Atsushi Takata

Identification of novel SNORD118 mutations in seven patients with leukoencephalopathy with brain calcifications and cysts

Leukoencephalopathy with brain calcifications and cysts (LCC) is neuroradiologically characterized by leukoencephalopathy, intracranial calcification, and cysts. Coats plus syndrome is also characterized by the same neuroradiological findings together with defects in retinal vascular development. Indeed, LCC and Coats plus were originally considered to be the same clinical entity termed cerebroretinal microangiopathy with calcifications and cysts, but evidence suggests that they are genetically distinct. Mutations in CTS telomere maintenance complex component 1 (CTC1) and small nucleolar RNA, C/D box 118 (SNORD118) genes have been found to cause Coats plus and LCC, respectively.

Novel biallelic SZT2 mutations in three cases of early-onset epileptic encephalopathy

The seizure threshold 2 (SZT2) gene encodes a large, highly conserved protein that is associated with epileptogenesis. In mice, Szt2 is abundantly expressed in the central nervous system. Recently, biallelic SZT2 mutations were found in 7 patients (from 5 families) presenting with epileptic encephalopathy with dysmorphic features and/or non‐syndromic intellectual disabilities. In this study, we identified by whole‐exome sequencing compound heterozygous SZT2 mutations in 3 patients with early‐onset epileptic encephalopathies. Six novel SZT2 mutations were found, including 3 truncating, 1 splice site and 2 missense mutations. The splice‐site mutation resulted in skipping of exon 20 and was associated with a premature stop codon. All individuals presented with seizures, severe developmental delay and intellectual disabilities with high variability. Brain MRIs revealed a characteristic thick and short corpus callosum or a persistent cavum septum pellucidum in each of the 2 cases. Interestingly, in the third case, born to consanguineous parents, had unexpected compound heterozygous missense mutations. She showed microcephaly despite the other case and previous ones presenting with macrocephaly, suggesting that SZT2 mutations might affect head size.

A novel mutation in SLC1A3 causes episodic ataxia

Episodic ataxias (EAs) are rare channelopathies characterized by recurrent ataxia and vertigo, having eight subtypes. Mutated genes were found in four of these eight subtypes (EA1, EA2, EA5, and EA6). To date, only four missense mutations in the Solute Carrier Family 1 Member 3 gene (SLC1A3) have been reported to cause EA6. SLC1A3 encodes excitatory amino-acid transporter 1, which is a trimeric transmembrane protein responsible for glutamate transport in the synaptic cleft. In this study, we found a novel missense mutation, c.383T>G (p.Met128Arg) in SLC1A3, in an EA patient by whole-exome sequencing. The modeled structural analysis suggested that p.Met128Arg may affect the hydrophobic transmembrane environment and protein function. Analysis of the pathogenicity of all mutations found in SLC1A3 to date using multiple prediction tools showed some advantage of using the Mendelian Clinically Applicable Pathogenicity (M-CAP) score. Various types of SLC1A3 variants, including nonsense mutations and indels, in the ExAC database suggest that the loss-of-function mechanism by SLC1A3 mutations is unlikely in EA6. The current mutation (p.Med128Arg) presumably has a gain-of-function effect as described in a previous report.

Loss-of-function and gain-of-function mutations in PPP3CA cause two distinct disorders

Calcineurin is a calcium (Ca2+)/calmodulin-regulated protein phosphatase that mediates Ca2+-dependent signal transduction. Here, we report six heterozygous mutations in a gene encoding the alpha isoform of the calcineurin catalytic subunit (PPP3CA). Notably, mutations were observed in different functional domains: in addition to three catalytic domain mutations, two missense mutations were found in the auto-inhibitory (AI) domain. One additional frameshift insertion that caused premature termination was also identified. Detailed clinical evaluation of the six individuals revealed clinically unexpected consequences of the PPP3CA mutations. First, the catalytic domain mutations and frameshift mutation were consistently found in patients with nonsyndromic early onset epileptic encephalopathy. In contrast, the AI domain mutations were associated with multiple congenital abnormalities including craniofacial dysmorphism, arthrogryposis and short stature. In addition, one individual showed severe skeletal developmental defects, namely, severe craniosynostosis and gracile bones (severe bone slenderness and perinatal fractures). Using a yeast model system, we showed that the catalytic and AI domain mutations visibly result in decreased and increased calcineurin signaling, respectively. These findings indicate that different functional effects of PPP3CA mutations are associated with two distinct disorders and suggest that functional approaches using a simple cellular system provide a tool for resolving complex genotype-phenotype correlations.

