一例经典型半乳糖血症患者的基因变异分析

目的 对1例Ⅰ型半乳糖血症患者及其家系成员进行致病基因GALT突变检测及结合患者表型进行生物信息学分析。方法 采集患者及其父母的外周血,应用高通量测序目标序列捕获技术进行家系全外显子组测序。应用Sanger测序技术对患者及其家系成员可疑致病突变位点进行验证。结果 Trio全外显子组测序结果显示,患者的GALT基因变异位点c.829T>C(p.S277P)和c.970C>G(p.P324A)为复合杂合变异,父亲为变异位点c.829T>C(p.S277P)的携带者,母亲为变异位点c.970C>G(p.P324A)的携带者。二者均为错义突变,目前未见文献报道,为未见报道的新突变。结论 通过检测1例Ⅰ型半乳糖血症患者及其家系,发现GALT基因2种未见报道的新突变,扩充了GALT基因致病突变谱,为疾病的临床诊断和遗传咨询提供依据。     

一个 MPV17基因变异引起线粒体DNA耗竭综合征家系的遗传学分析及产前诊断

目的探讨一例临床诊断为婴儿肝炎综合征的患儿的遗传学病因并对该家系进行产前诊断。方法对该家系先证者通过高通量测序进行代谢性肝病相关致病基因变异筛选, 并用Sanger测序验证其变异来源, 通过生物信息学方法对新报道变异致病性进行分析。结果高通量测序结果显示先证者为MPV17基因(NM₀02437)c.182T>C(p.F61S)与c.293C>T(p.P98L)复合杂合变异引起的线粒体DNA耗竭综合征患者, Sanger测序验证变异分别遗传自其父亲和母亲。其中c.182T>C(p.F61S)变异尚未报道, 经分析为可能致病性变异。结论 MPV17基因c.182T>C(p.F61S)与c.293C>T(p.P98L)复合杂合变异为该患儿的遗传学病因;本研究发现了MPV17基因的一个新变异, 拓宽了MPV17基因致病变异谱。     

一例Krabbe病患者家系的产前诊断和基因突变分析

目的探讨一例Krabbe病患者的遗传病因,并对家系成员进行基因突变分析及产前诊断。方法应用全外显子测序技术对Krabbe病患者进行致病突变筛查,结合临床表型,确定候选基因的致病位点,Sanger测序验证夫妻双方GALC基因,孕妇进行绒毛穿刺和羊水穿刺,对GALC基因测序并进行产前诊断。结果全外显子测序结果显示家系中Krabbe病患者存在GALC基因c.599C>A(p.Ser200*)和c.461C>A(p.Pro154His)位点复合杂合变异,其父亲为c.461C>A(p.Pro154His)位点杂合变异携带者,母亲为c.599C>A(p.Ser200*)杂合变异携带者。绒毛和羊水检测GALC基因为c.461C>A(p.Pro154His)位点杂合变异。结论 GALC基因c.599C>A(p.Ser200*)和c.461C>A(p.Pro154His)位点复合杂合变异是该家系中Krabbe病患者的发病原因,胎儿绒毛和羊水检测结果与先证者不同,为该家系遗传咨询和产前诊断提供有力证据,有效预防出生缺陷。     

一个婴儿神经轴索营养不良家系的基因变异分析

目的对1个临床拟诊婴儿神经轴索营养不良(infantile neuroaxonal dystrophy,INAD)家系的患儿及父母进行基因变异分析,明确其致病原因为遗传咨询及产前诊断提供依据。方法应用高通量测序方法对患儿相关致病基因进行初步筛查,再通过直接测序对患儿及父母可疑致病基因进行验证,寻找可能的致病突变位点,采用SIFT及PolyPhen-2生物信息学软件对变异位点进行致病性预测。结果高通量测序筛查显示患儿PLA2G6基因第5和第16外显子存在可疑致病突变;Sanger测序结果显示患儿PLA2G6基因第5外显子c.668C>A(p.Pro223Gln)和第16外显子c.2266C>T(p.Gln756Ter)复合杂合变异,父亲携带c.668C>A杂合变异,母亲携带c.2266C>T杂合变异。c.668C>A变异导致PLA2G6基因编码的第223位脯氨酸被谷氨酰胺替代,为已报道的致病变异;c.2266C>T变异导致编译第756位谷氨酰胺的密码子变为终止密码,使肽链合成提前终止,该变异尚未见文献报道,蛋白功能预测为有害变异;患儿的复合杂合变异分别来自父母。结论PLA2G6基因第5外显子c.668C>A(p.Pro223Gln)和第16外显子c.2266C>T(p.Gln756Ter)变异可能是患儿的致病原因,新变异的发现丰富了PLA2G6基因变异谱。     

