KCNQ1内含子1a中STR基因座遗传多态性及PIA分型

目的调查汉族群体KCNQ1基因内含子1a中STR基因座的遗传多态性,并采用PIA分型技术确定等位基因的亲代来源。方法用PCR-STR分型技术对230例武汉汉族无关个体样本进行KCNQ1基因内含子1a中STR基因座分型检测;同时选用两种甲基化敏感的限制酶（msRE）HhaI和HpaⅡ对家系中孩子的基因组DNA进行消化后,采用PIA分型技术检测父源等位基因。结果KCNQ1内含子1a中STR基因座在汉族人群中检出10个等位基因、24种基因型,其个体识别能力（PD）、多态性信息含量（PIC）和非父排除率（PE）分别为0．852、0．66和0．484。HhaI和HpaII可消化个体的母源等位基因,PIA分型仅能检测出单一的父源等位基因。结论KCNQ1内含子1a中STR基因座在汉族群体具有较高的遗传多态性,PIA分型技术可以确定个体等位基因的亲代来源,具有较高的法医学应用价值。     

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。     

H19基因上游差异性甲基化区SNPs多态性研究

目的获得H19基因上游差异性甲基化区中SNPs的群体遗传学信息。方法采用PCR和测序技术,对105例中国北方汉族健康无关个体H19上游启动子区检测;使用Haploview 4.1和PowerStats V12软件进行统计学分析。选用甲基化敏感的限制内切酶(msRE)HpaⅡ,检测5个家系样本H19等位基因的亲代来源。结果测序结果显示,H19启动子区含有13个SNPs,组成5种单倍型,13种单倍型组合,其个体识别能力为0.856、多态性信息含量为0.67、非父排除率为0.498。经msRE HpaⅡ消化母源等位基因后,进行PCR及测序分析,检测出父源等位基因,排除1例和肯定4例家系的亲缘关系。结论 DNA甲基化标记和SNPs多态性检测,可同时进行多态性分型并确定等位基因的亲代来源,具有较高的法医学应用价值。     

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.     

广东汉族人群H19基因上游差异甲基化区SNP

目的调查广东汉族人群中H19基因上游差异甲基化区(differentially methylated region,DMR)的单核苷酸多态性(SNP)及单倍型。方法应用PIA分型法,以限制性内切酶Mcr BC、HpaⅡ消化基因组DNA分别获得个体单亲源DNA模板链,经测序,分别获得个体H19基因上游DMR单亲源SNP等位基因、基因型及单倍型数据。结果共检出13个SNP(rs10840167、rs2525883、rs12417375、rs4930101、rs2525882、rs2735970、rs2735971、rs11042170、rs2735972、rs10732516、rs2071094、rs2107425、rs4930098)及1个突变点(g7351c)。所有位点经统计学分析均符合Hardy-Weinberg平衡定律(P0.05)。除rs12417375位点DP值为0.279,其余12个SNP DP值在0.446~0.614;g7351c突变点DP值为0.013,提示为南方汉族民族特异性位点。共检出8种单倍型(命名为单倍型1~8),其中有3种为新发现的单倍型,其DP、PIC、PE及H分别为0.891、0.714、0.524和0.758。结论 PIA分型法获得的H19基因上游DMR SNP位点及其单倍型遗传标记系统具有较高的鉴别能力,在法医学鉴定中具有较好的实用价值。     

Allele and genotype frequencies for the STR locus D18S51 in a western German population

Genetic marker typing based on DNA amplification by the polymerase chain reaction (PCR) increasingly is being employed in forensic casework and for paternity testing. Allele frequencies were determined using PCR for 102 unrelated Germans (Rhine area) for the locus D18S51. Twelve alleles were observed, with frequencies ranging from 0.005 (allele 11) to 0.191 (allele 14). The observed heterozygosity was 0.867, and the power of discrimination was 0.968. There was no deviation from expectations under Hardy-Weinberg assumptions (P = 0.451).     

