Analysis of phenotypes and genetic mutations in two pedigrees affected with hereditary protein C deficiency北大核心CSCD

Objective To explore the pathogenesis of protein C deficiency in two pedigrees through mutation detectionand model analysis. Methods Chromogenic substrate method and enzyme linked immunosorbent assay (ELISA) wereused to determine the plasma protein C activity (PC : A) and protein C antigen (PC : Ag) in the two probandsand their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCRand analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members werealso amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutationsand Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutationswere analyzed by Swiss-PdbViewer software. Results The PC : A and PC : Ag of proband 1 was 30% and 35%, whilePC : A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missensemutations were found in exons 7 and 5 of the PROC gene, namely c. 565 C>T (p. Argl47Trp) and c. 383 G >A (p. Gly86Asp). His father and aunt were carriers for c. 565 C>T, while his mother had carried c. 383 G>A. The PC : A of proband 2 and his son were 50% and 64% , respectively. And they were both positive for p. Argl47Trp. Analysis of PolyPhen-2 indicated that p. Argl47Trp was benign, while p. Gly86Asp was damaging. Clustal X analysis indicated that the p. Argl47Trp was non-conservative, while the p. Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trpl47 in p.Argl47Trp destroyed the two hydrogen bonds between Argl47-Lysl46 and Argl47-Lysl51, and steric hindranted withArgl78. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p.Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion. Conclusion Both probands hadhereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p.Argl47Trp and p. Gly86Asp were the main cause for PC : A activity decrease. Among these, p. Gly86Asp wasdiscovered for the first time.     

Streamlining the decision-making process for international DNA kinship matching using Worldwide allele frequencies and tailored cutoff log10LR thresholds

The identification of human remains belonging to missing persons is one of the main challenges for forensic genetics. Although other means of identification can be applied to missing person investigations, DNA is often extremely valuable to further support or refute potential associations. When reference DNA samples cannot be collected from personal items belonging to a missing person, a direct DNA identification cannot be carried out. However, identifications can be made indirectly using DNA from the missing person’s relatives. The ranking of likelihood ratio (LR) values, which measure the fit of a missing person for any given pedigree, is often the first step in selecting candidates in a DNA database. Although implementing DNA kinship matching in a national environment is feasible, many challenges need to be resolved before applying this method to an international configuration. In this study, we present an innovative and intuitive method to perform international DNA kinship matching and facilitate the comparison of DNA profiles when the ancestry is unknown or unsure and/or when different marker sets are used. This straightforward method, which is based on calculations performed with the DNA matching software BONAPARTE, Worldwide allele frequencies and tailored cutoff log₁₀LR thresholds, allows for the classification of potential candidates according to the strength of the DNA evidence and the predicted proportion of adventitious matches. This is a powerful method for streamlining the decision-making process in missing person investigations and DVI processes, especially when there are low numbers of overlapping typed STRs. Intuitive interpretation tables and a decision tree will help strengthen international data comparison for the identification of reported missing individuals discovered outside their national borders.     

8个家族性特发性基底节钙化家系的临床特点及遗传规律研究

【目的】探讨8个家族性特发性基底节钙化(FIBGC)家系的临床特点及遗传规律。【方法】收集8个 FIBGC 家系,根据临床表现分为运动受损组和精神症状组,绘制家系图,分析先证者及家系其他患者的发病年龄、临床表现、基底节区钙化的体积,总结遗传规律。【结果】8个家系均呈常染色体显性遗传,先证者的性别比：男/女=4/4;患者的性别比：男/女=18/17,两组先证者人数构成比：运动受损/精神症状=4/4,运动受损组与精神症状组性别比(男/女)无显著性差异[(3/1)vs (1/3),P >0.05]。两组先证者的发病年龄[(43.00±3.16)岁 vs(29.50±6.95)岁]和基底节区钙化的体积[(1.526±0.679)cm3 vs(0.233±0.114)cm3]比较差异具有统计学意义(P <0.05)。临床特点：患者均表现为一个系统损害的症状,运动系统受损或者精神症状。以运动受损的4个家系其他成员发病的症状也以运动受损为特点,精神症状为主的4个家系仅5人有精神症状,其他成员均没有临床症状。【结论】FIBGC 临床表现具有明显的异质性,以运动受损的患者其病情严重程度与基底节区钙化病灶的大小相关,且发病年龄较晚,家系成员临床症状具有遗传性;以精神症状为主的患者其基底节区钙化病灶小,发病年龄早,家系成员临床症状遗传性不明显。     

念珠状发家系Ⅱ型毛发角蛋白基因突变的研究

目的 探讨汉族念珠状发家系Ⅱ型毛发角蛋白(hHb)基因的突变情况.方法 采集该家系成员及50例毛发外观正常者外周血并提取基因组DNA.运用PCR扩增hHb1、hHb3、hHb6外显子1和外显子7,DNA直接测序检测突变位置.测序结果采用BLAST软件在互联网上进行比对.扫描电镜下观察毛干结构.结果 扫描电镜下,病发呈典型的念珠状表现,狭窄部见明显的纵嵴和纵沟,病发毛小皮厚薄不均,其中1例病发大部分皮质和髓质消失.经网上比对分析,该家系患者hHb6外显子7第1289位碱基鸟嘌呤(G)被腺嘌呤(A)取代,导致第430位精氨酸突变为谷氨酰胺(即R430Q).而该家系中未患病者和50位正常对照组均未发现该突变,hHb1、hHb3外显子1和外显子7及hHb6的外显子1未发现突变.结论 hHb6外显子7的R430Q错义突变是一种新的突变,可能与该家系的发病有关.     

