一个Gly341Arg纯合突变所致遗传性FⅫ缺陷症近亲婚配家系的分析CSCD

目的对一个姨表近亲婚配的遗传性凝血因子Ⅻ（coagulation factor FⅫ,Ⅻ）缺陷症家系进行实验室表型和F衄基因突变的分析,探讨其分子发病机制。方法检测先证者及9名家系成员活化部分凝血活酶时间（activated partial thromboplastin time,APTT）等凝血常规功能、FⅫ活性（FⅫactivity,FⅫ：c）和FⅫ抗原（FⅫantigen,FⅫ：Ag）含量,进行表型诊断;用DNA直接测序法分析先证者FⅫ基因所有14个外显子、侧翼、5’和3＇非翻译区及家系成员相应的突变位点区域,用反向测序证实突变。采用ClustalX-2．1-win软件分析突变氨基酸的保守性,并同时采用4个生物信息学评分软件（PolyPhen-2,PROVEAN,SIFT和Mutation Taster）分析突变对蛋白质功能的影响。结果先证者（IV2）和哥哥（Ⅳ1）APTT明显延长（分别为61．6s和68．6s）,FⅫ：C和FⅫ：Ag分别降低为12％、10％和11％、10％;先证者祖母（Ⅱ2）、外祖母（Ⅱ3）、父亲（Ⅲz）、母亲（Ⅲ6）、大姑（Ⅲ1）和大姨（Ⅲ5）APTT正常,FⅫ：C和FⅫ：Ag均降低约为正常值的一半;先证者小姑（Ⅲ3）和大舅（Ⅲ4）各项指标及家系成员其它指标均正常。基因测序发现先证者（IV。）和其哥哥（Ⅳ1）FⅫ基因第10外显子存在c．1078G〉A（P．Gly341Arg）纯合错义突变;先证者祖母（Ⅱ2）、外祖母（Ⅲ3）、父亲（Ⅲ2）、母亲（Ⅲ6）、大姑（Ⅲ1）和大姨（Ⅲ5）均存在P．Gly341Arg杂合错义突变;先证者小姑（Ⅲ3）和大舅（Ⅲ4）为正常野生型。ClustalX-2．1-win软件保守性分析结果表明,Gly341在同源物种间高度保守。4个生物信息学软件对该突变的预测结果一致：PolyPhen-2评分（0．934分）、PROVEAN评分（-6．214分）均预示为有害突变;Mutation Taster评分（0．976分）预示可引起相应疾病;SIFT评分（0．01分）预示可影响蛋白质功能。结论Gly341Arg纯合错义突变是该家系遗传性FⅫ缺陷症的分子发病机制,与先证者父母近亲结婚有关。     

Arg304Trp导致的遗传性凝血因子Ⅶ缺陷症家系分析

目的对1例姨表近亲结婚的遗传性凝血因子Ⅶ（coagulation factorⅦ,FⅦ）缺陷症家系进行表型与基因型分析。方法检测血浆中凝血酶原时间（prothrombin time,PT）、FⅦ促凝活性（FⅦprocoagulant activity,FⅦ：C）及其它凝血指标以明确诊断;用DNA直接测序法对先证者及家庭成员FⅦ基因的全部外显子及其侧翼、启动子区进行分析,寻找基因突变,用反向测序证实所发现的突变。结果先证者的PT和FⅦ：C明显异常,分别为35.1s和3%;其父亲、母亲、大儿子和小儿子的PT稍延长,FⅦ：C明显降低。先证者FⅦ基因外显子8存在11348C→T纯合突变导致Arg304Trp;其父亲为该突变位点的杂合子,其母亲、大儿子和小儿子均为Arg304Trp杂合突变和Arg353Gln杂合多态性。结论先证者纯合突变Arg304Trp遗传自近亲结婚且具有相同杂合突变位点的父母。Arg353Gln多态性可能不是影响该家系成员血浆FⅦ：C水平的主要因素。     

