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Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. We collected data from over 1.5 million genetic tests from 19 clinical laboratories across the United States and Canada from during 2020-2021. We found a lower rate of inconclusive results due to VUS on ES/GS tests compared to MGPs (22.5% vs. 32.6%; p

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Argl5G1n], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229deIC [p.G1n77Lys*11], c.399_400del [p.G1u133Aspfs*37], c.747G>T [p.G1n249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1 gamma 1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1 gamma 1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1 gamma 1 protein folding for missense variants, which was consistent with the observed altered AP1 gamma 1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1 gamma 1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out aplgl in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

Carpal and cubital tunnel syndrome (CTS, CuTS) are common among patients with hereditary neuropathy with liability to pressure-palsies (HNPP) and Charcot-Marie-Tooth type 1A (CMT1A) and may impact quality of life. We aimed to evaluate the utility of nerve decompression surgeries in these patients.Medical records were reviewed for patients with PMP22 mutations confirmed in Mayo Clinic laboratories from January 1999 to December 2020, who had CTS and CuTS and underwent surgical decompression.CTS occurred in 53.3% of HNPP and 11.5% of CMT1A, while CuTS was present in 43.3% of HNPP and 5.8% of CMT1A patients. CTS decompression occurred in 10-HNPP and 5-CMT1A patients, and CuTS decompression with/without transposition was performed in 5-HNPP and 1-CMT1A patients. In HNPP, electrodiagnostic studies identified median neuropathy at the wrist in 9/10 patients and ultrasound showed focal enlargements at the carpal and cubital tunnels. In CMT1A, median and ulnar sensory responses were all absent, and the nerves were diffusely enlarged. After CTS surgery, pain, sensory loss, and strength improved in 4/5 CMT1A, and 6/10 HNPP patients. Of clinical, electrophysiologic and ultrasound findings, only activity-provoked features significantly correlated with CTS surgical benefit in HNPP patients (odds ratio = 117.0:95% confidence interval, 1.94 999.99, p = 0.01). One CMT1A and one HNPP patient improved with CuTS surgery while 2 HNPP patients worsened.CTS symptom improvement post-surgery can be seen in CMT1A and (less frequent) in HNPP patients. CuTS surgery commonly worsened course in HNPP. Activity-provoked symptoms in HNPP best informed benefits from CTS surgery.

Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Purpose Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. Methods From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. Results The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. Conclusion Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

The hereditary ataxias are a heterogenous group of disorders with an increasing number of causative genes being described. Due to the clinical and genetic heterogeneity seen in these conditions, the majority of such individuals endure a diagnostic odyssey or remain undiagnosed. Defining the molecular etiology can bring insights into the responsible molecular pathways and eventually the identification of therapeutic targets. Here, we describe the identification of biallelic variants in the

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive distal myopathy with rimmed vacuoles that typically manifests in late adolescence/early adulthood. GNE encodes an enzyme that is the rate-limiting step in sialic acid biosynthesis which is necessary for proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study we aimed to address this knowledge gap by querying underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem cell (iPSC) model. Muscle and skin biopsies were acquired from two patients with GNE myopathy that presented with distinct histopathological features. Control and patient-specific iPSCs were derived from skin fibroblasts and differentiated down a skeletal muscle lineage in a three-stage process analogous to muscle development and muscle regeneration. Initial studies revealed: 1) the ability of patient-derived GNE iPSC clones to recapitulate key characteristics of the human pathology including TDP-43 accumulation and evidence of dysregulated autophagy, and 2) a striking defect in myogenic progression of the more severe GNE iPSC clone. Single-cell RNA sequencing time course studies were then performed to more rigorously explore myogenesis defects. Cluster-based bioinformatics analyses revealed clear differences between control and GNE iPSC-derived muscle precursor cells (iMPCs). On a transcriptional level, late stage GNE iMPCs resembled that of early stage control iMPCs, confirming stalled myogenic progression on a molecular level. Comparative expression and pathway studies revealed EIF2 signaling as a top signaling pathway altered in GNE iMPCs. In summary, we report a novel in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a likely novel contributing mechanism to the etiology of GNE myopathy.SUMMARY STATEMENTDevelopment of a novel cell-based model of GNE myopathy, utilizing GNE patient-derived samples, which recapitulates human disease characteristics, uncovered myogenic differentiation defects, and can elucidate possible mechanistic contributors to the disease.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disease. Affected infants manifest with severe respiratory distress and refractory pulmonary hypertension and uniformly die in the first month of life. Heterozygous point mutations or copy-number variant deletions involving FOXF1 and/or its upstream lung-specific enhancer on 16q24.1 have been identified in the vast majority of ACDMPV patients. We have previously described two unrelated families with a de novo pathogenic frameshift variant c.691_698del (p.Ala231Argfs*61) in the exon 1 of FOXF1. Here, we present a third unrelated ACDMPV family with the same de novo variant and propose that a direct tandem repeat of eight consecutive nucleotides GCGGCGGC within the ~ 4 kb CpG island in FOXF1 exon 1 is a novel mutation hotspot causative for ACDMPV.

