (St Vincent’s Hospital Sydney). Results: While 42.9% of respondents agreed that PG testing would benefit their patients and 73.1% agreed that PG testing can identify medications which are likely to cause side effects, the majority of respondents (31/42, 73.8%) have never had discussions with their patients regarding pharmacogenomic testing. Thirty-three percent of respondents thought that PG testing was too expensive for their patient. Over half, 54.8%, of respondents do not feel confident discussing PG results with their patients and 47.7% of respondents felt that there needed to be more evidence to support PG testing before it is clinically relevant. Conclusion: We have identified barriers to PG uptake and usage (including cost, knowledge, and evidence) as well as potential strategies to overcome such barriers. We propose a model of care to PG implementation with a goal of integrating PG in future routine psychiatric care.

Recurrent De Novo Missense Variants in Gnb2 can Cause Syndromic Intellectual Disability

Natalie B. Tan^1,2,3, Alistair T. Pagnamenta⁴, Matteo P. Ferla⁴, Jonathan Gadian⁵, Brian HY Chung⁶, Marcus CY Chan⁶, Jasmine LF Fung⁶, Edwin Cook⁷, Stephen Guter⁷, Felix Boschann⁸, Andre Heinen⁹, Jens Schallner¹⁰, Cyril Mignot¹¹, Boris Keren¹¹, Sandra Whalen¹², Catherine Sarret¹³, Dana Mittag¹⁴, Laurie Demmer¹⁴, Rachel Stapleton¹⁵, Ken Saida¹⁶, Naomichi Matsumoto¹⁶, Noriko Miyake¹⁶, Ruth Sheffer¹⁷, Hagar Mor-Shaked¹⁷, Christopher P. Barnett¹⁸, Alicia B. Byrne^19,20, Hamish S. Scott¹⁹, Alison Kraus^21,22, Gerarda Cappuccio^23,24, Nicola Brunetti-Pierri^23,24, Raffaele Iorio²³, Fabiola Di Dato²³, Lynn S. Pais²⁵, Alison Yeung^1,2,3, Tiong Y. Tan^1,2,3, Jenny C. Taylor⁴, John Christodoulou^1,2,3 and Susan M. White^1,2,3

¹Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia, ³Victorian Clinical Genetics Services, Melbourne VIC, Australia, ⁴NIHR Oxford BRC, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, ⁵Department of Paediatric Neurology, John Radcliffe Hospital, Oxford, United Kingdom, ⁶Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, ⁷Institute for Juvenile Research, Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois, United States, ⁸Institute of Medical Genetics and Human Genetics, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, ⁹Carl Gustav Carus Faculty of Medicine, Children’s Hospital, Technische Universität Dresden, Germany, ¹⁰Carl Gustav Carus Faculty of Medicine, Children’s Hospital, Department of Neuropediatrics, Technische Universität Dresden, Germany, ¹¹Département de Génétique, Hôpital Pitié-Salpêtrière, APHP.Sorbonne Université, Paris France, ¹²UF de Génétique Clinique, Centre de Référence Maladies Rares Anomalies du développement et syndromes malformatifs, APHP.Sorbonne Université, Hôpital Armand Trousseau, Paris, France, ¹³Service de génétique médicale, Hôpital Estaing, Centre hospitalo-universitaire de Clermont-Ferrand, Clermont-Ferrand, France, ¹⁴Division of Genetics, Levine Children’s Hospital, Carolinas Medical Center, Atrium Health, Charlotte, North Carolina, United States, ¹⁵Genetic Health Service NZ, Christchurch Hospital, Christchurch, New Zealand, ¹⁶Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan, ¹⁷Department of Human Genetics, Hadassah University Hospital, Jerusalem, Israel, ¹⁸South Australian Clinical Genetics Service, Women’s and Children’s Hospital, Adelaide, SA, South Australia, Australia, ¹⁹Department of Genetics and Molecular Pathology, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ²⁰UniSA Clinical and Health Sciences, University of South Australia, Adelaide, SA, Australia, ²¹Yorkshire Regional Genetics Service, Chapel Allerton Hospital, Leeds, United Kingdom, 22 Castle Hill Hospital, Cottingham, Hull 01482 622470, United Kingdom, ²³Department of Translational Medicine, Section of Pediatrics, Federico II University, Naples, Italy, ²⁴Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy and ²⁵Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, MA, USA

Background: GTP-binding proteins (G-proteins) mediate signalling pathways involved in diverse cellular functions and comprise Gα and Gβγ units. Human diseases have been reported for all five Gβ proteins. A de novo missense variant in GNB2 was recently reported in one individual with developmental delay/intellectual disability (DD/ID) and dysmorphism. We aimed to confirm GNB2 as a neurodevelopmental disease gene, and elucidate the GNB2-associated neurodevelopmental phenotype in a patient cohort. Methods: We discovered a GNB2 variant in an individual with syndromic intellectual disability via exome sequencing and sought individuals with GNB2 variants via international data-sharing initiatives. In silico modelling of the variants was assessed, along with multiple lines of evidence in keeping with ACMG guidelines for interpretation of sequence variants. Results: We identified 12 unrelated individuals with five de novo missense variants in GNB2, four of which are recurrent: p.(Ala73Thr), p.(Gly77Arg), p.(Lys89Glu), and p.(Lys89Thr) (K89T). All individuals have DD/ID with variable dysmorphism and extra-neurologic features. The variants are located at the universally conserved shared interface with the Gα subunit, which modelling suggests weaken this interaction. Conclusion: Missense variants in GNB2 cause a congenital neurodevelopmental disorder with variable syndromic features, broadening the spectrum of multisystem phenotypes associated with variants in genes encoding G-proteins.

Real World Outcomes of Clinical Exome Sequencing in an Adult Genetics Department

Maie Walsh, Kirsty West, Jessica A. Taylor, Adelaide Hopkins, Adrienne Sexton, Abiramy Ragunathan, Kunal Verma, Michael Fahey, Michael Christie, Bryony Thompson, Ingrid Winship, Alison Trainer and Paul A. James

Genomic Medicine, Royal Melbourne Hospital, Melbourne, VIC, Australia

Background; In addition to its technical advantages, falling testing costs and Victorian State Government subsidies have enabled whole exome sequencing (WES) to become a standard diagnostic test. There is extensive literature examining the yield and utility of WES in pediatric patients but no publications looking at WES in adult patients across all specialties written from a clinical perspective. Aim; To describe the clinical indications and quantify the diagnostic yield of WES in adults patients. A secondary aim was to examine the short-term clinical utility of WES in adults. Methods; Data including demographics, clinical indication, testing laboratory, cost, results, management changes, and cascade testing was collected for 250 consecutive patients who underwent WES through an adult genetics department between 2016 and 2021. Data were mainly analyzed using descriptive statistics. Testing where traditional gene panels were in standard usage, such as in heritable cancers, were excluded. Results; The average age at testing was 49 years (range 17–80). A diagnosis was identified in 27% patients and a likely diagnosis in a further 7.6%. Half of the patients with a diagnosis had immediate management changes. A third of patients with a diagnosis had at least one family member undergo cascade testing through our department. The results had reproductive implications for 16% of patients and 12% of family members. We also examined the clinical and age-related predictors of diagnostic outcomes. Conclusion: WES has a robust diagnostic rate and a clear clinical utility in adult patients across a range of phenotypes.

The Role of Whole Exome Sequencing in Interstitial Lung Disease of Childhoood

Suzanna Lindsey-Temple^1,2, Gladys Ho^3,4, Bruce Bennetts^3,4, Thet Gayagay³, Kirsten Boggs^5,6,7, Michael Buckley⁸, Tony Roscioli^7,8,9, Ying Zhu⁸, David Mowat^2,7, John Christodoulou^5,10, Andre Schultz¹¹, Nada Mirkovic⁵, Sebastian Lunke^5,10,12, Zornitza Stark^5,10,12 and Adam Jaffe^2,13

¹Department of Clinical Genetics, Liverpool Hospital, Sydney, NSW, Australia, ²School of Women’s and Children’s Health, Faculty of Medicine and Health, UNSW, Sydney, NSW, Australia, ³Sydney Genome Diagnostics; Western Sydney Genetics Program, The Children’s Hospital at Westmead, Sydney, NSW, Australia, ⁴Disciplines of Child & Adolescent Health and Genomic Medicine, University of Sydney, Sydney, NSW, Australia, ⁵Australian Genomics Health Alliance, Melbourne, VIC, Australia, ⁶Department of Clinical Genetics, Children’s Hospital Westmead, Sydney, NSW, Australia;, ⁷Centre for Clinical Genetics, Sydney Children’s Hospital Randwick, Sydney, NSW, Australia, ⁸Randwick Genomics Laboratory, NSW Health Pathology, Prince of Wales Hospital, Sydney, NSW, Australia, ⁹Neuroscience Research Australia (NeuRA), Sydney, NSW, Australia, ¹⁰University of Melbourne, Melbourne, VIC, Australia, ¹¹Perth Children’s Hospital, Perth, WA, Australia, ¹²Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Melbourne, VIC, Australiia and ¹³Sydney Children’s Hospital, Sydney, NSW, Australia

Background: Childhood interstitial lung disease (chILD) is a heterogeneous group of disorders often requiring life-long, complex care. Only a small proportion of cases have undergone genomic testing looking for a detectable monogenic cause. Most genomic testing has been based on a gene panel approach directed by the phenotypic presentation, with a reported diagnostic success rate of ∼12%. No data currently exist using whole exome sequencing (WES) as the standard clinical pipeline. Aim: To assess the diagnostic utility of using WES in chILD with a hypothesis that WES would lead to an increased diagnostic rate. Methods: The chILD research, Australia and New Zealand (chILDRANZ) flagship, part of Australian Genomics, prospectively enrolled children between 2016 and 2020 from Australian hospitals, if the treating physician suspected chILD using agreed clinical criteria. Concurrently some critically ill infants were prospectively enrolled through the Acute Care flagship to access rapid testing. Trio WES was performed with an initial gene panel approach followed by trio exome analysis. Results: A total of 42 patients were recruited from multiple states and territories. Four patients were found to have clinically significant pathogenic variants, and two patients had variants of uncertain significance suspected to be the cause of their clinical phenotype. Conclusion: This was the first study to perform WES to look for monogenic causes of ILD in childhood. The diagnostic yield from WES in this study was 9.5% which was lower than expected and highlights the complex underlying etiology and likely combination of multiple common variants in conjunction with environmental factors.

