Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

A KLHL40 3' UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism.

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure and neonatal death. Here, we describe a 26-year-old man with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles' contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound from age 13 after prolonged immobilization. Muscle magnetic resonance imaging at age 13 indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein, respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G > T) in the 3' untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G > T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G > T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3' UTR variants in ClinVar suggests variants that introduce aberrant 3' UTR splicing may be underrecognized in Mendelian disease. We encourage consideration of this mechanism during variant curation.

De novo variants in ATXN7L3 lead to developmental delay, hypotonia and distinctive facial features.

Deubiquitination is critical for the proper functioning of numerous biological pathways such as DNA repair, cell cycle progression, transcription, signal transduction, and autophagy. Accordingly, pathogenic variants in deubiquitinating enzymes (DUBs) have been implicated in neurodevelopmental disorders (ND) and congenital abnormalities. ATXN7L3 is a component of the DUB module of the SAGA complex, and two other related DUB modules, and serves as an obligate adaptor protein of 3 ubiquitin-specific proteases (USP22, USP27X or USP51). Through exome sequencing and GeneMatching, we identified nine individuals with heterozygous variants in ATXN7L3. The core phenotype included global motor and language developmental delay, hypotonia, and distinctive facial characteristics including hypertelorism, epicanthal folds, blepharoptosis, a small nose and mouth, and low-set posteriorly rotated ears. In order to assess pathogenicity, we investigated the effects of a recurrent nonsense variant [c.340C>T; p.(Arg114Ter)] in fibroblasts of an affected individual. ATXN7L3 protein levels were reduced, and deubiquitylation was impaired, as indicated by an increase in histone H2Bub1 levels. This is consistent with the previous observation of increased H2Bub1 levels in Atxn7l3-null mouse embryos, which have developmental delay and embryonic lethality. In conclusion, we present clinical information and biochemical characterization supporting ATXN7L3 variants in the pathogenesis of a rare syndromic ND.

Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

Genetics of inherited peripheral neuropathies and the next frontier: looking backwards to progress forwards.

Inherited peripheral neuropathies (IPNs) encompass a clinically and genetically heterogeneous group of disorders causing length-dependent degeneration of peripheral autonomic, motor and/or sensory nerves. Despite gold-standard diagnostic testing for pathogenic variants in over 100 known associated genes, many patients with IPN remain genetically unsolved. Providing patients with a diagnosis is critical for reducing their 'diagnostic odyssey', improving clinical care, and for informed genetic counselling. The last decade of massively parallel sequencing technologies has seen a rapid increase in the number of newly described IPN-associated gene variants contributing to IPN pathogenesis. However, the scarcity of additional families and functional data supporting variants in potential novel genes is prolonging patient diagnostic uncertainty and contributing to the missing heritability of IPNs. We review the last decade of IPN disease gene discovery to highlight novel genes, structural variation and short tandem repeat expansions contributing to IPN pathogenesis. From the lessons learnt, we provide our vision for IPN research as we anticipate the future, providing examples of emerging technologies, resources and tools that we propose that will expedite the genetic diagnosis of unsolved IPN families.

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.

Ablation of the carboxy-terminal end of MAMDC2 causes a distinct muscular dystrophy.

The extracellular matrix (ECM) has an important role in the development and maintenance of skeletal muscle, and several muscle diseases are associated with the dysfunction of ECM elements. MAMDC2 is a putative ECM protein and its role in cell proliferation has been investigated in certain cancer types. However, its participation in skeletal muscle physiology has not been previously studied. We describe 17 individuals with an autosomal dominant muscular dystrophy belonging to two unrelated families in which different heterozygous truncating variants in the last exon of MAMDC2 co-segregate correctly with the disease. The radiological aspect of muscle involvement resembles that of COL6 myopathies with fat replacement at the peripheral rim of vastii muscles. In this cohort, a subfascial and peri-tendinous pattern is observed in upper and lower limb muscles. Here we show that MAMDC2 is expressed in adult skeletal muscle and differentiating muscle cells, where it appears to localize to the sarcoplasm and myonuclei. In addition, we show it is secreted by myoblasts and differentiating myotubes into to the extracellular compartment. The last exon encodes a disordered region with a polar residue compositional bias loss of which likely induces a toxic effect of the mutant protein. The precise mechanisms by which the altered MAMDC2 proteins cause disease remains to be determined. MAMDC2 is a skeletal muscle disease-associated protein. Its role in muscle development and ECM-muscle communication remains to be fully elucidated. Screening of the last exon of MAMDC2 should be considered in patients presenting with autosomal dominant muscular dystrophy, particularly in those with a subfascial radiological pattern of muscle involvement.

Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

Bi-allelic loss-of-function OBSCN variants predispose individuals to severe recurrent rhabdomyolysis.

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

FXR1-related congenital myopathy: expansion of the clinical and genetic spectrum.

