Population genetics of the FRAXE and FRAXF GCC repeats,and a novel CGG repeat,in Xq28

Most of the rare folate sensitive fragile sites cloned to date arise from expansion of a CGG: CCG trinucleotide repeat array. Analysis of the CAG repeat at the Huntington Disease (HD) locus showed a positively skewed repeat distribution leading to the proposal that microsatellites are subject to a mutational bias toward expansion. Such a mutational bias predicts an increase in mean repeat size at all microsatellite loci. We present an analysis of repeats at two fragile site loci, FRAXE and FRAXF, and a novel CGG repeat in Xq28, in five different human populations, which suggests that these loci may also be subject to the same mutation process. The novel repeat array may represent the first evidence for the existence of a fourth fragile site in Xq27.3-28. Am. J. Med. Genet. 73:463–469, 1997. © 1997 Wiley-Liss, Inc.     

FRAXE mutation analysis in three spanish families

Very little is known about the phenotype of FRAXE-positive individuals and the relation between the genotype/phenotype and genotype/cytogenetic expression. We describe three families with normal and mildly affected individuals and a severely retarded male expressing fragility at the FRAXE locus or presenting different expansions at the CGG FRAXE triplet. In addition, we analyze the FRAXE mutation in sperm DNA from a retarded male carrier with a handicapped daughter expressing fragility at the FRAXE locus. Mental status in FRAXE individuals is highly variable and, although mild mental retardation is observed in most cases, several carrier males are apparently normal. It seems that methylation is not as strictly associated with size of CGG triplets in the FRAXE locus as in FRAXA, and it is possible that normal carrier individuals with fully methylated increments in lymphocytes have a certain proportion of unmethylated alleles in the critical (i.e., neural) tissues. FRAXE mutation is apparently similar to FRAXA in that males with somatic large methylated increments are carriers of small unmethylated ones in germinal cells. © 1996 Wiley-Liss, Inc.     

A new PCR assay useful for screening of FRAXE/FMR2 mental impairment among males

FRAXE (FMR2) is a fragile site associated with mental impairment located in Xq28, 600 kb distal to FRAXA (FMR1), the fragile X syndrome fragile site. The FRAXE mutation is an expansion of a CCG repeat that results in methylation of a nearby CpG island. FRAXE alleles could be divided into four categories: normal (6–30 CCG repeats), intermediate (31–60 CCG repeats), premutation (61–200 CCG repeats), and full mutation (over 200 repeats). We have developed a non‐isotopic polymerase chain reaction (PCR)‐based assay for the identification of FRAXE full mutation alleles among mentally impaired men. In this novel PCR test for the FRAXE locus, we used three primers to permit an amplification of a 223 bp monomorphic internal control fragment in addition to the amplification of a 419 bp (CCG)₁₆ FRAXE locus band. A linear series of 93 male patients referred for FRAXE testing but found to be negative for the (CCG)_n expansion in the FMR2 gene by Southern blotting analysis were retested by our PCR technique. In addition, we analyzed two positive controls consisting of a FRAXE fully mutated male and one male with a Xq terminal deletion. The developed PCR test showed accuracy of 100% in the normal individuals retested by PCR analysis, as well as in the two positive control samples utilized, in which the strategy of multiplex amplification worked as expected. Although not suitable for medical diagnosis of females and mosaics, it constitutes an important strategy for PCR typing and for FRAXE population screening. Hum Mutat 18:157–162, 2001. © 2001 Wiley‐Liss, Inc.     

Trinucleotide repeat expansion in the FRAXE locus is not common among institutionalized individuals with non-specific developmental disabilities

Expansion of a polymorphic GCC-repeat at the FRAXE locus has been associated with expression of chromosome fragility at this site and cognitive impairment in some individuals previously testing negative for CGG-repeat expansion in the fragile X mental retardation-1 (FMR1) gene. To determine the frequency of FRAXE triplet repeat expansion among persons with developmental disability, 396 individuals from two institutions were studied, all of whom were negative for FMR1 repeat expansion. Clinically, there was a wide range of mental impairment, with the majority (61.1%) being severely to profoundly affected. The distribution of FRAXE GCC-repeat numbers in the study population was 5– 38: 28 (5.6%) with 10–14 repeats; 366 (73.8%) with 15–19 repeats; 74 (14.9%) with 20–24 repeats; 20 (4.0%) with 25–29 repeats; and 5 (1.0%) with 30–38 repeats, with no individuals demonstrating repeat expansion. One profoundly retarded male was found to have a deletion of about 40 bp. Southern blots of HindIII-digested DNAs from individuals with ≥ 26 repeats all showed normal patterns. These results suggest that FRAXE GCC-repeat expansion is not a common cause of developmental disability in institutionalized persons with mild to profound mental retardation. © 1996 Wiley-Liss, Inc.     