Phenotype and Deep Sequencing in an Australian Tuberous Sclerosis Complex ‘No Mutations Identified’ Cohort

Clara WT Chung^1,2, John A Lawson^1,2, Sean E Kennedy^1,2, Vanessa Sarkozy^1,2, Edwin Kirk^1,2,3 and David Mowat^1,2

¹Sydney Children’s Hospital, Sydney, NSW, Australia, ²School of Women’s and Children’s Health, UNSW Medicine, UNSW Sydney, Sydney, NSW Australia and ³NSW Health Pathology Randwick Genomics Laboratory, Prince of Wales Hospital, Sydney, NSW, Australia

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant condition caused by pathogenic variants in the TSC1 or TSC2 gene with a prevalence of 8.8 per 100 000. TSC is associated with variable neurodevelopmental outcomes, seizures, and benign tumors in various organs. 10–15% of TSC patients have no pathogenic variants identified – the ‘no mutations identified’ (NMI) cohort. Previously, they have been reported to be phenotypically milder and likely have mosaic or intronic variants. Aim: To determine the phenotype of our NMI cohort, if testing multiple samples helps with diagnosis and to establish a testing strategy that may be viable in a diagnostic laboratory. Methods: We recruited 19 NMI patients from the TSC management clinic at Sydney Children’s Hospital. We compared their phenotype with those with heterozygous variants. We designed a custom target capture panel consisting of the whole genomic region of TSC1 and TSC2, and performed deep sequencing. A minimum of 2 samples was tested for each participant, including affected skin tissue (hypomelanotic macule, angiofibroma) where possible. Results: Our NMI cohort is not necessarily milder in phenotype. 17/19 (89%) of the patients had a likely causative variant found on deep sequencing, including 3 previously missed heterozygous variants, 10 likely germline pathogenic mosaic variants, and 5 suspicious mosaic variants. Conclusion: The majority of TSC NMI individuals are mosaic, but may not have a milder clinical phenotype. Testing multiple samples allows for the detection of germline mosaic variants on massively parallel sequencing without excessive cost and the need for specialized techniques.

Pathogenic Variants in Nucleoporin Tpr (Translocated Promoter Region, Nuclear Basket Protein) Cause a Neurological Phenotype in Humans

Nicole J Van Bergen^1,2, Katrina Bell^3,4, Kirsty Carey⁵, Russell Gear⁴, Sean Massey¹, Edward K Murrell¹, Lyndon Gallacher^2,4, Kate Pope⁴, Andrew Kornberg^2,6,7, Lynn Pais⁸, Marzena Walkiewicz⁹, Cas Simons^4,9, Vi Wickramasinghe^5,10, Susan M White^2,4 and John Christodoulou^1,2,11

¹Brain and Mitochondrial Research Group, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia, ²Department of Paediatrics, University of Melbourne, Melbourne, Australia, ³Bioinformatics, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia, ⁴Victorian Clinical Genetics Services, Royal Children’s Hospital, VIC, Australia, ⁵RNA Biology and Cancer Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia, ⁶Neurology Department, Royal Children’s Hospital, Melbourne, VIC, Australia, ⁷Neurosciences Research, Murdoch Children’s Research Institute, VIC, Australia, ⁸Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, MA, ⁹Translational Genomics Research Group, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia, ¹⁰Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia and ¹¹Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW, Australia

Background: The nuclear pore complex (NPC) is a nuclear envelope multi-protein complex that regulates the trafficking of macromolecules between the nucleus and cytoplasm. Mutations in other components of the NPC have been shown to cause human disease, including neurological disorders, intellectual disability, and microcephaly. TPR is a critical scaffolding element of the nuclear basket, facing the interior of the NPC. Aim: Here, we present two siblings with biallelic variants in the TPR gene who presented with microcephaly, ataxia, and severe intellectual disability. We undertook functional validation to confirm pathogenicity of the novel variants. Methods: Whole genome sequencing and RNAseq were used to identify potential pathogenic variants, and to assess splicing of TPR. Patient fibroblasts were used to determine TPR protein levels and determine TPR-containing nuclear pore density, global nuclear pore density, and RNA distribution. Results: The variants result in a premature truncation codon causing nonsense-mediated decay, and a splice variant leading to a 12-amino acid truncation in a critical alpha-helical domain that provides structural rigidity to the nuclear basket. Functional analyses in patient fibroblasts demonstrate significantly reduced TPR protein levels, and decreased TPR-containing NPC density. A compensatory increase in total NPC levels was observed, supporting previous TPR knockdown studies. RNA levels were significantly reduced in the nucleus of patient fibroblasts. Conclusion: The discovery of mutations that partly disable TPR function provide valuable insights into the role of this essential protein in human disease, and our findings suggest that TPR variants are the cause of the patients’ neurological disorder.

Novel Gene Discovery for Individuals with Rett Syndrome and Related Neurodevelopmental Disorders

Amber Condell^1,2, Simranpreet Kaur^1,2, Sean Massey¹, Nicole Van Bergen^1,2, Bruria Ben-Zeev^3,4,5, Martin Delatycki^1,6 and John Christodoulou^1,2,6

¹Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia, ³Paediatric Neurology Institute, The Edmond and Lily Safra Children’s Hospital, Israel, ⁴Chaim Sheba Medical Center, Tel HaShomer, Israel, ⁵Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel and ⁶Victorian Clinical Genetics Services, Melbourne, VIC, Australia

Background: Rett Syndrome (RTT) is a neurodevelopmental disorder (NDD) most often caused by mutations in the X-linked epigenetic regulator methyl-CpG-binding protein 2 (MECP2). However, RTT is genetically heterogenous, with approximately 2% of classic and 13% of atypical individuals without a genetic diagnosis. Aim: We analyzed sequencing data from MECP2 mutation-negative patients, curated variants, and performed functional validation to reveal novel genetic bases for RTT and other associated NDDs. Methods: Whole genome (n = 2), exome (n = 3), and custom targeted sequencing (n = 1) was used to screen MECP2-negative RTT individuals for pathogenic variants, followed by in silico pathogenicity prediction, population frequency evaluation, evaluation of protein expression, and pre-established disease associations. Predicted pathogenic variants were functionally validated in vitro using cultured fibroblasts, Western blotting to determine extracted protein levels, and immunohistochemistry. Results: We identified potentially novel RTT-associated genes in these patients; of notable interest is an individual with a predicted pathogenic heterozygous NUP188 missense [c.3922C>T, p.(Arg1308Cys)] on one allele, and a large (∼287kb) chromosomal deletion spanning NUP188 and SET on the other allele. NUP188 encodes a scaffolding component of the nucleopore for macromolecule exchange, while SET is involved in transcriptional silencing, chromatin remodelling, and inhibition of histone acetylation, and has previously been associated with intellectual disability, speech and developmental delay. Our preliminary studies found decreased NUP188 expression and enlarged nuclei in cultured fibroblasts, consistent with NUP188 dysfunction. Conclusion: These newly identified genes expand current knowledge of the RTT genetic spectrum, flagging potentially pathogenic variants to assist in diagnosing elusive MECP2-negative RTT patients.

Speech and Language Deficits are Central to SETBP1 Haploinsufficiency Disorder

Angela Morgan^1,2,3,4,5, Ruth Braden^1,2,5, Maggie M. K. Wong⁶, Estelle Colin⁷, David Amor^1,3,4, Frederique Liégeois⁸, Siddharth Srivastava⁹, Adam Vogel¹⁰, Varoona Bizaoui¹¹, Kara Ranguin¹¹, Simon E. Fisher^6,12 and Bregje W. van Bon¹³

¹Speech & Language, Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia, ³Royal Children’s Hospital, Melbourne, VIC, Australia, ⁴Victorian Clinical Genetics Service, Melbourne, VIC, Australia, ⁵Department of Audiology and Speech Pathology, University of Melbourne, Melbourne, VIC, Australia, ⁶Language and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands, ⁷Service de Génétique Médicale, Centre Hospitalier Universitaire d’Angers, Angers, France, ⁸UCL Great Ormond Street Institute of Child Health, London, UK, ⁹Boston Children’s, Harvard Medical Centre, Boston, MA, USA, ¹⁰Centre for Neuroscience of Speech, Department of Audiology and Speech Pathology, University of Melbourne, Melbourne, VIC, Australia, ¹¹Service de Génétique, Centre Hospitalier Universitaire Caen Normandie, Caen, France, ¹²Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands and ¹³Radboud University Medical centre, Nijmegen, The Netherlands

Background: Expressive communication impairment is associated with haploinsufficiency of SETBP1, as reported in small case series. Heterozygous pathogenic loss-of-function (LoF) variants in SETBP1 have also been identified in independent cohorts ascertained for childhood apraxia of speech (CAS), warranting further investigation of the roles of this gene in speech development. Aim: To prospectively examine the speech, language, and literacy phenotype of a cohort of children with pathogenic LoF variants in SETBP1, using standardized tools. Methods: Thirty-one participants (12 males, aged 0; 8–23; 2 years) were assessed for speech, language, and literacy abilities. Broader development was also examined with standardized motor, social, and daily life skills assessments. Results: Gross and fine motor deficits (94%) and intellectual impairments (68%) were common. Childhood apraxia of speech (CAS) was the most common speech disorder (80%), followed by phonological disorder. Language was typically low, to moderately impaired, with commensurate expression and comprehension ability. Children were sociable with a strong desire to communicate. Minimally verbal children (32%) augmented speech with sign language, gestures, or digital devices. Conclusion: Here we expand the linguistic phenotype associated with SETBP1 LoF syndrome (SETBP1 haploinsufficiency disorder), revealing a relatively homogeneous speech presentation implicating both motor (CAS, dysarthria) and language (phonological errors) systems. Overall, relative to general development, spoken language and literacy were poorer than social, daily living, motor and adaptive behavior skills. Findings show that poor communication is a central feature of SETBP1 haploinsufficiency disorder, confirming this gene as a strong candidate for speech and language disorders.

