Sequence characterization of microvariant alleles at DYS627 and DYS458

Y chromosome short tandem repeats (Y-STRs) have been widely used in genetic applications and forensic casework. Recently, we found two intermediate alleles, the DYS627 allele 24.1 and the DYS458 allele 15.3, from Chinese Han population. The two allelic variants have not been recorded by the YHRD database. We have examined the molecular structure of these allelic variants by Sanger sequencing. The results showed that this intermediate allele at DYS627 was confirmed as 24.1, the sequence of which showed a base “A” insertion in the 13th repeat unit, and the intermediate allele at DYS458 was confirmed as 15.3, the sequence of which showed a base “G” deletion in the 12th repeat unit. This may be important for individual identification and paternal kinship testing. Besides, more allelic variants detected can be enriched in the Y-STR database.     

A rare mutation in the primer binding region of the Amelogenin X homologue gene

Use of Amelogenin locus typing as a gender marker incorporated in STR multiplexes is common practice in forensic genetics analysis. Among 5534 Polish male individuals tested using the SGMPlus kit, one was found to lack the amelogenin X-specific homologue (0.02%). The same result was obtained with other commercial kits which also amplify the amelogenin locus, namely ProfilerPlus and PowerPlex16. When alternative amelogenin primers external to but encompassing the initial amplicon were applied, an X homologue product was seen. Sequencing of the X homologue amelogenin allele revealed C to G mutation located at the most 3′ base of the commonly used amelogenin reverse primer. To our knowledge, this mutation and failure to amplify the X homologue of the amelogenin gene has not been reported for the European population.     

Updated allelic structures of the DXS10135 and DXS10078 STR loci

DXS10135 and DXS10078 are two highly polymorphic STR loci situated in two different linkage groups on the short arm of the human X chromosome. Both loci comprise complex tetrameric repeat units which may partially explain their high degree of polymorphism. DXS10135 is relatively well characterized and is included in a commercially available kit, while DXS10078 has not been well described. We sequenced a large number of alleles of both loci to try and understand the allelic variation and as a prelude to construct allelic ladders from cloned alleles. Our data show interesting features and should encourage other workers to use these loci in forensic genetic investigations.     

Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis sativa L.) Cultivars

In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to <0.2% (%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35% B_T allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2% THC. The fact that 100% of the seized drug-type seeds in this study revealed at least one B_T allele, reflects that plant breeding efforts have resulted in a fixation of the B_T allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals.     

Variant allele 6.2 at locus D19S433 in Syrian family samples

Variant allele 6.2 at locus D19S433 was found in an immigration case involving Syrian family samples where 2 out of 4 children inherited this variant allele from their parents. This variant allele has not been reported previously in the Syrian population since locus D19S433 was not studied. However, records showed the presence of the variant allele 6.2 in Middle Eastern samples of Iraqi, Lebanese, Kuwaiti and Turkish origins. These findings indicate that variant allele 6.2 at locus D19S433 could be specific for the Middle Eastern populations and could be useful in forensic investigation for suspect search.     

Allele frequencies of 20 Y-chromosomal short tandem repeat loci in a tribal population of Deccan Plateau, India

Population: Eighty male individuals from a nomadic tribal population belonging to Dravidian and Indo-Caucasian ethnicities from Deccan Plateau, Andhra Pradesh, India, were analyzed in the present study.     

武汉汉族8个Y染色体双等位基因标记遗传多态性

目的筛选汉族群体中具有多态性的Y染色体双等位基因标记并研究其等位基因及单体群频率分布, 为法医学应用和群体进化研究提供基础数据。方法采用片段长度差异等位基因特异性PCR和PAGE技术对武汉地区160名男性汉族无关个体的8个Y染色体双等位基因标记(M9、M89、M111、M119、M122、M134、IMS-JST003305 和SRY+465)进行分型。结果 8个双等位基因标记在武汉汉族群体中均具有遗传多态性,其基因多样性(GD)范围为 0．0126-0．4830,共检出9种不同单体群(Hg1～9),其单体群多样性(HD)为0．7776。结论 8个Y染色体双等位基因标记组成的单体群在法医学应用和群体进化研究中具有一定的实用价值,可作为Y-STRs标记的有效补充。     

用FLDAS-PCR和PCR-RFLP检测汉族人群GPT的多态性

目的 建立片段长度差异等位基因特异性PCR(FLDAS-PCR)检测GPT基因型的新方法,并调查武汉地区汉族人群GPT多态性分布。方法 应用FLDAS-PCR、PCR-RFLP分别对武汉地区248例汉族个体进行GPT基因型分型。结果 FLDAS-PCR与PCR-RFLP分型结果完全一致,GPT的3种基因型分型明确,基因频率GPT*1=0.5423,GPT*2=0.4577,基因型分布符合Hardy-Weinberg平衡。结论 FLDAS-PCR和PCR-RFSP分型GPT在法医学鉴定中有实用价值。     

印记基因KCNQ1的遗传多态性及在亲权鉴定中的应用

目的为了调查印记基因KCNQ1的STR位点在中国汉族人群中的遗传多态性，利用亲源印记等位基因（parentally imprinting allele，PIA）分型法确定孩子的等位基因亲代来源，为亲权鉴定提供新的侯选STR位点。方法应用Chelex法提取153例佳木斯地区汉族健康无血缘关系个体DNA，用QIAamp Blood Kit（Qiagen）法提取3个家庭10个个体DNA，PCR扩增，凝胶电泳分型，ABIPRISM^TM 3730XL DNA测序仪测序；甲基化敏感性限制性内切酶消化孩子基因组DNA，PCR扩增，确定孩子等位基因的亲代来源。结果发现在中国佳木斯地区汉族人群中KCNQ1基因的STR有7个等位基因，多态信息含量为0．662，且KCNQ1基因的STR位点呈父源印记。结论印记基因KCNQ1的STR位点有很好的多态性，可为亲权鉴定提供新的侯选遗传标记，其亲源特异性甲基化标记有望应用于单亲鉴定中。     

Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC) and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58 and 0.322, respectively. For the H19FR2, two haplotypes and three genotyes were observed, and the Dp, PIC and PE were 0.626, 0.37 and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population, and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles.