河村 理恵

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

Maple syrup urine disease (MSUD) is a rare autosomal recessive inherited disorder of branched-chain amino acid metabolism caused by mutations in BCKDHA, BCKDHB, and DBT that encode the E1α, E1β, and E2 subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. Various MSUD-causing variants have been described; however, no structural rearrangements in BCKDHA have been reported to cause the classic MSUD phenotype. Here, we describe the classic patient with MSUD with compound heterozygous pathogenic variants in BCKDHA: a missense variant (NM_000709.3:c.757G > A, NP_000700.1:p.Ala253Thr) and a paracentric inversion disrupting Intron 1 of BCKDHA, which was identified by whole-genome sequencing and validated by fluorescence in situ hybridization. Using the sequence information of the breakpoint junction, we gained mechanistic insight into the development of this structural rearrangement. Furthermore, the establishment of junction-specific polymerase chain reaction could facilitate identification of the variant in case carrier or future prenatal/preimplantation tests are necessary.

Sex-chromosome discordant chimerism (XX/XY chimerism) is a rare chromosomal disorder in humans. We report a boy with ambiguous genitalia and hypospadias, showing 46,XY[26]/46,XX[4] in peripheral blood cells. To clarify the mechanism of how this chimerism took place, we carried out whole-genome genotyping using a SNP array and microsatellite analysis. The B-allele frequency of the SNP array showed a mixture of three and five allele combinations, which excluded mosaicism but not chimerism, and suggested the fusion of two embryos or a shared parental haplotype between the two parental cells. All microsatellite markers showed a single maternal allele. From these results, we concluded that this XX/XY chimera is composed of two different paternal alleles and a single duplicated maternal genome. This XX/XY chimera likely arose from a diploid maternal cell that was formed via endoduplication of the maternal genome just before fertilization, being fertilized with both X and Y sperm.

BACKGROUND: Female carriers of a balanced X; autosome translocation generally undergo selective inactivation of the normal X chromosome. This is because inactivation of critical genes within the autosomal region of the derivative translocation chromosome would compromise cellular function. We here report a female patient with bilateral retinoblastoma and a severe intellectual disability who carries a reciprocal X-autosomal translocation. CASE PRESENTATION: Cytogenetic and molecular analyses, a HUMARA (Human androgen receptor) assay, and methylation specific PCR (MSP) and bisulfite sequencing were performed using peripheral blood samples from the patient. The patient's karyotype was 46,X,t(X;13)(q28;q14.1) by G-banding analysis. Further cytogenetic analysis located the entire RB1 gene and its regulatory region on der(X) with no translocation disruption. The X-inactivation pattern in the peripheral blood was highly skewed but not completely selected. MSP and deep sequencing of bisulfite-treated DNA revealed that an extensive 13q region, including the RB1 promoter, was unusually methylated in a subset of cells. CONCLUSIONS: The der(X) region harboring the RB1 gene was inactivated in a subset of somatic cells, including the retinal cells, in the patient subject which acted as the first hit in the development of her retinoblastoma. In addition, the patient's intellectual disability may be attributable to the inactivation of the der(X), leading to a 13q deletion syndrome-like phenotype, or to an active X-linked gene on der (13) leading to Xq28 functional disomy.

PIEZO2 encodes a mechanically activated cation channel, which is abundantly expressed in dorsal root ganglion neuron and sensory endings of proprioceptors required for light touch sensation and proprioception in mice. Biallelic loss-of-function mutations in PIEZO2 (i.e., PIEZO2 deficiency) were recently found to cause an arthrogryposis syndrome. Sixteen patients from eight families have been reported to date. Herein we report a new case, including detailed clinical characteristics and courses as well as comprehensive neurological features. The patient was a 12-year-old girl presenting with congenital multiple contractures, progressive severe scoliosis, prenatal-onset growth impairment, motor developmental delay with hypotonia and myopathy-like muscle pathology, mild facial features, and normal intelligence. Her neurological features included areflexia, impaired proprioception, and decreased senses. Neurophysiological examination revealed decreased amplitude of sensory nerve action potentials, absent H reflex, and prolongation of central conduction times. Clinical exome sequencing revealed a novel homozygous frameshift mutation in PIEZO2 (NM_022068: c.4171_4174delGTCA: p.Val1391Lysfs*39) with no detectable mRNA expression of the gene. PIEZO2 deficiency represents a clinical entity involving characteristic neuromuscular abnormalities and physical features. Next generation sequencing-based comprehensive molecular screening and extensive neurophysiological examination could be valuable for diagnosis of the disorder.

