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KIF1A-related disorders (KRDs) encompass recessive and dominant variants with wide clinical variability. Recent genetic investigations have expanded the clinical phenotypes of heterozygous KIF1A variants. However, there have been a few long-term observational studies of patients with heterozygous KIF1A variants. A retrospective chart review of consecutive patients diagnosed with spastic paraplegia at Miyagi Children's Hospital from 2016 to 2020 identified six patients with heterozygous KIF1A variants. To understand the long-term changes in clinical symptoms, we examined these patients in terms of their characteristics, clinical symptoms, results of electrophysiological and neuroimaging studies, and genetic testing. The median follow-up period was 30 years (4-44 years). This long-term observational study showed that early developmental delay and equinus gait, or unsteady gait, are the first signs of disease onset, appearing with the commencement of independent walking. In addition, later age-related progression was observed in spastic paraplegia, and the appearance of axonal neuropathy and reduced visual acuity were characteristic features of the late disease phenotype. Brain imaging showed age-related progression of cerebellar atrophy and the appearance of hyperintensity of optic radiation on T2WI and FLAIR imaging. Long-term follow-up revealed a pattern of steady progression and a variety of clinical symptoms, including spastic paraplegia, peripheral neuropathy, reduced visual acuity, and some degree of cerebellar ataxia. Clinical variability between patients was observed to some extent, and therefore, further studies are required to determine the phenotype-genotype correlation., (© 2024 Wiley Periodicals LLC.)

Acid sphingomyelinase deficiency (ASMD) and Gaucher disease (GD) are lysosomal storage disorders associated with hepatosplenomegaly and thrombocytopenia. The incidences of ASMD and GD are known to be particularly high in the Ashkenazi Jewish population. Conversely, the number of reported patients with these diseases has been limited in Asian countries, including Japan. Here, we reviewed the allele frequencies of pathogenic variants causing ASMD and GD in the Japanese population and populations with various ancestry backgrounds using the Japanese Multi-Omics Reference Panel 54KJPN and the Genome Aggregation Database v4.0.0. The estimated carrier frequencies of ASMD- and GD-related variants were 1/180 and 1/154 in Japanese individuals, equivalent to disease occurrence frequencies of 1/128,191 and 1/94,791 individuals, respectively. These frequencies are much higher than previously expected. Our data also suggest that there are more patients with a milder form of ASMD and nonspecific clinical findings who have not yet been diagnosed., (© 2024. The Author(s).)

Hepatocyte nuclear factor 1β (HNF1β) is a transcription factor that plays a key role in the development and function of the liver, pancreas, and kidney. HNF1β plays a key role in early vertebrate development and the morphogenesis of these organs. In humans, heterozygous mutations in the HNF1B gene can result in organ dysplasia, making it the most common cause of developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. Pathogenic variants in the HNF1B gene are known to cause various diseases, including maturity-onset diabetes of the young and developmental renal diseases. This study presents the backbone resonance assignments of HNF1β POU S and POU HD domains, which are highly conserved domains required for the recognition of double-stranded DNA. Our data will be useful for NMR studies to verify the altered structures and functions of mutant HNF1B proteins that can induce developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. This study will provide the structural basis for future studies to elucidate the molecular mechanisms underlying how mutations in HNF1β cause diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by (epi)genetic alterations at 11p15. Because approximately 20% of patients test negative via molecular testing of peripheral blood leukocytes, the concept of Beckwith-Wiedemann spectrum (BWSp) was established to encompass a broader cohort with diverse and overlapping phenotypes. The prevalence of other overgrowth syndromes concealed within molecularly negative BWSp remains unexplored. Methods We conducted whole-exome sequencing (WES) on 69 singleton patients exhibiting molecularly negative BWSp. Variants were confirmed by Sanger sequencing or quantitative genomic PCR. We compared BWSp scores and clinical features between groups with classical BWS (cBWS), atypical BWS or isolated lateralised overgrowth (aBWS+ILO) and overgrowth syndromes identified via WES. Results Ten patients, one classified as aBWS and nine as cBWS, showed causative gene variants for Simpson-Golabi-Behmel syndrome (five patients), Sotos syndrome (two), Imagawa-Matsumoto syndrome (one), glycosylphosphatidylinositol biosynthesis defect 11 (one) or 8q duplication/9p deletion (one). BWSp scores did not distinguish between cBWS and other overgrowth syndromes. Birth weight and height in other overgrowth syndromes were significantly larger than in aBWS+ILO and cBWS, with varying intergroup frequencies of clinical features. Conclusion Molecularly negative BWSp encapsulates other syndromes, and considering both WES and clinical features may facilitate accurate diagnosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

