An intronic GAA repeat expansion in FGF14 causes the autosomal-dominant adult-onset ataxia SCA50/ATX-FGF14.

Adult-onset cerebellar ataxias are a group of neurodegenerative conditions that challenge both genetic discovery and molecular diagnosis. In this study, we identified an intronic (GAA) repeat expansion in fibroblast growth factor 14 (FGF14). Genetic analysis of 95 Australian individuals with adult-onset ataxia identified four (4.2%) with (GAA)>300 and a further nine individuals with (GAA)>250. PCR and long-read sequence analysis revealed these were pure (GAA) repeats. In comparison, no control subjects had (GAA)>300 and only 2/311 control individuals (0.6%) had a pure (GAA)>250. In a German validation cohort, 9/104 (8.7%) of affected individuals had (GAA)>335 and a further six had (GAA)>250, whereas 10/190 (5.3%) control subjects had (GAA)>250 but none were (GAA)>335. The combined data suggest (GAA)>335 are disease causing and fully penetrant (p = 6.0 × 10-8, OR = 72 [95% CI = 4.3-1,227]), while (GAA)>250 is likely pathogenic with reduced penetrance. Affected individuals had an adult-onset, slowly progressive cerebellar ataxia with variable features including vestibular impairment, hyper-reflexia, and autonomic dysfunction. A negative correlation between age at onset and repeat length was observed (R2 = 0.44, p = 0.00045, slope = -0.12) and identification of a shared haplotype in a minority of individuals suggests that the expansion can be inherited or generated de novo during meiotic division. This study demonstrates the power of genome sequencing and advanced bioinformatic tools to identify novel repeat expansions via model-free, genome-wide analysis and identifies SCA50/ATX-FGF14 as a frequent cause of adult-onset ataxia.

Genetic aetiologies for childhood speech disorder: novel pathways co-expressed during brain development.

Childhood apraxia of speech (CAS), the prototypic severe childhood speech disorder, is characterized by motor programming and planning deficits. Genetic factors make substantive contributions to CAS aetiology, with a monogenic pathogenic variant identified in a third of cases, implicating around 20 single genes to date. Here we aimed to identify molecular causation in 70 unrelated probands ascertained with CAS. We performed trio genome sequencing. Our bioinformatic analysis examined single nucleotide, indel, copy number, structural and short tandem repeat variants. We prioritised appropriate variants arising de novo or inherited that were expected to be damaging based on in silico predictions. We identified high confidence variants in 18/70 (26%) probands, almost doubling the current number of candidate genes for CAS. Three of the 18 variants affected SETBP1, SETD1A and DDX3X, thus confirming their roles in CAS, while the remaining 15 occurred in genes not previously associated with this disorder. Fifteen variants arose de novo and three were inherited. We provide further novel insights into the biology of child speech disorder, highlighting the roles of chromatin organization and gene regulation in CAS, and confirm that genes involved in CAS are co-expressed during brain development. Our findings confirm a diagnostic yield comparable to, or even higher, than other neurodevelopmental disorders with substantial de novo variant burden. Data also support the increasingly recognised overlaps between genes conferring risk for a range of neurodevelopmental disorders. Understanding the aetiological basis of CAS is critical to end the diagnostic odyssey and ensure affected individuals are poised for precision medicine trials.

A deep intronic variant in MME causes autosomal recessive Charcot-Marie-Tooth neuropathy through aberrant splicing.

BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AIMS: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. INTERPRETATION: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cß subunits of PKA during human development.

Heterozygous PNPT1 Variants Cause Spinocerebellar Ataxia Type 25.

