Published papers

© 2018 The Author(s). Insertions and deletions (indels) have been implicated in dozens of human diseases through the radical alteration of gene function by short frameshift indels as well as long indels. However, the accurate detection of these indels from next-generation sequencing data is still challenging. This is particularly true for intermediate-size indels (≥50 bp), due to the short DNA sequencing reads. Here, we developed a new method that predicts intermediate-size indels using BWA soft-clipped fragments (unmatched fragments in partially mapped reads) and unmapped reads. We report the performance comparison of our method, GATK, PINDEL and ScanIndel, using whole exome sequencing data from the same samples. False positive and false negative counts were determined through Sanger sequencing of all predicted indels across these four methods. The harmonic mean of the recall and precision, F-measure, was used to measure the performance of each method. Our method achieved the highest F-measure of 0.84 in one sample, compared to 0.56 for GATK, 0.52 for PINDEL and 0.46 for ScanIndel. Similar results were obtained in additional samples, demonstrating that our method was superior to the other methods for detecting intermediate-size indels. We believe that this methodology will contribute to the discovery of intermediate-size indels associated with human disease.

© 2018 Elsevier Masson SAS Among the many regulators of microRNA formation, Argonaute 1 (AGO1) plays critical roles in RNA interference, which controls a wide range of biological activities. Recent large-scale genomic studies have identified at least five patients with intellectual disability/autism spectrum disorder who had de novo mutations in AGO1, but detailed clinical information was not available. The recognizable clinical features that are associated with AGO1 mutations remain to be determined. The proposita was a 15-year-old girl with diffuse hypotonia, infrequent seizures, and intellectual disability with an intelligence quotient of 41. She had characteristic facial features consisting of telecanthus, wide nasal bridge with bulbous nasal tip, and a round face with downslanted palpebral fissures. Serial computed tomography scans showed progressive calcification in the globus pallidus that became evident during childhood. A whole exome analysis in trio revealed a de novo heterozygous mutation in AGO1, i.e., c.595G > A p.(Gly199Ser). The distinctive facial features, i.e., telecanthus, wide nasal bridge with bulbous nasal tip, and a round face with downslanted palpebral fissures, closely resembled previously reported patients who had a chromosomal microdeletion encompassing AGO1 locus. The combinatory phenotype of such characteristic facial features and radiographic features, i.e. progressive calcification in the globus pallidus, in the presently reported patient suggest that AGO1 mutations lead to a syndromic form of intellectual disability/autism spectrum disorder. Distinctive facial features with early and progressive calcification in the globus pallidus may be suggestive of the presence of AGO1 mutations.

© 2018 The Authors. Background Brugada syndrome (BS) is known to be 9 times more prevalent in males than females. However, little is known about the development of sick sinus syndrome in female members with familial BS. Methods and Results Familial BS patients and family members, both from our institutions and collaborating sites that specialize in clinical care of BS, participated in this study. We collected information on their clinical and genetic background, along with the inheritance patterns of BS. Detailed information on each case with familial BS is described. A total of 7 families, including 25 BS patients (12 females and 13 males), were included. Seven were probands and 18 were family members. Ten out of the 12 female patients and none of the 13 male patients developed sick sinus syndrome. Sudden death or spontaneous ventricular fibrillation occurred in 7 out of 13 male patients and 2 out of 12 female patients. Conclusions Familial BS existed in which female patients developed sick sinus syndrome but male patients did not. Some of those female patients with sick sinus syndrome had unrecognized BS. Information should be collected not only regarding a family history of sudden death or BS, but also whether a pacemaker was implanted in female members.

