Combining exome/genome sequencing with data repository analysis reveals novel gene-disease associations for a wide range of genetic disorders.

PURPOSE: Within this study, we aimed to discover novel gene-disease associations in patients with no genetic diagnosis after exome/genome sequencing (ES/GS). METHODS: We followed two approaches: (1) a patient-centered approach, which after routine diagnostic analysis systematically interrogates variants in genes not yet associated to human diseases; and (2) a gene variant centered approach. For the latter, we focused on de novo variants in patients that presented with neurodevelopmental delay (NDD) and/or intellectual disability (ID), which are the most common reasons for genetic testing referrals. Gene-disease association was assessed using our data repository that combines ES/GS data and Human Phenotype Ontology terms from over 33,000 patients. RESULTS: We propose six novel gene-disease associations based on 38 patients with variants in the BLOC1S1, IPO8, MMP15, PLK1, RAP1GDS1, and ZNF699 genes. Furthermore, our results support causality of 31 additional candidate genes that had little published evidence and no registered OMIM phenotype (56 patients). The phenotypes included syndromic/nonsyndromic NDD/ID, oral-facial-digital syndrome, cardiomyopathies, malformation syndrome, short stature, skeletal dysplasia, and ciliary dyskinesia. CONCLUSION: Our results demonstrate the value of data repositories which combine clinical and genetic data for discovering and confirming gene-disease associations. Genetic laboratories should be encouraged to pursue such analyses for the benefit of undiagnosed patients and their families.

Endoplasmic reticulum quality control of LDLR variants associated with familial hypercholesterolemia.

Loss-of-function mutations in the low-density lipoprotein receptor (LDLR) gene can cause familial hypercholesterolemia (FH), but detailed functional evidence for pathogenicity is limited to a few reported mutations. Here, we investigated the cellular pathogenic mechanisms of three mutations in LDLR causing FH, which are structurally identical to pathogenic mutations in the very low-density lipoprotein receptor (VLDLR). Similar to the VLDLR mutants, LDLR mutants D482H and C667F were found to be localized to the ER, while D445E, which is a conserved amino acid change, did not affect the trafficking of the receptor to the plasma membrane, as confirmed by the N-glycosylation profile. Although the ER-retained mutant proteins were soluble, induction of ER stress was observed as indicated by spliced X-box binding protein-1 (XBP-1) mRNA levels. The mutants were found to associate with ER quality control components, and their stability was enhanced by inhibitors of proteasome. Our results contribute to the growing list of transport-deficient class II LDLR variants leading to FH and provide evidence for the involvement of endoplasmic reticulum-associated degradation in their stability.

Homozygous missense variant in BMPR1A resulting in BMPR signaling disruption and syndromic features.

BACKGROUND: The bone morphogenetic protein (BMP) pathway is known to play an imperative role in bone, cartilage, and cardiac tissue formation. Truncating, heterozygous variants, and deletions of one of the essential receptors in this pathway, Bone Morphogenetic Protein Receptor Type1A (BMPR1A), have been associated with autosomal dominant juvenile polyposis. Heterozygous deletions have also been associated with cardiac and minor skeletal anomalies. Populations with atrioventricular septal defects are enriched for rare missense BMPR1A variants. METHODS: We report on a patient with a homozygous missense variant in BMPR1A causing skeletal abnormalities, growth failure a large atrial septal defect, severe subglottic stenosis, laryngomalacia, facial dysmorphisms, and developmental delays. RESULTS: Functional analysis of this variant shows increased chondrocyte death for cells with the mutated receptor, increased phosphorylated R-Smads1/5/8, and loss of Sox9 expression mediated by decreased phosphorylation of p38. CONCLUSION: This homozygous missense variant in BMPR1A appears to cause a distinct clinical phenotype.

A recessive truncating variant in thrombospondin-1 domain containing protein 1 gene THSD1 is the underlying cause of nonimmune hydrops fetalis, congenital cardiac defects, and haemangiomas in four patients from a consanguineous family.