Background and ObjectivesThere are limited population-based data on small fiber neuropathy (SFN). We wished to determine SFN incidence, prevalence, comorbid conditions, longitudinal impairments, and disabilities.MethodsTest-confirmed patients with SFN in Olmsted, Minnesota, and adjacent counties were compared 3:1 to matched controls (January 1, 1998–December 31, 2017).ResultsNinety-four patients with SFN were identified, with an incidence of 1.3/100,000/y that increased over the study period and a prevalence of 13.3 per 100,000. Average follow-up was 6.1 years (0.7–43 years), and mean onset age was 54 years (range 14–83 years). Female sex (67%), obesity (body mass index mean 30.4 vs 28.5 kg/m2), insomnia (86% vs 54%), analgesic-opioid prescriptions (72% vs 46%), hypertriglyceridemia (180 mg/dL mean vs 147 mg/dL), and diabetes (51% vs 22%, p < 0.001) were more common (odds ratio 3.8–9.0, all p < 0.03). Patients with SFN did not self-identify as disabled with a median modified Rankin Scale score of 1.0 (range 0–6) vs 0.0 (0–6) for controls (p = 0.04). Higher Charlson comorbid conditions (median 6, range 3–9) occurred vs controls (median 3, range 1–9, p < 0.001). Myocardial infarctions occurred in 46% vs 27% of controls (p < 0.0001). Classifications included idiopathic (70%); diabetes (15%); Sjögren disease (2%); AL-amyloid (1%); transthyretin-amyloid (1%); Fabry disease (1%); lupus (1%); postviral (1%); Lewy body (1%), and multifactorial (5%). Foot ulcers occurred in 17, with 71% having diabetes. Large fiber neuropathy developed in 36%, on average 5.3 years (range 0.2–14.3 years) from SFN onset. Median onset Composite Autonomic Severity Score (CASS) was 3 (change per year 0.08, range 0–2.0). Median Neuropathy Impairment Scale (NIS) score was 2 at onset (range 0–8, change per year 1.0, range −7.9 to +23.3). NIS score and CASS change >1 point per year occurred in only AL-amyloid, hereditary transthyretin-amyloid, Fabry, uncontrolled diabetes, and Lewy body. Death after symptom onset was higher in patients with SFN (19%) vs controls (12%, p < 0.001), 50% secondary to diabetes complications.DiscussionIsolated SFN is uncommon but increasing in incidence. Most patients do not develop major neurologic impairments and disability but have multiple comorbid conditions, including cardiovascular ischemic events, and increased mortality from SFN onsets. Development of large fiber involvements and diabetes are common over time. Targeted testing facilitates interventional therapies for diabetes but also rheumatologic and rare genetic forms.

Wolf-Hirschhorn syndrome (WHS) is a microdeletion syndrome characterized by distinctive facial features consisting of "Greek warrior helmet" appearance, prenatal and postnatal growth deficiency, developmental disability, and seizures. This disorder is caused by heterozygous deletions on chromosome 4p16.3 often identified by cytogenetic techniques. Many groups have attempted to identify the critical region within this deletion to establish which genes are responsible for WHS. Herein, clinical whole exome sequencing (WES) was performed on a child with developmental delays, mild facial dysmorphisms, short stature, failure to thrive, and microcephaly, and revealed a de novo frameshift variant, c.1676_1679del (p.Arg559Tfs*38), in WHSC1 (NSD2). While WHSC1 falls within the WHS critical region, individuals with only disruption of this gene have only recently been described in the literature. Loss-of-function de novo variations in WHSC1 were identified in large developmental delay, autism, diagnostic, and congenital cardiac cohorts, as well as recent case reports, suggesting that de novo loss-of-function WHSC1 variants may be related to disease. These findings, along with our patient suggest that loss-of-function variation in WHSC1 may lead to a mild form of Wolf-Hirschhorn syndrome, and also may suggest that the developmental delays, facial dysmorphisms, and short stature seen in WHS may be due to disruption of WHSC1 gene.

