論文 - 小崎　健次郎

IMSindel: An accurate intermediate-size indel detection tool incorporating de novo assembly and gapped global-local alignment with split read analysis

Shigemizu D., Miya F., Akiyama S., Okuda S., Boroevich K., Fujimoto A., Nakagawa H., Ozaki K., Niida S., Kanemura Y., Okamoto N., Saitoh S., Kato M., Yamasaki M., Matsunaga T., Mutai H., Kosaki K., Tsunoda T.

Scientific Reports （Scientific Reports）  8 （ 1 ） 5608 2018年04月

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© 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.

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Deep whole-genome sequencing reveals recent selection signatures linked to evolution and disease risk of Japanese

小崎 健次郎

Nature Communications （Nature Communications）  9 （ 1 ） 1631 2018年04月

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© 2018 The Author(s). Understanding natural selection is crucial to unveiling evolution of modern humans. Here, we report natural selection signatures in the Japanese population using 2234 high-depth whole-genome sequence (WGS) data (25.9×). Using rare singletons, we identify signals of very recent selection for the past 2000-3000 years in multiple loci (ADH cluster, MHC region, BRAP-ALDH2, SERHL2). In large-scale genome-wide association study (GWAS) dataset (n = 171,176), variants with selection signatures show enrichment in heterogeneity of derived allele frequency spectra among the geographic regions of Japan, highlighted by two major regional clusters (Hondo and Ryukyu). While the selection signatures do not show enrichment in archaic hominin-derived genome sequences, they overlap with the SNPs associated with the modern human traits. The strongest overlaps are observed for the alcohol or nutrition metabolism-related traits. Our study illustrates the value of high-depth WGS to understand evolution and their relationship with disease risk.

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Novel BICD2 mutation in a Japanese family with autosomal dominant lower extremity-predominant spinal muscular atrophy-2

小崎 健次郎

Brain and Development （Brain and Development）  40 （ 4 ） 343 - 347 2018年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03877604

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© 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.

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Tranilast inhibits the expression of genes related to epithelial-mesenchymal transition and angiogenesis in neurofibromin-deficient cells

小崎 健次郎

Scientific Reports （Scientific Reports）  8 （ 1 ） 6069 2018年04月

研究論文（学術雑誌）, 共著, 査読有り

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© 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.

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Large number of cutaneous neurofibromas beyond age-appropriate incidence in a patient with a large deletion of NF1.

小崎 健次郎

J Dermatol. 45 （ 3 ） 363 - 364 2018年03月

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Growth pattern of Rahman syndrome

小崎 健次郎

American Journal of Medical Genetics, Part A （American Journal of Medical Genetics, Part A）  176 （ 3 ） 712 - 714 2018年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15524825

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© 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.

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Biallelic mutations in NALCN: Expanding the genotypic and phenotypic spectra of IHPRF1

小崎 健次郎

American Journal of Medical Genetics, Part A （American Journal of Medical Genetics, Part A）  176 （ 2 ） 431 - 437 2018年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15524825

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© 2017 Wiley Periodicals, Inc. Loss-of function mutations in NALCN on chromosome 13q, a sodium leak channel that maintains baseline neuronal excitability, cause infantile hypotonia with psychomotor retardation and characteristic faces 1 (IHPRF1, OMIM #615419). Here, we document two individuals with early onset hypotonia with poor feeding and intellectual disability who were compatible with a diagnosis of IHPRF1. The two patients had bi-allelic mutations in NALCN through two different genetic mechanisms: Patient 1 had bi-allelic splice site mutations, that is c.1267-2A>G, derived from heterozygous parents, while Patient 2 had a partial maternal uniparental isodisomy that harbored a frameshift mutation, that is c.2022_2023delAT, in chromosome 13 that was detected through a dedicated algorithm for homozygosity data mapping in whole exome sequencing. The delineation of the exact pattern of inheritance provided vital information regarding the risk of recurrence. In animal models with Nalcn mutations, two behavioral phenotypes, that are, postnatal dyspnea and sleep disturbance, have been reported. Our observations of the two patients with postnatal dyspnea and one patient with sleep disturbance support an association between these two behavioral phenotypes and NALCN mutations in humans. The routine use of a detection algorithm for homozygosity data mapping might improve the diagnostic yields of next-generation sequencing.

