Identification and evaluation of 55 genetic variations in the <Emphasis Type="Italic">BRCA1</Emphasis> and the <Emphasis Type="Italic">BRCA2</Emphasis> genes of patients from 50 Japanese breast cancer families

We sequenced approximately 23 kb genomic regions containing all the coding exons and their franking introns of two breast cancer susceptibility genes, BRCA1 and BRCA2, of 55 individuals from 50 unrelated Japanese breast cancer families. We identified 55 single-nucleotide polymorphisms (SNPs) (21 in BRCA1 and 34 in BRCA2) containing nine pathogenic protein-truncating mutations (four in BRCA1 and five in BRCA2 from ten patients). Among the remaining 46 SNPs, allele frequencies of 40 were examined in both the breast cancer patients and 28 healthy volunteers with no breast cancer family history by PCR-RFLP or by direct DNA sequencing. Twenty-eight SNPs were common and were also found in the healthy volunteers and/or a SNP database. The remaining 18 were rare (allele frequency <0.05) and were not found in the healthy volunteers and/or the database. The pathogenic significance of these coding SNPs (cSNPs) remains to be clarified. The SNP information from this study will be useful in the future genetic testing of both BRCA1 and BRCA2 genes in the Japanese population.The first two authors contributed equally to this study.     

Direct or indirect association in a complex disease: the role of SLC22A4 and SLC22A5 functional variants in Crohn disease

A common haplotype spanning 250 kb on chromosome 5q31 is strongly associated with Crohn disease (CD). Recently, two functional variants within the SLC22A4 and SLC22A5 genes at this locus (IBD5), L503F (c.1507C > T) and G-207C (c.-207G > C), have been proposed to contribute directly to susceptibility to CD. However, extensive linkage disequilibrium at the IBD5 locus has complicated efforts to distinguish causal variants from association of the general risk haplotype. We genotyped the SLC22A4 and SLC22A5 variants and other polymorphisms across the risk haplotype in four populations of European origin, and applied regression-based haplotype analysis to over 1,200 fully genotyped case-control pairs, modeling case/control status on the presence of one or more SNPs to test for conditional association and to identify risk haplotypes. We found highly significant association of SNPs at the IBD5 locus with Crohn disease in all populations tested. However, the frequencies of L503F and G-207C in individuals who did not carry the general IBD5 risk haplotype were not significantly different in cases and controls, with associated disease odds ratios (ORs) of 0.90 (95% CI, 0.57-1.40) and 0.90 (95% CI, 0.65-1.23), respectively. Haplotype analysis showed that addition of the SLC22A4 and SLC22A5 variants to a null model that included the background risk haplotype did not significantly improve the model fit. In addition to the common risk haplotype, several rare haplotypes had an increased frequency in cases compared to controls. This study suggests that the molecular basis for Crohn disease susceptibility at the IBD5 locus remains to be defined, and highlights the challenge of the identification of causal variants in a complex disease in regions of extensive linkage disequilibrium.     

Association of the <Emphasis Type="Italic">HTRA1</Emphasis> gene variant with age-related macular degeneration in the Japanese population

The purpose of this investigation was to determine whether the high-temperature requirement A-1 (HTRA1) gene polymorphism is associated with age-related macular degeneration (AMD) in native, unrelated Japanese patients. A total of 123 patients with AMD and 133 control subjects without AMD were recruited for this study. The single-nucleotide polymorphism (SNP) rs11200638 in the HTRA1 gene was assessed using a TaqMan assay. The risk A allele frequencies in the AMD cases and control patients were 0.577 and 0.380, respectively, and were associated with a significant risk of developing AMD (p=7.75x10(-6)). The results were more significant in subtype analyses with wet AMD (p=5.96x10(-7)). We conclude that the rs11200638 variant in the HTRA1 gene is strongly associated with AMD in the Japanese population. This result supports the hypothesis that the HTRA1 gene may increase susceptibility to AMD development and can participate in a potential new molecular pathway for AMD pathogenesis by extending this association across diverse ethnicities.     