Non-immune hydrops fetalis (NIHF) is the abnormal accumulation of serous fluid in more than two fetal or neonatal interstitial spaces due to nonimmune causes. It is a serious condition that requires extensive medical care as it indicates severe fetal compromise. We clinically evaluated four patients from two branches of a highly consanguineous family from the UAE with NIHF using whole exome sequencing and in silico analysis. Fetal onset pleural and peritoneal effusions were detected in all four patients and were born with moderate to severe hydrops fetalis that resolved with age. Follow up showed relatively normal growth and development apart from mild ascites and haemangiomas in all affected children, recurrent hydrocele in all affected males, intestinal malabsorption in two patients, dysmorphic features in two patients, and congenital cardiac defects in three out of four patients. Molecular testing identified a homozygous eight nucleotide deletion in THSD1 gene (NM_199263:c.1163_1170delGGCCAGCC, p.Arg388Glnfs*66) as the underlying cause of this phenotype in the affected children. The novel variant cosegregates with the described phenotype in an autosomal recessive mode of inheritance and is predicted to be pathogenic as it leads to a truncated protein that lost important structural and functional domains. Thrombospondin-1 domain containing protein 1 gene THSD1 has been recently associated with of NIHF and embryonic lethality. Here, we report the novel truncating THSD1 variant, and describe new clinical features that have not been reported previously thus expanding the phenotype associate with loss-of-function mutations in THSD1 causing NIHF.

Clathrin heavy chain 22 contributes to the control of neuropeptide degradation and secretion during neuronal development.

The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active 'mature' forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway.

Defect in phosphoinositide signalling through a homozygous variant in PLCB3 causes a new form of spondylometaphyseal dysplasia with corneal dystrophy.

BACKGROUND: Bone dysplasias are a large group of disorders affecting the growth and structure of the skeletal system. METHODS: In the present study, we report the clinical and molecular delineation of a new form of syndromic autosomal recessive spondylometaphyseal dysplasia (SMD) in two Emirati first cousins. They displayed postnatal growth deficiency causing profound limb shortening with proximal and distal segments involvement, narrow chest, radiological abnormalities involving the spine, pelvis and metaphyses, corneal clouding and intellectual disability. Whole genome homozygosity mapping localised the genetic cause to 11q12.1-q13.1, a region spanning 19.32 Mb with ~490 genes. Using whole exome sequencing, we identified four novel homozygous variants within the shared block of homozygosity. Pathogenic variants in genes involved in phospholipid metabolism, such as PLCB4 and PCYT1A, are known to cause bone dysplasia with or without eye anomalies, which led us to select PLCB3 as a strong candidate. This gene encodes phospholipase C ß 3, an enzyme that converts phosphatidylinositol 4,5 bisphosphate (PIP2) to inositol 1,4,5 triphosphate (IP3) and diacylglycerol. RESULTS: The identified variant (c.2632G>T) substitutes a serine for a highly conserved alanine within the Ha2' element of the proximal C-terminal domain. This disrupts binding of the Ha2' element to the catalytic core and destabilises PLCB3. Here we show that this hypomorphic variant leads to elevated levels of PIP2 in patient fibroblasts, causing disorganisation of the F-actin cytoskeleton. CONCLUSIONS: Our results connect a homozygous loss of function variant in PLCB3 with a new SMD associated with corneal dystrophy and developmental delay (SMDCD).

Pharmaceutical Chaperones and Proteostasis Regulators in the Therapy of Lysosomal Storage Disorders: Current Perspective and Future Promises.

Different approaches have been utilized or proposed for the treatment of lysosomal storage disorders (LSDs) including enzyme replacement and hematopoietic stem cell transplant therapies, both aiming to compensate for the enzymatic loss of the underlying mutated lysosomal enzymes. However, these approaches have their own limitations and therefore the vast majority of LSDs are either still untreatable or their treatments are inadequate. Missense mutations affecting enzyme stability, folding and cellular trafficking are common in LSDs resulting often in low protein half-life, premature degradation, aggregation and retention of the mutant proteins in the endoplasmic reticulum. Small molecular weight compounds such as pharmaceutical chaperones (PCs) and proteostasis regulators have been in recent years to be promising approaches for overcoming some of these protein processing defects. These compounds are thought to enhance lysosomal enzyme activity by specific binding to the mutated enzyme or by manipulating components of the proteostasis pathways promoting protein stability, folding and trafficking and thus enhancing and restoring some of the enzymatic activity of the mutated protein in lysosomes. Multiple compounds have already been approved for clinical use to treat multiple LSDs like migalastat in the treatment of Fabry disease and others are currently under research or in clinical trials such as Ambroxol hydrochloride and Pyrimethamine. In this review, we are presenting a general overview of LSDs, their molecular and cellular bases, and focusing on recent advances on targeting and manipulation proteostasis, including the use of PCs and proteostasis regulators, as therapeutic targets for some LSDs. In addition, we present the successes, limitations and future perspectives in this field.