© 2018 Wiley Periodicals, Inc. Systemic lupus erythematosus (SLE) has been reported among patients with RASopathy. Five patients have been reported: three with SHOC2 variants, one with a PTPN11 variant, and one with a KRAS variant. SHOC2 variant might represent a relatively common predisposing factor for SLE among the RASopathy genes. However, the clinical details were only reported for two patients, while information on the remaining patient appeared only in a tabular format with minimal clinical description. Here, we report a patient with a SHOC2 variant and SLE. The proband was a 28-year-old male patient with intellectual disabilities, a short stature, dysmorphic facial features, and thin hair. He developed hypertrophic cardiomyopathy and afebrile generalized seizures at the ages of 7 and 18 years, respectively. At the age of 24 years, he presented with a 3-day history of intermittent fever accompanied by right chest pain and a malar butterfly rash. He fulfilled both the American College of Rheumatology (ACR) criteria and the Systemic Lupus International Collaborating Clinics (SLICC) criteria for SLE and was successfully treated with prednisolone. Medical exome sequencing identified a de novo SHOC2 variant (c.4A > G, p.S2G). The present report of a second patient who fulfills both the ACR criteria and the SLICC criteria of SLE. We suggest that the association between SHOC2 variant and SLE represents more than a chance association. In the event of fever of unknown origin in patients with constitutional SHOC2 pathogenic variant, SLE should be suspected.

© 2018 Wiley Periodicals, Inc. Timothy syndrome is characterized by a unique combination of a prolongation of the corrected QT interval of the electrocardiogram and bilateral cutaneous syndactyly of the fingers and the toes and is caused by heterozygous mutations in CACNA1C, a gene encoding a calcium channel. After the discovery of the CACNA1C gene as the causative gene for Timothy syndrome, patients with CACNA1C mutations with QT prolongation but without syndactyly were described. Here, we report a 5-year-old female patient with cutaneous syndactyly, developmental delay, and pulmonary hypertension. Exome analysis showed a previously undescribed de novo heterozygous mutation in the CACNA1C gene, p.Arg1024Gly. To our knowledge, this patient is the first to exhibit syndactyly and to carry a CACNA1C mutation but to not have QT prolongation, which has long been considered an obligatory feature of Timothy syndrome.

© 2018 The Authors. A 50-year-old woman visited the emergency department with a high fever due to pneumonia. Incessant monomorphic ventricular tachycardia occurred and was terminated by intravenous lidocaine. Her ECG during sinus rhythm demonstrated ST segment elevation suggestive of Brugada syndrome (BS). An intensive examination could not detect any structural disease, and typical coved-type ST elevation was unmasked by a pilsicainide injection leading to a diagnosis of BS. An ICD was implanted for secondary prevention of ventricular arrhythmia. The patient has been free from any recurrences of arrhythmia for 3 years.

© 2018 Wiley Periodicals, Inc. Somatic truncating variants of the WAC gene have been observed in patients with hematologic malignancies. Furthermore, de novo heterozygous constitutional pathogenic variants of WAC have recently been shown to cause a syndromic form of intellectual disability, DeSanto-Shinawi syndrome. It is unknown whether the constitutional pathogenic variants observed in the intellectual disability syndrome overlap with the somatic pathogenic variants observed in hematologic abnormalities. Herein, we report three patients with constitutional truncating variants of WAC in an attempt to address the above questions. All three of the patients had mild to moderate intellectual disability and dysmorphic features. We then reviewed the phenotypic features of 19 patients with DeSanto-Shinawi syndrome, including the three currently reported ones: eight and seven patients showed a bulbous nasal tip and short fingers, respectively. As for the pathogenetic mechanism, we demonstrated that the expression level of the mRNA derived from the wildtype allele was higher than that derived from the mutated allele, demonstrating nonsense-mediated mRNA decay. This observation makes a haploinsufficiency mechanism likely. Reviews of the constitutional and somatic pathogenic variants observed in patients with hematologic malignancies showed a significant overlap of the two. To date, no patients with DeSanto-Shinawi syndrome have been reported to have developed hematologic abnormalities, except for one of the three patients reported herein who developed leukopenia and thrombocytopenia at the age of 19 years. Larger data sets are required to determine hematologic prognosis of patients with constitutional WAC variants.

© 2018 Canadian Cardiovascular Society Patients with Mulibrey nanism (MUL) present with growth failure and multiple organ manifestations, and MUL is caused by mutations in TRIM37. In this article, we report on the first case series of Japanese patients with MUL who developed congestive heart failure due to constrictive pericarditis. Our case series suggests that early diagnosis and total pericardiectomy before adherence of the pericardium might provide clinical benefit and better prognosis for MUL.