Background Congenital myopathies due to ryanodine receptor (RYR1) mutations are increasingly identified and correlate with a wide range of phenotypes, most commonly that of malignant hyperthermia susceptibility and central cores on muscle biopsy with rare reports of distal muscle weakness, but in the setting of early onset global weakness. Methods We report a case of a patient presenting with childhood onset hand stiffness and adult onset progressive hand weakness and jaw contractures discovered to have two variants in the RYR1 gene. Results The patient manifested with distal upper limb weakness which progressed to involve the distal lower limb, proximal upper limb, as well as the face in addition to limited jaw opening. Creatine kinase was mildly elevated with EMG findings supporting a myopathy. Muscle biopsy showed features consistent with centronuclear myopathy. Whole exome sequencing revealed a novel heterozygous pathogenic variant in RYR1 (c.12315_12328delAGAAATCCAGTTCC, p.Glu4106Alafs*8), and a heterozygous missense variant (c.10648C>T, p.Arg3550Trp) of unknown significance in compound heterozygous state. Conclusion We expand the spectrum of RYR1-related myopathy with the description of a novel phenotype in an adult patient presenting with hand weakness and suggest considering RYR1 analysis in the diagnosis of distal myopathies.

Familial transthyretin (TTR) amyloidosis (FTA) is a serious autosomal dominant neuropathy caused by TTR germline mutations that lead to death on average 10 years from symptom onset. The mutant tetramer protein is produced predominantly in the liver, and liver transplantation is commonly performed to reduce the production of the mutant protein.1 The otherwise well-working explanted TTR liver can be transplanted into recipients who require liver transplantation but are unable to receive a liver in a timely fashion, the so-called domino liver transplant (DLTX). Recently, oligonucleotide drugs that reduce circulating TTR have been approved by the Food and Drug Administration (FDA), and these drugs use either antisense (inotersen) or RNA interference (patisiran), whereas earlier tetramer-stabilizing drugs (tafamidis and diflunisal) were less efficacious.2 Herein, we describe a domino liver recipient who died of respiratory failure from transplanted p.Val71Ala amyloidosis.

ObjectiveTo expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene–related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.MethodsWhole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays.ResultsCommon clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals.ConclusionsOur results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Actinopathy is a group of clinically and pathologically heterogeneous myopathies due to mutations in the skeletal muscle sarcomeric α-actin 1-encoding gene (ACTA1). Disease-onset spans from prenatal life to adulthood and weakness can preferentially affect proximal or distal muscles. Myopathological findings include a spectrum of structural abnormalities with nemaline rods being the most common. We report a daughter and father with prominent finger flexors and/or quadriceps involvement. Muscle biopsies revealed rimmed vacuoles in both patients, associated with type 1 fiber atrophy in the daughter, and nemaline rods in the father. Next generation sequencing identified a novel dominant ACTA1 variant, c.149G > A (p.Gly50Asp) in both individuals and no abnormal variants in vacuolar myopathy-associated genes. Our findings expand the clinico-pathological spectrum of actinopathy.

Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive-compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4's function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.Molecular Psychiatry advance online publication, 23 August 2016; doi:10.1038/mp.2016.135. ispartof: Molecular Psychiatry vol:23 issue:2 pages:222-230 ispartof: location:England status: published

Mutations in CRIPT encoding cysteine-rich PDZ domain-binding protein are rare, and to date have been reported in only two patients with autosomal recessive primordial dwarfism and distinctive facies. Here, we describe a female with biallelic mutations in CRIPT presenting with postnatal growth retardation, global developmental delay, and dysmorphic features including frontal bossing, high forehead, and sparse hair and eyebrows. Additional clinical features included high myopia, admixed hyper- and hypopigmented macules primarily on the face, arms, and legs, and syndactyly of 4-5 toes bilaterally. Using whole exome sequencing (WES) and chromosomal microarray analysis (CMA), we detected a c.8G>A (p.C3Y) missense variant in exon 1 of the CRIPT gene inherited from the mother and a 1,331 bp deletion encompassing exon 1, inherited from the father. The c.8G>A (p.C3Y) missense variant in CRIPT was apparently homozygous in the proband due to the exon 1 deletion. Our findings illustrate the clinical utility of combining WES with copy number variant (CNV) analysis to provide a molecular diagnosis to patients with rare Mendelian disorders. Our findings also illustrate the clinical spectrum of CRIPT related mutations. © 2016 Wiley Periodicals, Inc.