Endoplasmic reticulum retention of xylosyltransferase 1 (XYLT1) mutants underlying Desbuquois dysplasia type II.

Desbuquois syndrome is a heterogeneous rare type of skeletal dysplasia with a prevalence of less than 1 in 1,000,000 individuals. It is characterized by short-limbed dwarfism, dysmorphic facial features, and severe joint laxity. Two types have been recognized depending on the presence of distinctive carpal and phalangeal features. Mutations in the calcium activated nucleotidase 1 (CANT1) have been found to be responsible for type I and lately, for the Kim type of Desbuquois dysplasia. In addition, a number of Desbuquois dysplasia type II patients have been attributed to mutations in xylosyltransferase 1, encoded by the XYLT1 gene, an enzyme that catalyzes the transfer of UDP-xylose (a marker of cartilage destruction) to serine residues of an acceptor protein, essential for the biosynthesis of proteoglycans. We report here a patient with features consistent with Desbuquois dysplasia II including short long bones, flat face, mild monkey wrench appearance of the femoral heads. Whole exome sequencing revealed a novel homozygous duplication of a single nucleotide in XYLT1 gene (c.2169dupA). This variant is predicted to result in a frame-shift and stop codon p.(Val724Serfs*10) within the xylosyltransferase catalytic domain. Immunoflourescence staining of HeLa cells transfected with mutated XYLT1 plasmids constructs of the current as well as the previously reported missense mutations (c.1441C>T, p.(Arg481Trp) and c.1792C>T, p.(Arg598Cys)), revealed aberrant subcellular localization of the enzyme compared to wild-type, suggesting endoplasmic reticulum retention of these mutants as the likely mechanism of disease.

A novel aberrant splice site mutation in COL27A1 is responsible for Steel syndrome and extension of the phenotype to include hearing loss.

Steel syndrome is an autosomal recessive disease characterized by skeletal abnormalities and dysmorphic features. The first mutation associated with this syndrome was reported in Puerto Rican children. In this study, we identified a novel homozygous splice site variant in COL27A1 (c.3556-2A>G) in a consanguineous Emirati family with a child affected by Steel syndrome. In addition, the affected child had severe non-progressive sensorineural hearing loss not reported previously. The variant segregated in the family in an autosomal recessive manner and we show that the variant alters mRNA splicing. Furthermore, relative quantitative analysis revealed a marked reduction in gene expression in the proposita compared to healthy controls. Segregation analysis of heterozygous variants, related to hearing loss, identified by whole exome sequencing in the child (ILDR1: c.1159T>C, SYNE4: c.313G>C, and GPR98: c.18746T>G) excluded them from being responsible for the hearing loss in the proposita. In addition, the products of these genes are not interacting in the same pathway and have only been reported to cause deafness in an autosomal recessive manner. Therefore, we conclude that the novel splice-site variant identified in COL27A1 is the most likely cause for Steel syndrome in this family and that the hearing loss is part of this syndrome's phenotype.

Germline mutations in ABL1 cause an autosomal dominant syndrome characterized by congenital heart defects and skeletal malformations.

ABL1 is a proto-oncogene well known as part of the fusion gene BCR-ABL1 in the Philadelphia chromosome of leukemia cancer cells. Inherited germline ABL1 changes have not been associated with genetic disorders. Here we report ABL1 germline variants cosegregating with an autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. The variant c.734A>G (p.Tyr245Cys) was found to occur de novo or cosegregate with disease in five individuals (families 1-3). Additionally, a de novo c.1066G>A (p.Ala356Thr) variant was identified in a sixth individual (family 4). We overexpressed the mutant constructs in HEK 293T cells and observed increased tyrosine phosphorylation, suggesting increased ABL1 kinase activities associated with both the p.Tyr245Cys and p.Ala356Thr substitutions. Our clinical and experimental findings, together with previously reported teratogenic effects of selective BCR-ABL inhibitors in humans and developmental defects in Abl1 knockout mice, suggest that ABL1 has an important role during organismal development.