Speech and Language Phenotyping in KAT6A Variants

Miya St John^1,2, David Amor^1,3 and Angela Morgan^1,2

¹Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²Department of Audiology and Speech Pathology, University of Melbourne, VIC, Australia and ³Department of Paediatrics, University of Melbourne, VIC, Australia,

Background: Pathogenic KAT6A variants have recently been identified as a cause of syndromic neurodevelopmental disability. ‘Speech delay’ is commonly reported, yet no study has examined specific speech and language features in KAT6A syndrome. Aim: To expand the neurodevelopmental phenotype of KAT6A syndrome, with a focus on speech and language characteristics. Methods: Health, medical, and communication subdomains were assessed using descriptive and standardized measures, via online survey, and telehealth assessment. Results: 48 individuals with pathogenic KAT6A variants (1;8-31;10 years) were recruited. Truncating variants were most common (85%), with some missense and splicing variants. Individuals had intellectual disability (40% severe, 33% moderate, 19% mild) and 31% had autism. Other comorbidities included vision issues (76%), gastrointestinal problems (71%), sleep disturbance (66%), heart defects (46%), and microcephaly (52%). All displayed some form of communication impairment, with 75% relying primarily on nonverbal communication strategies. Verbal participants had a range of speech disorders, including speech apraxia (55%). Overall, most were severely impaired across adaptive functioning domains (daily living (91%), socialization (87%), communication (87%)) with late truncating variants consistently associated with greater difficulty. Conclusion: Severe communication difficulties are core in KAT6A syndrome, alongside a myriad of medical/neurodevelopmental comorbidities. Most are minimally verbal, and it can be difficult to delineate whether this relates to severe underlying motor deficits or severe linguistic impairment in the context of ID. Regardless, many rely on alternative means of communication beyond childhood (e.g. eye gaze, sign language). Alternative and augmentative options should be fostered as early as possible to allow for the best communication outcomes.

Somatic and Generational TTTTA/TTTCA Repeat Instability in Familial Adult Onset Myoclonic Epilepsy 2

Mark A. Corbett¹, Thessa Kroes¹, Brigid Regan², Saskia Molina¹, Jill Lai³, Alex Hastie³, Mark Bennett^4,5, Amy Schneider², Joshua Reid², Michael S. Hildebrand^2,6 FAME consortium, Melanie Bahlo^4,5, Lynette Sadleir⁷, Samuel F. Berkovic², Ingrid E. Scheffer^2,6,8,9 and Jozef Gecz^1,10

¹Adelaide Medical School and Robinson Research Institute, University of Adelaide, Adelaide, 5005, SA, Australia, ²Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, VIC, Australia, ³Bionano Genomics, San Diego, CA, USA, ⁴Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourn, VIC, Australia, ⁵Department of Medical Biology, the University of Melbourne, Melbourne, VIC, Australia, ⁶Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ⁷Department of Paediatrics and Child Health, University of Otago, Wellington; Wellington, New Zealand, ⁸Royal Children’s Hospital, Murdoch Children’s Research Institute and Florey Institute, Melbourne, VIC, Australia, ⁹Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, Melbourne, VIC, Australia and ¹⁰South Australian Health and Medical Research Institute, Adelaide, SA, Australia

Familial Adult Myoclonic Epilepsy (FAME) is characterized by cortical tremor, myoclonus, and myoclonic and/or generalized tonic-clonic seizures. There are six autosomal dominant forms of FAME each caused by expansion of an intronic TTTTA repeat, as well as insertion and expansion of a TTTCA repeat in one of six genes with unrelated molecular functions. FAME2 maps to chromosome 2 and is caused by repeat expansion in intron one of STARD7. We previously showed anticipation for earlier FAME2 disease onset over three generations of a large Australian/NZ family; however, it was not determined if this phenomenon was correlated with the length of the repeat expansion. Here, we measured the length of FAME2 repeats in blood DNA from 94 affected individuals over 10 FAME2 families using long-range PCR, agarose gels, and Oxford nanopore long-read sequencing. Additionally, Bionano optical mapping was performed for four of these individuals. The estimated length of repeats were consistent between PCR-based and PCR-free methods. The average length of repeats increased in successive generations in multiple FAME2 families and correlated with age of onset of myoclonus (r = -.329, p = .05). Over successive passages of patient-derived lymphoblastoid and fibroblast cell lines, we further showed dynamic changes in repeat lengths; potentially modelling somatic instability. Finally, we detected FAME2 repeats using a novel technique to selectively nanopore sequence regions corresponding only to known genomic loci implicated in Mendelian repeat expansion disorders. This cutting edge approach has the potential for future application in amplification-free detection and sequencing of any known repeat expansion.

Collaborative International Data Aggregation and Analysis for Rare Hematological Cancer Predisposition Syndromes Caused By Germline RUNX1, DDX41 and GATA2 Mutations

Claire C. Homan¹, Parvathy Venugopal¹, Michael W. Drazer², Kai Yu³, Jinghua Feng¹, Sarah L. King-Smith¹, David M. Lawrence¹, Peer Arts¹, NurHezrin Shahrin¹, James Andrews¹, Simone Feurstein², Lesley Rawlings¹, Mark Armstrong¹, Thuong Ha¹, Julia Dobbins¹, Csaba Bödör⁴, Alan Cantor⁵, Mario Cazzola⁶, Erin Degelman⁷, Courtney D. DiNardo⁸, Nicolas Duployez⁹, Remi Favier¹⁰, Stefan Fröhling¹¹, Jude Fitzgibbon¹², Jeffery M. Klco¹³, Alwin Krämer¹¹, Mineo Kurokawa¹⁴, Joanne Lee¹⁵, Luca Malcovati⁶, Neil V. Morgan¹⁶, Georges Natsoulis¹⁷, Carolyn Owen⁷, Keyur P. Patel⁸, Claude Preudhomme⁹, Hana Raslova¹⁸, Hugh Rienhoff¹⁷, Tim Ripperger¹⁹, Rachael Schulte²⁰, Kiran Tawana²¹, Elvira Velloso²², Benedict Yan¹⁵, Devendra Hiwase²³, Andreas W. Schreiber¹, Paul Liu³, Lucy A. Godley², Christopher N. Hahn¹, Hamish S. Scott¹ and Anna L. Brown¹

¹Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, Australia, ²The University of Chicago, Chicago, USA, ³National Institutes of Health, Bethesda, USA, ⁴Semmelweis University, Budapest, Hungary, ⁵Harvard Medical School, Boston, USA, ⁶University of Pavia, Pavia, Italy, ⁷Foothills Medical Centre, Calgary, Canada, ⁸University of Texas MD Anderson Cancer Center, Houston, USA, ⁹Universitaire de Lille, Lille, France, ¹⁰Armand Trousseau children’s Hospital, Paris, France, ¹¹University of Heidelberg, Heidelberg, Germany, ¹²Queen Mary University of London, London, UK, ¹³St Jude Children’s Research Hospital, Tennessee, USA, ¹⁴The University of Tokyo, Tokyo, Japan, ¹⁵National University Cancer Institute, National University Health System, Singapore, ¹⁶University of Birmingham, Birmingham, UK, ¹⁷Imago Biosciences, Inc., San Francisco, USA, ¹⁸Université Paris Sud, Villejuif, France, ¹⁹Hannover Medical School, Hannover, Germany, ²⁰Vanderbilt University Medical Center, Nashville, USA, ²¹Addenbrooke’s Hospital. Cambridge, UK, ²²University of Sao Paulo Medical School, Sao Paulo, Brazil and ²³SAHMRI, Adelaide, SA, Australia

Background: Hematological cancer predisposition syndromes (HCPS), caused by germline variants in known genes including RUNX1, DDX41, and GATA2, have been studied for decades; however, challenges remain to improve patient outcomes¹. Disease heterogeneity, small patient-cohorts, lack of identification/appropriate classification of variants, remains a significant challenge to our understanding of these syndromes. To help overcome these challenges, an international effort is required to collate and standardize disease-specific clinical and genomics data, accumulating enough data to make evidence-based clinical decisions. Method/Results: Collating phenotypic and genetic information extracted from peer-reviewed literature, and international colleagues, we created a standardized clinical resource for germline RUNX1 (gRUNX1) HCPS (RUNX1database:https://runx1db.runx1-fpd.org/snpdb/index). We created a registry of gRUNX1 variants expertly curated and classified according to ACMG/AMP gene-specific criteria, including 258 gRUNX1 probands/families and 164 unique gRUNX1 variants. Utilizing the collective wealth of NGS data generated from international laboratories, we created the largest gRUNX1 genomics cohort: 179 gRUNX1 NGS datasets, including both pre-leukemic(65) and malignancy(62) samples. Interrogating this dataset as a single cohort, we have identified novel somatic mutational patterns in pre-leukemic samples before progression to AML, including recurrent mutations in several clonal hematopoiesis genes including BCOR. We are currently expanding this initiative to both gGATA2 and gDDX41 HCPS. Conclusion: Aggregation of families, individuals, and disease stages into a centralized database where all data undergo rigorous quality control will aid in the exploration and discovery of the molecular progression of these syndromes. Providing a resource to inform evidence-based clinical decisions on molecular monitoring and a framework for the development of therapeutic interventions to prevent leukemia development.