The RNA-binding motif protein 10, RBM10, is an RNA splicing regulator essential for development. Loss-of-function RBM10 variants are associated with TARP syndrome, a severe X-linked recessive condition in males. We report a 3-year-old male with a mild phenotype, consisting of cleft palate, hypotonia, developmental delay, and minor dysmorphisms, associated with a missense RBM10 variant, c.943T>C, p.Ser315Pro, affecting the RRM2 RNA-binding domain. His clinical features were similar to a previously reported case associated with a missense variant. The p.Ser315Pro mutant protein was expressed normally in the nucleus, but its expression level and protein stability were slightly reduced. Nuclear magnetic resonance spectroscopy showed that the structure and the RNA-binding ability of the RRM2 domain with the p.Ser315Pro were unaffected. However, it affects the alternative splicing regulations of downstream genes, NUMB and TNRC6A, and its splicing alteration patterns were variable depending on target transcripts. In summary, a novel germline missense RBM10 p.Ser315Pro variant that causes functional changes in the expression of its downstream genes results in a non-lethal phenotype associated with developmental delays. The functional alteration effects depend on the residues affected by missense variants. Our findings are expected to bring broader insights into the RBM10-associated genotype-phenotype relationships by delineating the molecular mechanism of RBM10 functions., (© 2023. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

The SUZ12 gene encodes a subunit of polycomb repressive complex 2 (PRC2) that is essential for development by silencing the expression of multiple genes. Germline heterozygous variants in SUZ12 have been found in Imagawa-Matsumoto syndrome (IMMAS) characterized by overgrowth and multiple dysmorphic features. Similarly, both EZH2 and EED also encode a subunit of PRC2 each and their pathogenic variants cause Weaver syndrome and Cohen-Gibson syndrome, respectively. Clinical manifestations of these syndromes significantly overlap, although their different prevalence rates have recently been noted: generalized overgrowth, intellectual disability, scoliosis, and excessive loose skin appear to be less prevalent in IMMAS than in the other two syndromes. We could not determine any apparent genotype-phenotype correlation in IMMAS. The phenotype of neurofibromatosis type 1 arising from NF1 deletion was also shown to be modified by the deletion of SUZ12, 560 kb away. This review deepens our understanding of the clinical and genetic characteristics of IMMAS together with other overgrowth syndromes related to PRC2. We also report on a novel IMMAS patient carrying a splicing variant (c.1023+1G>C) in SUZ12. This patient had a milder phenotype than other previously reported IMMAS cases, with no macrocephaly or overgrowth phenotypes, highlighting the clinical variation in IMMAS., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Variants in the type IV collagen gene ( COL4A1/2 ) cause early-onset cerebrovascular diseases. Most individuals are diagnosed postnatally, and the prenatal features of individuals with COL4A1/2 variants remain unclear., Methods: We examined COL4A1/2 in 218 individuals with suspected COL4A1 /2-related brain defects. Among those arising from COL4A1/2 variants, we focused on individuals showing prenatal abnormal ultrasound findings and validated their prenatal and postnatal clinical features in detail., Results: Pathogenic COL4A1/2 variants were detected in 56 individuals (n=56/218, 25.7%) showing porencephaly (n=29), schizencephaly (n=12) and others (n=15). Thirty-four variants occurred de novo (n=34/56, 60.7%). Foetal information was available in 47 of 56 individuals, 32 of whom (n=32/47, 68.1%) had one or more foetal abnormalities. The median gestational age at the detection of initial prenatal abnormal features was 31 weeks of gestation. Only 14 individuals had specific prenatal findings that were strongly suggestive of features associated with COL4A1/2 variants. Foetal ventriculomegaly was the most common initial feature (n=20/32, 62.5%). Posterior fossa abnormalities, including Dandy-Walker malformation, were observed prenatally in four individuals. Regarding extrabrain features, foetal growth restriction was present in 16 individuals, including eight individuals with comorbid ventriculomegaly., Conclusions: Prenatal observation of ventriculomegaly with comorbid foetal growth restriction should prompt a thorough ultrasound examination and COL4A1/2 gene testing should be considered when pathogenic variants are strongly suspected., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Cytosolic PEPCK deficiency (PCKDC) is a rare autosomal recessive inborn error of metabolism, which can present with hypoglycemia, lactic acidosis, and liver failure. It is caused by biallelic pathogenic variants in the PCK1 gene. Only four PCK1 variants have been previously reported in seven patients with PCKDC, and their clinical course of this condition has not been well characterized. Here, we report a Hispanic male with novel biallelic PCK1 variants, p.(Gly430Asp) and p.(His496Gln), who had a unique clinical presentation. He presented with a new onset of growth failure, elevated blood lactate, transaminitis, and abnormal urine metabolites profile, but he has not had documented hypoglycemia. Growth restriction happened due to insufficient caloric intake, and it was improved with nutritional intervention. PCKDC is a manageable disorder and therefore appropriate nutritional and clinical suspicion from typical lab abnormalities which lead to molecular confirmation tests are essential to prevent poor clinical outcomes.

