Hironao Numabe

Comprehensive genetic analysis of relevant four genes in 49 patients with Marfan syndrome or Marfan‐related phenotypes

In order to evaluate the contribution of FBN1, FBN2, TGFBR1, and TGFBR2 mutations to the Marfan syndrome (MFS) phenotype, the four genes were analyzed by direct sequencing in 49 patients with MFS or suspected MFS as a cohort study. A total of 27 FBN1 mutations (22 novel) in 27 patients (55%, 27/49), 1 novel TGFBR1 mutation in 1 (2%, 1/49), and 2 recurrent TGFBR2 mutations in 2 (4%, 2/49) were identified. No FBN2 mutation was found. Three patients with either TGFBR1 or TGFBR2 abnormality did not fulfill the Ghent criteria, but expressed some overlapping features of MFS and Loeys–Dietz syndrome (LDS). In the remaining 19 patients, either of the genes did not show any abnormalities. This study indicated that FBN1 mutations were predominant in MFS but TGFBRs defects may account for approximately 5–10% of patients with the syndrome.

The majority of the marker chromosomes in Japanese patients with stigmata of Turner syndrome are derived from Y chromosomes

SummaryDNA analyses of 41 individuals with stigmata of Turner syndrome and a 45,X/46,X+mar or 46,X+mar karyotype were carried out. Southern-blot analysis employing 17 Y-specific probes was used to determine whether the marker chromosome was Y-chromosomal in origin. Of the 41 DNA samples from these patients, 23 contained detectable Y-chromosomal DNA. Points of chromosome breakage were distributed over the entire length of the Y long arm. Three individuals, who carry different portions of the Y chromosome, had developed gonadoblastoma. GBY (the gonadoblastoma locus on the Y chromosome) is mapped proximal to DYS132, midway between the 13 Yq loci that we have studied. We also used a polymerase chain reaction technique that could detect 7 loci over the length of the Y chromosome. This technique may be useful for the rapid assessment of marker chromosomes, especially for evaluating the risk of gonadoblastoma.

DNA analyses of XX and XX-hypospadiac males

Fourteen 46,XX “males” were analyzed by Southern blot hybridization with seventeen different Y chromosome-derived DNA probes and by the polymerase chain reaction for an additional two sites on the short arm of Y. Eight 46,XX males possessed various segments of the short arm of the Y chromosome, including the sex determining region. The detected segments ranged from the two most distal loci to nearly the entire length of the short arm, viz., 10 out of 11 loci. None of the eight patients had hypospadia. Five out of the six remaining cases had hypospadia and no Y sequence was detected, suggesting the presence of a causative difference between hypospadiac and non-hypospadiac groups.

GPC3 mutations in seven patients with simpson-golabi-behmel syndrome

We analyzed mutations of the GPC3gene in seven males with typical manifestations of Simpson–Golabi–Behmel syndrome (SGBS). Genomic DNA was PCR amplified for its all eight exons and exon–intron boundaries using designed set of primers, and PCR products were directly sequenced. All seven males studied had mutations: One patient had a large deletion spanning introns 6 and 7, four each had a C → T base substitution resulting in a stop codon formation in exons 2, 3, and 4, one had a single‐base insertion in exon 2, and the other had a six‐base deletion and a three‐base insertion in exon 3; all resulting in loss‐of‐function of the glypican‐3 protein. These results, together with previous studies of GPC3 mutations, indicate that there is no hot spot for GPC3 mutations or deletions in the patients with the syndrome. Also, no correlation has been noted between the location and nature of mutations and the phenotype of the patients studied, as is the case of the present study.

PCR detection of structurally abnormal Y chromosomes.

SummaryThree probes each detecting a locus on the proximal long arm of the Y chromosome were partially sequenced. Thus, 3 sets of novel primers were developed which enable PCR detection of these 3 loci. Five previously reported primer sets, 3 on the short arm and each one on the centromere and the distal long arm, were mapped along with the novel three using a mapping panel consisted of 8 patients each with different structural abnormality of the Y chromosome. Now, PCR detection of these 8 loci covering an entire length of the Y chromosome has become possible enabling rapid screening of patients with Y chromosome aberrations.

Comprehensive screening of CREB-binding protein gene mutations among patients with Rubinstein-Taybi syndrome using denaturing high-performance liquid chromatography.