Background: Fetal akinesia refers to a broad spectrum of disorders with reduced or absent fetal movements. There is no established approach for prenatal diagnosis of the cause of fetal akinesia. Chromosome 1p36 deletion syndrome is the most common subtelomeric terminal deletion syndrome, recognized postnatally from typical craniofacial features. However, the influence of chromosome 1p36 deletion on fetal movements remains unknown. Case Report: A 32-week-old fetus with akinesia showed multiple abnormalities, including fetal growth restriction, congenital cardiac defects, and ventriculomegaly. G-banding analysis using cultured amniocytes revealed 46,XY,22pstk+. Postnatal whole exome sequencing and subsequent chromosomal microarray identified a 3 Mb deletion of chromosomal region 1p36.33-p36.32. These results of molecular cytogenetic analyses were consistent with the fetal sonographic findings. Conclusion: Using the exome-first approach, we identified a case with fetal akinesia associated with chromosome 1p36 deletion. Chromosome 1p36 deletion syndrome may be considered for differential diagnosis in cases of fetal akinesia with multiple abnormalities.

Vascular Ehlers-Danlos syndrome (vEDS), a genetic disorder caused by mutations in procollagen type III gene (COL3A1), may lead to fatal vascular complication during peripartum period because of the arterial fragility. We experienced a case of vEDS with peripartum life-threatening arterial rapture diagnosed by next-generation sequencing (NGS) and successfully treated the vascular complications. A 25-year-old female in pregnancy at 34 weeks had sudden and acute pain in the left lower abdomen. After successful delivery, her computed tomography scan showed a dissecting aneurysm of the left common iliac artery (CIA). Four days after delivery, she presented in hemorrhagic shock induced by arterial rupture in the CIA. Since her clinical presentations inferred vEDS even in the absence of familial history, we performed NGS-based genetic screening for inherited connective tissue disorders including vEDS with informed consent. Even though we started intensive medication, her iliac aneurysm was progressively enlarging within 3 weeks. After an urgent molecular diagnosis for vEDS (a splice-site mutation), cautious endovascular therapy for her CIA aneurysm was successfully performed. This is the first report for pretreatment molecular diagnosis of vEDS using NGS in an emergent situation of severe vascular complications.

Management of children with trisomy 13 (T13) is controversial because of a paucity of evidence of the natural history, especially focusing on efficacy of treatment. There has been no report regarding natural history of children with T13 receiving intensive neonatal and pediatric treatment without cardiac surgery, although several reports have suggested efficacy of cardiac surgery. To describe the detailed and comprehensive natural history of children with T13 receiving intensive neonatal and pediatric treatment without cardiac surgery, we reviewed clinical information of 24 children with full T13 (15 boys, 9 girls) who were admitted to Nagano Children's Hospital from 1994 to 2016. Intensive neonatal and pediatric treatment without cardiac surgery was provided through careful discussion with the parents. We detailed accurate frequencies of complications, survival, underlying factors and the final modes of death, and psychomotor development of survivors. Unpublished complications including aortopulmonary window, pulmonary-ductus-descending aorta-trunk, biliary system abnormalities, eosinophilic enteritis, and neuroblastoma were described. Accurate frequencies of congenital heart defects (92%) and laryngomalacia and/or tracheomalacia (42%) were determined. The median survival time was 451 days and the 1-year survival rate was 54%. The major underlying factor associated with death was congenital heart defects and heart failure (63%) and the major final mode of death was heart failure (50%). Long-term survivors appeared to show slow but constant psychomotor development. Intensive neonatal and pediatric treatment without cardiac surgery for children with T13 is efficient for survival and psychomotor development, and could be a reasonable choice for parents having fetuses or children with T13.