Molecular analysis of HLA allele frequencies and haplotypes in Baloch of Iran compared with related populations of Pakistan

The extreme polymorphism in different loci of the human leukocyte antigen (HLA) system has been used as an invaluable tool for anthropological studies. Determination of HLA allele and haplotype frequencies in different ethnic groups is useful for population genetic analyses and the study of genetic relationships among them. In the present study, molecular analysis of HLA-A, -B, -C, -DQA1, -DQB1, and -DRB1 genes has been used to assign HLA allele and haplotype frequencies in 100 unrelated healthy individuals from the Baloch ethnic group of Iran. The results were compared with Baloch and other ethnic groups in the neighboring Pakistan. The results of this study showed that the most frequent HLA class I alleles were A*02011 (20.2%), B*4006 (11.1%), and C*04011 (28.6%). The most common HLA class II alleles were DQA1*0101/2 (42.5%), DQB1*0201 (32%), and DRB1*0301 (29%). Three-locus haplotype analysis revealed that A*11011-B*4006-C*15021 (5.8%) and DQA1*0501-DQB1*0201-DRB1*0301 (22.1%) were the most common HLA class I and II haplotypes, respectively, in this population. Neighbor-joining tree based on DA genetic distances and correspondence analysis according to HLA-A, -B, -DQB1, and -DRB1 allele frequencies showed that Baloch of Iran are genetically very close to Baloch and Brahui of Pakistan. This may reflect an admixture of Brahui and Baloch ethnic groups of Pakistan in the Balochistan province of Iran.     

隔离群体中3个Y-STR基因座的遗传多态性分析

目的 了解3个Y染色体上短串联重复序列（short tandem repeat on Y chromosome,Y-STR)基因座在隔离群体中的多态性，探讨用Y－STR分析群体亲缘关系的可能性。方法 在377测序仪上基因扫描技术对浙江省两个海岛隔离群体（80人和60人），和1个开放群体（36人）的DYS388、DYS390`DYS395进行检测。结果 DYS388、DYS390、DYS395在渔山岛、桃花岛、开放群体中的等位基因数分别为8、9、7；5、6、7；6、6、5。3个群体中每个Y－STR基因的基因多样性在0.80-0.80之间。隔离群体和开放群体在等位基因的频率分布、相同基因型数的分布差异无显著性。遗传距离最大的桃花岛与开放群体，其次是渔山岛与桃花岛群体，最小的是渔山岛和开放群体。结论 DYS388的常见等位基因为129，DYS390、DYS395分别为215和119。DYS388、DYS390、DYS395的基因频率、基因多样性、群体的相同基因型分布和遗传距离均不能说明隔离群体之间、以及与开放群体的亲缘关系程度，提示用Y－STR分析群体亲缘关系须在增加样本量的基础上进行继续深入的研究。     

FRAXA与FRAXE位点的快速基因筛查

Ｘ染色体长臂末端罕见脆性位点Ａ与Ｅ与分别由一个可遗传的三核苷酸重复序列的延长所致二种脆 性位占在细胞遗传学水平难以区别。为了建立快速筛查ＦＲＡＸＡ与ＦＲＡＸＥ位点的基因诊断法，本文采用毛细管ＰＣＲ－序列胶银染显示法对正常人，脆性Ｘ综合征家系及智力儿童三组样本进行了检测，结果显示正常人群的Ｐ（ＣＧＧ）ｎ与Ｐ（ＧＣＣ）ｎ均呈多态分面     

Slight instability of a FMR-1 allele over three generations in a family from the general population

We report on a family segregating a FMR-1 allele within the “grey zone” of triplet repeat length (n=51). The allele showed a 1-unit increment when transmitted through a female meiosis and a 1-unit increment when transmitted through a male of the next generation. At the following generation, a pregnant woman had amniocentesis performed. The latter showed she transmitted the allele unchanged (n = 53) to her male fetus. This family was not ascertained through an affected subject, and there was no family history of mental retardation. Thus our observation reflects the natural history of an unstable allele in the general population. Systematic analysis of such alleles may help refine our understanding of the grey zone of triplet repeat length. © 1996 Wiley-Liss, Inc.     

