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。     

印记基因5个SNP分型及亲代来源的检测

目的 采用复合PCR-Snapshot联合甲基化敏感酶切技术,检测印记基因中5个SNP的甲基化状态、印记亲代来源及分型.方法 选择15例亲子鉴定已证实为亲生关系的家系样本,采用单碱基延伸复合检测技术,检测家系样本IGF2AS rs1003483、SNURF rs220028、SNURF rs4906939、DLGAP2 rs6558478、SIM2 rs737380等5个SNP分型,同时选用核酸内切酶(McrBC)和甲基化敏感的限制酶(msRE) HhaⅠ、HpaⅡ消化子代DNA,验证印记基因的亲代来源.结果 经用本文方法检测,证实rs1003483为父源印记;rs220028、rs4906939为母源印记;rs6558478及rs737380未在差异甲基化区,不能确定其印记亲代来源.结论 复合PCR-Snapshot联合甲基化敏感酶切技术简单、高效,在检测多个SNP分型的同时可确定亲代来源,可在相关研究和实践中选用.     

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。     

Novel paternity testing by distinguishing parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene

Conventional PCR-based genotyping is useful for forensic testing but cannot be used to determine parental origins of alleles in DNA specimens. Here we describe a novel method of combined conventional genotyping and PIA typing (parentally imprinted allele typing) at a minisatellite region upstream from the H19 locus. The PIA typing uses two sets of primers and DNA digested with methylation-sensitive Hha I enzyme. The first amplification produces only the methylated fragment of paternal H19 allele, and the second detects polymorphism in the minisatellite. Hence, this distinguishes paternal and maternal alleles by difference in the DNA methylation. Furthermore, the polymorphism in this polymorphic locus was examined using 199 unrelated Japanese and 171 unrelated Germans, their polymorphism information content being 0.671 and 0.705, respectively. Feasibility of this typing is demonstrated for six families, and the usefulness is shown by application to paternity testing.     

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.     

Jordanian population data on the PCR-based loci: LDLR, GYPA, HBGG, D7S8 and GC.

Genotype and allele frequency distributions for PM polymerase chain reaction (PCR)-based genetic markers were determined in a Jordanian sample population. Results were obtained using the AmpliType PM PCR Amplification and typing kit. All loci were in agreement with the Hardy-Weinberg equilibrium expectations. The predominant alleles for LDLR, GYPA, HBGG, D7S8 and GC loci were B, A, B, A and C respectively. No statistically significant variation was detected in allele frequencies of these loci in Jordanians compared to that in Israeli Arab, U.S Caucasian and Japanese populations. Data presented here can be used to estimate the frequency of a specific DNA profile in the Jordanian population for forensic analyses and paternity testing.     

Two loci ‘exclusion’ of true paternity is due to genetic disorder in a child

We describe a paternity case with three genetic incompatibilities between a three-year-old boy and his putative father.STR analysis of 2 out of 25 markers revealed the absence of paternal alleles and presence of two maternal alleles at D2S441 and D2S1338 loci in the child. The rest 23 STR markers served to confirm paternity. In addition, we analyzed Y-STRs and determined the same haplotype in the child and his putative father.With massive parallel sequencing on HID Ion GeneStudio S5 System using Precision ID GlobalFiler NGS STR Panel v2 (Applied Biosystems) we confirmed the presence of two alleles of maternal origin at D2S441, D2S1338 loci and identified two maternal alleles at additional locus D2S1776 located on chromosome 2 in the child.Finally, we confirm paternity. Three loci ‘exclusion’ was due to maternal uniparental disomy of chromosome 2 in the child.     

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.     

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。     

SNRPN基因rs220030位点的分型及甲基化亲缘相关性

目的建立变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术与焦磷酸测序技术对小核核糖核蛋白多肽N(small nuclear ribonucleoprotein polypeptide N,SNRPN)基因rs220030位点的分型方法。建立应用焦磷酸测序技术分析CpG甲基化状态的方法,探讨rs220030位点用于亲缘等位基因判定的可行性。方法应用DGGE技术对97例上海地区汉族家系血样rs220030位点进行分型,同时应用焦磷酸测序技术对其中25例血液来源的家系样本的rs220030位点分型,并对两种方法在SNP分型结果上进行比较。通过重亚硫酸盐修饰联合焦磷酸测序技术分析随机2组家系样本rs220030位点上游CpG甲基化状态,判断甲基化是否有亲缘相关性。结果经DGGE检测97例家系血样rs220030位点分型结果为C纯合子20例,T纯合子29例,C/T杂合子48例。经焦磷酸测序检测25例血液来源的家系样本结果与DGGE检测结果一致。经重亚硫酸盐修饰联合焦磷酸测序技术分析,2组血液来源的家系子代的rs220030位点上游CpG甲基化状态均与母亲较相似。结论相比DGGE技术,焦磷酸测序技术更精确、方便,适合大样本、高通量SNP分型。重亚硫酸盐修饰联合焦磷酸测序技术可以精确分析CpG甲基化状态。rs220030位点可用于亲缘等位基因判定。     

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).     