Splice-Switching Oligonucleotide-Mediated Correction of a Deep Intronic Splice-Variant in Timmdc¹ in Cells of Patients with Severe Early Onset Neurodegenerative Disorder

Raman Kumar¹, Mark A. Corbett¹, Nicholas J. C. Smith², Zoya Kikhtyak³, Atsushi Morimoto¹, Sangmoon Lee⁴, Joseph G. Gleeson⁴, Eric A Haan⁵ and Jozef Gecz¹

¹Adelaide Medical School and the Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia, ²Department of Neurology, Women’s and Children’s Health Network, Adelaide, SA, Australia, ³Dame Roma Mitchell Cancer Research Laboratories, Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia, ⁴Neurogenetics Laboratory, Institute for Genomic Medicine and Departments of Neurosciences and Paediatrics, University of California, San Diego, CA, USA and ⁵Adult Genetics Unit, Royal Adelaide Hospital and Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia

Background: Combined analysis of genome sequencing (GS) and RNAseq data from patient-derived cells or disease-relevant tissues have been particularly effective in identifying disease variants with splicing or gene-regulatory effects. Splicing variants represent up to 13% of the known pathogenic variants and offer great opportunity for the development of treatments. Aim: To use Splice-Switching Oligonucleotides (SSOs) to correct perturbed mitochondrial function due to a deep-intronic, homozygous TIMMDC1 c.596+2146A>G, cryptic splice-site activating variant (allele frequency 1/10,000), in patients with severe early-onset, progressive, neurodegenerative disorder. Methods: We used GS and RNAseq (fibroblasts) analyses of two children and their consanguineous parents to identify the TIMMDC1 variant. We confirmed aberrant TIMMDC1 mRNA splicing and undetectable TIMMDC1 protein in two patient fibroblasts. We designed two different SSOs to target the TIMMDC1 deep intronic variant in patients’ cells. Results: TIMMDC1 encodes the Translocase of Inner Mitochondrial Membrane Domain-Containing protein 1 subunit of complex I of the electron transport chain responsible for ATP production. TIMMDC1 variant causes infantile-onset neurodegenerative disorder manifesting with a predominant sensorimotor axonal neuropathy, optic atrophy, and cognitive deficit. We showed that TIMMDC1 enhances aberrant splicing, leading to an insertion of a poison exon that introduces a premature stop codon (p.Gly199_Thr200ins5*) in the TIMMDC1 mRNA rendering it susceptible to NMD. This leads to undetectable TIMMDC1 protein and compromized mitochondrial complex I function in the patient fibroblasts. Using SSOs, we completely restored normal TIMMDC1 mRNA, protein, and mitochondrial function. Conclusion: We provide proof of principle correction of the molecular defect underlying this severe neurological disorder.

MBS Testing For Exomes: Review of the First 12 Months

Kathie Friend¹, Abhi Kulkarni¹, Evelyn Douglas¹, Louisa Sanchez¹, Song Gao¹, Julien Soubrier^1,2, Rosalie Kenyon³, Ming Lin³, Rob King³, Jinghua Feng^3,4, Andreas W Schreiber^2,3,4, David M. Lawrence³, Sarah King-Smith^1,4, Suzanne Sallevelt⁵, Jan Liebelt⁵, Lesley McGregor⁵, Christopher Barnett⁵, Sui Yu¹, Tristan Hardy^1,6, Janice Fletcher^1,7, Hamish S. Scott^1,4 and Karin S. Kassahn^1,4

¹Genetics and Molecular Pathology, Adelaide, SA, Australia, ²School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia, ³ACRF Cancer Genomics Facility, SA Pathology, Adelaide, SA, Australia, ⁴Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ⁵Paediatric and Reproductive Genetics Unit, Women’s and Children’s Hospital, Adelaide, SA, Australia, ⁶Repromed, Dulwich, SA, Australia and ⁷NSW Health Pathology, Sydney NSW, Australia

Background: SA Pathology has provided NATA-accredited exome testing since 2015 but uptake was limited due to cost of testing. In 2020, exome testing was added to the Medical Benefits Scheme (MBS). Aim: We review the outcomes of the first 12 months of MBS-funded exome testing. Methods: An audit of the number of referrals, diagnostic outcomes, and implementation challenges were performed. Results: Despite clinical and laboratory disruptions due to COVID-19, over 230 cases were referred (77% trios, 23% singletons). Trio requests were reported more rapidly and had fewer VUS (16% vs 30%) compared to singletons. Overall diagnostic rate was slightly higher in trios (32%) than singletons (27%). As expected, the majority of trio diagnoses were due to de novo variants (67%), with an additional 9% X-linked, 7% compound heterozygous, and 4% homozygous variants detected. Surprisingly, 11% of diagnoses involved inherited dominant disorders that were either mosaic or had variable expressivity in a parent. Detection of carrier status for unrelated conditions and VUS presented reporting challenges. Workforce training and managing the additional workload is proving to be a significant and ongoing challenge. Urgent requests have also increased in frequency. One such example was a pregnant couple with a 16-month-old child in which we established a diagnosis of Filippi syndrome and could confirm in a clinically relevant timeframe that the fetus also carried the variants. Conclusion: Our experience has highlighted a number of challenges for laboratories offering MBS-funded exome testing but has also corroborated the clinical utility of the testing.

GeneMatching Candidates From the Genomic Autopsy Study to Elevate Variant Classification for Prenatal Clinical Use

Thuong T. Ha^1,2, Peer Arts¹, Alicia B. Bryne^1,3, Jinghua Feng^1,2, David Lawrence^1,2, Milena Babic¹, Lynn Pais3Genomic Autopsy Study Research Network, Sarah L King Smith¹, Matilda R. Jackson^1,4, Andreas W Schreiber^2,5, Anne O’Donnell-Luria³, Christopher P. Barnett^6,7,# and Hamish S. Scott^1,2,4,6,#

¹Department of Genetics and Molecular Pathology, Centre for Cancer Biology, an alliance between SA Pathology and the University of South Australia, Adelaide Australia, ²ACRF Genomics Facility, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ³Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA, ⁴Australian Genomics, Melbourne, VIC, Australia, ⁵School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia, ⁶School of Medicine, University of Adelaide, Adelaide, SA, Australia and ⁷Paediatric and Reproductive Genetics Unit, South Australian Clinical Genetics Service, Women’s and Children’s Hospital, Adelaide, SA, Australia

Background: Trio analysis from the Genomic Autopsy Study revealed that a molecular diagnosis can be ascertained in half of clinically unresolved cases of early miscarriage or perinatal death (n = 89/170). However, only half (n = 41/89) of these putative disease genes can be used prenatally for assessing recurrence risk, despite compelling evidence from available animal models, gene constraints, and expression data. The ACMG guidelines are heavily weighted towards existing genotype to phenotype delineations in the post-natal setting, which can confound the interpretation of prenatal cases, with little to no hindsight on severe or lethal in utero presentations. Aim: Assess the proportion of cases with variants and/or genes of uncertain significance, which can be reclassified based on identifying additional kindreds from genematching. Methods: Between 2018 and 2021, we submitted 35 genes to the genotype matching platform, MatchMaker Exchange, to seek additional, unrelated kindreds with similar clinical phenotype and/or initiate collaborations with expert curators of genes or disease subgroups for each case². Results: We received matches for 33/34 gene submitted, with an average 7 participants responding per gene and contact initiated between 1 and 25 months after submission. The genematching identified additional kindreds with clinical overlap (10/33) and/or initiated functional validation (5/33) and diagnosis for publications (3/33). Conclusion: Phenotype matching is critical for clinical interpretation, which can be accelerated through the use of genotype sharing platforms like MatchMaker exchange. Additional kindreds can be identified in a fifth of cases with variants or genes of uncertain significance.

Routine RNA follow-up of Genomic Analysis to Prove Causality of Variants of Unknown Significance

Peer Arts¹, Thuong T. Ha^1,2, Andrew Dubowsky³, Parvathy Venugopal¹, Leila Eshraghi¹, Alicia B. Byrne^1,4, Oliver van Wageningen³, Duncan Holds³, Jinghua Feng^1,2, Paul Wang^1,2, David Lawrence^1,2, Nick Warnock², Tian Zhiyu², Song Gao³, John Toubia², Roula Ghaoui⁵, Sunita de Sousa^1,6,7, Milena Babic¹, Sarah L. King Smith¹, Matilda R. Jackson^1,8, Andreas W. Schreiber^2,9, Karin S. Kassahn³ and Hamish S. Scott^1,2,4,7,8

¹Department of Genetics and Molecular Pathology, Centre for Cancer Biology, an alliance between SA Pathology and the University of South Australia, Adelaide Australia, ²ACRF Genomics Facility, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ³Department of Genetics and Molecular Pathology, SA Pathology, Adelaide, SA, Australia, ⁴Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA, ⁵Department of Neurology, Royal Adelaide Hospital, Adelaide, South Australia, Australia, ⁶Endocrine and Metabolic Unit, Royal Adelaide Hospital, Adelaide, SA, Australia, ⁷Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia, ⁸Australian Genomics, Melbourne, VIC, Australia and ⁹School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia

Background: Mutations altering mRNA splicing (splice variants) contribute up to 9% of pathogenic variants underlying genetic diseases, which is likely an underrepresentation and biased towards canonical splice sites. Identifying splice variants among variants of unknown significance (VUS), outside of canonical splice sites is aided by bioinformatic predictions. However, clinical interpretation of these splice variants remains challenging without follow-up RNA experiments. Aim: Optimize RNA follow-up experiments, across various research and diagnostic projects, to validate and determine the pathogenicity of predicted splice variants. Methods: Patients from all ages with candidate variants predicted to cause aberrant splicing (multiple bioinformatics algorithms) were selected for our validation study. Based on tissue and RNA availability, and gene expression, we performed polyA RNAseq, RNAseq capture, or (M13 primer tagged) gene-specific RT-PCR coupled with Sanger (clinical testing) or long-read (Pacbio and Nanopore) sequencing. Results: Collectively, clinical evaluation of 99 different splice variants by RT-PCR and Sanger showed altered splicing for 53/99 variants, of which 38/53 were at canonical splice sites (+/-10bp). More recently, mRNA sequencing and long-read sequencing of RT-PCR products revealed altered splicing for 10 variants, upgrading their classification to (likely) pathogenic. Conclusions: RNA analysis provided evidence of aberrant splicing in 62/109 patients, guiding variant interpretation. For poorly expressed genes in available (proxy-) tissues, sequencing of RT-PCR products remains advantageous over RNAseq. In addition, the single molecular nature of recent (short and long-read) sequencing technologies allowed phasing of the variant or a heterozygous SNP to the observed splice effect.