HLA-A, -B and -DRB1 allele frequencies in the Bangladeshi population

Population genetic studies have become an invaluable tool because of the extreme polymorphism found at some of the loci of the human leukocyte antigen (HLA) system. In this study, we are reporting for the first time the genetic polymorphism of 141 healthy unrelated Bangladeshi Bangalees living in central region of Dhaka. We studied the HLA-A, -B and -DRB1 loci using polymerase chain reaction with sequence-specific primers. The allelic frequencies, two and three locus haplotype frequencies were statistically analyzed. A total of 16 HLA-A alleles, 26 HLA-B alleles and 14 HLA-DRB1 alleles were detected. A*33-B*44 (8.15%) was the most common two loci class 1 haplotype, whereas A*33-B*44-DRB1*07 (6.38%) was the most frequent three loci haplotype. The most common HLA-A, HLA-B and HLA-DRB1 alleles were A*33 (17.02%), B*15 (19.5%) and DRB1*15 (29.07%), respectively. Construction of phylogenetic tree using average linkage between groups and correspondence analysis showed close associations with Indian non-tribal random Dravidians, north Indian Hindus and some relations with Mongolian and Pakistani populations. We believe this data will provide useful information for bone marrow registry, legal medicine, disease association and anthropological studies.     

DNA diagnosis of FRAXA and FRAXE in Chinese children with neurodevelopmental disorders and fragile X syndrome

Fragile X (FraX) syndrome is the most common cause of inherited mental retardation. To see whether FRAXA or FRAXE can account for the etiology of some unexplained neurodevelopmental disorders in children, we screened for trinucleotide repeat expansion in a consecutive cohort of 73 Chinese children and their mothers seen in 1995 (group 1) referred for developmental assessment due to developmental delay, language delay, attention deficit hyperactivity disorder, autistic spectrum disorder, mental retardation and/or learning disability. We also screened DNA samples of all five previously diagnosed cytogenetically-positive FraX boys, their mothers and sisters (group 2). A control group of unrelated teenagers and adults were recruited from the community (group 3). In group 1, 3 families (2 mothers and a mother and her son) were found to carry a small premutation allele at FRAXA (premutation frequency = 2%, 3/153 independent X chromosomes), but none had any expansion at FRAXE. In group 2, all 5 FraX boys had full mutation at FRAXA and normal repeat length at FRAXE. In group 3, 1 male has a premutation allele out of 18 males and 59 females tested (premutation frequency of control = 0.7%, 1 out of 136 X chromosomes). For FRAXE screening in group 3, 2 females were carriers (1.5%, 2 out of 136 X chromosomes). Thus, FRAXA and FRAXE cannot account for the etiology of neurodevelopmental disorders in our cohort of Chinese children, and the prevalence of FRAXE mutation in normal Chinese population appears to be higher than reported in the Caucasians.     

蒙族和汉族人群 CYP1A1基因MspⅠ位点的多态性

目的研究内蒙古地区蒙族和汉族人群CYP1A1基因Msp Ⅰ位点的多态性。方法应用聚合酶链反应．限制性片段长度多态技术对无血缘关系的80名蒙族和120名汉族个体的基因型进行分析。结果内蒙古地区蒙族和汉族人群CYP1A1基因wt／wt、wt／vt和vt/vt 3种基因型的频率分布分别是：蒙族35．0％、48．7％、16．3％和汉族33．3％、52．5％、14．2％。两者之间经X^2检验差异无统计学意义（P〉0．05）。结论内蒙古地区蒙族和汉族人群CYP1A1 基因Msp Ⅰ的基因型频率分布无明显差异。     

APO E Polymorphism in Spanish and Moroccan populations

Apolipoprotein E (apoE) gene polymorphism was analyzed by polymerase chain reaction in one Moroccan and six Spanish populations, a total of 660 individuals. No significant differences were observed between samples, and the mean relative frequencies (with 95% confidence intervals) found were 0.104 (0.069–0.139) for the e4 allele, 0.855 (0.813–0.897) for e3 and 0.041 (0.015–0.067) for e2. Frequencies of the e4 allele were low in comparison to Northern European populations, but similar to those reported for other South-European populations. The presence of a rare mutation, E2 Christchurch, in one Basque individual was confirmed by sequence analysis.     