© 2017 Recently, 7 patients with de novo constitutional non-synonymous mutations in the CDK13 gene were ascertained through a trio exome analysis of a large cohort of 610 patients with congenital cardiac diseases. Despite another report describing 9 additional patients, the clinical spectrum of this condition has yet to be defined. Herein, we report 3 patients with heterozygous constitutional CDK13 mutations, who were ascertained through exome analysis of children with intellectual disability and minor anomalies, who lacked cardiac anomalies. Two patients had a c.2149G > A, p.Gly717Arg mutation, and one had a c.2525A > G, p. Asn842Ser mutation. A review of the previously described patients and those described herein has enabled the following points to be clarified. First, congenital heart diseases are not an essential feature (13/19). Second, nasal features may help syndromic recognition (14/16). Third, widely spaced and peg-shaped teeth may represent a previously unappreciated diagnostic clue for this newly identified syndrome. Here, we show that p.Gly717Arg represents a hotspot in addition to p.Asn842Ser. We suggest that this CDK13-related disorder may represent a clinically recognizable syndrome.

© 2018 The Author(s). Neurofibromatosis type 1 (NF1) is caused by germline mutations in the NF1 gene and is characterized by café au lait spots and benign tumours known as neurofibromas. NF1 encodes the tumour suppressor protein neurofibromin, which negatively regulates the small GTPase Ras, with the constitutive activation of Ras signalling resulting from NF1 mutations being thought to underlie neurofibroma development. We previously showed that knockdown of neurofibromin triggers epithelial-mesenchymal transition (EMT) signalling and that such signalling is activated in NF1-associated neurofibromas. With the use of a cell-based drug screening assay, we have now identified the antiallergy drug tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) as an inhibitor of EMT and found that it attenuated the expression of mesenchymal markers and angiogenesis-related genes in NF1-mutated sNF96.2 cells and in neurofibroma cells from NF1 patients. Tranilast also suppressed the proliferation of neurofibromin-deficient cells in vitro more effectively than it did that of intact cells. In addition, tranilast inhibited sNF96.2 cell migration and proliferation in vivo. Knockdown of type III collagen (COL3A1) also suppressed the proliferation of neurofibroma cells, whereas expression of COL3A1 and SOX2 was increased in tranilast-resistant cells, suggesting that COL3A1 and the transcription factor SOX2 might contribute to the development of tranilast resistance.

© 2017 The Japanese Society of Child Neurology Introduction: The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogenous and largely remain to be elucidated. We present a father and son with atrophy and weakness of the lower leg muscles since infancy. Genetic studies in this family revealed a novel BICD2 mutation causing autosomal dominant lower extremity-predominant SMA type 2. Patients: The proband was the father, aged 30, and the son was aged 3. Both of them were born uneventfully to nonconsanguineous parents. While the father first walked at the age of 19 months, the son was unable to walk at age 3 years. In both, knee and ankle reflexes were absent and sensation was intact. Serum creatine kinase levels were normal. The son showed congenital arthrogryposis and underwent orthopedic corrections for talipes calcaneovalgus. Investigation of the father at the age of 5 years revealed normal results on nerve conduction studies and sural nerve biopsy. Electromyography showed chronic neurogenic change, and muscle biopsy showed features suggestive of denervation. The father was diagnosed clinically with a sporadic distal SMA. Follow-up studies showed very slow progression. Investigations and results: Next-generation and Sanger sequencing revealed a deleterious mutation in BICD2: c.1667A>G, p.Tyr556Cys, in this family. Discussion: BICD2 is a cytoplasmic conserved motor-adaptor protein involved in anterograde and retrograde transport along the microtubules. Next-generation sequencing will further clarify the genetic basis of non-5q SMA.