From Vous to Gene Therapy: A Stem Cell Based Approach to Determining the Pathogenicity of Novel Ocular Genomic Variants

Benjamin Nash^1,2,3, To Ha Loi^1,2, Amin Sabri^1,2, Steven Eamegdool¹, Milan Fernando⁴, John Grigg¹, Anai Gonzalez-Cordero⁴ and Robyn Jamieson^1,2,5

¹Eye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney NSW, Australia, ²Specialty of Genomic Medicine, Children’s Hospital at Westmead Clinical School, Faculty of Medicine and Health, University of Sydney, Sydney NSW, Australia, ³Sydney Genome Diagnostics, Western Sydney Genetics Program, Sydney Children’s Hospitals Network, Sydney NSW, Australia, ⁴Stem Cell Medicine Group, Children’s Medical Research Institute, University of Sydney, Sydney NSW, Australia and ⁵Department of Clinical Genetics, Western Sydney Genetics Program, Sydney Children’s Hospitals Network, Sydney NSW, Australia

Background: Genomic studies of our Australian retinal dystrophy cohort in a clinical diagnostic setting are providing clear-cut molecular diagnoses in ∼50% of families examined; however, assessing pathogenicity of novel genomic variants can be challenging. This is especially relevant where there are strict inclusion criteria for emerging gene therapies. Patient-derived induced pluripotent stem cells (iPSCs) differentiated to retinal pigmented epithelial (RPE) cells or retinal organoids provide a valuable resource for investigation of genes with retinal-specific expression. Aim: The aim of this study was to determine the pathogenicity of a novel synonymous RPE65 variant of unknown significance detected in trans with an established pathogenic variant on diagnostic genomic testing, in siblings with early onset severe retinal degeneration. Method: iPSC cells were generated from the peripheral blood of a heterozygous RPE65 synonymous variant carrier. Gene expression studies using qRT-PCR and immunohistochemistry were performed to confirm pluripotency and subsequent differentiation to RPE. RNA was extracted from iPSC-RPE cells, with Sanger sequencing performed to investigate splicing aberrations. Results: Gene expression studies of iPSC and subsequent RPE lines confirmed pluripotency and presence of RPE-specific markers, respectively. SNP chromosome microarray studies of iPSCs confirmed genomic stability. Analysis of RNA from carrier parent iPSC-RPE demonstrated exon skipping in the RPE65 transcript, and a predicted mutant RPE65 allele of only 22 amino acids. Reduction in RPE65 expression was observed. Conclusion: These findings facilitated the variant’s reclassification to pathogenic, and further showcase the importance of synergistic stem cell studies and diagnostic genomics in the era of gene therapies.

Does Advanced Paternal Age Increase the Rate of Paternally Derived AneuploidaAs Detected by SNP Array?

Tanya Samarasekera¹, Tristan Hardy^1,7, Luk Rombauts^1,2,3, Mark P Green^1,4, Yanhe Liu^8,9,10,11, Deirdre Zander-Fox^1,3,5,6 and Elissa Willats¹

¹Monash IVF, Melbourne, VIC, Australia, ²Monash Health, Melbourne, VIC, Australia, ³Monash University, Melbourne, VIC, Australia, ⁴University of Melbourne, Melbourne, VIC, Australia, ⁵University of Adelaide, Adelaide, SA, Australia, ⁶University of South Australia, Adelaide, SA, Australia, ⁷Monash IVF Group, Adelaide, SA, Australia, ⁸Monash IVF Group, Gold Coast, Australia;, ⁹Bond University, Gold Coast, QLD, Australia, ¹⁰University of Western Australia, Perth, WA, Australia and ¹¹Edith Cowan University, Perth, WA, Australia

Background: Studies investigating the impact of paternal age on the incidence of embryo aneuploidy using young oocyte donors have reported conflicting results. This study takes a different approach, which is to look at the parental source of embryo aneuploidy as detected by SNP array. Aim: To determine whether the incidence and type of paternal-origin aneuploidy increases with advancing paternal age. Methods: A retrospective study of 1541 embryos with SNP array testing from 2012 to 2020 across three paternal age groups: <35 years (n = 410), 35–40 years (n = 789) and >40 years (n = 315). The primary outcome was frequency of paternally derived aneuploidy in each age bracket. The secondary endpoint was the comparison of rates of specific and complex aneuploidies of paternal origin. Results: There was no significant association between advancing paternal age and prevalence of paternal-origin aneuploidy (<35 years: 17.8%, 35–40 years:16.2%;>40 years:17.1%). Moreover, no significant differences were found between age cohorts in the occurrence of paternal-origin trisomy and paternal-origin monosomy. The rate of paternally derived complex abnormalities increased with advancing paternal age (<35 yrs: 2.9%, 35–40 years: 3.8%; >40 years: 4.4%) but did not reach statistical significance. Analysis of specific chromosomes showed the >40 yrs cohort to have a marked increase in rates of trisomy 4, 10, 12, and 19. Unlike previously reported, no clear difference was observed in the rate of trisomy 21 with advancing paternal age. Conclusion: We conclude that there is no association between paternal age and the overall rate of paternal-origin aneuploidy. However, our findings suggest a possible effect of advanced paternal age on the rate of paternally derived complex abnormalities, as well as increased rate of specific paternal-origin trisomies.

Integrated Analysis of Exome Case-Control and Tumor Sequencing Data from High-Grade Serous Ovarian Cancer Patients Reveals Potential Novel Predisposition Genes

Deepak Subramanian^1,2, Magnus Zethoven^1,2, Simone McInerny³, Simone Rowley¹, Prue Allan¹, Kylie Gorringe², Paul James^1,2,3 and Ian Campbel^l1,2

¹Peter MacCallum Cancer Centre, Melbourne, VIC, Australia, ²Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia and ³The Parkville Familial Cancer Centre, Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne, VIC, Australia

Background: High-grade serous ovarian carcinoma (HGSOC) has a significant hereditary component, half of which is currently unexplained. We recently reported enrichment for germline loss-of-function (LoF) variants in 43 candidate genes among 510 BRCA1/2-negative HGSOC patients. However, as the number of carriers for each gene was small, orthogonal approaches are needed to validate these findings, leading us to conduct tumor sequencing to seek molecular genetic evidence of biallelic inactivation of these genes in germline carriers. Methods: Whole exome, Sanger, and targeted bisulphite DNA sequencing were performed using archival HGSOC specimens from 89 patients who were heterozygous germline LoF variant carriers for candidate genes or previously proposed genes (e.g. PALB2). Data were analyzed for copy number loss, somatic point mutations, promoter methylation and mutational signatures. Results: Of the proposed genes, PALB2 and ATM displayed biallelic inactivation in nearly every tumor from germline carriers. For candidate genes, 19 of 38 studied demonstrated biallelic inactivation in at least one tumor from a germline carrier. One gene- LLGL2- emerged as a promising candidate, displaying biallelic inactivation in every sequenced sample and a distinctive mutational signature with low homologous recombination repair deficiency, similar to others’ and our own sequenced ATM tumors with biallelic inactivation. Conclusions: Our results for PALB2 demonstrate the utility of this approach for validating candidate familial cancer genes, providing further support that this is an HGSOC predisposition gene. Using this methodology, several candidate genes demonstrated evidence of tumor wildtype allelic inactivation to indicate a potential predisposing role, worthy of further investigation.

Clinical Utility of Next Generation Sequencing in the Diagnosis and Management of Myeloid Malignancies

Rishu Agarwal

Austin Pathology, Melbourne, VIC, Australia

Background: Next Generation Sequencing (NGS) has an increasingly recognized role in the diagnostic workup, management, and monitoring of patients with myeloid malignancies. At our institution, a NATA accredited myeloid NGS targeted panel is offered to all patients with clonal myeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPNs). To highlight its value, we sought to describe the real-world use and clinical utility of NGS panels. Methods: Patients’ samples were analyzed by a 31-gene custom-designed panel using Anchored Multiplexed PCR technology with molecular barcodes. Curation was done with standardized bioinformatics utilising error correction for highly sensitive and accurate variant calling. Results: 1102 samples were processed over a 3-year period. Mutations were found in 88% of AML (n = 189/216 cases), 86% of MDS (n = 74/86 cases), and 98% of MDS/MPN overlap patients (n = 39/40 cases) with morphologically confirmed diagnosis. 95% of MPN patients had mutations (n = 117/123 cases), majority of which were driver mutations (JAK2, CALR, MPL). Amongst cases of suspected MDS with indeterminate morphological features, 30% of cases had clonal mutations. Amongst cases of suspected MPN due to persistent abnormal blood counts, 13% had clonal mutations. Our data also uncovered novel and low allelic frequency variants that were missed by conventional assays and thus helped in supporting the diagnosis in few cases. Conclusion: NGS testing has a powerful impact in myeloid malignancies for conforming diagnosis, shape prognosis, and guide management choices including targeted therapies. However, challenges remain in routine availability, funding, and expertise needed for curation and variant interpretation.