用分子克隆技术构建D12S391基因座等位基因分型标准物及群体遗传学研究

目的 解决长期困扰短串联重复序列（short tandem repeat,STR）分型上存在的准确性和标准化问题。方法 先用PCR扩增出D12S391基因座的9个等位基因片段，将其插入pUC重组质粒中，经DNA测序分析证实插入片段的结构及大小，用国际标准将插入的等位基因片段进行命名，最后经转染、扩大培养、扩增及再鉴定后，制备出标准的D12S391等位基因分型标准物。结果 应用此法制备出大量的D12S391基因座等位基因分型标准物，并将其用于调查该基因座在德国Mainz地区、日本Miyazaki地区及中国成都汉族、北京汉族、新疆维吾尔族和甘肃回族6个群体中的基因型分布频率。D12S391基因座在各群体中均有较高的多态性，其非父排除概念及个人识别能力分别为0.609-0.786和0.940-0.952。结论 该法制备的STR基因座等位基因分型标准物在法医科学实践中应用价值极高，D12S391基因座是一个非常适合于群体遗传学研究和法医科学应用的遗传标记。     

中国四个人群中MICA基因多态性研究

目的：了解中国４个人群中ＭＩＣＡ基因第５外显子（ＧＣＴ）ｎ位点的遗传２多态性并从遗传学的角度提供白马藏族起源的初步证据。方法：４１１个无血缘关系个体的血标本或唾液标本分别采自四川平武县白巴藏族、西藏拉萨和云南中甸县藏族、成都地区汉族以及四川茂县羌族。以１对跨越ＭＩＣＡ基因第５外显子ＧＣＴ重复序列区的引物扩增基因组ＤＮＡ,产物用变性聚丙烯酰胺凝胶电泳分离,银染显色分析。结果：在４个群体中均检测出５种已报道的等位片段,并均以Ａ５最常见,在白马藏族、藏族、成都汉族和羌族中的频率分别为０．３２５、０．３４５、０．３９０和０．３１９。Ａ５．１在成都汉族和羌族中占第２位,频率分别为０．２３０和０．２９３,Ａ４和Ａ９在白马藏族和藏族中分别占第２位,频率分别为０．２５４和０．２７２。上述各等位基因分布在４个群体中存在差异,尤其     

A genomic analysis identifies a novel component in the genetic structure of sub-Saharan African populations

Studies of large sets of single nucleotide polymorphism (SNP) data have proven to be a powerful tool in the analysis of the genetic structure of human populations. In this work, we analyze genotyping data for 2841 SNPs in 12 sub-Saharan African populations, including a previously unsampled region of southeastern Africa (Mozambique). We show that robust results in a world-wide perspective can be obtained when analyzing only 1000 SNPs. Our main results both confirm the results of previous studies, and show new and interesting features in sub-Saharan African genetic complexity. There is a strong differentiation of Nilo-Saharans, much beyond what would be expected by geography. Hunter-gatherer populations (Khoisan and Pygmies) show a clear distinctiveness with very intrinsic Pygmy (and not only Khoisan) genetic features. Populations of the West Africa present an unexpected similarity among them, possibly the result of a population expansion. Finally, we find a strong differentiation of the southeastern Bantu population from Mozambique, which suggests an assimilation of a pre-Bantu substrate by Bantu speakers in the region.     

Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibitor gene (C1NH gene), including approximately 20% of partial deletions/duplications whose boundaries are usually within Alu repeats. To ensure complete molecular characterization of C1 inhibitor deficiencies a fluorescent multiplex assay was constructed to amplify simultaneously five exons of C1NH and an exon of the BRCA1 gene. PCR protocols were optimized for these amplicons (size range between 300 and 700 bp). Forward and reverse chimeric primers that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data were analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicons, rather than by calculating the ratios of peak areas. Tests on a collection of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were diagnosed by using this multiplex PCR approach: a deletion of exon 4, a deletion of exons 5 and 6, and an apparently complete gene deletion. Besides being suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted to complex genes for the screening of rearrangements.     