Study on the application of parent-of-origin specific DNA methylation markers to forensic genetics

In paternity test, especially in motherless cases, the allele inherited from father (obligatory gene, OG) often cannot be determined. The paternity exclusion probability (PE) of a genetic marker is reduced considerably. Therefore, it is necessary to develop a new technique, by which the parental origin of alleles can be determined without genealogical analysis. In this paper, we explored the possibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles, choosing the imprinted single nucleotide polymorphism (SNP) locus rs220028 (A/G) as a model system. We typed the SNP by mutagenically separated PCR (MS-PCR). The frequencies of alleles were A = 0.5085, G = 0.4915; the unbiased heterozygosity was 0.5020. In order to discriminate between the maternal allele and paternal allele, post-digestion MS-PCR, a novel PCR based methylation analysis and SNP typing technique was developed and performed on 18 heterozygous children, and the methylated maternal allele was detected specifically. As a pilot study on the use of epigenetic markers in forensic genetics, our results demonstrated the feasibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles.     

印记基因H19上游高甲基化区SNPs多态性研究

目的建立简单、高效的DNA甲基化标记和SNPs联合检测技术，并用于H19基因上游高甲基化区两组SNPs群体遗传学检测。方法用PCR—DGGE技术对232例武汉汉族无关个体H19基因上游启动子区H19FR1和H19FR2单倍型进行检测；同时选用两种甲基化敏感的限制酶（msRE）HpaⅡ和HhaⅠ，检测H19FR等位基因亲代来源。结果H19FR1区检出5种单倍型、9种表型组合，其个体识别能力（DP）、多态性信息含量（PIC）和非父排除率（PE）分别为0．803、0．58和0．322；H19FR2区检出2种单倍型、3种表型组合，其DP、PIC和PE值分别为0．626、0．37和0．162。测序结果显示，片段H19FR1含有a7342g、a7357g和g7547a3个SNPs与1个g7351c点突变；H19FR2仅含aS097g1个SNP。msREHpaⅡ或HhaⅠ可消化个体母源等位基因，PDP-DGGE分析仅能检测到父源等位基因。结论PDP-DGGE是一种简单、灵敏、高效的DNA甲基化标记和SNPs联合分析技术，其在进行多态性分型同时还可以确定等位基因的亲代来源，具有较高的法医学应用价值。     

Distinguishing minisatellite mutation from non-paternity by MVR-PCR

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was devised to map the interspersion pattern of subtle variant repeats along minisatellite tandem arrays. MVR-PCR has revealed enormous diversity of allele structures at several loci, far more than can be resolved by allele length analysis. We have reported the application of MVR-PCR at minisatellite MS32 (D1S8) and MS31A (D7S21) in a paternity case lacking a mother and showed that it resulted in higher paternity probabilities than for a set of 12 other DNA markers including six STRs. Hypervariable minisatellites like MS32 and MS3lA can however, show significant germline mutation rates to new length alleles which can generate false exclusions in paternity cases although paternity cases showing mutant paternal alleles at more than one locus will be rare when several MVR loci are examined. Detailed knowledge of mutation processes coupled with MVR analysis of allele structure can help distinguish mutation from non-paternity. We now show how similar mutant alleles are to their progenitors using both real and simulated data, and demonstrate how MVR-PCR can be used to identify mutant paternal allele in paternity cases showing apparent exclusions.     

AGCU Mini系统在法医学中的应用

目的考察及评价AGCU Mini系统在法医学实践中的应用价值。方法应用AGCU Mini系统检测12 775份陈旧血样,进行梯度DNA模板浓度分析,并与IdentifilerTM试剂盒检测结果进行比对,评价体系的检测成功率及方法的灵敏度。针对AGCU Mini系统中D19S253基因座,对699份浙江汉族无关个体进行多态性调查。结果 12 775份陈旧血样采用AGCU Mini试剂盒检测,12 885份(96.1%)分型成功,检测灵敏度为40pg(10μL体系),方法成功率及灵敏度均高于IdentifilerTM试剂盒。D19S253基因座共检出9个等位基因,频率范围为0.005 7～0.316 2,杂合度为0.814 0,多态性信息含量为0.772 9。结论 AGCU Mini系统可用于法医微量物证的STR分析,与相关试剂盒联合使用价值更高。     

Further characterization and population data for the pentanucleotide STR polymorphism D10S2325.