© 2018 Wiley Periodicals, Inc. Recently, in a cohort study with “overgrowth syndrome with intellectual disability,” five subjects were reported to have de novo heterozygous truncating variants in HIST1H1E, which encodes linker histone H 1.4. However, their growth pattern appeared complex that four out of five patients had a decreasing height percentile over time, and three of these patients began with above-average heights but exhibited reductions to average heights or below when they were older. Herein, we report a female patient with intellectual disability and distinctive facial features including a wide nasal bridge and prominent cheek bones. She did not exhibit skeletal overgrowth, but she had a short stature at 21 years of age. An exome analysis identified a de novo heterozygous 1-bp duplication in HIST1H1E, that is, c.433dup p.(Ala145Glyfs*51). The physical features of the proposita were essentially the same as those observed in patients with the aforementioned HIST1H1E-related overgrowth syndrome. Our review of the growth trajectories in seven patients showed that five of seven patients did not exhibit skeletal overgrowth. This “lack of overgrowth in overgrowth syndrome” is reminiscent of a subset of patients with a short stature who have Sotos syndrome, a prototypic overgrowth syndrome. Considering this complexity in growth, this newly identified condition should be referred to as Rahman syndrome.

© 2018 The Author(s). Background: Runt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models. Methods: Two cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis. Results: Mutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect. Conclusions: Taken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD.

© 2017 Japanese Dermatological Association Neurofibromatosis 1 (NF1) is characterized by cutaneous, neurological and osseous manifestations. Most NF1 patients develop cutaneous neurofibromas. However, time-dependent change with aging and the predilection site of cutaneous neurofibromas remain unclear. To clarify the natural course and characteristics of cutaneous neurofibromas, a retrospective study was conducted for 57 NF1 patients who were treated at the Department of Dermatology of Tottori University Hospital between January 2007 and April 2016. For each patient, we investigated the time-dependent changes and the numbers of cutaneous neurofibromas in four body surface regions. There was a positive correlation between age and number of cutaneous neurofibromas (r = 0.75, P < 0.001). Cutaneous neurofibromas were located on the trunk (60.2%), lower limbs (16.1%), upper limbs (14.4%), and head and neck (9.2%). There was no significant relationship between each body type (e.g. obese or thin) and cutaneous neurofibromas. With respect to the year-to-year percentage change in cutaneous neurofibromas, the average annual rate of increase was 0.21 (range, −0.71 to 1.2). The number of cutaneous neurofibromas had increased in approximately 61% of the patients 1 year later. Our data will enable physicians to estimate the overall state of cutaneous neurofibromas in NF1 and will be useful for handling cutaneous manifestations before they become a serious condition.

© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. Personalized healthcare (PHC) based on an individual's genetic make-up is one of the most advanced, yet feasible, forms of medical care. The Tohoku Medical Megabank (TMM) Project aims to combine population genomics, medical genetics and prospective cohort studies to develop a critical infrastructure for the establishment of PHC. To date, a TMM CommCohort (adult general population) and a TMM BirThree Cohort (birth+three-generation families) have conducted recruitments and baseline surveys. Genome analyses as part of the TMM Project will aid in the development of a high-fidelity whole-genome Japanese reference panel, in designing custom single-nucleotide polymorphism (SNP) arrays specific to Japanese, and in estimation of the biological significance of genetic variations through linked investigations of the cohorts. Whole-genome sequencing from >3,500 unrelated Japanese and establishment of a Japanese reference genome sequence from long-read data have been done. We next aim to obtain genotype data for all TMM cohort participants (>150,000) using our custom SNP arrays. These data will help identify disease-associated genomic signatures in the Japanese population, while genomic data from TMM BirThree Cohort participants will be used to improve the reference genome panel. Follow-up of the cohort participants will allow us to test the genetic markers and, consequently, contribute to the realization of PHC.

© 2019 Wiley Periodicals, Inc. Since 1961, all Japanese citizens have belonged to one of the available medical care insurance systems. This “universal care” system has contributed to the maintenance of health: the life expectancy at birth was 84 years in 2016, and the infant mortality rate (the number of infants dying before reaching 1 year of age) was 2.0 per 1,000 live births, which is one of the lowest rates in the world. The Japanese government initiated the National Program on Rare and Intractable Diseases in 1972. This program has promoted research and expanded support for patients with rare and intractable diseases. Registered patients are eligible for a subsidy scheme that helps to cover medical care costs. Among the 331 diseases that are currently included in this program, more than half of the diseases are Mendelian disorders. The National Program on Rare and Intractable Diseases has fostered research in medical genetics in Japan and many causative genes for Mendelian diseases have been identified by Japanese geneticists. Recently, the Japanese government has determined to support several genomic medicine initiatives including the undiagnosed disease program (Initiative on Rare and Undiagnosed Diseases) and pathogenic variant databases.