We report on a Japanese female infant as the fourth patient with the constitutional pure duplication 1q41-qter confirmed by chromosomal microarray and as the first who developed myelodysplastic syndrome (MDS) among those with the constitutional 1q duplication. Common clinical features of the constitutional pure duplication 1q41-qter include developmental delay, craniofacial characteristics, foot malformation, hypertrichosis, and respiratory insufficiency. The association between MDS and the duplication of the genes in the 1q41-qter region remains unknown.

BACKGROUND: Heterozygous mutations in CTCF have been reported in patients with distinct clinical features including intellectual disability. However, the precise pathomechanism underlying the phenotype remains to be uncovered, partly because of the diverse function of CTCF. Here we describe extensive clinical and genetic investigation for two patients with a microdeletion encompassing CTCF. METHODS: We performed genetic examination including comprehensive investigation of X chromosome inactivation and DNA methylation profiling at imprinted loci and genome-wide. RESULTS: Two patients showed comparable clinical features to those in a previous report, indicating that haploinsufficiency of CTCF was the major determinant of the microdeletion syndrome. Despite the haploinsufficiency of CTCF, X chromosome inactivation was normal. DNA methylation at imprinted loci was normal, but hypermethylation at CTCF binding sites was demonstrated, of which PRKCZ and FGFR2 were identified as candidate genes. CONCLUSIONS: This study confirms that haploinsufficiency of CTCF causes distinct clinical features, and that a microdeletion encompassing CTCF could cause a recognisable CTCF deletion syndrome. Perturbed DNA methylation at CTCF binding sites, not at imprinted loci, may underlie the pathomechanism of the syndrome.

Mutations in WDR45 are responsible for beta-propeller protein-associated neurodegeneration (BPAN), which is an X-linked form of neurodegeneration with brain iron accumulation. BPAN mainly affects females and is characterized by seizures and developmental delay or intellectual disability until adolescence or early adulthood, followed by severe dystonia, parkinsonism, and progressive dementia. However, rare male patients have recently been reported with hemizygous germline mutations in WDR45 and severe clinical manifestations, such as epileptic encephalopathies. We report here a 4-year-old boy presenting with profound developmental delay, non-syndromic epileptic encephalopathy, and early brain atrophy. The level of serum neuron specific enolase (NSE) was elevated, but the level of serum phosphorylated neurofilament heavy chain was not detectable. Targeted next-generation sequencing identified a de novo hemizygous splice donor site mutation, c.830+1G > A in WDR45, which resulted in a splicing defect evidenced by reverse transcriptase-PCR. Mutations in WDR45 should be considered as a cause for epileptic encephalopathies in males with profound developmental delay and brain atrophy. Furthermore, elevation of serum NSE may contribute to early diagnosis of BPAN.

The three-dimensional (3D) structure of the genome is organized non-randomly and plays a role in genomic function via epigenetic mechanisms in the eukaryotic nucleus. Here, we analyzed the spatial positioning of three target regions; the SNRPN, UBE3A, and GABRB3 genes on human chromosome 15q11.2-q12, a representative cluster of imprinted regions, in the interphase nuclei of B lymphoblastoid cell lines, peripheral blood cells, and skin fibroblasts derived from normal individuals to look for evidence of genomic organization and function. The positions of these genes were simultaneously visualized, and all inter-gene distances were calculated for each homologous chromosome in each nucleus after three-color 3D fluorescence in situ hybridization. None of the target genes were arranged linearly in most cells analyzed, and GABRB3 was positioned closer to SNRPN than UBE3A in a high proportion of cells in all cell types. This was in contrast to the genomic map in which GABRB3 was positioned closer to UBE3A than SNRPN. We compared the distances from SNRPN to UBE3A (SU) and from UBE3A to GABRB3 (UG) between alleles in each nucleus, 50 cells per subject. The results revealed that the gene-to-gene distance of one allele was longer than that of the other and that the SU ratio (longer/shorter SU distance between alleles) was larger than the UG ratio (longer/shorter UG distance between alleles). The UG distance was relatively stable between alleles; in contrast, the SU distance of one allele was obviously longer than the distance indicated by the genome size. The results therefore indicate that SNRPN, UBE3A, and GABRB3 have non-linear and non-random curved spatial positioning in the normal nucleus, with differences in the SU distance between alleles possibly representing epigenetic evidence of nuclear organization and gene expression.