BACKGROUND: Biallelic pathogenic variants in FXR1 have recently been associated with two congenital myopathy phenotypes: a severe form associated with hypotonia, long bone fractures, respiratory insufficiency and infantile death, and a milder form characterised by proximal muscle weakness with survival into adulthood. OBJECTIVE: We report eight patients from four unrelated families with biallelic pathogenic variants in exon 15 of FXR1. METHODS: Whole exome sequencing was used to detect variants in FXR1. RESULTS: Common clinical features were noted for all patients, which included proximal myopathy, normal serum creatine kinase levels and diffuse muscle atrophy with relative preservation of the quadriceps femoris muscle on muscle imaging. Additionally, some patients with FXR1-related myopathy had respiratory involvement and required bilevel positive airway pressure support. Muscle biopsy showed multi-minicores and type I fibre predominance with internalised nuclei. CONCLUSION: FXR1-related congenital myopathy is an emerging entity that is clinically recognisable. Phenotypic variability associated with variants in FXR1 can result from differences in variant location and type and is also observed between patients homozygous for the same variant, rendering specific genotype-phenotype correlations difficult. Our work broadens the phenotypic spectrum of FXR1-related congenital myopathy.

Partial loss-of-function variant in neuregulin 1 identified in family with heritable peripheral neuropathy.

Neuregulin 1 signals are essential for the development and function of Schwann cells, which form the myelin sheath on peripheral axons. Disruption of myelin in the peripheral nervous system can lead to peripheral neuropathy, which is characterized by reduced axonal conduction velocity and sensorimotor deficits. Charcot-Marie-Tooth disease is a group of heritable peripheral neuropathies that may be caused by variants in nearly 100 genes. Despite the evidence that Neuregulin 1 is essential for many aspects of Schwann cell development, previous studies have not reported variants in the neuregulin 1 gene (NRG1) in patients with peripheral neuropathy. We have identified a rare missense variant in NRG1 that is homozygous in a patient with sensory and motor deficits consistent with mixed axonal and de-myelinating peripheral neuropathy. Our in vivo functional studies in zebrafish indicate that the patient variant partially reduces NRG1 function. This study tentatively suggests that variants at the NRG1 locus may cause peripheral neuropathy and that NRG1 should be investigated in families with peripheral neuropathy of unknown cause.

Systemic AAV8-mediated delivery of a functional copy of muscle glycogen phosphorylase (Pygm) ameliorates disease in a murine model of McArdle disease.

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1-3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients.

Recent advances in our understanding of genetic rhabdomyolysis.

PURPOSE OF REVIEW: This review summarizes recent advances in our understanding of the genetics of rhabdomyolysis. RECENT FINDINGS: Rhabdomyolysis is the acute breakdown of myofibres resulting in systemic changes that can be life-threatening. Environmental triggers, including trauma, exercise, toxins and infections, and/or gene defects can precipitate rhabdomyolysis. A schema (aptly titled RHABDO) has been suggested for evaluating whether a patient with rhabdomyolysis is likely to harbour an underlying genetic defect. It is becoming increasingly recognized that defects in muscular dystrophy and myopathy genes can trigger rhabdomyolysis, even as the sole or presenting feature. Variants in genes not previously associated with human disease have been identified recently as causative of rhabdomyolysis, MLIP , MYH1 and OBSCN . Our understanding of the pathomechanisms contributing to rhabdomyolysis have also improved with an increased awareness of the role of mitochondrial dysfunction in LPIN1 , FDX2 , ISCU and TANGO2 -mediated disease. SUMMARY: An accurate genetic diagnosis is important for optimal clinical management of the patient, avoiding associated triggers and genetic counselling and cascade screening. Despite recent advances in our understanding of the genetics contributing to rhabdomyolysis, many patients remain without an accurate genetic diagnosis, suggesting there are many more causative genes, variants and disease mechanisms to uncover.

A TOR1AIP1 variant segregating with an early onset limb girdle myasthenia-Support for the role of LAP1 in NMJ function and disease.

Rare pathogenic variants in TOR1AIP1 (OMIM 614512), coding the inner nuclear membrane protein lamin-associated protein 1 (LAP1), have been associated with a spectrum of disorders including limb girdle muscular dystrophy with cardiac involvement and a severe multisystem phenotype. Recently, Cossins et al reported two siblings with limb girdle muscular dystrophy and impaired transmission of the neuromuscular synapse, demonstrating that defective LAP1 may lead to a congenital myasthenic syndrome. Herein, we describe the association of TOR1AIP1 deficiency with congenital myasthenic syndrome in three siblings.

Identification of a novel heterozygous DYSF variant in a large family with a dominantly-inherited dysferlinopathy.