© 2019 Japanese Society of Child Neurology. All Rights Reserved. Mutations in the pogo transposable element with zinc finger domain（POGZ）gene are associated with autism spectrum disorder （ASD）and intellectual disability. A total of 40 cases have been reported, and their clinical features include severe phenotypes such as severe speech and language delay, vision problems, microcephaly, and a predisposition towards obesity. Although 20% of these cases showed EEG abnormalities, epilepsy has not been reported. We present the case of a patient with refractory epilepsy and regression in which whole exome sequencing identified a de novo heterozygous POGZ mutation（c.2102del;p.Pro701Leufs ＊ 18）. He was diagnosed with autism spectrum disorder at 6 years of age. From 7 years and 8 months of age, he experienced epileptic seizures that involved falling backwards, head nodding and ocular deviation. An EEG showed a spike-and-slow-wave at F3 and F4. He was diagnosed with frontal lobe epilepsy, and treated with various anti-epileptic drugs （e.g. CZP, VPA, CLB, PHT, LTG, PB, CBZ and LEV）, which eventually reduced his epileptic seizures from over 10 to 2-3 times per day. Moreover, this patient had repetitive aspiration pneumonia from 14 years of age, necessitating a tracheotomy and laryngo-tracheal separation. Phenotypes associated with a POGZ mutation might, therefore, have a wider spectrum than previously recognized, and include not only ASD, but various neurological problems such as progressive epilepsy syndrome.

© 2019 Wiley Periodicals, Inc. Cleft palate can be classified as either syndromic or nonsyndromic. SATB2-associated syndrome is one example of a syndromic cleft palate that is accompanied by intellectual disability, and various dental anomalies. SATB2-associated syndrome can be caused by several different molecular mechanisms including intragenic mutations and deletions of SATB2. Here, we report two patients with SATB2 truncating mutations (p.Arg239* and p.Asp702Thrfs*38) and one with a 4.4 megabase deletion including the SATB2 locus. All three patients had cleft palate and other dysmorphic features including macrodontia wide diastema. None of the three patients had acquired any meaningful words at the age of 5 years. In a review of the linguistic natural history of presently reported three patients and 30 previously reported patients, only two patients had attained verbal skills beyond speaking a few words. This degree of delayed speech contrasts with that observed in the prototypic form of syndromic cleft palate, 22q11.2 deletion syndrome. The recognition of SATB2-associated syndrome prior to palatoplasty would be important for plastic surgeons and the families of patients because precise diagnosis should provide predictive information regarding the future linguistic and intellectual abilities of the patients. Macrodontia with a wide diastema and cleft palate is a helpful and highly suggestive sign for the diagnosis of SATB2-associated syndrome.

© 2018 Wiley Periodicals, Inc. Schuurs-Hoeijmakers syndrome is a rare disease characterized by intellectual disability and dysmorphic facial features among various physical abnormalities due to PACS1 mutation. To date, 28 patients with a recurrent de novo PACS1 mutation (c.607C > T) have been reported, primarily in Western populations. Here, we describe two Japanese patients with Schuurs-Hoeijmakers syndrome with a recurrent PACS1 mutation. In addition to the typical clinical symptoms, each patient presented novel clinical phenotypes. One patient presented with involuntary movements and was treated with trihexyphenidyl hydrochloride. We hypothesized that the PACS1 mutation leads to an inherent dopaminergic insufficiency that underlies the developing symptoms along with the neurodevelopmental processes. The second patient was diagnosed with lipomyelomeningocele during an examination for severe constipation at the age of 2 years and 8 months. The diagnosis of lipomyelomeningocele in this patient was delayed due to the lack of cutaneous lesions. As the majority of patients with PACS1 mutation present constipation, underdiagnosis of lipomyelomeningocele is a possibility. As the phenotypic expansion of the patients with Schuurs-Hoeijmakers syndrome was not fully recognized, additional studies are needed to clarify the clinical spectrum.