Evidence Sharing Across Australian Clinical Genetic Testing Laboratories Via Shariant Platform Aids Variant Interpretation

Emma Tudini^1,2, James Andrews^1,3, David Lawrence³, Hamish Scott^1,3,4, Amanda B. Spurdle² and on behalf of Australian Genomics

¹Australian Genomics (NHMRC 1113531, 200001 and the Medical Research Future Fund), ²QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia, ³ACRF Cancer Genome Facility, Centre for Cancer Biology, Adelaide, SA, Australia and ⁴Genetics and Molecular Pathology, SA Pathology, Adelaide, SA, Australia

Background: Sharing genomic variant clinical interpretations across laboratories is recognized to improve diagnostic accuracy and patient management¹. Australian Genomics initiated an implementation project to improve variant interpretation in the Australian setting. Methods: In consultation with Australian public and private clinical genetic testing laboratories, Shariant was designed as a controlled access platform to allow inter-laboratory automated sharing of structured evidence for clinically curated variants. Shariant was also developed to support a process to automatically identify and promote resolution of variant interpretation discrepancies. Results: To date, ten clinical genetic testing laboratories across four states have contributed >7000 prospective variant interpretations to Shariant. Connection of another nine laboratories from three states is in progress. Approximately 10% (15) of variants with classifications from multiple laboratories have been identified as discrepant across the tiers (Likely) Pathogenic, Variant of Uncertain Significance, (Likely) Benign; 73% (11) are medically significant differences. Seven discrepancies have already been resolved, attributing changes to review of structured evidence (2), segregation evidence from one laboratory (1), new published functional evidence (1), review of an out-of-date classification (1) and a low penetrance/risk allele (1). Assessment of ACMG-AMP² code usage and weight for (Likely) Pathogenic and Uncertain Significance variants identified inter-laboratory variability for application of PS4 (prevalence in affected individuals over controls, ∼25% of records), and PS3 (functional analysis, ∼16% of records). Conclusion: These findings demonstrate the feasibility of implementing a national variant sharing platform, highlight advantages of using structured evidence to resolve inter-laboratory discrepancies, and present possible areas for variant curation standardization across Australia.

Systematic Follow-Up to Redefine Recurrence Risk of De Novo Mutations Leading to Perinatal Death

Peer Arts¹, Thuong T. Ha^1,2, Alicia B. Byrne^1,3, Matilda R. Jackson^1,7, Milena Babic¹, Tristan S.E. Hardy^4,5, David Lawrence², Sarah L. King-Smith¹, AlanMcGovern¹, Thessa Kroes⁶, Mark Corbett6Genomic Autopsy Study Research Network, Jozef Gecz⁶, Karin S. Kassahn^4,9, Andreas W. Schreiber^2,9, Christopher P. Barnett^8,10,# and Hamish S. Scott^1,2,7,9,#

¹Department of Genetics and Molecular Pathology, Centre for Cancer Biology, an alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ²ACRF Genomics Facility, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA, Australia, ³Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA, ⁴Department of Genetics and Molecular Pathology, SA Pathology, Adelaide, SA, Australia, ⁵Repromed, Dulwich, SA, Australia, ⁶Adelaide Medical School and Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia, ⁷Australian Genomics, Melbourne, VIC, Australia, ⁸School of Medicine, University of Adelaide, Adelaide, SA, Australia, ⁹School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia and ¹⁰Paediatric and Reproductive Genetics Unit, South Australian Clinical Genetics Service, Women’s and Children’s Hospital, Adelaide, SA, Australia

Background: The Genomic Autopsy Study investigates the genetic underpinnings of perinatal death; the loss of a fetus or neonate. To date, exome and genome sequencing in 170 families has yielded 47 (27.6%) causative variants and 41 (24.1%) candidate variants, of which 42 (48%) were called de novo. Follow-up of these de novo mutations is not routinely performed in genetic pathology laboratories; with recurrence risks counselled at 1%. Aim: To systematically assess parental mosaicism for de novo variants involved in perinatal death, to allow more accurate counseling on actual recurrence risk. Methods: We performed phasing for 42 de novo variants using existing genomic sequencing data (N = 17) or Nanopore sequencing (N = 25) to identify the parental origin. In addition, we applied custom droplet digital PCR (ddPCR) assays to determine presence of the specific variant in parental blood (31), saliva (11), and sperm (4). Results: Similar to earlier reports, 80% of autosomal de novo mutations were phased to the paternal allele. Follow-up by ddPCR revealed paternal mosaicism for 4/29 (14%) variants tested to date, with variant allele frequencies ranging between 0.05% and 10% in blood samples; the latter corresponding to 20% in sperm. While the variant was not called by the GATK haplotypecaller, the (10%) mosaicism could be visualized using IGV. Conclusion: Autosomal de novo mutations underlying perinatal death occur on the paternal allele in 80% of cases. Using somatic variant callers, manual inspection in IGV and assessment of low-level mosaicism by ddPCR facilitates more accurate counseling regarding recurrence risks for future pregnancies.

What is the Impact of Brca1/2 Status on Young Women’s Reproduction and Relationships after Predictive Testing? An Australian Case-Control Study

Laura Forrest^1,2, Rowan Forbes Shepherd¹, Louise Keogh³, Mary-Anne Young⁴, Lucinda Salmon⁵, Linda Warwick⁶, Jo Burke^7,8, Rachel Williams^9,10,11, Catherine Beard¹, Rebecca D’Souza¹² and Paul James^1,2

¹Parkville Familial Cancer Centre, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, VIC, Australia, ²Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia, ³Melbourne School of Population and Global Health, The University of Melbourne, VIC, Australia, ⁴Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney.,NSW, Australia, ⁵Clinical Genetics, Austin Health, Melbourne, VIC, Australia, ⁶ACT Genetics Service, ACT Health, Canberra, Australian Capital Territory, Australia, ⁷Tasmanian Clinical Genetics Service, Royal Hobart Hospital, Hobart, TAS, Australia, ⁸School of Medicine, University of Tasmania, Hobart, TAS, Australia, ⁹Hereditary Cancer Centre, Prince of Wales, Sydney, New South Wales, Australia, ¹⁰Hereditary Cancer Centre, St George Hospital, Sydney, NSW, Australia, ¹¹Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney. NSW, Australia and ¹²Genetics Services of Western Australia, King Edward Memorial Hospital, Perth, WA, Australia

Background: Predictive testing for the high-risk cancer predisposition genes BRCA1/2 in young women has unique implications for relationships and reproductive choices. Although explored thematically in the past, the impact of these issues has not been quantified. Aim: To investigate the impact of BRCA1/2 status on women’s reproduction and partnering. Methods: Data were collected using an online survey with a case-control design from June 2019 to May 2021. Unaffected women aged 18−40 years, who had predictive BRCA1/2 testing were recruited from eight Australian clinical genetics services. Outcomes included childbearing, relationship status, and intimacy. Descriptive and inferential statistics were used. Open text responses were collected, and content analysis was employed. Results: 559 participants responded: 62.4% BRCA positive; 37.6% BRCA negative, with no demographic differences observed between groups. Women were more likely to have children after genetic testing if they were BRCA positive (OR 3.5, 95% CI [1.1, 5.6], p = .03) and less likely if they had children pretest (OR 0.01, 95% CI [0.01, 0.4], p < .001). Of the 131 unpartnered participants, 21% described that forming relationships was impacted by the burdensome nature of their BRCA status. Intimacy subscales were not associated with BRCA status (pleasure p = .9; discomfort p = .9). However, intimacy (pleasure) was positively associated with body image (OR 2.4, 95% CI [1.3, 4.2], p = .004). Further, women who had a bilateral prophylactic mastectomy had lower body image than women who had not (p = .02). Conclusion: Findings from this large multicentre study extend the evidence to develop services better tailored to the needs of younger high-risk women.

Reproductive Carrier Screening Participants Want to Know Chance of Having a Deaf Child But Don’t Know How They’d Use It

Lucinda Freeman^1,2, Martin Delatycki^3,4, Jackie Leach Scully⁵ and Edwin Kirk^1,2,6

¹Centre for Clinical Genetics, Sydney Children’s Hospital, Sydney, NSW, Australia, ²School of Women’s & Children’s Health, University of New South Wales, Sydney, NSW, Australia, ³Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ⁴Victorian Clinical Genetics Services, Melbourne, VIC, Australia, ⁵Disability Innovation Institute, University of New South Wales, Sydney, NSW, Australia and ⁶NSW Health Pathology East Genomics Laboratory, Sydney, NSW, Australia

Background: While many commercial companies include nonsyndromic deafness genes in reproductive genetic carrier screening (RGCS) panels, there is little research exploring the acceptability and utility of including these genes. Although some couples have made decisions to avoid having a child who is deaf, there are effective interventions available for children who are deaf, and there is no consensus on whether deafness is a disabling condition. Aim: This study explores views of people who have had RCGS regarding inclusion of genes for deafness in expanded carrier screening. Methods: Surveys were sent to people who had undergone RCGS three months following receipt of results. Results: 930 people were invited to take part, and 278 completed the survey (30% participation rate). When compared to other conditions screened in RGCS, 34% thought deafness was not a severe health condition, 30% thought it was, while 36% were unsure. Most participants (86%) indicated they would want to know their chance of having a child who is deaf. However, only 35% indicated they would make different reproductive decisions if they had an increased chance of having a child who is deaf; 31% said they would not change their reproductive plans and the remainder (34%) were unsure what they would do with this information. Conclusion: While the majority of respondents favored screening for deafness, there was no consensus about whether it represents a health condition that warrants changing reproductive decision making. Whether hypothetical views of those who favor it would translate into actual reproductive decision making remains uncertain.

