云南纳西族10个X-STR基因座遗传多态性研究

为研究云南纳西族人群10个位于X染色体的短串联重复序列基因座及单倍型的遗传多态性, 采用PCR扩增, 变性聚丙烯酰胺凝胶电泳结合银染显色分型技术, 对98名云南纳西族无关男性个体X染色体的10个STR基因座进行基因分型。结果显示, 98名无关男性个体中, DXS7423、DXS7424、DXS6799、DXS7133、DXS6804、DXS8378、HPRTB、DXS7130、DXS7132 和DXS6789分别检出4、7、6、3、6、5、5、7、6和8个等位基因, 等位基因频率分布在0.0102(DXS7132、DXS6789)~0.7347(DXS7133)之间。由DXS8378与DXS7130基因座组成的单倍型共检出20种, 由DXS6789、DXS6799和DXS7424基因座组成的单倍型共检出56种, 单倍型多样性分别为0.8553和0.9649, 说明所选的10个X-STR位点有较高的多态性信息, 在基因组多样性研究、法医学个体识别、亲权鉴定中具有重要应用价值。     

The relevance of paternity analysis in Romanian population using the D1S80 locus

At present, DNA fingerprinting for human identification and paternity testing is a necessary and usual procedure. D1S80 is one of the best known polymorphic loci showing a VNTR, and exhibiting a high heterozygosity. This genetic locus, with a Tsp 509 I polymorphism of its 5' flanking sequence (1, 9), have been successfully amplified from human genomic DNA isolated from blood. The Tsp 509 I polymorphism was detected by restriction after PCR amplification. We tested the relevance of paternity analysis using the D1S80 locus considering the allele frequency distribution characteristic for our country. Paternal and maternal bands were compared with the children's DNA patterns. Our data include a comparison between D1S80 alleles amplified from mother, child and the supposed father for three tested families. This study was the first of this type made in Romania. We concluded a good power of discrimination and exclusion for this locus. It can be used successfully in the case of subtypes with low frequencies, and this is frequent for our population because of the high heterozygosity of D1S80 subtypes in Romanian population. We recommend the D1S80 use for exclusion paternity tests in Romanian population, as a very useful molecular tool, but we also recommend a complete set of molecular markers for confirmation paternity test in the same population.     

Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.     

Genetic mapping of two new DNA markers in Xq26-q28 relative to the fragile-X syndrome locus.

We have characterized and genetically mapped two new DNA markers (DXS311 and DXS312) with respect to 10 existing loci in Xq26----Xq28 in a set of 15 families in which the fragile-X [fra(X)] syndrome was segregating. Two-point and multipoint linkage analyses were performed taking into account the incomplete penetrance of the fra(X) mutation. The most likely order on the basis of these data is centromere-DXS79-DXS10-DXS311-DXS86-(F9-DXS99 )-(DXS98-DXS312)-fra(X)-DXS52- DXS15-F8C-telomere. DXS98 and one of the new loci, DXS312, were found to be the proximal markers closest to the fra(X) locus. The order F9-(DXS98-DXS312)-fra(X) was found to be 5.9 x 10(4) times more likely than the order (DXS98-DXS312)-F9-fra(X).     

Gene of X-chromosomal congenital stationary night blindness is closely linked to DXS7 on Xp

Summary Congenital stationary night blindness is characterized by disturbed or absent night vision that is always present at or shortly after birth and nonprogressive. The X-linked form of the disease (CSNBX; McKusick catalog no. 31050) differs from the autosomal types in that the former is frequently associated with myopia. X-chromosome-specific polymorphic DNA markers were used to carry out linkage analysis in three European families segregating for CSNBX. Close linkage without recombination was found between the disease locus and the anonymous locus DXS7, mapped to Xp11.3, assigning the mutation to the proximal short arm of the X chromosome. Linkage data obtained with markers flanking DXS7 provided further support for this localization of the gene locus. Thus, in addition to retinitis pigmentosa and Norrie disease, CSNBX represents the third well-known hereditary eye disease the locus of which is mapped on the proximal Xp and closely linked to DXS7.     