We describe two patients with Pallister-Hall syndrome (PHS) with genital abnormalities: a female with hydrometrocolpos secondary to vaginal atresia and a male with micropenis, hypoplastic scrotum, and bilateral cryptorchidism. Nonsense mutations in GLI3 were identified in both patients. Clinical and molecular findings of 12 previously reported patients who had GLI3 mutations and genital abnormalities were reviewed. Genital features in the male patients included hypospadias, micropenis, and bifid or hypoplastic scrotum, whereas all the females had hydrometrocolpos and/or vaginal atresia. No hotspot for GLI3 mutations has been found. The urogenital and anorectal abnormalities associated with PHS might be related to dysregulation of SHH signaling caused by GLI3 mutations rather than hormonal aberrations. We recommend that clinical investigations of genital abnormalities are considered in patients with PHS, even those without hypopituitarism.

Ehlers-Danlos syndrome (EDS) is a heterogeneous connective tissue disorder involving skin and joint laxity and tissue fragility. A new type of EDS, similar to kyphoscoliosis type but without lysyl hydroxylase deficiency, has been investigated. We have identified a homozygous CHST14 (carbohydrate sulfotransferase 14) mutation in the two familial cases and compound heterozygous mutations in four sporadic cases. CHST14 encodes dermatan 4-O-sulfotransferase 1 (D4ST1), which transfers active sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetyl-D-galactosamine (GalNAc) residues of dermatan sulfate (DS). Transfection experiments of mutants and enzyme assays using fibroblast lysates of patients showed the loss of D4ST1 activity. CHST14 mutations altered the glycosaminoglycan (GAG) components in patients' fibroblasts. Interestingly, DS of decorin proteoglycan, a key regulator of collagen fibril assembly, was completely lost and replaced by chondroitin sulfate (CS) in the patients' fibroblasts, leading to decreased flexibility of GAG chains. The loss of the decorin DS proteoglycan due to CHST14 mutations may preclude proper collagen bundle formation or maintenance of collagen bundles while the sizes and shapes of collagen fibrils are unchanged as observed in the patients' dermal tissues. These findings indicate the important role of decorin DS in the extracellular matrix and a novel pathomechanism in EDS.

We previously described two unrelated patients showing characteristic facial and skeletal features, overlapping with the kyphoscoliosis type Ehlers-Danlos syndrome (EDS) but without lysyl hydroxylase deficiency [Kosho et al. (2005) Am J Med Genet Part A 138A:282-287]. After observations of them over time and encounter with four additional unrelated patients, we have concluded that they represent a new clinically recognizable type of EDS with distinct craniofacial characteristics, multiple congenital contractures, progressive joint and skin laxity, and multisystem fragility-related manifestations. The patients exhibited strikingly similar features according to their age: craniofacial, large fontanelle, hypertelorism, short and downslanting palpebral fissures, blue sclerae, short nose with hypoplastic columella, low-set and rotated ears, high palate, long philtrum, thin vermilion of the upper lip, small mouth, and micro-retrognathia in infancy; slender and asymmetric face with protruding jaw from adolescence; skeletal, congenital contractures of fingers, wrists, and hips, and talipes equinovarus with anomalous insertions of flexor muscles; progressive joint laxity with recurrent dislocations; slender and/or cylindrical fingers and progressive talipes valgus and cavum or planus, with diaphyseal narrowing of phalanges, metacarpals, and metatarsals; pectus deformities; scoliosis or kyphoscoliosis with decreased physiological curvatures of thoracic spines and tall vertebrae; cutaneous, progressive hyperextensibility, bruisability, and fragility with atrophic scars; fine palmar creases in childhood to acrogeria-like prominent wrinkles in adulthood, recurrent subcutaneous infections with fistula formation; cardiovascular, cardiac valve abnormalities, recurrent large subcutaneous hematomas from childhood; gastrointestinal, constipation, diverticula perforation; respiratory, (hemo)pneumothorax; and ophthalmological, strabismus, glaucoma, refractive errors.