Perineuriomas are rare nerve sheath tumors, divided into intraneural and extraneural (soft tissue) types. Intraneural perineuriomas frequently contain TRAF7 mutations, and rarely, chr22q12 deletions. While chr22q losses can occur in soft tissue perineuriomas, comprehensive high-resolution molecular profiling has not been reported in these tumors and TRAF7 status is unknown. We used whole-exome sequencing and OncoScan single nucleotide polymorphism (SNP) array to evaluate 14 soft tissue perineuriomas. Thirteen cases showed 2 or more chromosomal abnormalities, composed primarily of large deletions. Recurrent chr22q deletions, containing the NF2 locus (n=6) and the previously unreported finding of chr17q deletions, with the NF1 locus (n=4) were frequent events and were mutually exclusive in all but1 case. In addition, 5 cases had varying chr2 deletions; and 4 cases had chr6 deletions. A chr10 deletion (previously reported in the sclerosing variant of soft tissue perineurioma) was observed in one case and another case had chr7 chromothripsis as the sole chromosomal abnormality. No TRAF7 mutations or alterations were identified in any case and no other evaluated gene (MAF0.0001) had recurrent, deleterious mutations in2 cases. The molecular genetic profiles showed no association with patient sex, age, tumoral histology or anatomic site. OncoScan SNP array analysis was performed on 10 cases and showed high concordance with the whole exome data, validating the large-scale deletions, duplications, and chr7 chromothripsis findings. In soft tissue perineuriomas, recurrent 22q12 deletions (with NF2) and 17q11 deletions (with NF1) appear to be mutually exclusive events, and alterations in NF1 or NF2 likely contribute to perineurioma pathogenesis, similar to other nerve sheath tumors. Moreover, the lack of TRAF7 mutations in soft tissue perineuriomas indicates divergent pathogenetic mechanisms from those of intraneural perineuriomas.

X-linked adrenoleukodystrophy is a peroxisomal disorder caused by a mutation in ABCD1 gene. The three main phenotypes of X-linked adrenoleukodystrophy include cerebral adrenoleukodystrophy, adrenomyeloneuropathy, and isolated primary adrenal insufficiency. More than 750 non-recurrent mutations exist throughout the coding region of the ABCD1 gene. We report a 62-year-old man with a 17-year history of progressive gait imbalance and numb feet. He had noted difficulty rising from a chair for 3 years. Examination revealed proximal lower limb weakness and length-dependent sensory loss with preservation of reflexes and unilateral Babinski sign. Electrodiagnostic evaluation confirmed a length-dependent sensorimotor peripheral neuropathy and proximal myopathy. Family history was remarkable for similar symptoms in 6 siblings. A targeted gene approach for 102 known peripheral neuropathy genes led to discovery of ABCD1 mutation confirmed by kindred evaluation and biochemical assay. This case highlights the importance of combining targeted gene approaches with functional assay confirmation especially for atypical clinical presentations.

Background Rare de novo mutations have been implicated as a significant cause of idiopathic intellectual disability. Large deletions encompassing 10p11.23 have been implicated in developmental delay, behavioural abnormalities and dysmorphic features, but the genotype–phenotype correlation was not delineated. Mutations in WAC have been recently reported in large screening cohorts of patients with intellectual disability or autism, but no full phenotypic characterisation was described. Methods Clinical and molecular characterisation of six patients with loss-of-function WAC mutations identified by whole exome sequencing was performed. Clinical data were obtained by retrospective chart review, parental interviews, direct patient interaction and formal neuropsychological evaluation. Results Five heterozygous de novo WAC mutations were identified in six patients. Three of the mutations were nonsense, and two were frameshift; all are predicted to cause loss of function either through nonsense-mediated mRNA decay or protein truncation. Clinical findings included developmental delay (6/6), hypotonia (6/6), behavioural problems (5/6), eye abnormalities (5/6), constipation (5/6), feeding difficulties (4/6), seizures (2/6) and sleep problems (2/6). All patients exhibited common dysmorphic features, including broad/prominent forehead, synophrys and/or bushy eyebrows, depressed nasal bridge and bulbous nasal tip. Posteriorly rotated ears, hirsutism, deep-set eyes, thin upper lip, inverted nipples, hearing loss and branchial cleft anomalies were also noted. Conclusions Our case series show that loss-of-function mutations in WAC cause a recognisable genetic syndrome characterised by a neurocognitive phenotype and facial dysmorphism. Our data highly suggest that WAC haploinsufficiency is responsible for most of the phenotypic features associated with deletions encompassing 10p11.23.