Pentanucleotide tandem repeat markers are interesting for forensic sciences, because they may present less stutter on the electrophoretic pattern. We focused on the analysis of the DNA sequence for each allele at the pentanucleotide STR locus D10S2325 in order to understand their structures in the human genome and to construct human allelic ladder, which is necessary for forensic DNA typing. In order to evaluate the forensic applicability of D10S2325 and to construct a preliminary database, the genotype distributions and allele frequencies in three major ethnic groups were investigated. The population samples included Caucasians (Germans), Africans (African Americans), and Asians (Chinese). A total of 520 samples from unrelated individuals was analyzed by Amp-FLP. An example of each allele and new alleles were sequenced. Allele determination was carried out by comparison with a sequenced human allelic ladder made in-house. This pentanucleotide STR provided easily interpretable results. A total of 15 alleles was found in our population samples. Three new alleles were observed and named as alleles 19 and 21 based on the number of repeat motifs, while allele 19 can be divided further into two alleles, 19a and 19 according to analysis of the sequence. No evidence of deviation from Hardy-Weinberg equilibrium was observed. In 64 confirmed father/mother/child triplets no mutation event was observed. Using a maximum likelihood method, the mutation rate was indirectly estimated as 2.5 x 10(-5). These results suggest that D10S2325 is a useful marker for forensic casework and paternity analysis.     

A study on polymerase chain reaction-based HLA DQ alpha locus in Elaziğ, Turkey

The polymerase chain reaction (PCR) was used for genetic characterization of 45 samples taken from the city of Elazi? in Turkey. The polymorphism at the human leukocyte antigen DQalpha locus was detected. Allele and genotype frequencies were determined for unrelated individuals at this locus. Laboratory analyses were done by PCR amplification of DNA. Hybridization to allele specific oligonucleotide probes was performed using a reversed dot-blot typing method. The collected genotype and allele frequencies have been tested, and a comparison was made with other population surveys of this locus. Allele frequencies ranged from 3.3% (allele 1.3) to 36.7% (allele 4), with a discrimination power of 0.92. No deviation was seen from Hardy-Weinberg equilibrium in the findings.     

A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci

In this study a proposal for the allele nomenclature of six polymorphic short tandem repeat (STR) loci (PEZ3, PEZ6, PEZ8, PEZ10, FHC2161, and FHC2328) for canine genotyping (Canis lupus familiaris) is presented. The nomenclature is based on the sequence data of the polymorphic region of the microsatellite markers as recommended by the DNA commission of the International Society of Forensic Haemogenetics (ISFH) in 1994 for human DNA typing. To cover commonly and rarely occurring alleles, a selection of homozygous and heterozygous animals were analyzed and subjected to sequence studies. The alleles consisted of simple tri- and tetra-nucleotide repeat patterns as well as compound and highly complex repeat patterns. Several alleles revealing the same fragment size but different repeat structures were found. The allele designation described here was adopted to the number of repeats, including all variable regions within the amplified fragment. In a second step the most commonly occurring alleles were added to an allelic ladder for each marker allowing a reliable typing of all alleles differing in size. A total number of 142 unrelated dogs from surrounding municipal animal homes, private households, and canines in police duty were analyzed. The data were added to a population database providing allele frequencies for each marker.     

武汉地区汉族人群CSF1PO座位多态性分析

应用Ｙｏｓｈｉｄａ设计的ＣＳＦ１ＰＯ新引物对武汉地区３１２例汉族个体作了分型调查,获得了武汉地区汉族人ＣＳＦ１ＰＯ座位的基因频率资料。与Ｈａｍｍｏｎｄ等提出的传统引物扩增效果相比较,本文所选用的引物更适用于法医检案,尤其对严重降解ＤＮＡ材料的分型效果有明显的优越性     

D16S539 microvariant or D2S1338 off-ladder allele? A case report about a range overlapping between two loci

All forensic laboratories routinely use commercial kits and softwares for automated typing; in rare cases genotyping misinterpretations or mislabellings occur. This study refers to the investigation on a D2S1338 off-ladder allele mislabelling observed in DNA profile of murdered woman.The Identifiler^® revealed heterozygosity in the range of D16S539, with a presumptive microvariant allele “14.2”, based on assigned size, while PowerPlex^®16 resulted in a homozygosity of allele “11”. Singleplex amplification of D16S539 locus confirmed homozigosity. D2S1338 locus, the closest to D16S1338 in Identifiler^®, genotyped as homozigote “19”, was singleplex amplified. The off-ladder peak was gel-isolated, sequenced and designed as a rare “11” allele variant [(TGCC)6(TTCC)5]. Genotype was finally designed as D16S539 “11,11” and D2S1338 “11,19”.To avoid genotyping misinterpretations or mislabelling, ambiguous genotypes should be established by two commercial kits at least. Furthermore, off ladder alleles as well as allele microvariants should be assigned by direct sequencing. This issue should be considered in Criminal DNA database requirements, that is still under debate in Italy.     