片段长度差异等位基因特异性PCR——一种改良的SNP分型新方法

目的建立一种基于等位基因特异性PCR原理的改良SNP分型新方法:片段长度差异等位基因特异性PCR,并考察特异性引物的3'端第3位、第4位碱基错配对特异性延伸的影响。方法以SNP位点rs759117和rs760887为例,设计两条长度不同、3'末端分别与SNP两个等位基因碱基配对的上游引物,同时在两个等位基因特异性引物3'端第3或第4位碱基引入错配以增加特异性,下游为公用引物。PCR产物经聚丙烯酰胺凝胶电泳、银染显带后确定样本的基因型。结果不同SNP纯合子为长度不同的单一谱带,杂合子则为两条带,其结果与直接测序完全一致。两条特异性上游引物3'端第3或第4位碱基引入错配后非特异性延伸显著减少,且对PCR反应条件的严格性要求明显降低。结论片段长度差异等位基因特异性PCR是一种简单快速而有效的SNP分型新方法;两条特异性引物3'端第3、第4位碱基引入错配可使特异性显著增加     

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.     

Investigation of the methylation status around parent-of-origin detectable SNPs in imprinted genes

Methylation of CpG dinucleotides was investigated in five regions by bisulphite treatment of gDNA, PCR and cloning/sequencing. The gDNA was prepared from peripheral blood, saliva, semen, nails and hair from the head. In gDNA from peripheral blood, three regions were investigated in 16, 23 and 24 individuals, respectively (Fig. 2). In gDNA from other sources, three or five regions were investigated in five individuals (Fig. 3). In many of the sequenced fragments, all the CpG dinucleotides were either methylated or not, which support the idea that the parental origin of an allele may be determined by the methylation status of the allele. However, the methylation of CpG dinucleotides varies across the fragment in some of the sequenced fragments, especially from semen samples, which indicate that it may be difficult to determine the parental origin from some gDNA sources by restriction-enzyme analysis (DMPA method).     

The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.     

Parentage of Hydatidiform Moles

We were presented with the STR (short tandem repeat) profiles from two separate paternity trios. Each trio consisted of a mother, an alleged father, and products of conception (POC) that contained a hydatidiform mole but no visible fetus. In both cases , antecedent pregnancies had followed alleged sexual assaults. Mole classification and pathogenesis are described in order to explain the analyses and statistical reasoning used in each case. One mole exhibited several loci with two different paternal alleles, indicating it was a dispermic (heterozygous) mole. Maternal decidua contaminated the POC, preventing the identification of paternal obligate alleles (POAs) at some loci. The other mole exhibited only one paternal allele/locus at all loci and no maternal alleles, indicating it was a diandric and diploid (homozygous) mole. In each case, traditional calculations were used to determine paternity indices (PIs) at loci that exhibited one paternal allele/locus. PIs at mole loci with two different paternal alleles/locus were calculated from formulas first used for child chimeras that are always dispermic. Combined paternity indices in both mole cases strongly supported the paternity of each suspect.     

DNA甲基化标记法医学应用探讨

CpG的胞嘧啶在DNA复制后多数被甲基化,5-甲基胞嘧啶的分布是贮存表遗传信息的主要形式。近年来研究表明,DNA甲基化标记具有信息含量丰富、相对稳定、检测和结果处理方便、可与SNP联合分析等优点,是一种新的强有力的遗传分析工具。基因组的甲基化差异可用甲基化敏感性限制酶、重亚硫酸盐转化、Maxam-Gilbert裂解等技术来分析。人类基因组甲基化谱有时空特异性、亲源特异性、病理特异性等特征,在法医亲子鉴定、个人识别等方面有潜在应用价值。     

SNPs and MALDI-TOF MS: tools for DNA typing in forensic paternity testing and anthropology

DNA markers used for individual identification in forensic sciences are based on repeat sequences in nuclear DNA and the mitochondrial DNA hypervariable regions 1 and 2. An alternative to these markers is the use of single nucleotide polymorphisms (SNPs). These have a particular advantage in the analysis of degraded or poor samples, which are often all that is available in forensics or anthropology. In order to study the potential of SNP analysis in these fields, 41 SNPs were selected on the basis of following criteria: conservation, lack of phenotypic expression, and frequency of occurrence in populations. Thirty-six autosomal SNPs were used for genotyping 21 inclusionary and 3 exclusionary paternity cases. The behavior of 5 X-chromosome SNPs was analyzed in a French representative population. Our approach to SNP typing is a multiplex PCR based amplification followed by simultaneous detection by primer extension (PEX) analyzed by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The selected autosomal SNPs showed independent inheritance and gave clear results in paternity investigation. All X-SNPs were useful as both paternity and identification markers. PEX and MALDI-TOF MS, with their high sensitivity, precision and speed, gave a powerful method for forensic and anthropological exploitation of biallelic markers.     

基于等位基因特异性PCR原理建立的SNP分型新方法

目的建立一种新方法,对多个单核苷酸多态性(singlenucleotidepolymorphism,SNP)位点进行分型。方法基于等位基因特异性PCR原理,采用荧光标记复合扩增和毛细管电泳技术,根据PCR片段长度差异进行分型。选择SNP位点共11个,每个SNP位点设计两条长度不同、3’末端分别与SNP两个等位基因碱基配对的上游引物,同时为了增加特异性,在两条等位基因上游引物的3’末端第3或第4位碱基人为引入错配。在距离上游引物100～300bp范围内的合适位置,设计下游共用引物,并进行荧光标记。所有位点经过复合扩增后,PCR产物经ABIPrismTM310型遗传分析仪电泳分离,确定每个SNP的基因型。结果每个SNP位点纯合子为单一产物峰,杂合子则为长度不同的两个产物峰。不同的SNP位点扩增产物长度不同,根据产物长度和产物峰的数量进行SNP分型,一次完成11个SNP位点分型,其结果与直接测序完全一致。结论荧光标记复合扩增片段长度差异等位基因特异性PCR法是一种简单快速而有效的SNP分型新方法。