一例皮肤松弛症患儿的临床与遗传学分析CSCD

目的采用遗传病相关基因外显子测序的方法确定1例皮肤松弛症患儿的致病基因,并对其相关的临床表型及基因型进行总结。方法收集先证者及其父母的临床资料,首先对先证者进行遗传病相关基因的外显子测序,确定了可能的致病突变,对先证者及其父母进行Sanger测序验证,确定基因突变位点。结果先证者存在ATP6VOA2基因C．187C〉T（P．R63X）杂合突变和C．1189G〉C（P．A397P）杂合突变,先证者父母分别携带ATP6VOA2基因c．1189G〉c（P．A397P）杂合突变和C．187C〉T（P．R63X）杂合突变。结合患儿临床表型,诊断为常染色体隐性遗传皮肤松弛症2A型（autosomal recessive cutis laxa type2A,ARCL2A）。结论通过遗传学方法确诊了ARCL2A型患儿,总结了ARCL2A患儿的典型临床特征,新突变的发现扩大了ATP6VOA2基因的突变谱。外显子测序可作为诊断复杂遗传病致病基因的重要工具。     

一例巨轴索神经病患者中发现GAN基因两种新型致病突变北大核心CSCD

目的寻找1例疑似巨轴索神经病患者的致病突变位点,为疾病诊断提供科学资料。方法应用目标序列捕获测序技术对患者进行遗传筛查,找到巨轴突蛋白基因（Gigaxonin,GAIN）的可疑致病突变位点,并提取患者父母的外周血DNA,进行Sanger测序验证。同时对200名正常人的400个GAN基因进行Sanger测序。此外,应用生物信息学软件Polyphen对突变巨轴突蛋白进行了功能预测。结果在患者GAN基因中鉴定出2种新型突变：c．778G〉T（p．Glu260Ter）和c．277G〉A（p．Gly93Arg）,为复合杂合突变;Sanger测序证实c．778G〉T（p．Glu260Ter）突变源于父方,c．277G〉A（P．Gly93Arg）突变则与母亲相同。在200名正常人GAN基因中未检测到同样的突变,排除其为多态性。生物信息学分析预测这两种新型GAN突变均可引发巨轴突蛋白功能异常。结论研究鉴定出了患者GAN的两种新型突变,它们均可能引起巨轴突蛋白结构和功能的异常,从而导致疾病的发生,为日后类似疾病的诊断提供了依据。     

一个遗传性凝血因子Ⅹ缺陷症家系的临床特征与基因突变分析

目的 探讨一个遗传性凝血因子Ⅹ（FⅩ）缺陷症家系的临床特征与基因突变关系。方法 调查家系，采用Sanger测序法分析先证者F10基因8个外显子及其侧翼序列，并对家系成员相同突变位点进行检测。通过凝血酶生成试验评估家系成员FⅩ的促凝活性。ClustalX-2.1-win和在线生物信息学软件（PolyPhen-2、SIFT和Mutation Taster）分别用于分析突变位点氨基酸的保守性和致病性；Swiss-PdbViewer软件用于蛋白质空间结构和突变氨基酸相互作用分析。结果 先证者FⅩ活性（FⅩ:C）和FⅩ抗原（FⅩ:Ag）分别降低至7%和14%（参考范围分别为：90%～114%和90%～110%）；先证者平素无自发性出血现象，外伤后也无明显出血异常。基因测序发现先证者F10基因第4号外显子存在c.361T>C(p.Cys121Arg)杂合错义突变及第8号外显子存在c.979C>T(p.Arg327Trp)杂合错义突变；其母亲和女儿为p.Arg327Trp的杂合子，二姐和儿子为p.Cys121Arg的杂合子。先证者凝血酶生成潜力比值和峰高比值下降，而其延迟时间和达峰时间延...     