OBJECTIVE: Dominant spinocerebellar ataxias (SCA) are characterized by genetic heterogeneity. Some mapped and named loci remain without a causal gene identified. Here we applied next generation sequencing (NGS) to uncover the genetic etiology of the SCA25 locus. METHODS: Whole-exome and whole-genome sequencing were performed in families linked to SCA25, including the French family in which the SCA25 locus was originally mapped. Whole exome sequence data were interrogated in a cohort of 796 ataxia patients of unknown etiology. RESULTS: The SCA25 phenotype spans a slowly evolving sensory and cerebellar ataxia, in most cases attributed to ganglionopathy. A pathogenic variant causing exon skipping was identified in the gene encoding Polyribonucleotide Nucleotidyltransferase PNPase 1 (PNPT1) located in the SCA25 linkage interval. A second splice variant in PNPT1 was detected in a large Australian family with a dominant ataxia also mapping to SCA25. An additional nonsense variant was detected in an unrelated individual with ataxia. Both nonsense and splice heterozygous variants result in premature stop codons, all located in the S1-domain of PNPase. In addition, an elevated type I interferon response was observed in blood from all affected heterozygous carriers tested. PNPase notably prevents the abnormal accumulation of double-stranded mtRNAs in the mitochondria and leakage into the cytoplasm, associated with triggering a type I interferon response. INTERPRETATION: This study identifies PNPT1 as a new SCA gene, responsible for SCA25, and highlights biological links between alterations of mtRNA trafficking, interferonopathies and ataxia. ANN NEUROL 2022;92:122-137.

Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma.

We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.

Mosaicism in tuberous sclerosis complex: Lowering the threshold for clinical reporting.

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%-15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling.

Clinical impact of genomic testing in patients with suspected monogenic kidney disease.

PURPOSE: To determine the diagnostic yield and clinical impact of exome sequencing (ES) in patients with suspected monogenic kidney disease. METHODS: We performed clinically accredited singleton ES in a prospectively ascertained cohort of 204 patients assessed in multidisciplinary renal genetics clinics at four tertiary hospitals in Melbourne, Australia. RESULTS: ES identified a molecular diagnosis in 80 (39%) patients, encompassing 35 distinct genetic disorders. Younger age at presentation was independently associated with an ES diagnosis (p < 0.001). Of those diagnosed, 31/80 (39%) had a change in their clinical diagnosis. ES diagnosis was considered to have contributed to management in 47/80 (59%), including negating the need for diagnostic renal biopsy in 10/80 (13%), changing surveillance in 35/80 (44%), and changing the treatment plan in 16/80 (20%). In cases with no change to management in the proband, the ES result had implications for the management of family members in 26/33 (79%). Cascade testing was subsequently offered to 40/80 families (50%). CONCLUSION: In this pragmatic pediatric and adult cohort with suspected monogenic kidney disease, ES had high diagnostic and clinical utility. Our findings, including predictors of positive diagnosis, can be used to guide clinical practice and health service design.

Lymphedema distichiasis syndrome may be caused by FOXC2 promoter-enhancer dissociation and disruption of a topological associated domain.

Lymphedema distichiasis syndrome (LDS) is a rare autosomal dominant condition characterized by lower limb lymphedema, distichiasis, and variable additional features. LDS is usually caused by heterozygous sequence variants in the FOXC2 gene located at 16q24, but in one previous instance LDS has resulted from a balanced reciprocal translocation with a breakpoint at 16q24, 120 kb distal to the FOXC2 gene suggesting a position effect. Here, we describe a second family with LDS caused by a translocation involving 16q24. The family were ascertained after detection of a paternally inherited balanced reciprocal translocation t(16;22)(q24;q13.1) in a pregnancy complicated by severe fetal hydrops. There was a past history of multiple miscarriages in the father's family, and a personal and family history of lymphedema and distichiasis, consistent with the diagnosis of LDS. Using whole genome amplified DNA from single sperm of the male proband, bead array analysis demonstrated that the FOXC2 gene was intact and the chromosome 16 breakpoint mapped to the same region 120Kb distal to the FOXC2 gene. This case highlights the clinical consequences that can arise from a translocation of genomic material without dosage imbalance, and that it is increasingly feasible to predict and characterize possible effects with improved access to molecular techniques.

Paediatric genomic testing: Navigating medicare rebatable genomic testing.