A homozygous splicing mutation in ELAC2 suggests phenotypic variability including intellectual disability with minimal cardiac involvement.

BACKGROUND: The group of ELAC2-related encephalomyopathies is a recent addition to the rapidly growing heterogeneous mitochondrial disorders. RESULTS: We describe a highly inbred consanguineous Pakistani family with multiple affected children in 2 branches exhibiting moderately severe global developmental delay. Using homozygosity mapping, we mapped the phenotype in this family to a single locus on chromosome 17. In addition, whole-exome sequencing identified a homozygous splicing mutation (c.1423 + 2 T > A) in ELAC2 gene that disrupted the canonical donor splice site of intron 15 of all known isoforms. A noticeable reduction in ELAC2 expression was observed in patients compared to controls. In addition, patients exhibited significantly increased levels of 5' end unprocessed mt-RNAs compared to the control fibroblast cells. CONCLUSIONS: The only three previously reported families with defects in ELAC2 gene exhibited infantile hypertrophic cardiomyopathy and complex I deficiency. In contrast, our patients exhibited intellectual disability as the main feature with minimal cardiac involvement. Therefore our findings expand the phenotypic spectrum of ELAC2- associated disorders illustrating clinical heterogeneity of mutations in this gene. In addition, ELAC2 mutations should be considered when evaluating patient with mainly intellectual disability phenotypes.

Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes.

BACKGROUND: Ciliopathies are an extensive group of autosomal recessive or X-linked disorders with considerable genetic and clinical overlap, which collectively share multiple organ involvement and may result in lethal or viable phenotypes. In large numbers of cases the genetic defect remains yet to be determined. The aim of this study is to describe the mutational frequency and phenotypic spectrum of the CEP120 gene. METHODS: Exome sequencing was performed in 145 patients with Joubert syndrome (JS), including 15 children with oral-facial-digital syndrome type VI (OFDVI) and 21 Meckel syndrome (MKS) fetuses. Moreover, exome sequencing was performed in one fetus with tectocerebellar dysraphia with occipital encephalocele (TCDOE), molar tooth sign and additional skeletal abnormalities. As a parallel study, 346 probands with a phenotype consistent with JS or related ciliopathies underwent next-generation sequencing-based targeted sequencing of 120 previously described and candidate ciliopathy genes. RESULTS: We present six probands carrying nine distinct mutations (of which eight are novel) in the CEP120 gene, previously found mutated only in Jeune asphyxiating thoracic dystrophy (JATD). The CEP120-associated phenotype ranges from mild classical JS in four patients to more severe conditions in two fetuses, with overlapping features of distinct ciliopathies that include TCDOE, MKS, JATD and OFD syndromes. No obvious correlation is evident between the type or location of identified mutations and the ciliopathy phenotype. CONCLUSION: Our findings broaden the spectrum of phenotypes caused by CEP120 mutations that account for nearly 1% of patients with JS as well as for more complex ciliopathy phenotypes. The lack of clear genotype-phenotype correlation highlights the relevance of comprehensive genetic analyses in the diagnostics of ciliopathies.

A recessive syndrome of intellectual disability, moderate overgrowth, and renal dysplasia predisposing to Wilms tumor is caused by a mutation in FIBP gene.