一个遗传性凝血因子Ⅻ缺陷症家系的表型与基因型分析

目的对1个姨表近亲婚配的遗传性凝血因子Ⅻ(FⅫ)缺陷症家系进行实验室表型和F12基因突变分析,探讨其分子发病机制。方法检测先证者及其家系成员(共3代6人)血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血因子Ⅷ活性(FⅧ:C)、凝血因子Ⅸ活性(FⅨ:C)、凝血因子Ⅺ活性(FⅪ:C)、FⅫ活性(FⅫ:C)和FⅫ抗原(FⅪ:Ag)等指标,明确临床表型诊断。采用DNA直接测序法分析先证者F12基因所有外显子、侧翼、启动子区及家系成员相应的突变位点区域,并用反向测序证实所发现的突变。用ClustalX-2.1-win软件分析突变氨基酸的保守性;突变对蛋白质功能的影响则应用四个生物信息学评分软件(PolyPhen-2,PROVEAN,SIFT和MutationTaster)进行分析。结果先证者APTT(102.8s)、FⅫ:C(2%)和FⅪ:Ag(2%)明显异常;家系其他成员APTT正常,其姐姐、大女儿、二女儿和外孙的FⅫ:C和FⅫ:Ag均减低为正常对照的一半左右,表现为交叉反应物质(CRM)阴性型。基因测序发现先证者F12基因第13号外显子存在c.1556TG纯合突变,导致p.Leu519Arg;其姐姐、大女儿、二女儿和外孙均存在c.1556TG杂合突变。保守性分析结果显示Leu519在同源物种间呈高度保守;四个生物信息学软件对该突变预测的评分结果均显示为有害突变。结论F12基因13号外显子区p.Leu519Arg突变是导致该家系遗传性FⅫ缺陷症发病的原因;推测先证者纯合p.Leu519Arg突变基因分别遗传自近亲结婚的父母。     

双重杂合性突变Arg304Gln和Arg304Trp导致的遗传性凝血因子Ⅶ缺陷症

目的 探讨1例遗传性凝血因子Ⅶ(coagulation factorⅦ，FⅦ)缺陷症及其家系基因突变的类型。方法 检测凝血指标以明确诊断；用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子和其侧翼以及启动子进行分析，寻找基因突变；将含突变序列克隆人pGEM T—easy质粒载体中，对所得两条染色体相应序列分别测序，以确定不同突变在染色体上的分布。应用限制性内切酶Msp Ⅰ对先证者及家系成员相应基因片段进行酶切分析，证实测序所发现的突变。结果 先证者在第8外显子上有两种基因突变：11348位C→T突变和11349位G→A突变。pGEM T—easy质粒克隆测序结果显示上述两种突变位于不同的染色体上。为不同染色体同一编码区Arg(CGG)304Trp(TGG)和Arg(CGG)304Gln(CAG)双重杂合性突变。其父亲、母亲分别为11349位G→A和11348位C→T杂合突变；其弟弟FⅦ基因为正常野生型；其哥哥和先证者的3个子女均为杂合性突变。聚合酶链反应辅助限制性酶切证实了先证者及其家系成员的基因突变。结论 先证者FⅦ基因突变为不同染色体同一编码区Arg304Trp和Arg304Gln双重杂合性突变，此种突变类型的组合尚属首例。     

双重杂合性突变Arg304Gln和Arg304Trp导致的遗传性凝血因子缺陷症

目的　探讨 1例遗传性凝血因子 (coagulation factor ,F )缺陷症及其家系基因突变的类型。方法　检测凝血指标以明确诊断 ;用 DNA直接测序法对先证者及其家庭成员 F 基因的全部外显子和其侧翼以及启动子进行分析 ,寻找基因突变 ;将含突变序列克隆入 p GEM T- easy质粒载体中 ,对所得两条染色体相应序列分别测序 ,以确定不同突变在染色体上的分布。应用限制性内切酶 Msp 对先证者及家系成员相应基因片段进行酶切分析 ,证实测序所发现的突变。结果　先证者在第 8外显子上有两种基因突变 :11348位 C→ T突变和 11349位 G→ A突变。 p GEM T- easy质粒克隆测序结果显示上述两种突变位于不同的染色体上。为不同染色体同一编码区 Arg(CGG) 30 4 Trp(TGG)和 Arg(CGG) 30 4 Gln(CAG)双重杂合性突变。其父亲、母亲分别为 11349位 G→ A和 11348位 C→ T杂合突变 ;其弟弟 F 基因为正常野生型 ;其哥哥和先证者的 3个子女均为杂合性突变。聚合酶链反应辅助限制性酶切证实了先证者及其家系成员的基因突变。结论　先证者 F 基因突变为不同染色体同一编码区 Arg30 4 Trp和 Arg30 4 Gln双重杂合性突变 ,此种突变类型的组合尚属首例。     