GJB2基因p.V37I突变致病性及家系临床表型分析

目的探讨GJB2基因突变耳聋家系p.V37I(c.109GA)突变致病性及分析家系患者临床表型。方法收集6个GJB2基因p.V37I(c.109GA)突变耳聋患者及家系的临床资料及外周血样本,运用PCR产物直接测序技术对家系耳聋患者进行GJB2、GJB3基因编码区、SLC26A4基因外显子7和8、以及线粒体m.1494CT、m.1555AG位点检测分析,另对家系5先证者行高通量全外显子序列检测。结果 6个家系均检出GJB2基因p.V37I突变,其中家系3、5为纯合突变,家系1、2、6先证者另复合GJB2基因c.235del C杂合突变,家系4患者另检出c.299-300del AT杂合突变;全部家系均未检出GJB3基因编码区、SLC26A4基因外显子7和8、以及线粒体m.1494CT、m.1555AG位点突变,家系5先证者高通量全外显子序列检测结果亦提示GJB2基因p.V37I纯合突变为其致聋原因。结论 GJB2基因p.V37I突变具有一定致病性,该位点纯合突变可导致轻度至中度听力损失,而p.V37I突变复合c.299-300del AT、c.235del C杂合突变可导致中度至重度感音神经性耳聋。     

一个遗传性凝血因子Ⅴ缺乏症家系的临床表型及基因分析

目的探讨一个遗传性凝血因子V缺乏症家系的表型特征及其分子致病机制。方法综合分析患儿及其家系成员的临床表现及辅助检查结果,并应用靶向捕获高通量测序及Sanger测序进行变异位点分析和家系验证。结果患儿凝血酶原时间、活化部分凝血活酶时间延长,Ⅴ因子活性仅为0.1%,但无任何出血征象;基因检测结果显示患儿F5基因上携带父源性c.653T>C(p.F218S)杂合变异和母源性c.3642_3643del(p.P1215Rfs*175)杂合变异,患儿哥哥携带父源性的c.653T>C(p.F218S)杂合变异,符合常染色体隐性遗传规律。其中c.653T>C(p.F218S)为已报道的致病性变异,c.3642_3643del(p.P1215Rfs*175)为国际上未见报道的可疑致病性变异。结论明确了F5基因为该患儿的致病基因,靶向捕获高通量测序结合Sanger测序可以快速准确的对该病进行基因变异检测。     

遗传性耳聋家系的致病原因分析及产前诊断

目的 对3个先天性耳聋家系进行遗传性耳聋基因检测及突变类型分析,为再生育的家庭提供遗传咨询及产前诊断。方法 对在宁波市妇女儿童医院就诊的3例先天性耳聋患儿抽取静脉血,提取DNA。先应用目标基因捕获和高通量测序技术对先证者进行耳聋基因相关的全外显子和相邻内含子测序,利用生物信息学技术对测序数据进行分析,筛选相关变异基因并对其进行致病性分析。再应用Sanger测序法对先证者及其父母做基因突变位点验证。最后对第二胎需要进行产前诊断的孕妇采集羊水进行位点验证。结果 家系1先证者中检出肌球蛋白XVA(myosinXVA,MYO15A)基因c.2075C>A、c.4520G>A、c.6668C>T和c.10250＿10252del杂合突变。家系2先证者中检出MYO15A基因c.5964+3G>A和c.7395+6T>G复合杂合突变。家系3先证者中检出缝隙连接蛋白β2(gap junction protein beta 2,GJB2)基因c.299＿300delAT和c.109G>A复合杂合突变。产前诊断结果显示家系1和家系2胎儿的基因型均与先证者的不同,听力表型...     

一个琥珀酸半醛脱氢酶缺陷病家系ALDH5A1基因突变分析

目的 对1例临床怀疑且经尿筛查诊断为琥珀酸半醛脱氢酶缺陷病的患儿及家系进行ALDH5A1基因突变分析,以进一步明确诊断和辅助遗传咨询.方法 应用聚合酶链反应及DNA直接测序技术对1例琥珀酸半醛脱氢酶缺陷病患儿及其父母的ALDH5A1基因进行突变位点检测,同时检测100名健康对照者的ALDH5A1基因以排除多态性变异.结果 患儿携带有ALDH5A1基因编码区序列第3外显子c.527G＞A(p.Gly176Glu)和第4外显子c.691G＞A(p.Glu231Lys)两种杂合突变,其母亲为c.527G＞A杂合突变携带者,父亲为c.691G＞A杂合突变携带者.另外患儿还存在两种已报道的核酸多态性改变:c.545C＞T杂合突变和c.538C＞T纯合突变.患儿的c.545C＞T突变来源于父亲,c.538C＞T突变分别来源于父母.100名健康对照者中未检测到c.527G＞A和c.691G＞A突变.结论 c.527G＞A(p.Gly176Glu)和c.691G＞A(p.Glu231 Lys)错义突变可能是该患儿的致病突变.     