Cold-induced sweating syndrome (CISS) is a rare autosomal recessive disorder caused by mutations in CRLF1 (cytokine receptor-like factor 1), characterized by profuse sweating in cold environmental temperature and craniofacial and skeletal features. Mutations in CRLF1 also cause Crisponi syndrome (CS), characterized by neonatal-onset paroxysmal muscular contractions as well as craniofacial and skeletal manifestations and abnormal functions of the autonomic nerve system. To date, it is an unresolved problem whether the two conditions are distinct clinical entities or a single clinical entity with variable expressions or with different presentations depending on the patients' age at diagnosis. We report on a 30-year-old Japanese woman with CISS and homozygous out-of-frame 23-base deletion of CRLF1. In infancy, she did not show paroxysmal muscular contractions, but showed feeding difficulty, hyperthermia, and facial characteristics including thick and arched eyebrows, a short nose with anteverted nostrils, full cheeks, an inverted upper lip, and a small mouth, resembling those observed in CS. Profuse sweating was noticed at 3 years of age. Cold-induced sweating was recognized in her elementary school days. In adolescence to adulthood, she showed a Marfanoid habitus with progressive kyphoscoliosis and craniofacial characteristics including dolichocephaly, a slender face with poor expression, a distinctive nose with hypoplastic nares, malar hypoplasia, prognathism, and a small mouth. This is the first report of detailed longitudinal observation of a patient with CRLF1 abnormalities, compatible with the notion that CISS and CS may be a single clinical entity.

Loeys-Dietz Syndrome (LDS) is an autosomal dominant aortic aneurysm syndrome with multisystem involvement, caused by heterozygous mutations of transforming growth factor beta receptor type 1 (TGFBR1) or type 2 (TGFBR2) genes. We report on a neonate with the disorder caused by a known TGFBR2 mutation, who developed neonatal-onset progressive dilation of the aortic valve and aneurysms of the aortic root and main pulmonary artery (PA) associated with a large left-to-right shunt via a ventricular septal defect (VSD) and an atrial septal defect. He also had skeletal features (flexion contractures of the fingers, talipes equinovarus, a cleft palate, and joint laxity), mild facial dysmorphisms, and developmental delay. The dilation and aneurysms progressed after PA banding at age 12 days; and the patient received an intracardiac repair of the defects and PA plasty at age 42 days, followed by no further progression of the dilation and the aneurysms. Neonates with generalized hypotonia, a cleft palate, inguinal herniae, musculoskeletal features such as camptodactyly and talipes equinovarus, and a cardiac murmur should be suspected to have LDS, and extensive cardiovascular evaluation and testing of TGFBR1 and TGFBR2 are recommended. LDS patients with cardiac defects that lead to a large left-to-right shunt and congestive heart failure such as VSD should be considered for intracardiac repair even in early infancy.

Monosomy 1p36 is a common subtelomeric microdeletion syndrome, characterized by craniofacial dysmorphisms, developmental delay, mental retardation, hypotonia, epilepsy, cardiovascular complications, and hearing impairment; deleted regions have been mapped within 10.0 Mb from the telomere in most documented cases. We report on a girl with a 10.5-11.1 Mb terminal deletion of 1p36 shown by fluorescence in situ hybridization (FISH). She had three distinct structural abnormalities: bilateral perisylvian polymicrogyria, periventricular nodular heterotopia, and left ventricular noncompaction. She died in early infancy with intractable epilepsy, progressive congestive heart failure and pulmonary hypertension. To date, this is the first case with monosomy 1p36, complicated by this combination of manifestations; she is also the first who had possibly a simple terminal deletion of 1p36 and died in early infancy. An atypically large deletion in this patient might be the basis for the development of these features and the severe clinical course.