Clinical classification of overgrowth syndromes represents a challenge since a wide spectrum of disorders result in marked overgrowth. Therefore, there is a continuous effort to identify the genetic basis of these disorders that will eventually facilitate their molecular classification. Here, we have identified the genetic etiology and the pathogenetic mechanism underlying a rare autosomal recessive overgrowth syndrome in three affected siblings. The overgrowth phenotype in the patients was accompanied by developmental delay, learning disabilities, and variable congenital abnormalities. To elucidate the genetic etiology of the disorder, whole-genome genotyping and whole-exome sequencing were used. The disease was mapped to 3p21.1-p14.2 and 11q13.1-q13.4, where an in-frame insertion (c.175_176insTAA) in FIBP gene was revealed. The resulting indel (p.H59LN) was predicted to change the protein conformation with likely deleterious effect on its function as one of the fibroblast growth factor signaling mediators. In vitro cellular proliferation assay and in situ hypridization in vivo were then performed to understand the pathophysiology of the disease. The patients' skin fibroblasts showed an increased proliferation capacity compared to the controls' explaining the observed overgrowth phenotype. In addition, we detected Fibp expression most notably in the brains of mice embryos suggesting a possible effect on cognitive functions early in development. To date, only one patient has been reported with a homozygous nonsense mutation in FIBP exhibiting an overgrowth syndrome with multiple congenital abnormalities. Taken all together, these findings provide convincing evidence implicating FIBP aberrations in the newly recognized overgrowth syndrome and expand the associated phenotypes to include possible Wilms tumor predisposition. © 2016 Wiley Periodicals, Inc.

EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.

Vici syndrome is a progressive neurodevelopmental multisystem disorder due to recessive mutations in the key autophagy gene EPG5. We report genetic, clinical, neuroradiological, and neuropathological features of 50 children from 30 families, as well as the neuronal phenotype of EPG5 knock-down in Drosophila melanogaster. We identified 39 different EPG5 mutations, most of them truncating and predicted to result in reduced EPG5 protein. Most mutations were private, but three recurrent mutations (p.Met2242Cysfs*5, p.Arg417*, and p.Gln336Arg) indicated possible founder effects. Presentation was mainly neonatal, with marked hypotonia and feeding difficulties. In addition to the five principal features (callosal agenesis, cataracts, hypopigmentation, cardiomyopathy, and immune dysfunction), we identified three equally consistent features (profound developmental delay, progressive microcephaly, and failure to thrive). The manifestation of all eight of these features has a specificity of 97%, and a sensitivity of 89% for the presence of an EPG5 mutation and will allow informed decisions about genetic testing. Clinical progression was relentless and many children died in infancy. Survival analysis demonstrated a median survival time of 24 months (95% confidence interval 0-49 months), with only a 10th of patients surviving to 5 years of age. Survival outcomes were significantly better in patients with compound heterozygous mutations (P = 0.046), as well as in patients with the recurrent p.Gln336Arg mutation. Acquired microcephaly and regression of skills in long-term survivors suggests a neurodegenerative component superimposed on the principal neurodevelopmental defect. Two-thirds of patients had a severe seizure disorder, placing EPG5 within the rapidly expanding group of genes associated with early-onset epileptic encephalopathies. Consistent neuroradiological features comprised structural abnormalities, in particular callosal agenesis and pontine hypoplasia, delayed myelination and, less frequently, thalamic signal intensity changes evolving over time. Typical muscle biopsy features included fibre size variability, central/internal nuclei, abnormal glycogen storage, presence of autophagic vacuoles and secondary mitochondrial abnormalities. Nerve biopsy performed in one case revealed subtotal absence of myelinated axons. Post-mortem examinations in three patients confirmed neurodevelopmental and neurodegenerative features and multisystem involvement. Finally, downregulation of epg5 (CG14299) in Drosophila resulted in autophagic abnormalities and progressive neurodegeneration. We conclude that EPG5-related Vici syndrome defines a novel group of neurodevelopmental disorders that should be considered in patients with suggestive features in whom mitochondrial, glycogen, or lysosomal storage disorders have been excluded. Neurological progression over time indicates an intriguing link between neurodevelopment and neurodegeneration, also supported by neurodegenerative features in epg5-deficient Drosophila, and recent implication of other autophagy regulators in late-onset neurodegenerative disease.

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport.

The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.

Defective cellular trafficking of the bone morphogenetic protein receptor type II by mutations underlying familial pulmonary arterial hypertension.