Haplotype architecture of the norepinephrine transporter gene <Emphasis Type="Italic">SLC6A2</Emphasis> in four populations

The norepinephrine transporter (NET) regulates levels of monoamine neurotransmitters integral to a variety of behaviors and autonomic functions. Two SLC6A2 polymorphisms have been used in genetic association studies, generating intriguing but nondefinitive results on traits such as hypertension and mood. One of these SLC6A2 variants is functional but rare. The other is common but not informative over the entire 48 kb SLC6A2 region and is insufficient to capture the functional diversity potentially contained within any SLC6A2 region. To elucidate SLC6A2 haplotype structure and define markers sufficient to capture haplotype diversity within detected haplotype blocks, 26 single-nucleotide polymorphisms (SNPs) were genotyped in 384 individuals evenly divided across Finnish Caucasian, US Caucasian, Plains American Indian, and African American populations. Three conserved blocks, 13.6, 12.5, and 25 kb in size and showing little evidence for historical recombination were observed in all populations. Haplotype diversity in block 1 and numbers of common haplotypes were highest in African Americans, among whom 5–6 optimal markers were sufficient to maximize diversity of each block. For other populations, 2–3 markers/block sufficed, but the optimal markers differed across populations. The SLC6A2 haplotype map and 25-marker panel (excluding the monomorphic one) is a comprehensive tool for genetic linkage studies on phenotypes related to NET function.Parts of this work were presented at the 53rd Annual Meeting of American Society of Human Genetics, Los Angeles, CA, USA, November 2003.     

Catalog of 86 single-nucleotide polymorphisms (SNPs) in three uridine diphosphate glycosyltransferase genes: <Emphasis Type="Italic">UGT2A1</Emphasis>, <Emphasis Type="Italic">UGT2B15</Emphasis>, and <Emphasis Type="Italic">UGT8</Emphasis>

We report here three high-density maps of variations found among 48 Japanese individuals in three uridine diphosphate glycosyltransferase (UGT) genes, UGT2A1, UGT2B15, and UGT8. A total of 86 single-nucleotide polymorphisms (SNPs) were identified through systematic screening of genomic regions containing these genes: 8 in 5′ flanking regions, 7 in coding regions, 67 in introns, 3 in 3′ untranslated regions, and 1 in a 3′ flanking region. We also discovered 14 variations of other types. Of the 86 SNPs, 63 (73%) were considered to be novel on the basis of comparison of our data with the Database of SNPs (dbSNP) of the National Center for Biotechnology Information. Among the seven SNPs identified in exonic sequences, five were non-synonymous changes that would result in amino-acid substitutions. The collection of SNPs derived from this study will serve as an additional resource for studies of complex genetic diseases and responsiveness to drug therapy. Received: June 12, 2002 / Accepted: June 13, 2002     

Association and interaction analyses of <Emphasis Type="Italic">NRG1</Emphasis> and <Emphasis Type="Italic">ERBB4</Emphasis> genes with schizophrenia in a Japanese population

Neuregulin 1 (NRG1) is one of the most promising candidate genes for schizophrenia. A number of replication studies have been conducted, although the results were inconsistent and no susceptible variant has yet been identified. The inconsistency might be attributed to the ethnic difference in allele and haplotype frequencies. However, it is equally possible that one or more genes interacting with NRG1 may also be implicated in schizophrenia and attribute to the inconsistency. To test the hypothesis, we conducted an interaction analysis between NRG1 and one of its receptor’s (ERBB4) polymorphisms as well as the association analysis of the two genes associated with schizophrenia in Japanese. We observed no significant difference between patients and controls in allele frequencies or genotypic distributions of the 18 polymorphisms of the genes. The permutation test showed no significant differences in estimated haplotype frequencies between patients and controls, including the haplotype HAP_ICE. In the interaction analysis, significant interaction was observed between rs2919381 in NRG1 and rs7560730 in ERBB4 (P = 0.047, corrected). Thus, our results suggest the possibility that interaction between variants in NRG1 and ERBB4 might contribute to susceptibility for schizophrenia in a Japanese population. Further investigation may be necessary to confirm our results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

High-density single-nucleotide polymorphism (SNP) map of the 150-kb region corresponding to the human ATP-binding cassette transporter A1 (<Emphasis Type="Italic">ABCA1</Emphasis>) gene

Highly dense catalogs of human genetic variations, in combination with high-throughput genotyping technologies, are expected to clarify individual genetic differences in pharmacological responsiveness and predispositions to common diseases. Here we report single-nucleotide polymorphisms (SNPs) present among 48 Japanese individuals at the locus for the human ATP-binding cassette transporter A1 (ABCA1) gene. ABCA1 plays a key role in apolipoprotein-mediated cholesterol transport, and mutations in this gene are responsible for Tangier disease and familial high-density lipoprotein deficiency associated with reduced cholesterol efflux. We identified a total of 162 SNPs, 149 of which were novel, within the 150-kb region encompassing the entire ABCA1 gene. Eight of the SNPs lie within coding elements, two in 5′ flanking regions, 147 in introns, and five in 3′ untranslated regions, but none were found in 5′ untranslated or 3′ flanking regions. The ratio of transitions to transversions was approximately 2.37 to 1. Our dense SNP map of this region could serve as a powerful resource for studies of complex genetic diseases that may be associated with ABCA1 and of individual responses to drug therapy. Received: May 22, 2001 / Accepted: June 12, 2001     