‘A More Complicated Scenario Than What I Had Ever Realized’: Exploring the Experiences of People Who Received an Increased Risk Result for Sex Chromosome Aneuploidy Through NonInvasive Prenatal Testing (NIPT)

Miranda Lewit-Mendes^1,2, Hazel Robson³, Joanne Kelley⁴, Justine Elliott⁵, Erica Brown⁶, Melody Menezes^1,3 and Alison Archibald⁵

¹University of Melbourne, Melbourne, VIC, Australia, ²Monash Genetics, Clayton, VIC, Australia, ³Monash Ultrasound for Women, Melbourne, VIC, Australia, ⁴Mercy Hospital for Women, Heidelberg, VIC, Australia, ⁵Victorian Clinical Genetics Services, Melbourne, VIC, Australia and ⁶The Royal Women’s Hospital, Melbourne, VIC, Australia

Background: NIPT offers prenatal screening for common chromosome conditions: trisomy 21, trisomy 18, trisomy 13, as well as sex chromosome aneuploidies (SCA). Yet, SCAs have mild and variable features compared to the autosomal aneuploidies for which prenatal screening has been traditionally offered. Little is known about the experiences of people who receive an increased risk SCA result through NIPT. Aim: This study aimed to explore the experiences of people who received this result and their perspective on the information, care, and support they received from healthcare practitioners regarding their result. Methods: Semistructured interviews were conducted with 18 women and two male partners, who received an increased risk SCA result through NIPT and continued their pregnancy. Transcribed data were analyzed using rigorous thematic analysis to identify important patterns and themes. Results: Participants described embarking on NIPT, primarily based on advice from their healthcare professional and without much consideration. Consequently, participants expressed feeling unprepared for the unanticipated complexity of an increased risk SCA result and were faced with making a time-sensitive decision about a condition they had not previously considered. While more pre-test information was desired, timely access to genetic counseling and transparency from clinicians post-test assisted with adjustment to the result. Conclusion: These findings suggest that routinization of NIPT may be compromising informed decision-making and resulting in unpreparedness for an increased risk result. Given the likelihood of increasing uptake and expanding the scope of NIPT, resources should be dedicated to educating NIPT providers and ensuring timely access to genetic counseling post-result.

Knowledge, Views and Expectations for Cancer Polygenic Risk Testing in Clinical Practice: A Cross-Sectional Survey of Health Professionals

Amelia K. Smit^1,2, Ashleigh R. Sharman¹, David Espinoza³, Courtney Wallingford⁴, Mary-Anne Young⁵, Kate Dunlop¹, Jane Tiller⁶, Ainsley J. Newson⁷, Bettina Meiser⁸, Anne E. Cust^1,2 and Tatiane Yanes⁴

¹The University of Sydney, Faculty of Medicine and Health, Sydney School of Public Health, Cancer Epidemiology and Prevention Research, Australia, ²Melanoma Institute Australia, The University of Sydney, NSW, Australia, ³NHMRC Clinical Trials Centre, The University of Sydney, NSW, Australia; , ⁴The University of Queensland Diamantina Institute, Dermatology Research Centre, University of Queensland, Brisbane, Australia, ⁵Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia, ⁶Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC, Australia, ⁷The University of Sydney, Faculty of Medicine and Health, Sydney School of Public Health, Sydney Health Ethics, Sydney, NSW, Australia and ⁸Psychosocial Research Group, Prince of Wales Clinical School, University of NSW, Sydney, NSW, Australia

Background: Polygenic risk scores (PRS) are becoming increasingly available in clinical practice to evaluate cancer risk. However, little is known about health professionals’ knowledge, attitudes, and expectations of PRS. Aim: To explore knowledge, views, and expectations around the use of PRS in clinic for health professionals who provide cancer risk assessments. Methods: An online questionnaire was distributed by relevant health professional organizations predominately in Australia, Canada and the US to evaluate health professionals’ knowledge, views, and expectations of PRS. Eligible participants were health professionals who provide cancer risk assessments. Results from the questionnaire were analyzed descriptively and content analysis was undertaken of free-text responses. Results: In total, 105 health professionals completed the questionnaire (genetic counselors 84%; oncologists 6%; clinical geneticists 4%; other 7%). Although responses differed between countries, most participants (61%) had discussed PRS with patients, 20% had ordered a test, and 14% had returned test results to a patient. Confidence and knowledge around interpreting PRS were low. Although 69% reported that polygenic testing would certainly or likely influence patient care in the future, most felt unprepared for this. If scaled up to the population, 49% expect that general practitioners would have a primary role in the provision of PRS, supported by genetic health professionals. Conclusions: These findings will inform the development of resources to support health professionals offering polygenic testing, currently and in the future.

Opinions and Experiences of Recontacting Patients: A Survey of Genetic Health Professionals in Australasia

Helen Mountain^1,2, Bhavya Vora¹, Cassandra Nichols¹ and Lyn Schofield¹

¹Genetic Services of Western Australia, Subiaco, WA, Australia and ²Graduate School of Health, UTS, Sydney, NSW, Australia

The issue of recontacting past patients is becoming more relevant, particularly in the familial cancer setting. Next-generation sequencing is providing more information on the pathogenicity and prevalence of genetic variants, often leading to the need to recontact patients. Various international genetic societies have position statements and recommendations to help genetic health professionals (GHPs) navigate the legal, ethical, and practical issues of recontacting. In the absence of a standardized Australasian protocol, we explored the organizational policies, experiences, and opinions of Australasian GHPs regarding patient follow-up and recontacting practices. Forty-five respondents completed an online survey of multiple choice and open-ended questions. The majority of respondents indicated that recontacting occurred on an ad hoc basis but most genetic services relied on patients (or family) to initiate the recontact. The idea of implementing a routine recontacting system was widely dismissed by 73% of respondents, citing lack of resources, limited information on legal responsibility, and setting unrealistic expectations as common barriers. However, if recontact were to be initiated, E-communication was believed to be an ideal first step in re-establishing contact with patients. An established position statement or a protocol may help the provision of equitable services, however, patient individuality and circumstances means that a ‘one size fits all’ approach may not be achievable.

Family Communication About Genomic Testing for Early Onset Dementia

Alice Poulton^1,9, Lisette Curnow^2,8, Dhamidhu Eratne^3,4,5 and Adrienne Sexton^6,7

¹Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia, ²Victorian Clinical Genetics Services, Royal Childrens Hospital, Melbourne, VIC, Australia, ³Neuropsychiatry, Royal Melbourne Hospital, Melbourne VIC, Australia, ⁴Melbourne Neuropsychiatry Centre, University of Melbourne and NorthWestern Mental Health, Melbourne, VIC, Australia, ⁵Melbourne Genomics, Melbourne, VIC, Australia. , ⁶Genomic Medicine, The Royal Melbourne Hospital, Melbourne VIC, Australia, ⁷Department of Medicine (Royal Melbourne Hospital), The University of Melbourne, Melbourne, VIC, Australia, ⁸Murdoch Children’s Research Institute, Melbourne, VIC, Australia and ⁹Monash Ultrasound for Women, Melbourne, VIC, Australia

Early-onset dementia (EOD) refers to onset <65 years of age and may be associated with genetic cause. Family communication surrounding any genetic risk is complex, and this process may be further complicated in an EOD context due to effects on cognition and behavior, and associated psychosocial consequences. This study aimed to investigate how individuals experience family communication about potential genetic risk and testing for EOD. Thematic analysis was performed on verbatim transcripts of 10 semistructured interviews undertaken with family members who attended a neurogenetics clinic due to a relative diagnosed with EOD. Interviews explored the participants’ experiences of learning EOD might be inherited, and the ensuing family communication about genetic testing. Four key themes emerged: (1) a clinical diagnostic odyssey was common and could be a motivator for genomic testing, (2) preexisting family tension and/or disconnection was a common barrier, (3) family members’ autonomy was considered, and (4) avoidant coping strategies influenced communication. Communication regarding potential EOD genetic risk is a complicated process, and may be influenced by pre-existing family dynamics, individual coping mechanisms and a desire to promote autonomy in relatives. Findings emphasise the importance of health professionals having awareness of the psychological impact of the clinical diagnostic odyssey and providing appropriate support. To promote effective risk communication, genetic counselors should pre-emptively address family tensions that may emerge in an EOD context and offer psychosocial support to facilitate coping with this tension in an adaptive way. Findings also indicated the importance of extending genetic counseling support to relatives.