Detection of copy number variations in epilepsy using exome data

Epilepsies are common neurological disorders and genetic factors contribute to their pathogenesis. Copy number variations (CNVs) are increasingly recognized as an important etiology of many human diseases including epilepsy. Whole‐exome sequencing (WES) is becoming a standard tool for detecting pathogenic mutations and has recently been applied to detecting CNVs. Here, we analyzed 294 families with epilepsy using WES, and focused on 168 families with no causative single nucleotide variants in known epilepsy‐associated genes to further validate CNVs using 2 different CNV detection tools using WES data. We confirmed 18 pathogenic CNVs, and 2 deletions and 2 duplications at chr15q11.2 of clinically unknown significance. Of note, we were able to identify small CNVs less than 10 kb in size, which might be difficult to detect by conventional microarray. We revealed 2 cases with pathogenic CNVs that one of the 2 CNV detection tools failed to find, suggesting that using different CNV tools is recommended to increase diagnostic yield. Considering a relatively high discovery rate of CNVs (18 out of 168 families, 10.7%) and successful detection of CNV with <10 kb in size, CNV detection by WES may be able to surrogate, or at least complement, conventional microarray analysis.

A novel missense SNAP25b mutation in two affected siblings from an Israeli family showing seizures and cerebellar ataxia

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.

A novel homozygous DPH1 mutation causes intellectual disability and unique craniofacial features

Biallelic mutations of the gene encoding diphthamide biosynthesis 1 (DPH1, NM_001383.3) cause developmental delay, dysmorphic features, sparse hair, and short stature (MIM *603527). Only two missense DPH1 mutations have been reported to date. Here, we describe a consanguineous family with two siblings both showing developmental delay, agenesis of the corpus callosum, dysmorphic facial features, sparse hair, brachycephaly, and short stature. By wholeexome sequencing, a homozygous frameshift mutation in DPH1 (c.1227delG, p.[Ala411Argfs*91]) was identified, which is likely responsible for the familial condition. The unique clinical features of the affected siblings are cleft palate and absent renal findings.

Confirmation of SLC5A7-related distal hereditary motor neuropathy 7 in a family outside Wales

To the Editor: Distal hereditary motor neuropathy 7 (dHMN7 [MIM: 158580]) is a disease characterized by distal motor neuropathy and vocal cord paralysis. Variants in the solute carrier family 5 member 7 gene (SLC5A7; MIM: 608761) presumably cause dHMN7, although only 1 variant has been reported in 2 families from Wales. Here, we report a Japanese dHMN7 family with a novel SLC5A7 variant. The proband was a 52 year old male with motor neuropathy (Figure 1A) who has been a slow runner since childhood and stumbles frequently. He experienced muscle weakness in his fingers from his 20s, and difficulty in standing up since his 40s. Dysphagia was observed in his 50s. At present, other observations include: muscle weakness in the distal part of all 4 limbs and the proximal part of lower limbs, muscle atrophy especially of the thenar eminence (Figure 1B) and lower limbs, as well as pes cavus (Figure 1C). Deep tendon reflexes were absent, and the Babinski sign was negative. Superficial and deep sensations were normal. He can only walk short distances with a stick and leg braces. Rhino-laryngo fiberscopic examination revealed that his left vocal cord was paralyzed and fixed in the middle although he did not have a hoarse voice. A nerve conduction study (NCS) implied axonal motor neuropathy because of diminished complex motor action potential (CMAP) amplitudes in median and tibial nerves but not the ulnar nerve, normal motor nerve conduction velocities (MCV) in all nerves, and normal sensory NCSs in median, ulnar, and sural nerves. His father, sister, and 2 sons have pes cavuslike feet with no obvious muscle weakness (Figure 1D). NCS of their tibial nerves showed normal MCVs, but CMAP amplitude was extremely low in his sister and at the lower limit of normal in his father. Having obtained written informed consent from the proband, his parents, and sister, and ethical approval from the Yokohama City University School of Medicine institutional review board, we performed whole exome sequencing of the proband and identified a heterozygous truncating SLC5A7: c.1526del (p.Pro509Leufs*3) (NM_021 815.2). Sanger sequencing confirmed the presence of the variant in the proband, his father, and his sister (Figure 1A). The variant was absent from Exome Aggregation Consortium, Human Gene Mutation Database, and ClinVar databases. SLC5A7 encodes a presynaptic choline transporter (CHT), which plays a critical role in synaptic acetylcholine synthesis and release at the neuromuscular junction. Biallelic loss-of-function SLC5A7 variants cause congenital myasthenic syndrome, while a monoallelic truncating variant in the last exon of SLC5A7 was reported in 2 unrelated dHMN7 families from Wales (Figure 1E). The truncating mutant had a dominant-negative effect on CHT activity and was hypothesized to cause neuropathy. Our dHMN7 case with a novel SLC5A7 variant, truncating the C terminus, provides further evidence for this phenotype-genotype correlation. The present family showed intra-familial variable expressivity (Figure 1A), consistent with previous reports, and which could be a characteristic of dHMN7.

A novel GFI1B mutation at the first zinc finger domain causes congenital macrothrombocytopenia

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A case of atypical Kabuki syndrome arising from a novel missense variant in HNRNPK

A novel causative variant (c. 464T>C, p.Leu155Pro) in the heterogeneous nuclear ribonucleoprotein K (HNRNPK) gene.