Satomi Mitsuhashi

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.

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.176Gu2009>u2009C, p.Arg59Pro) inherited from their unaffected father. Two SNAP25 isoforms are known, SNAP25a and SNAP25b, which each contain a different exon 5. The c.176Gu2009>u2009C 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.

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.

Robust detection of tandem repeat expansions from long DNA reads

Tandemly repeated sequences are highly mutable and variable features of genomes. Tandem repeat expansions are responsible for a growing list of human diseases, even though it is hard to determine tandem repeat sequences with current DNA sequencing technology. Recent long-read technologies are promising, because the DNA reads are often longer than the repetitive regions, but are hampered by high error rates. Here, we report robust detection of human repeat expansions from careful alignments of long (PacBio and nanopore) reads to a reference genome. Our method (tandem-genotypes) is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we can prioritize pathological expansions within the top 10 out of 700000 tandem repeats in the genome. This may help to elucidate the many genetic diseases whose causes remain unknown.

A novel SLC9A1 mutation causes cerebellar ataxia

The mammalian Na+/H+ exchanger isoform one (NHE1), encoded by Solute Carrier Family 9, member 1 (SLC9A1), consists of 12 membrane domains and a cytosolic C-terminal domain. NHE1 plays an important role in maintaining intracellular pH homeostasis by exchanging one intracellular proton for one extracellular sodium ion. Mice with a homozygous null mutation in Slc9a1 (Nhe1) exhibited ataxia, recurrent seizures, and selective neuronal cell death. In humans, three unrelated patients have been reported: a patient with a homozygous missense mutation in SLC9A1, c.913G>A (p.Gly305Arg), which caused Lichtenstein–Knorr syndrome characterized by cerebellar ataxia and sensorineural hearing loss, a patient with compound heterozygous mutations, c.1351A>C (p.Ile451Leu) and c.1585C>T (p.His529Tyr), which caused a neuromuscular disorder, and a patient with de novo mutation, c.796A>C (p.Asn266His) which associated multiple anomalies. In this study, using whole exome sequencing, we identified a novel homozygous SLC9A1 truncating mutation, c.862del (p.Ile288Serfs*9), in two affected siblings. The patients showed cerebellar ataxia but neither of them showed sensorineural hearing loss nor a neuromuscular phenotype. The main clinical feature was similar to Lichtenstein–Knorr syndrome but deafness may not be an essential phenotypic feature of SLC9A1 mutation. Our report expands the knowledge of clinical features of SLC9A1 mutations.

Novel recessive mutations in MSTO1 cause cerebellar atrophy with pigmentary retinopathy

Misato 1, mitochondrial distribution and morphology regulator (encoded by the MSTO1 gene), is involved in mitochondrial distribution and morphology. Recently, MSTO1 mutations have been shown to cause clinical manifestations suggestive of mitochondrial dysfunction, such as muscle weakness, short stature, motor developmental delay, and cerebellar atrophy. Both autosomal dominant and recessive modes of inheritance have been suggested. We performed whole-exome sequencing in two unrelated patients showing cerebellar atrophy, intellectual disability, and pigmentary retinopathy. Three novel mutations were identified: c.836 Gu2009>u2009A (p.Arg279His), c.1099-1 Gu2009>u2009A (p.Val367Trpfs*2), and c.79u2009Cu2009>u2009T (p.Gln27*). Both patients had compound heterozygous mutations with a combination of protein-truncation mutation and missense mutation, the latter shared by them both. This survey of two patients with recessive and novel MSTO1 mutations provides additional clinical and genetic information on the pathogenicity of MSTO1 in humans.

Expanding the phenotype of IBA57 mutations: related leukodystrophy can remain asymptomatic

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.

A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4

&NA; Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient‐derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2‐derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS‐induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS‐induced DUX4 increase and identified ataxia‐telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS‐induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.

A novel CYCS mutation in the α-helix of the CYCS C-terminal domain causes non-syndromic thrombocytopenia

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.