RBM10, is an RNA binding protein that is important for development by regulating the expression of multiple genes. RBM10 is on the X chromosome, and nonsense and frameshift RBM10 variants cause TARP syndrome in males. In a 4-year-old male, we identified a novel maternally inherited missense RBM10 variant in the RRM2 RNA binding domain, c.965C>T, p.Pro322Leu. His clinical features included intellectual disability, developmental delay, growth restriction, hypotonia, and craniofacial malformations. These features were much milder than those described in previously reported cases of TARP syndrome. By in vitro assays, we found that the mutant p.Pro322Leu RBM10 protein retained its specific RNA binding capacity, while gaining a low-affinity nonspecific RNA binding. It was normally localized to the nucleus, but its expression level was significantly reduced with a significantly short half-life. These results indicated that the p.Pro322Leu missense variant causes a developmental disorder in humans through a unique loss-of-function mechanism., (© 2020 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Background: Citrullinemia type I (CTLN1, MIM #215700) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate synthase (ASS). CTLN1 is characterized by life-threatening hyperammonemia and risk for resulting neurocognitive impairments. The diagnosis of CTLN1 is confirmed by the identification of biallelic pathogenic variants in the ASS1 gene. However, there are a small percentage of CTLN1 patients with a characteristic biochemical phenotype without identifiable variants in ASS1 . We describe the molecular characterization of two related Romani children with biochemically diagnosed CTLN1, whose clinical genetic testing failed to detect any pathogenic variant in ASS1 ., Methods: Genomic DNA was extracted from peripheral blood lymphocytes collected from both patients. Sanger sequencing was performed after PCR amplifications of 5'- and 3'-untranslated regions of the ASS1 gene. A luciferase reporter assay was performed using the human malignant melanoma A2058 cell line and the human liver cancer cell line HepG2., Results: We interrogated the non-coding regions of ASS1 by targeted PCR amplification and identified a homozygous 477-bp microdeletion in the promoter region of the ASS1 gene in both patients. Heterozygosity of the variant was confirmed in their parents. Sanger sequencing confirmed the microdeletion contained the entire sequence of the non-coding exon 1 of ASS1 that includes promoter elements of GC-box, E-box, AP2-binding site, and TATA-box. Luciferase reporter assay using an expression plasmid containing the wild-type or mutant ASS1 sequences showed robust reporter expression from the wild-type sequence and significantly reduced expression driven by the mutant insert (3.6% in A2058 cells and 3.3% in HepG2 cells). These findings were consistent with the hypothesis that the microdeletion identified in the patients disrupted an essential promoter element and resulted in deficiency of ASS1 mRNA expression., Conclusions: This is the first report of CTLN1 patients caused by a Romani microdeletion variant affecting the non-coding upstream sequence of ASS1 . Ablation of the promoter sequence can cause CTLN1 by the reduction of ASS1 expression. Currently available clinical sequencing methods usually do not cover the promoter sequence including the non-coding exon of ASS1 , highlighting the importance of evaluating this region in genetic testing for CTLN1., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 The Authors.)

Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10 -8 ; exome-wide threshold: 2.5 × 10 -6 ). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 × 10 -13 ) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Coffin-Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6 were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1 in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.