The Role of Genomics in Diagnosing Inherited Cardiac Diseases

Fergus Stafford^1,3, Neesha Krishnan^1,3, Ebony Richardson^1,3, Charlotte Burns^4,5, Belinda Gray^4,6, Caroline Medi⁵, Natalie Nowak^4,5, Hariharan Raju⁶, David Richmond^5,6, Mark Ryan⁵, Emma S. Singer^4,6, Raymond Sy^5,6, Laura Yeates^1,6, Richard D. Bagnall^4,6, Christopher Semsarian^4,6 and Jodie Ingles^1,6

¹Cardio Genomics Program at Centenary Institute, The University of Sydney, Sydney, NSW, Australia, ²Centre for Population Genomics, Garvan Institute of Medical Research, and UNSW Sydney, Sydney, NSW, Australia, ³Centre for Population Genomics, Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ⁴Agnes Ginges Centre for Molecular Cardiology at Centenary Institute, The University of Sydney, Sydney, NSW, Australia, ⁵Department of Cardiology, Royal Prince Alfred Hospital, Sydney, NSW, Australia and ⁶Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia

Background: The diagnostic yield of genetic testing for inherited cardiac diseases is ∼40%. Many do not meet accepted clinical diagnostic criteria, or a genetic diagnosis can indicate a clinical misdiagnosis. For a large proportion, there is growing evidence of a polygenic basis. We sought to evaluate the role of cardiac genomics in clarifying the underlying diagnosis. Methods: We performed a retrospective audit of consecutive, unrelated probands attending a specialized, multidisciplinary clinic between 2002-20. Individuals were considered ‘solved at baseline’, ‘solved on review’, or ‘unsolved’, based on reaching a clinical diagnosis and concordant genetic diagnosis. We developed a scoring system to identify those most likely to have monogenic disease, based on phenotype, positive family history, young age at onset and severe presentations (e.g., cardiac arrest, transplant). Scores ≤1.5 indicated low, 1.5-3.0 moderate, and >3.0 a high chance of a monogenic disease. Results: There were 1807 probands, with 916 excluded (missing data, no genetic testing). 891 probands were included, with a clinical-genetic diagnosis achieved in 361 (40%) at baseline, 50 (6%) solved on review and 480 (54%) remain unsolved. Diagnostic yield was 32% in the low (n = 533, 60%), 60% in the moderate (n = 177; 20%) and 73% in the high chance of monogenic disease group (n = 181; 20%). Those solved on review were due to: research-based exome/genome sequencing (48; 96%), clinical re-review (18; 36%), new knowledge (19; 38%), segregations (12; 24%) and/or functional studies (5; 10%). Conclusion: We highlight an important role of cardiac genomics in clarifying diagnosis and propose a framework for prioritising families with a high likelihood of monogenic disease with the goal to solve their underlying cause of disease.

Psychological Wellbeing of Patients and Their Families After Genetic Testing for Dilated Cardiomyopathy

Bridget Douglas^1,3, Chris Jacobs¹, Diane Fatkin^2,3 and Renee Johnson^2,3

¹University of Technology Sydney, NSW, Australia, ²University of NSW, Sydney, NSW, Australia and ³Victor Chang Cardiac Research Institute, Sydney, NSW, Australia

Background: Genetic testing for individuals with dilated cardiomyopathy (DCM) has become increasingly available; however, there is little research that has assessed the impact of genetic testing on DCM patients’ psychosocial wellbeing. Aim: This study investigates the psychological impact of genetic testing for DCM and assesses satisfaction and unmet needs. Methods: Validated questionnaires were emailed to 167 participants who had received genetic test results for DCM in the past 10 years either through research or through clinical genetic testing. Survey outcomes were coded to a numerical outcome and examined statistically. Results: Fifty-three participants responded (response rate 32%) with 33 genotype-positive, 18 genotype negative, and 2 unclear findings (variants of unknown significance or indeterminate results). Motivation for DCM genetic testing was high and was directed primarily by concerns about risk to children and personal health. The overall psychological impact from genetic testing was low and did not differ between participants receiving results within 6 months compared to longer time periods. However, concern about genetic results was significantly higher in the first six months after receiving a genetic test result (p = .0122). Genotype negative respondents showed less tolerance to uncertainty (p = .0396) and had lower trust in their health care providers (p = .0153). Conclusion: Genetic testing for DCM was well tolerated and had little overall impact on participants’ psychological wellbeing. Participants were highly motivated to access genetic testing and overall satisfaction was high. However, genotype-negative patients may benefit from further support following genetic testing.

Preparing Nongenetic Medical Specialists for Genomic Medicine: The Melbourne Genomics Workforce Development Program

M. Martyn^1,2,3, E. Lynch^2,3,4, F. Maher^2,3,5, T. Charles^1,3,4, C. McEwan^1,2, R. Tytherleigh^1,2, B. McClaren^1,3, M. Janinski^1,2, F. Cunningham^2,6, R. McNeil^1,2, A. Nisselle^1,2,3 and C. Gaff^1,2,3

¹Murdoch Children’s Research Institute, Melbourne, VIC, Australia, ²Melbourne Genomics Health Alliance, Melbourne, VIC, Australia, ³The University of Melbourne, Melbourne, VIC, Australia, ⁴Victorian Clinical Genetics Services, Melbourne, VIC, Australia, ⁵Walter and Eliza Hall Institute, Melbourne, VIC, Australia and ⁶Monash Genetics, Monash Health, Melbourne, VIC, Australia

To address the challenge of preparing nongenetic medical specialists for genomic medicine, Melbourne Genomics established a multifaceted workforce development strategy. Informed by adult learning and implementation science theories, this aimed to improve genomic expertise amongst specialists, ranging from basic familiarity to speciality experts. Immersive and structured learning activities (workshops and blended course) were offered and evaluated using mixed methods. All 22 immersive-learning participants completed post-program interviews. 375 specialists participated in structured learning with surveys completed at 4 time-points: baseline (n = 265/375, 71%); post-online modules (blended-learning only, n = 40/85, 47%); post-workshop (n = 277/361, 77%); long-term (>6 month) follow-up (n = 48/291, 16%). Quantitative data were analyzed using descriptive statistics; survey comments and interview data were analyzed using inductive content analysis. Immersive-learning participants reported improved genomic capability and changed genomic medicine practice. They reported being recognized as credible genomic experts within their specialty, acting as a resource for peers and providing speciality expertise to geneticists. Structured learning activities improved participants’ self-rated and objective genomic knowledge, skills, and confidence; confidence in knowing referral pathways and demonstrated ability to correctly interpret a report were maintained over time. Structured learning participants reported changes to practice at follow-up (39/48, 81%) including: referring to/consulting Genetics; requesting an exome; educating others. We conclude that structured learning supports long-term improvements in genomic capability, while immersion enables development of speciality genomic experts who, in turn, support peer learning and champion genomics. A workforce development strategy that incorporates both these approaches contributes to sustained behavior change and development of a workforce with a spectrum of genomic expertise.

The Evolving Multidisciplinary Delivery of Ultra-Rapid Genomic Sequencing in Acute Care

Fiona Lynch^1,2,3, Belinda McClaren^1,2,3, Amy Nisselle^1,2,3 and Clara Gaff^1,2,3

¹Australian Genomics Health Alliance, Melbourne, VIC, Australia, ²Genomics in Society, Murdoch Children’s Research Institute, Melbourne, VIC, Australia and ³The University of Melbourne, Melbourne, VIC, Australia

Background: Rapid and ultra-rapid genomic sequencing (rGS) is being established as a first-tier diagnostic test for critically unwell infants and children across Australia. This service represents an overlap in practice between the disciplines of genetics and acute care; multidisciplinary teamwork will be crucial for its successful delivery. Aim: To explore the evolution of the multidisciplinary delivery of rGS in acute care in Australia. Methods: In-depth qualitative interviews were conducted with 11 health professionals with experience in the delivery of rGS: seven genetic counselors (GCs), in 2018 and again in 2020; and four acute care clinicians (‘intensivists’) at a single timepoint in 2020-21. Interviews were audio-recorded, transcribed and analyzed using content analysis, and compared over time for GCs. Results: Interviews reveal an evolving establishment of multidisciplinary practice in acute care. While there were initial challenges, both disciplines appear to be benefiting from workplace learning, facilitating the movement towards truly integrated practice. Intensivists and GCs are working together to support parents’ informed decision making about rGS. Irrespective of the outcome of rGS, participants report that families need post-test support for a variety of reasons. Conclusion: This study demonstrates maturation in the delivery of rGS over recent years and provides insight into the future delivery of genomic medicine in acute care. Practices reported around decision making are still putting undue pressure on parents to consent to rGS. A wider range of post-test needs were reported by health professionals than families undergoing rGS, making all perspectives essential for informing service delivery.

Outcomes of a Four-Year Laboratory-Based Genetic Counseling Role

Yana Smagarinsky^1,2,3, Lisette Curnow^1,2,3, Helen Savoia¹ and Susan M. White^2,3

¹The Royal Children’s Hospital, Melbourne, VIC, Australia, ²Victorian Clinical Genetics Services, Melbourne, VIC, Australia and ³Murdoch Children’s Research Institute, Melbourne, VIC, Australia

The Royal Children’s Hospital (RCH) has employed a laboratory-based genetic counselor (LBGC) since mid-2017. The LBGC aims to facilitate access to high-quality, cost-effective, clinically indicated genetic and genomic testing hospital wide. This is achieved by overseeing the test request review process and providing ongoing education and support to nongenetics clinicians within the hospital. Data collected via hospital systems as well as a LBGC-designed REDCap database allowed for evaluation of the role. Analysis showed the number of referral laboratories decreased dramatically over the initial 3 year period, from >100 referral laboratories in 2015, to <30 in 2020. This is despite a steady increase in test requests being observed, with orders increasing by 52% from 2017-2020. The LBGC role enabled management of the increased test requests in a financially sustainable way, where identification of and access to alternative funding sources for appropriate requests represented a 62% cost saving for RCH in 2018/2019. As test requests continue to increase and more genomic tests become available, the number of declined or modified requests are expected to rise from an average of 9%. A monthly genetic review meeting allowed for strong relationships to be built with frequent test requestors as well as the implementation of specific processes for expert reviewers in different departments. Although departmental ordering trends and diagnostic yield improved, ultimately the review process confirmed that nongenetics clinicians would benefit from the ongoing support of this role. The role continues to be supported by a clinical geneticist and a recently employed laboratory medical technician.