用复合PCR检测DXS7132和DXS6804的单倍型

建立X染色体短串联重复基因座DXS7132和DXS6804的复合扩增系统,并用聚丙烯酰胺凝胶电泳和银染技术进行分型,调查广东汉族人群该二基因座的多态性。结果发现DXS7132有7个等位基因,扩增产物的生长在276-300bp;DXS6804有6个等位基因,扩增产物的长度在177-201bp。序列分析揭示这两个基因座的重复单位均为四核苷酸重复。由于基因组作图信息显示DXS7132和DXS6804之间存在紧密连锁,故用家系分析法确定女性的单体型。在827条染色体（男性211,女性616）中,共发现了33种单体型,女性的二倍体数据符合Hardy-Weinberg平衡。基因多样性、多态性信息量和女孩非父排除率分别达0.9999、0.9440和0.9411。对308个三联体的家系调查表明这两个基因座符合X染色体共显性遗传,未发现突变。结果表明,DXS7231和DXS6804在女孩的亲权认定,特别是缺乏双亲检验的案件中有重要的应用价值。     

湖南土家族群体X染色体10个STR位点的遗传多态性及法医学应用价值评估

对198名湖南土家族健康无关个体抽取静脉血, 提取DNA, 经PCR扩增, 变性聚丙烯酰胺凝胶电泳, 银染进行等位基因分型; 软件SPSS13.0计算各位点基因型频率和等位基因频率, 进行Hardy-Weinberg平衡检验, 并检验基因分布差异有无统计学意义; Fstat软件计算基因多态性及固定指数; Powerstats软件计算各种法医学应用指标。在湖南土家族群体中, 共检出65个等位基因, 频率分布在0.0048～0.6170之间; 10个X-STR位点中DXS6789、DXS6799、HPRTB位点的多态性和分化程度较低; DXS7133、DXS7423位点的法医学应用价值较低; 位点比较中, 湖南土家族群体与德国、意大利群体的差异最明显。DXS6804、DXS7132、DXS7130、DXS8378、DXS6789、DXS6799、DXS7424、HPRTB等8个位点在湖南土家族群体的个体识别和女孩的亲权鉴定中有应用价值, 对疾病相关研究有实际意义; 差异性检验结果揭示高加索人种与蒙古人种间存在着较大的差异性。     

An extremely polymorphic locus on the short arm of the human X chromosome with homology to the long arm of the Y chromosome.

A genomic DNA clone named CRI-S232 reveals an array of highly polymorphic restriction fragments on the X chromosome as well as a set of non-polymorphic fragments on the Y chromosome. Every individual has multiple bands, highly variable in length, in every restriction enzyme digest tested. One set of bands is found in all males, and co-segregates with the Y chromosome in families. These sequences have been regionally localized by deletion mapping to the long arm of the Y chromosome. Segregation analysis in families shows that all of the remaining fragments co-segregate as a single locus on the X chromosome, each haplotype consisting of three or more polymorphic fragments. This locus (designated DXS278) is linked to several markers on Xp, the closest being dic56 (DXS143) at a distance of 2 cM. Although it is outside the pseudoautosomal region, the S232 X chromosome locus shows linkage to pseudoautosomal markers in female meiosis. In determining the X chromosome S232 haplotypes of 138 offspring among 19 families, we observed three non-parental haplotypes. Two were recombinant haplotypes, consistent with a cross-over among the S232-hybridizing fragments in maternal meiosis. The third was a mutant haplotype arising on a paternal X chromosome. The locus identified by CRI-S232 may therefore be a recombination and mutation hotspot.     

Population genetic study of four closely-linked X-STR trios in Koreans

We investigated four X chromosomal short tandem repeat (X-STR) markers (DXS10079, DXS10103, DXS10146, and DXS10148) in 450 unrelated Koreans (300 males and 150 females), and evaluated their forensic usage in relation to the four X-STR linkage groups. Forensic statistical parameters for these X-STR markers indicated that they are highly informative for forensic application in Koreans. No significant deviations from Hardy-Weinberg equilibrium were observed in any of the four X-STR markers. In addition, we present haplotypes and their frequency data for four linkage groups each comprised of three X-STRs (DXS10148-DXS10135-DXS8378, DXS7132-DXS10079-DXS10074, DXS10103-HPRTB-DXS10101, and DXS10146-DXS10134-DXS7423) in 300 males. Haplotype diversity values in the four linkage trios were all higher than 0.98, and 77.1% of all haplotypes showed a frequency less than 0.01. Therefore, the four closely-linked X-STR trios will contribute to complex kinship testing in Koreans.     

Fluorescent Multiplex Amplification of Three X-STR Loci

This study was carried out to evaluate the value of three X-STR loci (DXS6803, DXS981and DXS6809) in forensic application and thereby investigate their polymorphism. The primer for each locus was labeled with fluorochrome 6-FAM. A fluorescent multiplex PCR for simultaneously amplifying three X-STR loci was set up. The PCR products that were obtained were analyzed using capillary electrophoresis and ABI PRISM 3100 Genetic Analyzer, with GENESCAN Analysis Software. When 340 male and 195 female individuals of Han population in China were tested, 13, 12, and 11 alleles were observed for DXS6803, DXS981 and DXS6809, respectively. One hundred and eighty three haplotypes were detected in the male individuals. The haplotype diversity reached 0.9926. The results show that the three loci of the multiplex system provide significant information on polymorphism for forensic identification and paternity testing, particularly for complicated paternity deficient cases.     