Disorders of sex development (DSDs) are defined as congenital conditions in which chromosomal, gonadal or anatomical sex is atypical. In many DSD cases, genetic causes remain to be elucidated. Here, we performed a case-control exome sequencing study comparing gene-based burdens of rare damaging variants between 26 DSD cases and 2625 controls. We found exome-wide significant enrichment of rare heterozygous truncating variants in the MYRF gene encoding myelin regulatory factor, a transcription factor essential for oligodendrocyte development. All three variants occurred de novo. We identified an additional 46,XY DSD case of a de novo damaging missense variant in an independent cohort. The clinical symptoms included hypoplasia of Müllerian derivatives and ovaries in 46,XX DSD patients, defective development of Sertoli and Leydig cells in 46,XY DSD patients and congenital diaphragmatic hernia in one 46,XY DSD patient. As all of these cells and tissues are or partly consist of coelomic epithelium (CE)-derived cells (CEDC) and CEDC developed from CE via proliferaiton and migration, MYRF might be related to these processes. Consistent with this hypothesis, single-cell RNA sequencing of foetal gonads revealed high expression of MYRF in CE and CEDC. Reanalysis of public chromatin immunoprecipitation sequencing data for rat Myrf showed that genes regulating proliferation and migration were enriched among putative target genes of Myrf. These results suggested that MYRF is a novel causative gene of 46,XY and 46,XX DSD and MYRF is a transcription factor regulating CD and/or CEDC proliferation and migration, which is essential for development of multiple organs., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: The diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20-40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants., Methods: Aberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES., Results: We identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases., Conclusion: These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.

Although there are many known Mendelian genes linked to epileptic or developmental and epileptic encephalopathy (EE/DEE), its genetic architecture is not fully explained. Here, we address this incompleteness by analyzing exomes of 743 EE/DEE cases and 2366 controls. We observe that damaging ultra-rare variants (dURVs) unique to an individual are significantly overrepresented in EE/DEE, both in known EE/DEE genes and the other non-EE/DEE genes. Importantly, enrichment of dURVs in non-EE/DEE genes is significant, even in the subset of cases with diagnostic dURVs (P = 0.000215), suggesting oligogenic contribution of non-EE/DEE gene dURVs. Gene-based analysis identifies exome-wide significant (P = 2.04 × 10 -6 ) enrichment of damaging de novo mutations in NF1, a gene primarily linked to neurofibromatosis, in infantile spasm. Together with accumulating evidence for roles of oligogenic or modifier variants in severe neurodevelopmental disorders, our results highlight genetic complexity in EE/DEE, and indicate that EE/DEE is not an aggregate of simple Mendelian disorders.

Potocki-Shaffer syndrome (PSS) is a contiguous gene syndrome caused by 11p11.2 deletions. PSS is clinically characterized by intellectual disability, craniofacial anomalies, enlarged parietal foramina, and multiple exostoses. PSS occasionally shows autism spectrum disorder, epilepsy, and overgrowth. Some of the clinical features are thought to be associated with haploinsufficiency of two genes in the 11p11.2 region; variants affecting the function of ALX4 cause enlarged parietal foramina and EXT2 lead to multiple exostoses. However, the remaining clinical features were still yet to be linked to specific genetic alterations. In this study, we identified de novo truncating variants in an 11p11.2 gene, PHF21A, in three cases with intellectual disability and craniofacial anomalies. Among these three cases, autism spectrum disorder was recognized in one case, epilepsy in one case, and overgrowth in two cases. This study shows that PHF21A haploinsufficiency results in intellectual disability and craniofacial anomalies and possibly contributes to susceptibility to autism spectrum disorder, epilepsy, and overgrowth, all of which are PSS features.