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Biallelic mutations in LARS2 can cause Perrault syndrome type 2 with neurologic symptoms

小崎 健次郎

American Journal of Medical Genetics, Part A （American Journal of Medical Genetics, Part A）  176 （ 2 ） 404 - 408 2018年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15524825

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© 2017 Wiley Periodicals, Inc. Perrault syndrome represents a genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and ovarian dysfunction in females. Causative genes include HARS2, HSD17B4, CLPP, C10orf2, and LARS2. Some patients with Perrault syndrome exhibit neurologic features including learning disability, cerebellar ataxia, and peripheral neuropathy and are classified as type 2 and are clinically separate from those without neurological symptoms other than a hearing loss (type 1). To date, all reported patients with LARS2 mutations (15 patients in 8 families) have been classified as type 1. Here, we report female siblings with biallelic mutations in LARS2, p.Glu294Lys, and p.Thr519Met, who were classified as type 2. The proposita developed progressive sensorineural hearing loss at 18 months and pervasive developmental disorder at 8 years, with repetitive behavior, insistence on sameness, attention deficit, tic, irritability, and an ataxic gait. At age 15 years, she was diagnosed as having primary amenorrhea with elevated FSH and LH and a decreased estradiol; ultrasound and magnetic resonance imaging examinations revealed a small uterus and no detectable ovaries. The proposita's younger sister presented with neonatal sensorineural hearing loss and a mild delay in motor and speech development. She was diagnosed as having primary amenorrhea with endocrinologic and radiographic findings that were comparable to those of her sister. She had difficulty with reading comprehension, and had trouble with open-ended test questions at 12 years of age. We concluded that Perrault syndrome patients with LARS2 mutations are at risk for neurologic problems, despite previous notions otherwise.

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Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model

小崎 健次郎

Stem Cell Research and Therapy （Stem Cell Research and Therapy）  9 （ 1 ） 12 2018年01月

研究論文（学術雑誌）, 共著, 査読有り

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© 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.

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Natural course and characteristics of cutaneous neurofibromas in neurofibromatosis 1

小崎 健次郎

Journal of Dermatology （Journal of Dermatology）  45 （ 1 ） 53 - 57 2018年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03852407

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© 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.

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CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations.

小崎 健次郎

Elife. 2017年11月

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A Novel SCN5A Mutation Found in a Familial Case of Long QT Syndrome Complicated by Severe Left Ventricular Dysfunction

小崎 健次郎

Can J Cardiol. 33 （ (4) ） 554 2017年

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A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly.

小崎 健次郎

BMC Med Genet. 18 （ (1) ） 4 2017年

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Diagnostic Use of Computational Retrotransposon Detection: Successful Definition of Pathogenetic Mechanism in Ciliopathy Phenotype

小崎 健次郎

Am J Med Genet A 173 （ (5) ） 1353 - 1357 2017年

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Mitochondrial mutations in maternally inherited hearing loss.

小崎 健次郎

BMC Med Genet. 18 （ (1) ） 32 2017年

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Novel MCA/ID syndrome with ASH1L mutation.

小崎 健次郎

Am J Med Genet A. 173 （ (6) ） 1644 - 1648 2017年

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Further evidence that a blepharophimosis syndrome phenotype is associated with a specific class of mutation in the ADNP gene.

小崎 健次郎

Am J Med Genet A. 173 （ (6) ） 1631 - 1634 2017年

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Initiating an undiagnosed diseases program in the Western Australian public health system.

小崎 健次郎

Orphanet J Rare Dis. 12 （ (1) ） 83 2017年

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A novel missense mutation in the HECT domain of NEDD4L identified in a girl with periventricular nodular heterotopia, polymicrogyria and cleft palate.

小崎 健次郎

J Hum Genet 2017年

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YY1 Haploinsufficiency Causes an Intellectual Disability Syndrome Featuring Transcriptional and Chromatin Dysfunction.

小崎 健次郎

Am J Hum Genet. 100 （ (6) ） 907 - 925 2017年

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