F11基因复合杂合变异导致遗传性凝血因子Ⅺ缺陷症一个家系的分析

目的探讨1个F11基因新变异导致复合杂合性遗传性凝血因子Ⅺ(FⅪ)缺陷症家系的分子致病机制。方法选取2020年11月30日因"尿路结石"就诊于温州医科大学附属第一医院的1例遗传性凝血因子Ⅺ缺陷症男性先证者及其家系成员(3代7人)作为研究对象, 收集先证者的临床资料, 检测先证者及其家系成员的相关凝血指标。提取外周血基因组DNA进行PCR扩增, 用DNA直接测序法分析先证者F11基因的全部外显子、侧翼序列、5′和3′端非翻译区序列及家系成员相应的变异位点区域。用生物信息学软件分析氨基酸变异位点的保守性, 分析变异对蛋白质功能的影响。根据美国医学遗传学与基因组学学会(ACMG)相关变异评级指南对变异位点进行评级。结果先证者为36岁男性, 其活化部分凝血活酶时间(APTT)为89.2 s, 明显延长, FⅪ活性(FⅪ:C)和FⅪ抗原(FⅪ:Ag)分别为2.0%和3.5%, 均极度降低。基因测序发现先证者和其姐姐的F11基因第7外显子存在父源c.689G>T(p.Cys230Phe)杂合错义变异, 第13外显子存在母源c.1556G>A(p.Trp519*)杂合无义变异。保守性分析...     

一个Waardenburg综合征Ⅱ型家系SOX10基因的突变分析CSCD

目的对1例Waardenburg综合征Ⅱ型先证者及其家系成员进行SOX10基因的突变分析,探讨其可能的分子生物学致病原因。方法抽提先证者及其家系成员的外周血基因组DNA,芯片捕获高通量测序方法对MITF、PAX3、SOX10、SNA12、END3和ENDRB基因的全部外显子及其侧翼序列进行检测。根据高通量测序结果,对先证者及其父母进行突变位点的Sanger测序验证分析。结果Sanger测序结果显示先证者存在SOX10C．127c〉T（P．R43X）杂合突变,导致SOX10基因第43位编码精氨酸的密码子（CGA）突变为终止密码子（UGA）,产生截短蛋白,影响蛋白质功能的正常发挥。经检索人类基因突变数据库,该突变为未报道过的新突变。患儿父母未检测到该突变。结论先证者SOX10基因C．127c〉T（P．R43X）杂合突变可能是其分子生物学致病原因。     

七个鸟氨酸氨甲酰基转移酶缺陷症家系基因变异分析及产前诊断

目的对7个鸟氨酸氨甲酰基转移酶缺陷症(ornithine transcarbamylase deficiency,OTCD)家系进行OTC基因变异检测,明确其致病原因并为家系的遗传咨询和产前诊断提供依据。方法应用靶向高通量测序(next-generation sequencing,NGS)技术对7例经串联质谱筛查或临床诊断可疑OTCD的患儿或其母亲进行遗传代谢病相关基因panel检测,发现可疑致病变异位点后,应用PCR扩增和Sanger测序进行变异验证分析。在患儿母亲再次妊娠时抽取绒毛或羊水细胞进行相应基因变异检测,用于产前诊断。结果7个家系中共检测到7种OTC基因变异,分别为c.583G>A(p.Glyl95Arg).c.6260 T(p.Ala209Val)、c.6740 T(p.Pro225Leu)、c.482A>G(p.Asnl61Ser)、IVS1-2A>G、c.116G>T(p.Gly39Val).c.898delT(p.300Phefs*22),其中IVSl-2A>G、c・116G>T(p.Gly39Val)和c.898delT(p.300Phefs*22)为未报道过的新变异。产前诊断家系中3例胎儿基因测序均发现携带OTC基因变异半合子,性别为男性,孕妇选择终止妊娠,胎儿流产组织基因变异分析结果与产前诊断一致;另1例胎儿为OTC基因杂合变异,性别为女性,出生后新生儿筛查结果阴性,随访12个月,生长发育未见异常。结论OTC基因变异为7个OTCD家系的致病原因,致病变异的检出为家系的遗传咨询和产前诊断提供了依据。     