We report a patient with thrombocytopenia from a Japanese family with hemophilia A spanning four generations. Various etiologies of thrombocytopenia, including genetic, immunological, and hematopoietic abnormalities, determine the prognosis for this disease. In this study, we identified a novel heterozygous mutation in a gene encoding cytochrome c, somatic (CYCS, MIM123970) using whole exome sequencing. This variant (c.301_303del:p.Lys101del) is located in the α-helix of the cytochrome c (CYCS) C-terminal domain. In silico structural analysis suggested that this mutation results in protein folding instability. CYCS is one of the key factors regulating the intrinsic apoptotic pathway and the mitochondrial respiratory chain. Using the yeast model system, we clearly demonstrated that this one amino acid deletion (in-frame) resulted in significantly reduced cytochrome c protein expression and functional defects in the mitochondrial respiratory chain, indicating that the loss of function of cytochrome c underlies thrombocytopenia. The clinical features of known CYCS variants have been reported to be confined to mild or asymptomatic thrombocytopenia, as was observed for the patient in our study. This study clearly demonstrates that thrombocytopenia can result from CYCS loss-of-function variants., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Biallelic mutations in IBA57 cause a mitochondrial disorder with a broad phenotypic spectrum that ranges from severe intellectual disability to adolescent-onset spastic paraplegia. Only 21 IBA57 mutations have been reported, therefore the phenotypic spectrum of IBA57-related mitochondrial disease has not yet been fully elucidated. In this study, we performed whole-exome sequencing on a Sepharadi Jewish and Japanese family with leukodystrophy. We identified four novel biallelic variants in IBA57 in the two families: one frameshift insertion and three missense variants. The three missense variants were predicted to be disease-causing by multiple in silico tools. The 29-year-old Sepharadi Jewish male had infantile-onset optic atrophy with clinically asymptomatic leukodystrophy involving periventricular white matter. The 19-year-old younger brother, with the same compound heterozygous IBA57 variants, had a similar clinical course until 7 years of age. However, he then developed a rapidly progressive spastic paraparesis following a febrile illness. A 7-year-old Japanese girl had developmental regression, spastic quadriplegia, and abnormal periventricular white matter signal on brain magnetic resonance imaging performed at 8 months of age. She had febrile convulsions at the age of 18 months and later developed epilepsy. In summary, we have identified four novel IBA57 mutations in two unrelated families. Consequently, we describe a patient with infantile-onset optic atrophy and asymptomatic white matter involvement, thus broadening the phenotypic spectrum of biallelic IBA57 mutations.

Objective: Approximately 5% of cerebral small vessel diseases are hereditary, which include COL4A1/COL4A2-related disorders. COL4A1/COL4A2 encode type IV collagen α1/2 chains in the basement membranes of cerebral vessels. COL4A1/COL4A2 mutations impair the secretion of collagen to the extracellular matrix, thereby resulting in vessel fragility. The diagnostic yield for COL4A1/COL4A2 variants is around 20 to 30%, suggesting other mutated genes might be associated with this disease. This study aimed to identify novel genes that cause COL4A1/COL4A2-related disorders., Methods: Whole exome sequencing was performed in 2 families with suspected COL4A1/COL4A2-related disorders. We validated the role of COLGALT1 variants by constructing a 3-dimensional structural model, evaluating collagen β (1-O) galactosyltransferase 1 (ColGalT1) protein expression and ColGalT activity by Western blotting and collagen galactosyltransferase assays, and performing in vitro RNA interference and rescue experiments., Results: Exome sequencing demonstrated biallelic variants in COLGALT1 encoding ColGalT1, which was involved in the post-translational modification of type IV collagen in 2 unrelated patients: c.452 T > G (p.Leu151Arg) and c.1096delG (p.Glu366Argfs*15) in Patient 1, and c.460G > C (p.Ala154Pro) and c.1129G > C (p.Gly377Arg) in Patient 2. Three-dimensional model analysis suggested that p.Leu151Arg and p.Ala154Pro destabilized protein folding, which impaired enzymatic activity. ColGalT1 protein expression and ColGalT activity in Patient 1 were undetectable. RNA interference studies demonstrated that reduced ColGalT1 altered COL4A1 secretion, and rescue experiments showed that mutant COLGALT1 insufficiently restored COL4A1 production in cells compared with wild type., Interpretation: Biallelic COLGALT1 variants cause cerebral small vessel abnormalities through a common molecular pathogenesis with COL4A1/COL4A2-related disorders. Ann Neurol 2018;84:843-853., (© 2018 American Neurological Association.)

SUZ12 is a core component of polycomb repressive complex 2 (PRC2) along with EZH2 and EED. Recently, germline mutations in the SUZ12, EZH2 and EED genes have been reported in Weaver syndrome (WS) or Weaver-like syndrome, suggesting a functional link between PRC2 deficits and WS. However, only one case of a SUZ12 mutation presenting with Weaver-like syndrome has been reported. Here, we report a missense and a frameshift mutation in SUZ12 (c.1797A>C; p.Gln599His and c.844_845del; p.Ala282Glnfs*7), both of which are novel, in two individuals. Their clinical features included postnatal overgrowth, increased bifrontal diameter, large ears, round face, horizontal chin crease and skeletal anomalies, but did not fulfill the WS diagnostic criteria. These data provide strong evidence that SUZ12 mutations cause Weaver-like syndrome., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