3个X-STR基因座荧光标记复合扩增

为研究DXS6803、DXS981和DXS68093个基因座多态性及其在法医学中的应用,建立X染色体基因座（DXS6803、DXS981和DXS6809）的荧光复合扩增体系。用荧光标记引物PCR技术复合扩增3个基因座,并用ABI PRISM 3100毛细管电泳及其软件进行基因分型。结果在中国汉族340名无关男性个体及195名无关女性个体中,DXS6803、DXS981和DXS6809三个基因座分别发现了13、12、11个等位基因,男性个体共检出183种单倍型,单倍型多样性为0．9926。结果表明这3个基因座有较高的多态性信息,在个体识别和亲权鉴定（特别是在缺失双亲的特殊检案）中有重要的应用价值。     

Examination of X chromosome markers in Rett syndrome: exclusion mapping with a novel variation on multilocus linkage analysis.

Rett syndrome is a neurologic disorder characterized by early normal development followed by regression, acquired deceleration of head growth, autism, ataxia, and stereotypic hand movements. The exclusive occurrence of the syndrome in females and the occurrence of a few familial cases with inheritance through maternal lines suggest that this disorder is most likely secondary to a mutation on the X chromosome. To address this hypothesis and to identify candidate regions for the Rett syndrome gene locus, genotypic analysis was performed in two families with maternally related affected half-sisters by using 63 DNA markers from the X chromosome. Maternal and paternal X chromosomes from the affected sisters were separated in somatic cell hybrids and were examined for concordance/discordance of maternal alleles at the tested loci. Thirty-six markers were informative in at least one of the two families, and 25 markers were informative in both families. Twenty loci were excluded as candidates for the Rett syndrome gene, on the basis of discordance for maternal alleles in the half-sisters. Nineteen of the loci studied were chosen for multipoint linkage analysis because they have been previously genetically mapped using a large number of meioses from reference families. Using the exclusion criterion of a lod score less than -2, we were able to exclude the region between the Duchenne muscular dystrophy locus and the DXS456 locus. This region extends from Xp21.2 to Xq21-q23. The use of the multipoint linkage analysis approach outlined in this study should allow the exclusion of additional regions of the X chromosome as new markers are analyzed. This in turn will result in a defined region of the X chromosome that should be searched for candidate sequences for the Rett syndrome gene in both familial and sporadic cases.     

Founder effect in a Belgian-Dutch fragile X population

For many years, the high prevalence of the fragile X syndrome was thought to be caused by a high mutation frequency. The recent isolation of the FMR1 gene and identification of the most prevalent mutation enable a more precise study of the fragile X mutation. As the vast majority of fragile X patients show amplification of an unstable trinucleotide repeat, DNA studies can now trace back the origin of the fragile X mutation. To date, de novo mutations leading to amplification of the CGG repeat have not yet been detected. Recently, linkage disequilibrium was found in the Australian and US populations between the fragile X mutation and adjacent polymorphic markers, suggesting a founder effect of the fragile X mutation. We present here a molecular study of Belgian and Dutch fragile X families. No de novo mutations could be found in 54 of these families. Moreover, we found significant (P < 0.0001) linkage disequilibrium in 68 unrelated fragile X patients between the fragile X mutation and an adjacent polymorphic microsatellite at DXS548. This suggests that a founder effect of the fragile X mutation also exists in the Belgian and Dutch populations. Both the absence of new mutations and the presence of linkage disequilibrium suggest that a few ancestral mutations are responsible for most of the patients with fragile X syndrome.     

X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DXS43 at Xp22

Summary Two families with X-linked dominant hypophosphatemia (McKusick No. ^*30780) were investigated for linkage of the disease locus with several marker genes defined by cloned, single-copy DNA sequences derived from defined regions of the X chromosome. Close linkage was found with DNA markers DXS41 (p99-6) and DXS43 (pD2) at Xp22, suggesting a location of the HPDR gene on the distal short arm of the X chromosome.     

New distal marker closely linked to the fragile X locus

Summary We have isolated II-10, a new X-chromosomal probe that identifies a highly informative two-allele TaqI restriction fragment length polymorphism at locus DXS466. Using somatic cell hybrids containing distinct portions of the long arm of the X chromosome, we could localize DXS466 between DXS296 and DXS304, both of which are closely linked distal markers for fragile X. This regional localization was supported by the analysis, in fragile X families, of recombination events between these three loci, the fragile X locus and locus DXS52, the latter being located at a more distal position. DXS466 is closely linked to the fragile X locus with a peak lod score of 7.79 at a recombination fraction of 0.02. Heterozygosity of DXS466 is approximately 50%. Its close proximity and relatively high informativity make DXS466 a valuable new diagnostic DNA marker for fragile X.     