Investigation of citrullinemia type I variants by in vitro expression studies

Mild citrullinemia is an allelic variant of classical citrullinemia type I also caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). Affected patients comprise a biochemical but no clinical phenotype. However, there is no reliable parameter allowing conclusions regarding the course of the disorder or its type of manifestation. The aim of this study was to test the importance of varying levels of ASS residual activities for the severity at diagnosis. Bacterial in vitro expression studies allowed the enzymatic analysis of purified wild-type and the mutant ASS proteins p.Ala118Thr (c.352G>A), p.Trp179Arg (c.535T>C), p.Val263Met (c.787G>A), p.Arg265Cys (c.793C>T), p.Met302Val (c.904A>G), p.Gly324Ser (c.970G>A), p.Gly362Val (c.1085G>T), and p.Gly390Arg (c.1168G>A). In the chosen system, classical mutations do not show any significant enzymatic activity, whereas mutations associated with a mild course yield significant ASS activity levels. The mutation p.Ala118Thr (c.352G>A) impresses by a high residual activity (62%) but a severe reduction of affinity toward the substrates citrulline and aspartate. This mutation was identified in a hitherto healthy female adult with no history of known citrullinemia who had died during the postpartum period from hyperammonemic coma. The results of this study suggest that even a high level of residual ASS activity is not a reliable prognostic marker for an uneventful clinical course. Determination of ASS residual activities, therefore, cannot help in anticipating the risk of metabolic derangement. This study should guide clinicians as well as patients with mild citrullinemia toward a lifelong awareness of the disorder.     

Large-scale population-based metabolic phenotyping of thirteen genetic polymorphisms related to one-carbon metabolism

Several polymorphisms of genes involved in one-carbon metabolism have been identified. The reported metabolic phenotypes are often based on small studies providing inconsistent results. This large-scale study of 10,601 population-based samples was carried out to investigate the association between a panel of biochemical parameters and genetics variants related to one-carbon metabolism. Concentrations of total homocysteine (tHcy), folate, vitamin B(12) (cobalamin), methylmalonic acid (MMA), vitamin B(2) (riboflavin), vitamin B(6) (PLP), choline, betaine, dimethylglycine (DMG), cystathionine, cysteine, methionine, and creatinine were determined in serum/plasma. All subjects were genotyped for 13 common polymorphisms: methylenetetrahydrofolate reductase (MTHFR) c.665C>T (known as 677C>T; p.Ala222Val) and c.1286A>C (known as 1298A>C; p.Glu429Ala); methionine synthase (MTR) c.2756A>G (p.Asp919Gly); methionine synthase reductase (MTRR) c.66A>G (p.Ile22Met); methylenetetrahydrofolate dehydrogenase (MTHFD1) c.1958G>A (p.Arg653Gln); betaine homocysteine methyltransferase (BHMT) c.716G>A (known as 742G>A; p.Arg239Gln); cystathionine beta-synthase (CBS) c.844_845ins68 and c.699C>T (p.Tyr233Tyr); transcobalamin-II (TCN2) c.67A>G (p.Ile23Val) and c.776C>G (p.Pro259Arg); reduced folate carrier-1 (SLC19A1) c.80G>A (p.Arg27His); and paraoxonase-1 (PON1) c.163T>A (p.Leu55Met) and c.575A>G (p.Gln192Arg). The metabolic profile in terms of the measured vitamins and metabolites were investigated for these 13 polymorphisms. We confirmed the strong associations of MTHFR c.665C>T with tHcy and folate, but also observed significant (P<0.01) changes in metabolite concentrations according to other gene polymorphisms. These include MTHFR c.1286A>C (associations with tHcy, folate and betaine), MTR c.2756A>G (tHcy), BHMT c.716G>A (DMG), CBS c.844_845ins68 (tHcy, betaine), CBS c.699C>T (tHcy, betaine, cystathionine) and TCN2 c.776C>G (MMA). No associations were observed for the other polymorphisms investigated.     

Low frequency of deafness-associated GJB2 variants in Kenya and Sudan and novel GJB2 variants