ABSTRACT  Mutations in the CREBBP (CREB‐binding protein gene) cause Rubinstein‐Taybi syndrome (RSTS). At present, however, genetic testing of CREBBP is not commonly applied in clinical settings because the currently available assays are technically and financially demanding, mainly because of the size of the gene. In the present study, we took advantage of a highly sensitive and specific, automated denaturing high‐performance liquid chromatography (DHPLC) technique. First, we developed a DHPLC‐based protocol to analyze the entire coding region of CREBBP. Second, we analyzed genetic samples from 21 RSTS patients using DHPLC. The coding region was amplified by 41 primer pairs, all of which have the same cycling conditions, aliquoted on a 96‐well format PCR plate. In this manner, all the exons were simultaneously amplified using a single block in a PCR machine. We then wrote a computer script to analyze all the PCR amplicons generated from various portions of the CREBBP gene in a serial manner at optimized conditions determined individually for each amplicon. Heterozygous CREBBP mutations were identified in 12 of the 21 patients: five frameshift mutations, three nonsense mutations, two splice‐site mutations, and two missense mutations. The resulting detection rate of 57% was comparable to the outcome of previous studies. The relatively high detection rate in the present study demonstrates the enhanced sensitivity of the DHPLC‐based mutation analysis, as exemplified by mutation analyses of other genes. The implementation of similar methodologies for other dysmorphic syndromes will help medical geneticists to confirm their clinical impressions and to provide accurate genetic counseling for patients and their families.

Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

Microarray analysis of 50 patients reveals the critical chromosomal regions responsible for 1p36 deletion syndrome-related complications

OBJECTIVE Monosomy 1p36 syndrome is the most commonly observed subtelomeric deletion syndrome. Patients with this syndrome typically have common clinical features, such as intellectual disability, epilepsy, and characteristic craniofacial features. METHOD In cooperation with academic societies, we analyzed the genomic copy number aberrations using chromosomal microarray testing. Finally, the genotype-phenotype correlation among them was examined. RESULTS We obtained clinical information of 86 patients who had been diagnosed with chromosomal deletions in the 1p36 region. Among them, blood samples were obtained from 50 patients (15 males and 35 females). The precise deletion regions were successfully genotyped. There were variable deletion patterns: pure terminal deletions in 38 patients (76%), including three cases of mosaicism; unbalanced translocations in seven (14%); and interstitial deletions in five (10%). Craniofacial/skeletal features, neurodevelopmental impairments, and cardiac anomalies were commonly observed in patients, with correlation to deletion sizes. CONCLUSION The genotype-phenotype correlation analysis narrowed the region responsible for distinctive craniofacial features and intellectual disability into 1.8-2.1 and 1.8-2.2 Mb region, respectively. Patients with deletions larger than 6.2 Mb showed no ambulation, indicating that severe neurodevelopmental prognosis may be modified by haploinsufficiencies of KCNAB2 and CHD5, located at 6.2 Mb away from the telomere. Although the genotype-phenotype correlation for the cardiac abnormalities is unclear, PRDM16, PRKCZ, and RERE may be related to this complication. Our study also revealed that female patients who acquired ambulatory ability were likely to be at risk for obesity.

HRAS mutants identified in Costello syndrome patients can induce cellular senescence: possible implications for the pathogenesis of Costello syndrome

Costello syndrome (CS) is a congenital disease that is characterized by a distinctive facial appearance, failure to thrive, mental retardation and cardiomyopathy. In 2005, we discovered that heterozygous germline mutations in HRAS caused CS. Several studies have shown that CS-associated HRAS mutations are clustered in codons 12 and 13, and mutations in other codons have also been identified. However, a comprehensive comparison of the substitutions identified in patients with CS has not been conducted. In the current study, we identified four mutations (p.G12S, p.G12A, p.G12C and p.G12D) in 21 patients and analyzed the associated clinical manifestations of CS in these individuals. To examine functional differences among the identified mutations, we characterized a total of nine HRAS mutants, including seven distinct substitutions in codons 12 and 13, p.K117R and p.A146T. The p.A146T mutant demonstrated the weakest Raf-binding activity, and the p.K117R and p.A146T mutants had weaker effects on downstream c-Jun N-terminal kinase signaling than did codon 12 or 13 mutants. We demonstrated that these mutant HRAS proteins induced senescence when overexpressed in human fibroblasts. Oncogene-induced senescence is a cellular reaction that controls cell proliferation in response to oncogenic mutation and it has been considered one of the tumor suppression mechanisms in vivo. Our findings suggest that the HRAS mutations identified in CS are sufficient to cause oncogene-induced senescence and that cellular senescence might therefore contribute to the pathogenesis of CS.

Squamous cell carcinoma-related antigen in children with atopic dermatitis.

dermatitis (AD) has increased. Children who show exanthema over the entire body with severe itching are not rare. However, the severity of AD is not completely correlated with the levels of IgE and eosinophil counts. Soluble E-selection is now considered a good marker.1 However, the assay is not easy to carry out. There is no clinical good marker reflecting the severity of atopic dermatitis. Squamous cell carcinoma (SCC)-related antigen has been purified from human uterine cervical SCC and developed to be a tumor marker, mainly for cervical squamous cell cancer.2 The antigen is also present and expressed in normal epithelium and epithelial tissues. Squamous cell carcinomarelated antigen has been reported to be elevated in several benign lesions, including lung and skin diseases.3 In the present study, we have shown that SCC-related antigen is useful to evaluate the severity of AD in children.