The polymorphic marker DXS304 is within 5 centimorgans of the fragile X locus

The fragile X syndrome, which is the most common cause of inherited mental retardation, poses important diagnostic problems for genetic counseling. The development of diagnostic strategies based on DNA analysis has been impaired by the lack of polymorphic markers very close to the disease locus. Here we report that the polymorphic probe U6.2 (locus DXS304) is much closer to the fragile X locus than all the previously reported markers. A recombination fraction of 0.02 between DXS304 and the fragile X locus was estimated by multipoint linkage analysis (confidence interval 0.002 to 0.05). Our data suggest that DXS304 is distal to the fragile X locus. This marker thus represents a major improvement for carrier detection and prenatal diagnosis in fragile X families.     

Refinement of linkage of human severe combined immunodeficiency (SCIDX1) to polymorphic markers in Xq13.

The most common form of human severe combined immunodeficiency (SCID) is inherited as an X-linked recessive genetic defect, MIM 300400. The disease locus, SCIDX1, has previously been placed in Xq13.1-q21.1 by demonstration of linkage to polymorphic markers between DXS159 and DXS3 and by exclusion from interstitial deletions of Xq21.1-q21.3. We report an extension of previous linkage studies, with new markers and a total of 25 SCIDX1 families including female carriers identified by nonrandom X chromosome inactivation in their T lymphocytes. SCIDX1 was nonrecombinant with DXS441, with a lod score of 17.96. Linkage relationships of new markers in the SCIDX1 families were consistent with the linkage map generated in the families of the Centre d'Etude du Polymorphisme Humain (CEPH) and with available physical map data. The most likely locus order was DXS1-(DXS159,DXS153)-DXS106-DXS132-DXS4 53-(SCIDX1,PGK1, DXS325,DXS347,DXS441)-DXS447-DXS72-DXYS 1X-DXS3. The SCIDX1 region now spans approximately 10 Mb of DNA in Xq13; this narrowed genetic localization will assist efforts to identify gene candidates and will improve genetic management for families with SCID.     

Genetic analysis of the 10 ChrX STRs loci in Chinese Han nationality from Guangdong province

This study is to explore the polymorphic nature of X-Chromosome short tandem repeats (ChrX STRs) loci, and to determine its application in kinship tests for forensic cases. A new fluorescent multiplex PCR that simultaneously amplifies the 10 ChX STRs loci in the same PCR reaction had been set up. DXS7132, DXS981, DXS6801, DXS6809, DXS6789, DXS7424, DXS101, DXS7133, GATA165B12 and GATA31E08 were analyzed in a sample of 511 (399 males and 112 females) unrelated individuals from Guangdong Han nationality in China. One hundred and one alleles were observed in all the loci. Here, we investigated the allele frequencies and mutation rates of the ten loci, and then made the comparison of allele frequencies distribution among different populations. The results show the ten loci in the multiplex systems may provide high polymorphism information for kinship testing and relationship investigations, and it is necessary to gain allele frequency and mutation rate of different population for forensic application.     

Linkage analysis of families with fragile-X mental retardation, using a novel RFLP marker (DXS 304).

A new polymorphic DNA marker U6.2, defining the locus DXS304, was recently isolated and mapped to the Xq27 region of the X chromosome. In the previous communication we describe a linkage study encompassing 16 fragile-X families and using U6.2 and five previously described polymorphic markers at Xq26-q28. One recombination event was observed between DXS304 and the fragile-X locus in 36 informative meioses. Combined with information from other reports, our results suggest the following order of the examined loci on Xq: cen-F9-DXS105-DXS98-FRAXA-DXS304-(DXS52-F8 -DXS15). The locus DXS304 is closely linked to FRAXA, giving a peak lod score of 5.86 at a corresponding recombination fraction of .00. On the basis of the present results, it is apparent that U6.2 is a useful probe for carrier and prenatal diagnosis in fragile-X families.     

Linkage studies in X-linked Alport's syndrome

Summary Four kindreds segregating for Alport's syndrome (ASLN) compatible with a X-linked inheritance were studied for linkage with polymorphic markers of the human X chromosome. No recombinant was observed between the ASLN locus and the DXS101 and DXS94 loci, the maximum lod scores were z=3.93 and 3.50 respectively. Linkage data between the ASLN locus and the other genetic markers used in the present study are in keeping with the assignment of the mutation to the proximal Xq arm.