A large proportion of non-syndromic autosomal recessive deafness (NSARD) in many populations is caused by variants of the GJB2 gene. Here, the frequency of GJB2 variants was studied in 406 and 183 apparently unrelated children from Kenya and Sudan, respectively, with mostly severe to profound non-syndromic deafness. Nine (2.2 %) Kenyan and 12 (6.6 %) of the Sudanese children only were carriers of variants within the coding sequence of the GJB2 gene. Variants in the 5'-adjacent region were detected in further 115 individuals. A total of 10 novel variants was recognized, among them four variants in the adjacent 5'-region of the GJB2 coding exon 2 (g.3318-6T>A, g.3318-15C>T, g.3318-34C>T, g.3318-35T>G), a 6 base-pair deletion (g.3455_3460del [p.Asp46_Gln48delinsGlu]), a variant leading to a stop codon (g.3512C>A [p.Tyr65X]), synonymous variants (g.3395C>T [p.Thr26], g.3503C>T [p.Asn62], g.3627A>C [p.Arg104]), and one non-synonymous variant (g.3816C>A [p.Val167Met]). In addition, the previously described variants g.3352delG (commonly designated 30delG or 35 delG), g.3426G>A [p.Val37Ile], g.3697G>A [p.Arg127His], g.3774G>A [p.Val153Ile], and g.3795G>A [p.Gly160Ser] were identified. With the exception of g.3318-34C>T and g.3352delG, all variants occurred heterozygously. For most of the variants identified in the Kenyan and Sudanese study population, a causative association with NSARD appears to be unlikely. Compared to many other ethnic groups, deafness-associated variants of the coding region of GJB2 are rare in Sudan and Kenya, suggesting a role of other genetic, or epigenetic factors as a cause for deafness in these countries.     

Novel CLDN14 mutations in Pakistani families with autosomal recessive non-syndromic hearing loss

Mutations in the CLDN14 gene are known to cause autosomal recessive (AR) non-sydromic hearing loss (NSHL) at the DFNB29 locus on chromosome 21q22.13. As part of an ongoing study to localize and identify NSHL genes, the ARNSHL segregating in four Pakistani consanguineous families were mapped to the 21q22.13 region with either established or suggestive linkage. Given the known involvement of CLDN14 gene in NSHL, DNA samples from hearing-impaired members from the four families were sequenced to potentially identify causal variants within this gene. Three novel CLDN14 mutations, c.167G>A (p.Trp56*), c.242G>A (p.Arg81His), and c.694G>A (p.Gly232Arg), segregate with hearing loss (HL) in three of the families. The previously reported CLDN14 mutation c.254T>A (p.Val85Asp) was observed in the fourth family. None of the mutations were detected in 400 Pakistani control chromosomes and all were deemed damaging based on bioinformatics analyses. The non-sense mutation c.167G>A (p.Trp56*) is the first stop codon mutation in CLDN14 gene to be identified to cause NSHL. The c.242G>A (p.Arg81His) and c.694G>A (p.Gly232Arg) mutations were identified within the first extracellular loop and the carboxyl-tail of claudin-14, respectively, which highlights the importance of the extracellular domains and phosphorylation of cytoplasmic tail residues to claudin function within the inner ear. The HL due to novel CLDN14 mutations is prelingual, severe-to-profound with greater loss in the high frequencies.     

MSH2 missense mutations and HNPCC syndrome: pathogenicity assessment in a human expression system

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is associated with germline mutations in one of several MisMatch Repair (MMR) genes. An increasing proportion (20-25%) of the reported MSH2 variants consists of single amino-acid substitution with uncertain disease-causing significance. The present study was undertaken to functionally characterize 3 MSH2 nontruncating variants: p.Gly162Arg (c.484G>C), p.Asp167His (c.499G>C) and p.Arg359Ser (c.1077A>T). Missense alterations, were assessed in a human system for expression/stability and for the ability to heterodimerize with MSH6 and correctly localize into the nucleus. Functional assays results were correlated with clinical and genetic features indicative of HNPCC as MicroSatellite-Instability (MSI), abnormalities of MMR gene expression in tumour tissue (IHC) and familial history. p.Gly162Arg and p.Arg359Ser variants showed a clearly decreased expression level of the MutSá complex and were associated with an abnormal subcellular localization pattern, which can be suggestive of an incorrect MSH2/MSH6 heterodimerization. Functional analysis results were supported by MSI and IHC data and by familial cancer history. The subcellular localization assay, performed in a human expression system, classifies as pathogenetic two MSH2 nontruncating alterations providing a useful tool in genetic testing programs.     