SNAP25 is a core component of the soluble N-ethylmaleimide-sensitive factor attachment receptor complex, which plays a critical role in synaptic vesicle exocytosis. To date, six de novo SNAP25 mutations have been reported in patients with neurological features including seizures, intellectual disability, severe speech delay, and cerebellar ataxia. Here, we analyzed an Israeli family with two affected siblings showing seizures and cerebellar dysfunction by whole-exome sequencing, and identified a novel missense SNAP25 mutation (c.176G > C, p.Arg59Pro) inherited from their unaffected father. Two SNAP25 isoforms are known, SNAP25a and SNAP25b, which each contain a different exon 5. The c.176G > C mutation found in this study was specific to SNAP25b, while five previously reported mutations were identified in exons common to both isoforms. Another was previously reported to be specific to SNAP25b. Comparing clinical features of reported patients with SNAP25 mutations, the current patients demonstrated apparently milder clinical features with normal intelligence, and no magnetic resonance imaging abnormality or facial dysmorphism. Our results expand the clinical spectrum of SNAP25 mutations.

SOX9 is responsible for campomelic dysplasia (CMPD). Symptoms of CMPD include recurrent apnea, upper respiratory infection, facial features, and shortening of the lower extremities. The variant acampomelic CMPD (ACMPD) lacks long bone curvature. A patient showed macrocephaly (+3.9 standard deviations [SD]) and minor anomalies, such as hypertelorism, palpebronasal fold, small mandible, and a cleft of soft palate without long bone curvature. From three months of age, he required tracheal intubation and artificial respiration under sedation because of tracheomalacia. Cranial magnetic resonance imaging was normal at one month of age but showed ventriculomegaly, hydrocephaly, and the corpus callosum thinning at two years of age. Exome sequencing revealed a de novo novel mutation, c. 236A>C, p (Q79P), in SOX9. Sox9 is thought to be crucial in neural stem cell development in the central and peripheral nervous system along with Sox8 and Sox10 in mice. In humans, neuronal abnormalities have been reported in cases of CMPD and ACMPD, including relative macrocephaly in 11 out of 22 and mild lateral ventriculomegaly in 2 out of 22 patients. We encountered a two-year old boy with ACMPD presenting with tracheomalacia and macrocephaly with a SOX9 mutation. We described for the first time an ACMPD patient with acquired diminished white matter and corpus callosal thinning, indicating the failure of oligodendrocyte/astrocyte development postnatally. This phenotype suggests that SOX9 plays a crucial role in human central nervous system development. Further cases are needed to clarify the relationship between human neural development and SOX9 mutations., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Weaver syndrome (WS) is a rare congenital overgrowth disorder caused by heterozygous mutations in EZH2 (enhancer of zeste homolog 2) or EED (embryonic ectoderm development). EZH2 and EED are core components of the polycomb repressive complex 2 (PRC2), which possesses histone methyltransferase activity and catalyzes trimethylation of histone H3 at lysine 27. Here, we analyzed eight probands with clinically suspected WS by whole-exome sequencing and identified three mutations: a 25.4-kb deletion partially involving EZH2 and CUL1 (individual 1), a missense mutation (c.707G>C, p.Arg236Thr) in EED (individual 2), and a missense mutation (c.1829A>T, p.Glu610Val) in SUZ12 (suppressor of zeste 12 homolog) (individual 3) inherited from her father (individual 4) with a mosaic mutation. SUZ12 is another component of PRC2 and germline mutations in SUZ12 have not been previously reported in humans. In vitro functional analyses demonstrated that the identified EED and SUZ12 missense mutations cause decreased trimethylation of lysine 27 of histone H3. These data indicate that loss-of-function mutations of PRC2 components are an important cause of WS., (© 2017 Wiley Periodicals, Inc.)

Congenital scoliosis (CS) occurs as a result of vertebral malformations and has an incidence of 0.5-1/1,000 births. Recently, TBX6 on chromosome 16p11.2 was reported as a disease gene for CS; about 10% of Chinese CS patients were compound heterozygotes for rare null mutations and a common haplotype defined by three SNPs in TBX6. All patients had hemivertebrae. We recruited 94 Japanese CS patients, investigated the TBX6 locus for both mutations and the risk haplotype, examined transcriptional activities of mutant TBX6 in vitro, and evaluated clinical and radiographic features. We identified TBX6 null mutations in nine patients, including a missense mutation that had a loss of function in vitro. All had the risk haplotype in the opposite allele. One of the mutations showed dominant negative effect. Although all Chinese patients had one or more hemivertebrae, two Japanese patients did not have hemivertebra. The compound heterozygosity of null mutations and the common risk haplotype in TBX6 also causes CS in Japanese patients with similar incidence. Hemivertebra was not a specific type of spinal malformation in TBX6-associated CS (TACS). A heterozygous TBX6 loss-of-function mutation has been reported in a family with autosomal-dominant spondylocostal dysostosis, but it may represent a spectrum of the same disease with TACS., (© 2017 WILEY PERIODICALS, INC.)