Introduction: A calpain-3 (CAPN3) gene heterozygous deletion (c.643_663del21) was recently linked to autosomal dominant (AD) limb girdle muscular dystrophy. However, the possibility of digenic disease was raised. We describe three families with AD calpainopathy carrying this isolated mutation. Methods: Probands heterozygous for CAPN3 c.643_663del21 were identified by targeted next generation or whole exome sequencing. Clinical findings were collected for probands and families. Calpain-3 muscle western blots were performed in three unrelated individuals. Results: Probands reported variable weakness in their 40s-50s with myalgia, back pain or hyperlordosis. Pelvic girdle muscles were affected with adductor and hamstring sparing. CK was normal-1,800 U/L independent of weakness severity. Imaging demonstrated lumbar paraspinal muscle atrophy. EMG and muscle biopsies were normal to mildly myopathic. Muscle calpain-3 expression was reduced. Discussion: This study provides further evidence for AD calpainopathy associated with CAPN3 c.643_663del21. No pathogenic variants in other genes known to cause myopathy were detected. This article is protected by copyright. All rights reserved.

Background and purpose CACNA1S encodes Cav 1.1, a voltage sensor for muscle excitation-contraction coupling, which activates the ryanodine receptor 1 (RYR1) leading to calcium release from the sarcoplasmic reticulum. CACNA1S mutations cause hypokalemic periodic paralysis, malignant hyperthermia and congenital myopathy. RYR1 mutations result in congenital myopathy, malignant hyperthermia and rhabdomyolysis. Methods The aim was to describe a novel phenotype associated with a CACNA1S variant at a site previously linked to hypokalemic periodic paralysis. Results The patient presented with fluctuating asymptomatic creatine kinase elevation after an episode of rhabdomyolysis but has no history of periodic paralysis. His muscle biopsy showed core-like structures occurring mainly in type 2 fibers. He carries a novel Cav 1.1 variant (p.Arg528Leu) affecting a highly conserved amino acid. Different mutations at the same location cause hypokalemic periodic paralysis. Conclusion This case underscores the similarity between the phenotypes caused by mutations in two functionally linked proteins, RYR1 and Cav 1.1.

Bosch–Boonstra–Schaaf optic atrophy syndrome (BBSOAS) is a recently described autosomal dominant disorder caused by mutations in the NR2F1 gene. There are presently 28 cases of BBSOAS described in the literature. Its common features include developmental delay, intellectual disability, hypotonia, optic nerve atrophy, attention deficit disorder, autism spectrum disorder, seizures, hearing defects, spasticity, and thinning of the corpus callosum. Here we report two unrelated probands with novel, de novo, missense variants in NR2F1. The first is a 14-yr-old male patient with hypotonia, intellectual disability, optic nerve hypoplasia, delayed bone age, short stature, and altered neurotransmitter levels on cerebrospinal fluid testing. The second is a 5-yr-old female with severe developmental delay, motor and speech delay, and repetitive motion behavior. Whole-exome sequencing identified a novel missense NR2F1 variant in each case, Cys86Phe in the DNA-binding domain in Case 1, and a Leu372Pro in the ligand-binding domain in Case 2. The presence of clinical findings compatible with BBSOAS along with structural analysis at atomic resolution using homology-based molecular modeling and molecular dynamic simulations, support the pathogenicity of these variants for BBSOAS. Short stature, abnormal CNS neurotransmitters, and macrocephaly have not been previously reported for this syndrome and may represent a phenotypic expansion of BBSOAS. A review of published cases along with new evidence from this report support genotype–phenotype correlations for this disorder.