2型糖尿病家系对氧磷酯酶2 C311S基因多态性研究

目的:探讨对氧磷酯酶2(PON2)Cys 311 Ser(C311S)基因多态性与2型糖尿病(T2DM)的关系。方法:应用荧光偏振-模板依赖的染料掺入反应法(TDI-FP)技术,对135例的T2DM家系成员和45例正常人的PON2 C311S基因多态性进行研究。结果:测序结果与TDI-FP方法检测结果比较,符合率100%。2型糖尿病家系中T2DM组、NDR组的基因型频率和等位基因频率与正常对照比较无显著差异(P>0.05)。结论:应用TDI-FP技术能准确敏感地检测PON2 C311S基因多态性。未发现PON2 C311S基因多态性与T2DM的发生存在相关性。     

单纯型家族性发作性运动诱发性运动障碍二家系基因连锁分析

目的 对2个中国汉族单纯型发作性运动诱发性运动障碍(PKD)大家系进行排除定位,为PKD发病机制的探讨及疾病基因的克隆奠定基础.方法 抽取2个家系27名成员外周血,选取16号染色体上覆盖目前已知PKD位点的微卫星标记,进行参数及非参数连锁分析,并进行单体型分析.结果 通过对2个显性遗传的PKD大家系的参数及非参数连锁分析发现,家系A的最大LOD值以及NPL值均为负值,P＞0.05,排除与已知的PKD位点连锁;家系B的最大LOD值＜1,最大NPL值0.77,P＞0.05,不支持致病基因与已知PKD位点连锁.进一步对2个家系进行单体型分析,排除其致病基因位点与已知位点连锁,提示存在新的PKD疾病基因位点.结论 PKD具有遗传异质性,单纯型PKD家系存在新的疾病基因位点.     

糖尿病家族遗传特性与肾虚证的相关性探讨

目的:探讨糖尿病遗传特性和肾虚证的相关性。方法:根据自编糖尿病四诊症状调查表对2269例2型糖尿病(Type 2 Diabetes Mellitus,T2DM)患者及3个典型T2DM家系进行调查,通过肾虚主症和四诊症状总计分的比较,分析肾虚证程度与T2DM家族史背景的关系及肾虚证在T2DM家系中的分布特征。结果:2269例患者中,有家族史者有444例,占总调查人数的19.57%;阳性T2DM家族史的患者的四诊症状定性总分明显高于阴性家族史患者(P0.01);3个典型T2DM家系中肾虚证成员占总调查成员的比例分别为66.7%、50%和75%。结论:肾虚证在T2DM家系中呈家族聚集性趋势,且和T2DM有伴发倾向,肾虚在T2DM的遗传特性中起重要作用。     

*常染色体显性先天性缝合状白内障家系致病基因的定位

目的对我国一常染色体显性先天性缝合状白内障家系进行致病基因的定位。方法采集家系成员外周静脉血提取基因组DNA。选用美国Applied Biosystems公司提供的约400个遗传标记物进行基因扫描。数据经Linkage软件包进行连锁分析,初步确定致病基因所在染色体区域。在阳性区域内选取更高密度的荧光标记物进行精细扫描,用Cyrillic软件进行单体型分析。结果两点间连锁分析在D3S1279处获得最大对数优势记分（LOD）值Zmax=2．32（θmax=0．00）。通过精细扫描和单体型分析将致病基因定位于D3S1267和D3S1614之间约38．6厘摩（cM）区域内。结论先天性缝合状白内障家系的致病基因位于3号染色体3q21．1-q26．2约38．6cM区域内。（中华腥科杂志,2006,42：908—912）     

先天性无虹膜症家系的基因突变位点研究

目的探讨先天性无虹膜症家系的基因突变位点。方法抽取家系成员的外周血2~5ml,提取DNA;先合成2个多态性微卫星遗传标记(D11S904和D11S935)的引物进行聚合酶链反应(PCR),PCR产物变性后用变性聚丙烯酰胺(PAGE)胶分离,根据带型和家系成员间的关系进行单体型连锁分析,判断家系无虹膜表型是否与PAX6基因相关;PCR扩增PAX6基因的所有外显子,所有PCR产物分别进行单链构象多态性(SSCP)分析,通过患者与正常人带型的差异确定突变发生的外显子,对有差异SSCP带型的PCR产物进行直接DNA测序,找到突变位点。结果该家系先天性无虹膜表型明显与PAX6基因连锁;SSCP分析PAX6基因第9外显子PCR产物,显示患者均有异常带型出现,而家系正常人均无此异常带;测序结果显示突变位点为PAX6基因第9外显子c1080核苷酸C突变为T,使编码精氨酸的密码子突变为终止密码子。结论PAX6基因突变可导致先天性无虹膜。