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing technologies enable comprehensive genetic analyses in monogenic and oligonenic diseases. Especially whole-exome sequencing(WES)targeting exonic regions of protein coding genes is mainly used for Mendelian diseases. Here we describe a case of Hand-foot-genital syndrome arising from a HOXA13 mutation[c.1102A>T(p.Ile368Phe)]as one of successful examples in our successful WES analyses.

Background: Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial dysfunction. LS is characterised by elevated lactate and pyruvate and bilateral symmetric hyperintense lesions in the basal ganglia, thalamus, brainstem, cerebral white matter or spinal cord on T2-weighted MRI. LS is a genetically heterogeneous disease, and to date mutations in approximately 40 genes related to mitochondrial function have been linked to the disorder., Methods: We investigated a pair of female monozygotic twins diagnosed with LS from consanguineous healthy parents of Indian origin. Their common clinical features included optic atrophy, ophthalmoplegia, spastic paraparesis and mild intellectual disability. High-blood lactate and high-intensity signal in the brainstem on T2-weighted MRI were consistent with a clinical diagnosis of LS. To identify the genetic cause of their condition, we performed whole exome sequencing., Results: We identified a homozygous nonsense mutation in C12orf65 (NM_001143905; c.346delG, p.V116*) in the affected twins. Interestingly, the identical mutation was previously reported in an Indian family with Charcot-Marie Tooth disease type 6, which displayed some overlapping clinical features with the twins., Conclusions: We demonstrate that the identical nonsense mutation in C12orf65 can result in different clinical features, suggesting the involvement of unknown modifiers., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Cataract is defined as opacity in the crystalline lens and congenital cataract occurs during the first year of life. Until now, mutations of more than 50 genes in congenital cataract have been reported with various modes of inheritance. Among them, HSF4 mutations have been reported in autosomal dominant, autosomal recessive and age-related forms of cataract. The inheritance patterns of these mutations depend on their mutational positions in HSF4: autosomal dominant or recessive mutations are respectively found either in a DNA-binding domain or in (or downstream of) hydrophobic repeats. Here we report a novel homozygous HSF4 mutation (c.521T>C, p.Leu174Pro) in two affected sibs of an Iranian consanguineous family using whole exome sequencing. The mutation is predicted as highly pathogenic by in silico analysis (SIFT, Polyphen2 and MutationTaster) and is not found in any of control databases. This mutation is located in a hydrophobic repeat of the HSF4 protein, which is consistent with the mode of inheritance as an autosomal recessive trait.

Warburg micro syndrome is an autosomal recessive disease where patients present with optic, neurologic and genital symptoms. Until now, four disease genes for Warburg micro syndrome, RAB3GAP1, RAB3GAP2, RAB18 and TBC1D20, have been identified. Here, we report two novel homozygous RAB3GAP1 mutations (c.22G>T, p.Glu8* and c.1353delA, p.Pro452Hisfs*5) in two consanguineous families by whole-exome sequencing.

Using whole exome sequencing, we confirmed a diagnosis of biotin-responsive basal ganglia disease (BBGD) accompanied by possible Kawasaki Disease. BBGD is an autosomal-recessive disease arising from a mutation of the SLC19A3 gene encoding the human thiamine transporter 2 protein, and usually manifests as subacute to acute encephalopathy. In this case, compound heterozygous mutations of SLC19A3, including a de novo mutation in one allele, was the cause of disease. Although a large number of genetic neural diseases have no efficient therapy, there are several treatable genetic diseases, including BBGD. However, to achieve better outcome and accurate diagnosis, therapeutic analysis and examination for disease confirmation should be done simultaneously. We encountered a case of possible Kawasaki disease, which had progressed to BBGD caused by an extremely rare genetic condition. Although the prevalence of BBGD is low, early recognition of this disease is important because effective improvement can be achieved by early biotin and thiamine supplementation.