Familial pulmonary arterial hypertension (FPAH) is a relatively rare but fatal disorder characterized by elevated arterial pressure caused by abnormal proliferation of endothelial cells of the arteries, which eventually leads to heart failure and death. FPAH is inherited as an autosomal dominant trait and is caused by heterozygous mutations in the BMPR2 gene encoding the bone morphogenetic protein type II receptor (BMPR2). BMPR2 belongs to the TGF ß/BMP super-family of receptors involved in a signal transduction cascade via the SMAD signaling pathway. The BMPR2 polypeptide is composed of 1038 amino acids and consists of a ligand binding domain, a kinase domain and a cytoplasmic tail. To investigate the cellular and functional consequence of BMPR2 mutations, C-terminally FLAG-tagged constructs of eighteen pathogenic BMPR2 missense mutants were generated by site directed mutagenesis and expressed in HeLa and HEK-293T cell lines. The subcellular localizations of the mutant proteins were investigated using immunostaining and confocal microscopy. Post-translational modifications of the proteins were analyzed by Endoglycosidase H deglycosylation assay. Our results indicated that mutations in the ligand binding domain affecting highly conserved cysteine residues resulted in retention of the mutant proteins in the endoplasmic reticulum (ER), as evident from their co-localization with the ER resident protein calnexin. The kinase domain mutants showed both ER and plasma membrane (PM) distributions, while the cytoplasmic tail domain variants were localized exclusively to the PM. The subcellular localizations of the mutants were further confirmed by their characteristic glycosylation profiles. In conclusion, our results indicate that ER quality control (ERQC) is involved in the pathological mechanism of several BMPR2 receptor missense mutations causing FPAH, which can be explored as a potential therapeutic target in the future.

Improved plasma membrane expression of the trafficking defective P344R mutant of muscle, skeletal, receptor tyrosine kinase (MuSK) causing congenital myasthenic syndrome.

Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine-rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein will misfold, get retained in the endoplasmic reticulum (ER) and lose its biological function through degradation by the highly conserved ER associated degradation (ERAD) machinery. We report that P344R-MuSK mutant is trafficking-deficient when expressed at 37°C in HeLa, COS-7 and HEK293 cell lines. It colocalized with the ER marker calnexin in contrast to wild-type MuSK which localized to the plasma membrane. The N-glycosylation status of P344R-MuSK is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant's half-life is shorter than wild-type MuSK protein. Proteasomal inhibition resulted in the stabilization of the mutant protein. The mutant protein is highly ubiquitinated compared to wild-type confirming targeting for proteasomal degradation. The mutant showed around 50% of its in vivo autophosphorylation activity. P344R-MuSK mutant's trafficking defect is correctable by culturing the expressing cells at 27°C. Moreover, chemical compounds namely 2.5% glycerol, 1% dimethyl sulfoxide, 10 µM thapsigargin and 1 µM curcumin improved the maturation and exit of the mutant protein from the ER. These findings open perspectives for potential therapeutic intervention for patients with CMS harboring the P344R-MuSK mutation.

Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.

Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 6.3.5.4), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.

A novel whole exon deletion in WWOX gene causes early epilepsy, intellectual disability and optic atrophy.

Recent studies have implicated the WW domain-containing oxidoreductase encoding gene (WWOX) in a severe form of autosomal recessive neurological disorder. This condition showed an overlapping spectrum of clinical features including spinocerebellar ataxia associated with generalized seizures and delayed psychomotor development to growth retardation, spasticity, and microcephaly. We evaluated a child from a consanguineous Emirati family that presented at birth with growth retardation, microcephaly, epileptic seizures, and later developed spasticity and delayed psychomotor development. Screening for deletions and duplications using whole-chromosomal microarray analysis identified a novel homozygous microdeletion encompassing exon 5 of the WWOX gene. Analysis of parental DNA indicated that this deletion was inherited from both parents and lies within a large region of homozygosity. Sanger sequencing of the cDNA showed that the deletion resulted in exon 5 skipping leading to a frame-shift and creating a premature stop codon at amino acid position 212. Quantification of mRNA revealed striking low level of WWOX expression in the child and moderate level of expression in the mother compared to a healthy control. To the best of our knowledge, this is the first homozygous germline structural variation in WWOX gene resulting in truncated transcripts that were presumably subject to NMD pathway. Our findings extend the clinical and genetic spectrum of WWOX mutations and support a crucial role of this gene in neurological development.