Genomic testing for a genetic diagnosis is becoming standard of care for many children, especially those with a syndromal intellectual disability. While previously this type of specialised testing was performed mainly by clinical genetics teams, it is increasingly being 'mainstreamed' into standard paediatric care. With the introduction of a new Medicare rebate for genomic testing in May 2020, this type of testing is now available for paediatricians to order, in consultation with clinical genetics. Children must be aged less than 10 years with facial dysmorphism and multiple congenital abnormalities or have global developmental delay or moderate to severe intellectual disability. This rebate should increase the likelihood of a genetic diagnosis, with accompanying benefits for patient management, reproductive planning and diagnostic certainty. Similar to the introduction of chromosomal microarray into mainstream paediatrics, this genomic testing will increase the number of genetic diagnoses, however, will also yield more variants of uncertain significance, incidental findings, and negative results. This paper aims to guide paediatricians through the process of genomic testing, and represents the combined expertise of educators, clinical geneticists, paediatricians and genomic pathologists around Australia. Its purpose is to help paediatricians navigate choosing the right genomic test, consenting patients and understanding the possible outcomes of testing.

Cryopyrin-associated periodic syndrome: a treatable genetic inflammatory condition.

A 20-year-old man presented with recurrent subdural haemorrhages on a background of progressive sensorineural hearing loss, juvenile idiopathic arthritis and intracranial hypertension of unknown cause. His mother had a similar previous history. They both had a persistently mildly elevated serum C reactive protein. Repeat lumbar punctures identified persistently elevated intracranial pressure and mild pleocytosis. A dural biopsy showed necrotising pachymeningitis with granulomatous vasculitis. The underlying cause in both patients was a cryopyrin-associated periodic syndrome. We discuss its varied phenotype and how clinicians need to be aware of this treatable genetic condition to facilitate early treatment and to prevent accumulation of disability.

Rapid Diagnosis of Spinocerebellar Ataxia 36 in a Three-Generation Family Using Short-Read Whole-Genome Sequencing Data.

BACKGROUND: Spinocerebellar ataxias are often caused by expansions of short tandem repeats. Recent methodological advances have made repeat expansion (RE) detection with whole-genome sequencing (WGS) feasible. OBJECTIVES: The objective of this study was to determine the genetic basis of ataxia in a multigenerational Australian pedigree with autosomal-dominant inheritance. METHODS AND RESULTS: WGS was performed on 3 affected relatives. The sequence data were screened for known pathogenic REs using 2 RE detection tools: exSTRa and ExpansionHunter. This screen provided a clear and rapid diagnosis (<5 days from receiving the sequencing data) of spinocerebellar ataxia 36, a rare form of ataxia caused by an intronic GGCCTG RE in NOP56. CONCLUSIONS: The diagnosis of rare ataxias caused by REs is highly feasible and cost-effective with WGS. We propose that WGS could potentially be implemented as the frontline, cost-effective methodology for the molecular testing of individuals with a clinical diagnosis of ataxia. © 2020 International Parkinson and Movement Disorder Society.

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size.

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.

A novel approach to offering additional genomic findings-A protocol to test a two-step approach in the healthcare system.

Internationally, the practice of offering additional findings (AFs) when undertaking a clinically indicated genomic test differs. In the USA, the recommendation is to include analysis for AFs alongside diagnostic analysis, unless a patient opts-out, whereas European and Canadian guidelines recommend opt-in models. These guidelines all consider the offer of AFs as an activity concurrent with the offer of diagnostic testing. This paper describes a novel two-step model for managing AFs within the healthcare system in Victoria, Australia and presents the study protocol for its evaluation. Adults who have received results of diagnostic whole exome sequencing undertaken within the healthcare system are invited to attend a genetic counseling appointment to consider reanalysis of their stored genomic data for AFs. The evaluation protocol addresses uptake, decision-making, understanding, counseling challenges, and explores preferences for future models of care. Recruitment commenced in November 2017 and will cease when 200 participants have been approached. When the study is concluded, the evaluation results will contribute to the evidence base guiding approaches to counseling and models of care for AFs.

A craniosynostosis massively parallel sequencing panel study in 309 Australian and New Zealand patients: findings and recommendations.