Whole-exome sequencing (WES) is becoming a standard tool for detecting nucleotide changes, and determining whether WES data can be used for the detection of copy-number variations (CNVs) is of interest. To date, several algorithms have been developed for such analyses, although verification is needed to establish if they fit well for the appropriate purpose, depending on the characteristics of each algorithm. Here, we performed WES CNV analysis using the eXome Hidden Markov Model (XHMM). We validated its performance using 27 rare CNVs previously identified by microarray as positive controls, finding that the detection rate was 59%, or higher (89%) with three or more targets. XHMM can be effectively used, especially for the detection of >200 kb CNVs. XHMM may be useful for deletion breakpoint detection. Next, we applied XHMM to genetically unsolved patients, demonstrating successful identification of pathogenic CNVs: 1.5-1.9-Mb deletions involving NSD1 in patients with unknown overgrowth syndrome leading to the diagnosis of Sotos syndrome, and 6.4-Mb duplication involving MECP2 in affected brothers with late-onset spasm and progressive cerebral/cerebellar atrophy confirming the clinical suspect of MECP2 duplication syndrome. The possibility of an 'exome-first' approach for clinical genetic investigation may be considered to save the cost of multiple investigations.

We report on a girl with absent nails, short/absent distal phalanges of the second to fifth fingers and toes, short thumbs, absent halluces, and carpo-tarsal coalition who also had genitourinary malformations. Trio-based whole exome sequencing identified a novel de novo mutation (c.1102A>T, p.Ile368Phe) in the HOXA13 gene. Heterozygous HOXA13 mutations have been previously reported in hand-foot-genital syndrome and Guttmacher syndrome, which are variably associated with small nails, short distal and middle phalanges, short thumbs and halluces, but not absent nails. Considering the molecular data, the phenotype in the present patient was defined as the severe end of hand-foot-genital and Guttmacher syndrome spectrum. Our observation expands the clinical spectrum caused by heterozygous HOXA13 mutations and reinforces the difficulty of differential diagnosis on clinical grounds for the disorders with short distal phalanges, short thumbs, and short halluces., (© 2014 Wiley Periodicals, Inc.)

Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder characterized by unique, bilateral neuropathological findings in brainstem, basal ganglia, cerebellum and spinal cord. LS is genetically heterogeneous, with the majority of the causative genes affecting mitochondrial malfunction, and many cases still remain unsolved. Here, we report male sibs affected with LS showing ketonemia, but no marked elevation of lactate and pyruvate. To identify their genetic cause, we performed whole exome sequencing. Candidate variants were narrowed down based on autosomal recessive and X-linked recessive models. Only one hemizygous missense mutation (c.665G>C, p.W222S) in glycogenin-2 (GYG2) (isoform a: NM_001079855) in both affected sibs and a heterozygous change in their mother were identified, being consistent with the X-linked recessive trait. GYG2 encodes glycogenin-2 (GYG2) protein, which plays an important role in the initiation of glycogen synthesis. Based on the structural modeling, the mutation can destabilize the structure and result in protein malfunctioning. Furthermore, in vitro experiments showed mutant GYG2 was unable to undergo the self-glucosylation, which is observed in wild-type GYG2. This is the first report of GYG2 mutation in human, implying a possible link between GYG2 abnormality and LS.

Since first being described in 1998, de novo autoimmune hepatitis (AIH) after liver transplantation has been reported in several cases suffering from non-autoimmune liver diseases and primary biliary cirrhosis (PBC). Glutathione S-transferase (GST) T1 genotype mismatches between donor and recipient have also been suggested to constitute a risk factor for de novo AIH. Here, we report a 33-yr-old woman who presented complaining of marked fatigue and jaundice four yr after living-donor liver transplantation for PBC. On examination, transaminase levels were highly elevated and ANA and antimitochondrial antibody M2 were positive. Histological findings showed zonal necrosis with lymphoplasmacytic infiltration closely resembling AIH. She had pretreatment AIH score of 16 and 19 points after relapse of de novo AIH. Two color fluorescence in situ hybridization with X and Y chromosome-specific probes clearly revealed that the hepatocytes were of donor origin and lymphocytes were of patient origin. The GSTT1 genotype of the patient and the donor were the same null type, suggesting that mechanisms other than GSTT1 mismatches may exist in de novo AIH development. In conclusion, recipient immune cells attacked the allogeneic transplanted liver of the patient via de novo AIH, although the exact participation of autoimmune mechanisms is unclear.