Pathogenic variants in EBF3 were recently described in three back-to-back publications in association with a novel neurodevelopmental disorder characterized by intellectual disability, speech delay, ataxia, and facial dysmorphisms. In this report, we describe an additional patient carrying a de novo missense variant in EBF3 (c.487C>T, p.(Arg163Trp)) that falls within a conserved residue in the zinc knuckle motif of the DNA binding domain. Without a solved structure of the DNA binding domain, we generated a homology-based atomic model and performed molecular dynamics simulations for EBF3, which predicted decreased DNA affinity for p.(Arg163Trp) compared with wild-type protein and control variants. These data are in agreement with previous experimental studies of EBF1 showing the paralogous residue is essential for DNA binding. The conservation and experimental evidence existing for EBF1 and in silico modeling and dynamics simulations to validate comparable behavior of multiple variants in EBF3 demonstrates strong support for the pathogenicity of p.(Arg163Trp). We show that our patient presents with phenotypes consistent with previously reported patients harboring EBF3 variants and expands the phenotypic spectrum of this newly identified disorder with the additional feature of a bicornuate uterus.

Gardner syndrome describes a variant phenotype of familial adenomatous polyposis (FAP), primarily characterized by extracolonic lesions including osteomas, dental abnormalities, epidermal cysts, and soft tissue tumors. We describe a 2-yr-old boy presenting with a 2-cm soft tissue mass of the forehead. Pathologic evaluation revealed a nuchal-type/Gardner-associated fibroma. Sequencing of the APC gene revealed a pathologic variant c.4666dupA. Parental sequencing of both blood and buccal tissue supported the de novo occurrence of this pathologic variant. Further imaging revealed a number of additional lesions including a large lumbar paraspinal desmoid, a 1-cm palpable lesion posterior to the left knee, firm lesions on bilateral heels, and multiple subdermal lesions. Colonoscopy was negative. This case illustrates a genetic variant of Gardner syndrome resulting in an aggressive early childhood phenotype and highlights the need for an individualized approach to treatment.

Whole-exome sequencing is a diagnostic approach for the identification of molecular defects in patients with suspected genetic disorders.We developed technical, bioinformatic, interpretive, and validation pipelines for whole-exome sequencing in a certified clinical laboratory to identify sequence variants underlying disease phenotypes in patients.We present data on the first 250 probands for whom referring physicians ordered whole-exome sequencing. Patients presented with a range of phenotypes suggesting potential genetic causes. Approximately 80% were children with neurologic phenotypes. Insurance coverage was similar to that for established genetic tests. We identified 86 mutated alleles that were highly likely to be causative in 62 of the 250 patients, achieving a 25% molecular diagnostic rate (95% confidence interval, 20 to 31). Among the 62 patients, 33 had autosomal dominant disease, 16 had autosomal recessive disease, and 9 had X-linked disease. A total of 4 probands received two nonoverlapping molecular diagnoses, which potentially challenged the clinical diagnosis that had been made on the basis of history and physical examination. A total of 83% of the autosomal dominant mutant alleles and 40% of the X-linked mutant alleles occurred de novo. Recurrent clinical phenotypes occurred in patients with mutations that were highly likely to be causative in the same genes and in different genes responsible for genetically heterogeneous disorders.Whole-exome sequencing identified the underlying genetic defect in 25% of consecutive patients referred for evaluation of a possible genetic condition. (Funded by the National Human Genome Research Institute.).

Haploinsufficiency of CHD7 (OMIM# 608892) is known to cause CHARGE syndrome (OMIM# 214800). Molecular testing supports a definitive diagnosis in approximately 65–70% of cases. Most CHD7 mutations arise de novo, and no mutations affecting exon-7 have been reported to date. We report on an 8-year-old girl diagnosed with CHARGE syndrome that was referred to our laboratory for comprehensive CHD7 gene screening. Genomic DNA from the subject with a suspected diagnosis of CHARGE was isolated from peripheral blood lymphocytes and comprehensive Sanger sequencing, along with deletion/duplication analysis of the CHD7 gene using multiplex ligation-dependent probe amplification (MLPA), was performed. MLPA analysis identified a reduced single probe signal for exon-7 of the CHD7 gene consistent with potential heterozygous deletion. Long-range PCR breakpoint analysis identified a complex genomic rearrangement (CGR) leading to the deletion of exon-7 and breakpoints consistent with a replicative mechanism such as fork stalling and template switching (FoSTeS) or microhomology-mediated break-induced replication (MMBIR). Taken together this represents the first evidence for a CHD7 intragenic CGR in a patient with CHARGE syndrome leading to what appears to be also the first report of a mutation specifically disrupting exon-7. Although likely rare, CGR may represent an overlooked mechanism in subjects with CHARGE syndrome that can be missed by current sequencing and dosage assays. © 2013 Wiley Periodicals, Inc.