Tatsuya Kishino

Heterozygous TGFBR2 mutations in Marfan syndrome

Marfan syndrome is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton and cardiovascular systems associated with defects in the gene encoding fibrillin (FBN1) at 15q21.1 (ref. 1). A second type of the disorder (Marfan syndrome type 2; OMIM 154705) is associated with a second locus, MFS2, at 3p25–p24.2 in a large French family (family MS1). Identification of a 3p24.1 chromosomal breakpoint disrupting the gene encoding TGF-β receptor 2 (TGFBR2) in a Japanese individual with Marfan syndrome led us to consider TGFBR2 as the gene underlying association with Marfan syndrome at the MSF2 locus. The mutation 1524G→A in TGFBR2 (causing the synonymous amino acid substitution Q508Q) resulted in abnormal splicing and segregated with MFS2 in family MS1. We identified three other missense mutations in four unrelated probands, which led to loss of function of TGF-β signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2, a putative tumor-suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders.

ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspaserecruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associatedspeck-like protein containing a CARD). TheASC gene was mapped on chromosome 16p11.2–12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.

A novel GATA4 mutation completely segregated with atrial septal defect in a large Japanese family

cause Holt-Oram syndrome with ASD or ventricular septal defect (VSD), non-syndromic CHD including ASD and atrioventricular conduction abnormal- ities, Ellis-van Creveld syndrome with ASD, and a familial isolated ASD, respectively. We encountered a large family of four generations where 11 members were affected with ASD, and where disease transmission was consistent with an autosomal dominant mode of inheritance. Here we report a novel mutation of GATA4 in this family. METHODS Subjects A large Japanese family composed of a total of 29 members across four generations contained 11 members with ASD (I-1, II-2, II-6, II-7, III-1, III-2, III-4, IV-1, IV-2, IV-5, and IV-6; fig 1A). ASD in five patients (II-7, III-1, III-2, IV-1, and IV-2) was surgically repaired, and two patients (II-2 and IV-1) also had pulmonary stenosis (PS). The heart defects in eight subjects (II-2, II-7, III-1, III-2, IV-1, and IV-2) including two diseased subjects (I-1 and II-6) had been clinically diagnosed by one of co-authors (BK) on the basis of their past histories, operation records, 12-lead electrocardiograms, and echocar- diograms with colour Doppler apparatus (fig 1A), while those in three other subjects (III-4, IV-5, and IV-6) were retro- spectively found using information provided by the mutation analysis described below. None of the 11 affected members had any other abnormalities in the cardiac conduction system or other organs. After informed consent was obtained, DNA was extracted from peripheral blood leukocytes of 22 family members.

LRP5, low-density-lipoprotein-receptor-related protein 5, is a determinant for bone mineral density.

AbstractOsteoporosis is a multifactorial trait with low bone mineral density (BMD). We report results of an association study between BMD and nine candidate genes (TGFB1, TGFBR2, SMAD2, SMAD3, SMAD4, IFNB1, IFNAR1, FOS and LRP5), as well as of a case-control study of osteoporosis. Samples for the former association study included 481 general Japanese women. Among the nine candidate genes examined, only LRP5 showed a significant association with BMD. We identified a strong linkage disequilibrium (LD) block within LRP5. Of five LPR5 single nucleotide polymorphisms (SNPs) that are located in the LD block, three gave relatively significant results: Women with the C/C genotype at the c.2220C>T SNP site had higher adjusted BMD (AdjBMD) value compared to those with C/T and T/T (p=0.022); and likewise, G/G at IVS17-30G>A and C/C women at c.3989C>T showed higher AdjBMD than those with G/A or A/A (p=0.039) and with C/T or T/T (p=0.053), respectively. The case-control study in another series of samples consisting of 126 osteoporotic patients and 131 normal controls also gave a significant difference in allele frequency at c.2220C>T (χ2=6.737, p=0.009). These results suggest that LRP5 is a BMD determinant and also contributes to a risk of osteoporosis.

Nonaka myopathy is caused by mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase gene (GNE)

AbstractThis is the first report on mutations of the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase gene (GNE) in Nonaka myopathy or distal myopathy with rimmed vacuoles (OMIM 605820), an autosomal recessive neuromuscular disorder. Sequence and haplotype analyses of GNE in two siblings with Nonaka myopathy from a Japanese family revealed that both patients were compound heterozygotes for a C→T transition (A460V) in exon 8 and a G→C transition (V572L) in exon 10. Their parents and a normal elder brother were all carriers for one or the other of the mutations. GNE mutations are known to cause two other disorders: sialuria (OMIM #269921) and autosomal recessive inclusion body myopathy (IBM2, OMIM #600737). Mutations associated with sialuria are located in the epimerase domain, and those associated with IBM2 are in the epimerase or the kinase domain or both, whereas the mutations we observed in the Nonaka myopathy patients were located in the sugar kinase domain of the gene. Thus, Nonaka myopathy is the third disease known to be caused by GNE mutations.

A catalog of 106 single-nucleotide polymorphisms (SNPs) and 11 other types of variations in genes for transforming growth factor-β1 (TGF-β1) and its signaling pathway

AbstractTransforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that is produced in the platelet, bone, placenta, and other tissues. It acts as a growth inhibitor in many types of cells, and also mediates extracellular matrix production and immunosuppression. Mutations in the specific domain of its gene (TGFB1) cause Camurati-Engelmann disease, a bone-sclerosing disorder, and those in other domains may be associated with osteoporosis. We identified 106 single-nucleotide polymorphisms and 11 other types of variations in TGFB1 >and six other genes. These genes were TGF-β type I receptor gene (TGFBR1), TGF-β type II receptor gene (TGFBR2), SMAD2 gene (SMAD2), SMAD3 gene (SMAD3), SMAD4 gene (SMAD4), and SMAD7 gene (SMAD7), all of which compose the TGF-β1 signaling pathway. We also estimated allele frequencies of these DNA polymorphisms among 48 Japanese individuals. Our data will provide a useful resource for the study of disease susceptibility.