<Emphasis Type="Italic">SCN1A,</Emphasis><Emphasis Type="Italic">SCN1B</Emphasis>, and <Emphasis Type="Italic">GABRG2</Emphasis> gene mutation analysis in Chinese families with generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+; MIM#604233) is a familial epilepsy syndrome characterized by phenotypic and genetic heterogeneity. It was associated with mutations in the neuronal voltage-gated sodium channel subunit gene (SCN1A, SCN2A, SCN1B) and ligand-gated gamma aminobutyric acid receptors genes (GABRG2, GABRD). We investigated the roles of SCN1A, SCN1B, and GABRG2 mutations in the etiology of Chinese GEFS+ families. Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood lymphocytes of 23 probands and their family members. The sequences of SCN1A, SCN1B, and GABRG2 genes were analyzed by polymerase chain reaction (PCR) and direct sequencing. The major phenotypes of affected members in the 23 GEFS+ families exhibited FS and FS+, whereas rare phenotypes afebrile generalized tonic-clonic seizures (AGTCS), myoclonic-astatic epilepsy (MAE), and partial seizures were also observed. A novel SCN1A mutation, p.N935H, was identified in one family and another novel mutation in GABRG2, p.W390X, in another family. However, no SCN1B mutation was identified. The combined frequency of SCN1A, SCN1B, and GABRG2 mutations was 8.7% (2/23), extending the distribution of SCN1A and GABRG2 mutations to Chinese GEFS+ families. There were still unidentified genes contributing to the pathogenesis of GEFS+.     

<Emphasis Type="Italic">MECP2 </Emphasis>and <Emphasis Type="Italic">CDKL5</Emphasis> gene mutation analysis in Chinese patients with Rett syndrome

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this study, the MECP2 sequences in 121 unrelated Chinese patients with classical or atypical RTT were screened for deletions and mutations. In all, we identified 45 different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation (15.7%) was the most common, followed in order of frequency by p. R168X (11.8%), p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p. R306C (3.9%). In addition, we identified five novel MECP2 mutations: three missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare (0.9%). The remaining cases without MECP2 mutations were screened for the cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with homozygous mutations and normal female karyotypes. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases.     

An autophagy gene, <Emphasis Type="Italic">MgATG5</Emphasis>, is required for cell differentiation and pathogenesis in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Autophagy is a conserved degradation pathway that is involved in the maintenance of normal cell differentiation and development. The Saccharomyces cerevisiae ATG5 gene is an important component of the autophagy process. In this study, we identified MgATG5 as an autophagy-related gene in Magnaporthe oryzae that is homologous to ATG5. Using targeted gene replacement, an Mgatg5∆ mutant was generated and fungal autophagy was blocked. Cytological analysis revealed that the mutant had poor fungal morphogenic development, including a shortened aerial hyphae lifespan, decreased conidiation and perithecia formation, delayed conidial germination and appressorial formation, postponement of conidial cytoplasm transfer during appressorium formation, and reduction in formation of the penetration peg. Turnover of endogenous matter in the Mgatg5∆ mutant was also affected, as demonstrated by defects in the formation of conidial lipid droplets, and in the degradation of conidial glycogen deposits during appressorium formation. Lipid droplets and glycogen are necessary to generate adequate turgor in appressoria for invading the host surface. As a result of the decreased appressorium turgor and differentiation in the penetration peg, Mgatg5∆ pathogenicity was deficient in two host plants tested. The developmental and pathogenic phenotypes were restored by the introduction of an intact copy of MgATG5 into Mgatg5∆, demonstrating that the MgATG5 deletion was responsible for the cellular defects. Taken together, these findings suggest that autophagy promotes cell differentiation through turnover of endogenous matter during fungal development, and is thus essential for the pathogenicity of the rice blast fungus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J.-P. Lu and X.-H. Liu contributed equally to this work and are regarded joint first authors.     

Tracing origin of serrated adenomas with <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">KRAS</Emphasis> mutations

Serrated neoplasm of the colorectum raised many as-yet unanswered issues. To characterize serrated neoplasia pathway, we investigated BRAF and KRAS mutations in 35 traditional serrated adenomas. BRAF exons 11 and 15, and KRAS exon 2 were amplified by polymerase chain reaction and directly sequenced. BRAF V599E mutation was found in 27 serrated adenomas (77.1%), and KRAS mutations were found in 3 (8.6%) of 35 traditional serrated adenomas. In 13 cases, mixed polyps composed of traditional serrated adenomas and hyperplastic (serrated) polyps were observed, and seven of them showed the same BRAF mutations in both components. Somatic mutations of BRAF and KRAS genes were mutually exclusive. These findings suggest that BRAF mutations are early and a critical event in the serrated adenomas, and most serrated adenomas in both sides of colon may progress from microvesicular hyperplastic polyps via BRAF mutations, and some left-sided serrated adenomas develop via KRAS mutations.     

Hypermethylation of<Emphasis Type="Italic"> Chfr</Emphasis> and<Emphasis Type="Italic"> hMLH1</Emphasis> in gastric noninvasive and early invasive neoplasias

Human tumors are genetically unstable, and the instability exists at two distinct levels—the chromosomal level and the nucleotide level. Chfr and hMLH1 hypermethylation, which may lead to chromosomal instability (CIN) and microsatellite instability (MSI), respectively, was analyzed in gastric noninvasive neoplasias (NIN, Padova international classification) and submucosal invasive adenocarcinomas and in their corresponding non-neoplastic gastric epithelia. Results were compared with microsatellite status, p53 immunoreactivity, and cellular phenotype. Hypermethylation of Chfr and hMLH1 was observed in: 10% (1/10) and 0% (0/10) of low-grade NIN (L-NIN); 63% (5/8) and 63% (5/8) of high-grade NIN, including suspicion for carcinoma without invasion (H-NIN); 36% (5/14) and 57% (8/14) of high-grade NIN, including carcinoma without invasion; and 35% (7/20) and 25% (5/20) of submucosal invasive adenocarcinomas, respectively. Hypermethylation was less frequent in L-NIN than H-NIN (P<0.05) for Chfr and was also less frequent in L-NIN than the others (P<0.05) for hMLH1. We failed to find a significant correlation between Chfr hypermethylation and chromosomal loss of heterozygosity, although hypermethylation of hMLH1 was significantly associated with high-frequency MSI (P<0.01). Expression of p53 was not associated with Chfr or hMLH1 methylation. As for cellular phenotype, hypermethylation of Chfr and hMLH1 was frequent in tumors exhibiting the foveolar epithelial phenotype (50%, 2/4 and 75%, 3/4, respectively) and the ordinary phenotype (40%, 16/40 and 38%, 15/40, respectively), but never in those with the complete-type intestinal metaplastic phenotype (0%, 0/8 for both). In addition, hypermethylation of Chfr and hMLH1 occurred concurrently (P<0.01); methylation was more frequent in patients over 70 years of age (P<0.01), and it was also present in some samples of non-neoplastic gastric epithelia from elderly patients. Thus, some gastric tumors with the foveolar or ordinary phenotype may develop as a result of age-related methylation of Chfr and hMLH1, although Chfr methylation was not associated with CIN.     

Expression of <Emphasis Type="Italic">HSPF1</Emphasis> and <Emphasis Type="Italic">LIM</Emphasis> in the lymphoblastoid cells derived from patients with bipolar disorder and schizophrenia

We have previously reported the altered expressions of HSPF1 and LIM in the lymphoblastoid cell lines (LCLs) derived from Japanese patients with bipolar disorder (bipolar I disorder). The altered expression at the LCL level would be useful for developing diagnostic markers as well as a cellular model for bipolar disorder. In this study, we extended our previous study by measuring their expressions using the following samples: (1) larger number of LCLs from Japanese subjects, (2) LCLs from Caucasian subjects, and (3) LCLs from patients with bipolar II disorder or schizophrenia. We confirmed the increased expression of HSPF1 (P=0.009) and decreased expression of LIM (P=0.001) in the LCLs from patients with Japanese bipolar I disorder. These altered expressions were also observed in those from patients with Japanese bipolar II disorder (P=0.002 for HSPF1 and P=0.072 for LIM). We also found the altered expressions of HSPF1 in LCLs from Caucasian patients with bipolar II disorder (P=0.011) and LIM in those from patients with schizophrenia (P=0.001).