AIMS: Dysferlinopathy is an autosomal recessive muscular dystrophy, caused by bi-allelic variants in the gene encoding dysferlin (DYSF). Onset typically occurs in the second to third decade and is characterised by slowly progressive skeletal muscle weakness and atrophy of the proximal and/or distal muscles of the four limbs. There are rare cases of symptomatic DYSF variant carriers. Here, we report a large family with a dominantly inherited hyperCKaemia and late-onset muscular dystrophy. METHODS AND RESULTS: Genetic analysis identified a co-segregating novel DYSF variant [NM_003494.4:c.6207del p.(Tyr2070Metfs*4)]. No secondary variants in DYSF or other dystrophy-related genes were identified on whole genome sequencing and analysis of the proband's DNA. Skeletal muscle involvement was milder and later onset than typical dysferlinopathy presentations; these clinical signs manifested in four individuals, all between the fourth and sixth decades of life. All individuals heterozygous for the c.6207del variant had hyperCKaemia. Histological analysis of skeletal muscle biopsies across three generations showed clear dystrophic signs, including inflammatory infiltrates, regenerating myofibres, increased variability in myofibre size and internal nuclei. Muscle magnetic resonance imaging revealed fatty replacement of muscle in two individuals. Western blot and immunohistochemical analysis of muscle biopsy demonstrated consistent reduction of dysferlin staining. Allele-specific quantitative PCR analysis of DYSF mRNA from patient muscle found that the variant, localised to the extreme C-terminus of dysferlin, does not activate post-transcriptional mRNA decay. CONCLUSIONS: We propose that this inheritance pattern may be underappreciated and that other late-onset muscular dystrophy cases with mono-allelic DYSF variants, particularly C-terminal premature truncation variants, may represent dominant forms of disease.

Biallelic hypomorphic variants in ALDH1A2 cause a novel lethal human multiple congenital anomaly syndrome encompassing diaphragmatic, pulmonary, and cardiovascular defects.

This study shows a causal association between ALDH1A2 variants and a novel, severe multiple congenital anomaly syndrome in humans that is neonatally lethal due to associated pulmonary hypoplasia and respiratory failure. In two families, exome sequencing identified compound heterozygous missense variants in ALDH1A2. ALDH1A2 is involved in the conversion of retinol (vitamin A) into retinoic acid (RA), which is an essential regulator of diaphragm and cardiovascular formation during embryogenesis. Reduced RA causes cardiovascular, diaphragmatic, and associated pulmonary defects in several animal models, matching the phenotype observed in our patients. In silico protein modeling showed probable impairment of ALDH1A2 for three of the four substitutions. In vitro studies show a reduction of RA. Few pathogenic variants in genes encoding components of the retinoic signaling pathway have been described to date, likely due to embryonic lethality. Thus, this study contributes significantly to knowledge of the role of this pathway in human diaphragm and cardiovascular development and disease. Some clinical features in our patients are also observed in Fryns syndrome (MIM# 229850), syndromic microphthalmia 9 (MIM# 601186), and DiGeorge syndrome (MIM# 188400). Patients with similar clinical features who are genetically undiagnosed should be tested for recessive ALDH1A2-deficient malformation syndrome.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

A novel RFC1 repeat motif (ACAGG) in two Asia-Pacific CANVAS families.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive late-onset, neurological disease. Recently, a pentanucleotide expansion in intron 2 of RFC1 was identified as the genetic cause of CANVAS. We screened an Asian-Pacific cohort for CANVAS and identified a novel RFC1 repeat expansion motif, (ACAGG)exp, in three affected individuals. This motif was associated with additional clinical features including fasciculations and elevated serum creatine kinase. These features have not previously been described in individuals with genetically-confirmed CANVAS. Haplotype analysis showed our patients shared the same core haplotype as previously published, supporting the possibility of a single origin of the RFC1 disease allele. We analysed data from >26 000 genetically diverse individuals in gnomAD to show enrichment of (ACAGG) in non-European populations.

A Maori specific RFC1 pathogenic repeat configuration in CANVAS, likely due to a founder allele.

Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) is a recently recognized neurodegenerative disease with onset in mid- to late adulthood. The genetic basis for a large proportion of Caucasian patients was recently shown to be the biallelic expansion of a pentanucleotide (AAGGG)n repeat in RFC1. Here, we describe the first instance of CANVAS genetic testing in New Zealand Maori and Cook Island Maori individuals. We show a novel, possibly population-specific CANVAS configuration (AAAGG)10-25(AAGGG)exp, which was the cause of CANVAS in all patients. There were no apparent phenotypic differences compared with European CANVAS patients. Presence of a common disease haplotype among this cohort suggests this novel repeat expansion configuration is a founder effect in this population, which may indicate that CANVAS will be especially prevalent in this group. Haplotype dating estimated the most recent common ancestor at ∼1430 ce. We also show the same core haplotype as previously described, supporting a single origin of the CANVAS mutation.