中国人p33.6位点的扩增片段长度多态性

用PCR、小型聚丙烯酰胺凝胶电泳和银染法对小卫星区域p33.6(D1S111)位点的扩增片段长度多态性(Amp—FLP)进行分析和对100例无关中国人p33.6位点的等位基因频率进行调查及数据处理,发现该位点核心序列重复数从9到22之间的全部14个等位基因,片段长度分布于435～925bp之间,基因频率为0.5～35.5％,杂合度为76％。对6个家系共22名相关个体进行分析,符合孟德尔遗传定律;对人体各种不同组织DNA进行该位点的分析,显示出高度的一致性。该位点适用于法医学上的个人识别以及亲子鉴定。     

中国成都汉族及泰国群体D7S2846基因座的遗传多态性

研究STR基因座D7S2 846的遗传多态性 ,为法科学应用提供基础数据。应用PCR及PAG电泳技术 ,对376名中国成都汉族无关个体及 131名泰国无关个体进行了调查。两群体分别检出 8个和 7个等位基因 ,首次获得该基因座基因在两群体中的频率分布。两群体基因型频率分布均符合Hardy Weinberg平衡。家系调查证实了等位基因的传递遵循孟德尔遗传规律。该基因座在两群体中的个人识别能力 (Dp)分别为 0 85 70、 0 86 0 2 ,杂合度 (H )分别为0 6 915、 0 6 870 ,多态性信息含量 (PIC)分别为 0 6 445、 0 6 5 5 3 ,非父排除率 (PE )分别为 0 415 2、 0 40 85。D7S2 846基因座在法医学个人识别及亲子鉴定中具有较高的实用价值。     

成都地区汉族人群D2S441位点的遗传多态性研究

为研究 STR位点 D2S441的遗传多态性,为法医学应用提供基础数据,应用 PCR及 PAG电泳技术对 260名成都地区汉族无关个体进行了调查,共检出 9个等位基因及 26种基因型,首次获得汉族群体频率分布 ,其等位基因片段大小范围为 131～ 155bp。该位点基因型频率分布符合 Hardy－ Weinberg平衡。家系调查证实了等位基因的传递遵循孟德尔遗传规律。其个人识别能力（ Dp）、杂合度（ H）、多态性信息含量（ PIC）和非父排除率（ PE）分别为 0.9084、 0.7885、 0.7390和 0.5778,表明该位点在法医学个人识别及亲子鉴定中具有较高的实用价值。     

Population studies and validation of paternity determinations by six microsatellite loci

A single locus system of 6 microsatellite markers was evaluated for paternity testing. A nonradioactive method based on peroxidase labeling of a DNA probe was used to estimate the allele frequency of markers D1S216, D3S1217, D7S480, D9S157, D13S153, and D16S422 by genotyping 1134-1698 chromosomes. The number of detected alleles were 22, 15, 23, 10, 16, and 19, respectively, and the allele frequency varied from 0.001 to 0.317. The genotype of 87 families, consisting of mother, father, and child was determined. The probability that a random individual will give a positive paternity was evaluated. We conclude that the markers can be reliably typed and give sufficient and reliable information for paternity testing.     

用等位基因特异扩增技术检测人类补体第八成份C8A DNA多态性的研究

建立补体第八成份蛋白质多态性的DNA检测方法。根据导致C8A多态性的DNA点突变核苷酸差异 ,建立一个检测C8A基因型的特异性扩增方法。采用该法 ,对 10 0份血样本进行检测 ,并同时用传统的检测C8A蛋白质多态性的SDS 凝胶电泳方法进行对照检测 ,结果显示新建立的C8A多态性PCR分型方法不仅快速、灵敏、稳定 ,而且分型结果清晰 ,容易判读 ,适用于法医学中多种检材的C8A多态性分型