SgD-CNV, a database for common and rare copy number variants in three Asian populations

Copy number variants (CNVs) extend our understanding of the genetic diversity in humans. However, the distribution and characteristics of CNVs in Asian populations remain largely unexplored, especially for rare CNVs that have emerged as important genetic factors for complex traits. In the present study, we performed an in-depth investigation of common and rare CNVs across 8,148 individuals from the three major Asian ethnic groups: Chinese (n = 1,945), Malays (n = 2,399), and Indians (n = 2,217) in Singapore, making this investigation the most comprehensive genome-wide survey of CNVs outside the European-ancestry populations to date. We detected about 16 CNVs per individual and the ratio of loss to gain events is ～2:1. The majority of the CNVs are of low frequency (<10%), and 40% are rare (<1%). In each population, ～20% of the CNVs are not previously catalogued in the Database of Genomic Variants (DGV). Contrary to findings from European studies, the common CNVs (>5%) in our populations are not well tagged by SNPs in Illumina 1M and 610K arrays, and most disease-associated common CNVs previously reported in Caucasians are rare in our populations. We also report noticeable population differentiation in the CNV landscape of these Asian populations, with the greatest diversity seen between the Indians and the Chinese.     

Genetic variation and evolutionary stability of the FMR1 CGG repeat in six closed human populations

In an attempt to understand the allelic diversity and mutability of the human FMR1 CGG repeat, we have analyzed the AGG substructure of this locus within six genetically-closed populations (Mbuti pygmy, Baka pygmy, R. surui, Karitiana, Mayan, and Hutterite). Most alleles (61/92 or 66%) possessed two AGG interspersions occurring with a periodicity of one AGG every nine or ten CGG repeats, indicating that this pattern is highly conserved in all human populations. Significant differences in allele distribution were observed among the populations for rare variants possessing fewer or more AGG interruptions than the canonical FMR1 CGG repeat sequence. Comparisons of expected heterozygosity of the FMR1 CGG repeat locus with 30 other microsatellite loci, demonstrated remarkably similar levels of polymorphism within each population, suggesting that most FMR1 CGG repeat alleles mutate at rates indistinguishable from other microsatellite loci. A single allele (1 out of 92) was identified with a large uninterrupted tract of pure repeats (42 pure CGG triplets). Retrospective pedigree analysis indicated that this allele had been transmitted unstably. Although such alleles mutate rapidly and likely represent evolving premutations, our analysis suggests that in spite of the estimated frequency of their occurrence, these unstable alleles do not significantly alter the expected heterozygosity of the FMR1 CGG repeat in the human population. © 1996 Wiley-Liss, Inc.     

New polymorphic microsatellite markers in the human MHC class II region

The human major histocompatibility complex (MHC) class II region spans approximately 1.1 Mb and presently contains over 30 functional genes Susceptibility loci to numerous diseases, mainly of autoimmune nature are known to map to the this region, as assessed by associations with particular HLA class II alleles. However, it has been difficult to precisely localize these susceptibility loci to a single gene, for example DQB1 or DRB1, due to the tight linkage disequilibrium observed in the HLA class II region. To facilitate disease mapping within this region, we have analyzed 2 to approximately 5 bases short tandem repeats (microsatellites) in this same region. A total of 494 microsatellites were identified from the genomic sequence of the HLA class II region. These consist of 158 di-, 65 tri-, 163 tetra-, and 108 pent-nucleotide repeats, out of which four were located within the coding sequence of expressed genes (Daxx, BING1, RXRB and COL11A2). Twenty-two repeats were selected as polymorphic markers due to their high (average) number of alleles (8.9) as well as their high polymorphic content value (PIC) (0.58). These novel polymorphic microsatellites will provide useful genetic markers in HLA-related research, such as genetic mapping of HLA class II-associated diseases, transplantation matching, population genetics, identification of recombination hot spots as well as linkage disequilibrium studies.     

浙南畲族群体3个STR基因座遗传多态性研究

目的对浙南畲族群体的D18S865、D18S535和D18S1364三个短串联重复序列基因座的遗传多态性进行调查分析,获得相应多态性基因座的群体遗传学数据。方法从无血缘关系的110名浙南地区畲族个体的抗凝血中提取DNA,进行PCR扩增,聚丙烯酰胺凝胶垂直电泳（PAGE）并银染。结果D18S865、D18S535和D18S1364基因座的等位基因分别为6,9,11个,基因型分别为15,27,38种。基因型频率的分布符合Hardy-Weinberg平衡,三个基因座的观测杂合度分别为0.773、0.773、0.891,多态信息含量分别为0.730、0.800、0.850,三个基因座联合个人识别力为0.9998。结论所得到的等位基因频率数据可为浙南畲族人群法医个体识别,亲子鉴定及遗传学研究提供更多依据。