Complex low-copy repeats associated with a common polymorphic inversion at human chromosome 8p23

To characterize a submicroscopic, common 8p23 polymorphic inversion, we constructed a complete BAC/PAC-based physical map covering the entire 4.7-Mb inversion and its flanking regions. Two low-copy repeats (LCRs), REPD (approximately 1.3 Mb) and REPP (approximately 0.4 Mb), were identified at each of the inversion breakpoints. Comparison of the REPD and REPP sequences revealed that REPD showed high homology to REPP, with complex direct and inverted orientations. REPD and REPP contain six and five olfactory receptor gene-related sequences, respectively. LCRs at 8p23 showed multiple FISH signals from an Old World monkey to the human. Thus, multiplication of the LCR may have occurred at least 21-25 million years ago. We also investigated the frequency of the 4.7-Mb inversion in the general Japanese population and found that the allele frequency for the 8p23 inversion was estimated to be 27%.

BAC array CGH reveals genomic aberrations in idiopathic mental retardation

Array using 2,173 BAC clones covering the whole human genome has been constructed. All clones spotted were confirmed to show a unique signal at the predicted chromosomal location by FISH analysis in our laboratory. A total of 30 individuals with idiopathic mental retardation (MR) were analyzed by comparative genomic hybridization using this array. Three deletions, one duplication, and one unbalanced translocation could be detected in five patients, which are likely to contribute to MR. The constructed array was shown to be an efficient tool for the detection of pathogenic genomic rearrangements in MR patients as well as copy number polymorphisms (CPNs).

Decreased Tonic Inhibition in Cerebellar Granule Cells Causes Motor Dysfunction in a Mouse Model of Angelman Syndrome

Ube3a deficiency causes a decrease in cerebellar tonic inhibition by preventing degradation of GABA transporter 1 in a mouse model of Angelman syndrome. Elucidating the Mechanism of Ataxia in Angelman Syndrome Angelman syndrome is a neurodevelopmental disorder caused by loss of the ubiquitin E3 ligase Ube3a. A variety of symptoms, including severe developmental delay, speech impairment, epilepsy, and movement and balance problems, are associated with Angelman syndrome. In a new study, Egawa et al. investigate electrophysiological features of cerebellar dysfunction in a mouse model of Angelman syndrome. The authors report that extrasynaptic γ-aminobutyric acid type A (GABAA) receptor–mediated tonic inhibition decreases in cerebellar granule cells from the Ube3a-deficient Angelman mice. They then show that, normally, Ube3a binds to GABA transporter 1, a key modulator of extrasynaptic GABA, and controls its degradation in the mouse cerebellum. The Ube3a-deficient mice, however, showed a surplus of GABA transporter 1, resulting in decreased GABA concentrations in the extrasynaptic space and thus decreased tonic inhibition of cerebellar granule cells. Pharmacological compensation of decreased tonic inhibition by administering low doses of THIP, a selective agonist of extrasynaptic GABAA receptors, alleviated impaired motor function in the Ube3a-deficient mice. Thus, attenuated GABA transporter 1 degradation and a consequent decrease in tonic inhibition of cerebellar granule cells play a key role in the cerebellar dysfunction characteristic of Angelman syndrome. Pharmacologically increasing tonic inhibition with THIP may be a useful strategy for alleviating movement and balance problems in patients with this disease. Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the UBE3A gene encoding a ubiquitin E3 ligase. Motor dysfunction is a characteristic feature of Angelman syndrome, but neither the mechanisms of action nor effective therapeutic strategies have yet been elucidated. We report that tonic inhibition is specifically decreased in cerebellar granule cells of Ube3a-deficient mice, a model of Angelman syndrome. As a mechanism underlying this decrease in tonic inhibition, we show that Ube3a controls degradation of γ-aminobutyric acid (GABA) transporter 1 (GAT1) and that deficiency of Ube3a induces a surplus of GAT1 that results in a decrease in GABA concentrations in the extrasynaptic space. Administering low doses of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP), a selective extrasynaptic GABAA receptor agonist, improves the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in Ube3a-deficient mice in vivo. These results suggest that pharmacologically increasing tonic inhibition may be a useful strategy for alleviating motor dysfunction in Angelman syndrome.

Molecular characterization of del(8)(p23.1p23.1) in a case of congenital diaphragmatic hernia

A 36‐week‐old fetus was referred to the medical center because of his cystic mass and fluid in left thoracic cavity, and was delivered by cesarean section to manage neonatal problems at 37 weeks of gestation. Emergent surgical repair of the left diaphragmatic hernia was performed, but severe hypoxia persisted, and he expired on the following day. Chromosome analysis of cultured amniotic fluid cells indicated 46,XY,del(8)(p23.1p23.1). This is the fourth case of 8p23.1 deletion associated with diaphragmatic hernia. Microarray comparative genomic hybridization analysis using DNA of cultured amniotic fluid cells showed that six clones were deleted, which were mapped to the region between two low copy repeats (LCRs) at 8p23.1 previously described. Microsatellite analysis revealed that the deletion was of paternal origin, and his parents did not carry 8p23.1 polymorphic inversion. These data strongly suggested that the 8p23.1 interstitial deletion should have arisen through a different mechanism from that of inv dup del(8p) whose structural abnormality is always of maternal origin and accompanies heterozygous 8p23.1 polymorphic inversion in mother.