PURPOSE: The craniosynostoses are characterized by premature fusion of one or more cranial sutures. The relative contribution of previously reported genes to craniosynostosis in large cohorts is unclear. Here we report on the use of a massively parallel sequencing panel in individuals with craniosynostosis without a prior molecular diagnosis. METHODS: A 20-gene panel was designed based on the genes' association with craniosynostosis, and clinically validated through retrospective testing of an Australian and New Zealand cohort of 233 individuals with craniosynostosis in whom previous testing had not identified a causative variant within FGFR1-3 hot-spot regions or the TWIST1 gene. An additional 76 individuals were tested prospectively. RESULTS: Pathogenic or likely pathogenic variants in non-FGFR genes were identified in 43 individuals, with diagnostic yields of 14% and 15% in retrospective and prospective cohorts, respectively. Variants were identified most frequently in TCF12 (N = 22) and EFNB1 (N = 8), typically in individuals with nonsyndromic coronal craniosynostosis or TWIST1-negative clinically suspected Saethre-Chotzen syndrome. Clinically significant variants were also identified in ALX4, EFNA4, ERF, and FGF10. CONCLUSION: These findings support the clinical utility of a massively parallel sequencing panel for craniosynostosis. TCF12 and EFNB1 should be included in genetic testing for nonsyndromic coronal craniosynostosis or clinically suspected Saethre-Chotzen syndrome.

Dual genetic diagnoses: Atypical hand-foot-genital syndrome and developmental delay due to de novo mutations in HOXA13 and NRXN1.

We describe a male patient with dual genetic diagnoses of atypical hand-foot-genital syndrome (HFGS) and developmental delay. The proband had features of HFGS that included bilateral vesicoureteric junction obstruction with ectopic ureters, brachydactyly of various fingers and toes, hypoplastic thenar eminences, and absent nails on both 4th toes and right 5th toe. The atypical features of HFGS present were bilateral hallux valgus malformations and bilateral preaxial polydactyly of the hands. Chromosomal microarray analysis identified a de novo 0.5 Mb deletion at 2p16.3, including the first four exons of the NRXN1 gene. Whole exome sequencing and subsequent Sanger sequencing identified a de novo missense mutation (c.1123G>T, p.Val375Phe) in exon 2 of the HOXA13 gene, predicted to be damaging and located in the homeobox domain. The intragenic NRXN1 deletion is thought to explain his developmental delay via a separate genetic mechanism.

Cancer Risks Associated With TP53 Pathogenic Variants: Maximum Likelihood Analysis of Extended Pedigrees for Diagnosis of First Cancers Beyond the Li-Fraumeni Syndrome Spectrum.

PURPOSE: Establishing accurate age-related penetrance figures for the broad range of cancer types that occur in individuals harboring a pathogenic germline variant in the TP53 gene is essential to determine the most effective clinical management strategies. These figures also permit optimal use of cosegregation data for classification of TP53 variants of unknown significance. Penetrance estimation can easily be affected by bias from ascertainment criteria, an issue not commonly addressed by previous studies. MATERIALS AND METHODS: We performed a maximum likelihood penetrance estimation using full pedigree data from a multicenter study of 146 TP53-positive families, incorporating adjustment for the effect of ascertainment and population-specific background cancer risks. The analysis included pedigrees from Australia, Spain, and United States, with phenotypic information for 4,028 individuals. RESULTS: Core Li-Fraumeni syndrome (LFS) cancers (breast cancer, adrenocortical carcinoma, brain cancer, osteosarcoma, and soft tissue sarcoma) had the highest hazard ratios of all cancers analyzed in this study. The analysis also detected a significantly increased lifetime risk for a range of cancers not previously formally associated with TP53 pathogenic variant status, including colorectal, gastric, lung, pancreatic, and ovarian cancers. The cumulative risk of any cancer type by age 50 years was 92.4% (95% CI, 82.2 to 98.3) for females and 59.7% (95% CI, 39.9 to 81.3) for males. Females had a 63.3% (95% CI, 35.6 to 90.1) cumulative risk of developing breast cancer by age 50 years. CONCLUSION: The results from maximum likelihood analysis confirm the known high lifetime risk for the core LFS-associated cancer types providing new risk estimates and indicate significantly increased lifetime risks for several additional cancer types. Accurate cancer risk estimates will help refine clinical recommendations for TP53 pathogenic variant carriers and improve TP53 variant classification.