A Gly1609Arg missense mutation in the vWF gene in a Korean patient with von Willebrand disease type 2A

We describe a case of a c.4825G>A (p.Gly1609Arg [Gly846Arg]) missense mutation in the gene encoding von Willebrand factor (vWF) in a Korean patient with von Willebrand disease (vWD) type 2A. The proband is a 37-year-old female who suffers from dysmenorrhea and menorrhagia. On laboratory testing, we found a low (0.01) vWF:RCo/Ag ratio, a decrease in high and intermediate molecular weight multimers from plasma, and abnormalities in the collagen binding capacity of plasma vWF, all of which were indicative of vWD type 2. Family studies revealed that her sister, son, and daughter also had a low vWF:RCo/ Ag ratio and a decrease in high molecular weight multimers from plasma. Genetic analyses showed that she and her three family members had the same heterozygous c.4825G>A (p.Gly1609Arg [Gly846Arg]) missense mutation. To our knowledge, this is the first report of the c.4825G>A (p.Gly1609Arg [Gly846Arg]) heterozygote mutation in Korean family members with vWD type 2A.     

Identification of eight novel glucokinase mutations in Italian children with maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a clinically heterogeneous group of disorders characterized by early onset non-insulin-dependent diabetes mellitus, autosomal dominant inheritance, and primary defect in the function of the beta cells of the pancreas. Mutations in the glucokinase (GCK) gene account for 8%-56% of MODY, with the highest prevalences being found in the southern Europe. While screening for GCK mutations in 28 MODY families of Italian origin, we identified 17 different mutations (corresponding to 61% prevalence), including eight previously undescribed ones. The novel sequence variants included five missense mutations (p.Lys161Asn c.483G>C in exon 4, p.Phe171Leu c.511T>C in exon 5 and p.Thr228Ala c.682A>G, p.Thr228Arg c.683C>G, p.Gly258Cys c.772G>T in exon 7), one nonsense mutation (p.Ser383Ter c.1148C>A in exon 9), the splice site variant c.1253+1G>T in intron 9, and the deletion of 12 nucleotides in exon 10 (p.Ser433_Ile436del c.1298_1309del12). Our study indicates that mutations in the GCK/MODY2 gene are a very common cause of MODY in the Italian population and broadens our knowledge of the naturally occurring GCK mutation repertoire.     

Women heterozygous for NALP7/NLRP7 mutations are at risk for reproductive wastage: report of two novel mutations

Familial recurrent hydatidiform moles are a rare recessive condition in which molar tissues have biparental contribution to their genome. One maternal locus responsible for this condition has been mapped to 19q13.4 and the causative gene, NALP7, identified (HUGO-approved nomenclature is now NLRP7). Here we report a first stop codon, c.295G>T (p.Glu99X) and a missense mutation, c.1970A>T (p.Asp657Val) in NLRP7 in two sisters with RHMs. We found these two mutations and a previously reported one, c.2078G>C (p.Arg693Pro) in a homozygous state, in males with normal reproductive outcomes. This suggests that NLRP7 is not required for normal spermatogenesis. The mother of the patients is heterozygous for Glu99X and had one stillbirth and three normal pregnancies. Our data on this new family and on heterozygous women from previously reported families indicate that women heterozygous for NLRP7 mutations are at risk for reproductive wastage without the manifestation of molar phenotype.     

Mutation analysis in Costello syndrome: functional and structural characterization of the HRAS p.Lys117Arg mutation

Costello syndrome is a mental retardation syndrome characterized by high birth weight, postnatal growth retardation, coarse face, loose skin, cardiovascular problems, and tumor predisposition. De novo heterozygous missense mutations in HRAS codon 12 and 13 disturbing the intrinsic GTP hydrolysis cause Costello syndrome. We report a patient with typical Costello syndrome and a novel heterozygous missense mutation in codon 117 (c.350A>G, p.Lys117Arg) of the HRAS gene, resulting in constitutive activation of the RAS/MAPK pathway similar to the typical p.Gly12Ser and p.Gly12Ala mutations. Recombinant HRAS p.Lys117Arg demonstrates normal intrinsic GTP hydrolysis and responsiveness to GTPase-activating proteins, but the nucleotide dissociation rate is increased 80-fold. Consistent with the biochemical data, the crystal structure of the p.Lys117Arg mutant indicates an altered interaction pattern of the side chain that is associated with unfavorable nucleotide binding properties. Together, these data show that a RAS mutation that only perturbs guanine nucleotide binding has similar functional consequences as mutations that impair GTP hydrolysis and causes human disease.