四个遗传性耳聋家系的TMC1基因变异分析及产前诊断

目的对4个耳聋家系进行遗传性耳聋基因变异的筛查,为家系遗传咨询与产前诊断提供依据。方法应用二代测序技术对家系先证者进行耳聋基因检测,并对可疑基因变异采用Sanger双向测序对先证者及家系成员进行验证,确定可疑致病变异后,对3个家系高危胎儿进行产前诊断。结果家系1先证者检测到TMC1基因c.100C>T(p.R34X)和c.642+4A>C复合杂合变异,家系2先证者检测到TMC1基因c.582G>A(p.W194X)和c.589G>A(p.G197R)复合杂合变异,家系3先证者检测到TMC1基因c.1396_1398delAAC和c.1571T>C(p.F524S)复合杂合变异,家系4先证者检测到TMC1基因c.2050G>C(p.D684H)纯合变异,4个家系先证者父母均为携带者,其中c.642+4A>C、c.1571T>C(p.F524S)变异位点既往未见报道;产前诊断结果显示3个家系胎儿均不是患者,出生后随访至2019年9月,听力未见异常。结论TMC1基因变异是4个耳聋家系的可能致病原因,分子生物学的发现增加了对TMC1基因功能的认识并丰富了人类基因变异数据库,为家系遗传咨询和产前诊断提供了依据。     

一例戊二酸血症Ⅰ型患儿的 GCDH基因变异分析

目的:明确1例经串联质谱筛查诊断为戊二酸血症Ⅰ型患儿的遗传学病因。方法:提取患儿及其双亲外周血基因组DNA,采用基因捕获技术进行高通量测序,对可疑基因变异位点进行Sanger测序验证和生物信息学预测。结果:患儿存在 GCDH基因c.523G>A与c.1190T>C位点复合杂合变异,c.523G>A来源于父亲...     

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.     

两个有血缘关系的常染色体显性非综合性耳聋家系基因突变分析

目的 对两个有血缘关系的常染色体显性非综合性耳聋家系进行基因定位及突变分析,确定其致病基因.方法 通过家系调查和临床检查,鉴定了两个有血缘关系的常染色体显性非综合性耳聋大家系.并对已知位点及基因进行连锁分析,对致病基因在染色体上进行定位.PCR扩增候选基因MYH14基因的所有外显子和外显子-内含子交界区,直接测序法进行突变检测.结果 将这两个家系的致病基因定位于DFNA4位点,最大连锁值为4.94.具有统计学意义.突变检测发现MYH14基因的杂合突变c.359T>C(p.S120L),DNA直接测序确证两家系的所有患者均携带该突变,而家系中正常人则均不携带该突变.结论 第1次在中国非综合性耳聋家系中发现MYH14基因的突变,表明MYH14基因突变也是导致中国人非综合性耳聋的原因.     

Heterologous expression and characterization of a rare Gaucher disease mutation (c.481C > T) from a Canadian aboriginal population using archival tissue samples.

Gaucher disease is an inherited sphingolipidosis resulting from deleterious mutations in the glucocerebrosidase gene. Through direct sequence analysis of genomic DNA from whole blood, fibroblast cultures, and formalin-fixed archival tissue samples, we have identified a rare homozygous C > T transition at cDNA nucleotide 481 of the glucocerebrosidase gene that results in a proline to serine amino acid substitution (p.P122S) in an aboriginal family of Cree descent in northern Alberta, Canada. A 13-month-old boy (JB) presented with severe visceral Gaucher disease and was treated with enzyme replacement. Currently, at 11 years he is developmentally delayed, with oculomotor apraxia. A cousin (MS) had previously died at age 7 from complications of severe Gaucher disease, before enzyme replacement therapy was available. She was also developmentally delayed. Heterologous expression of this allele using a baculovirus expression system revealed 19.2% of normal enzyme activity on the artificial substrate 4-methylumbelliferyl beta-d-glucopyranoside (4MUGP). Genotype/phenotype correlation is complicated by incomplete clinical details, enzyme replacement therapy, and the difficulty in excluding other genetic and environmental causes of developmental delay. However the development of oculomotor apraxia in JB suggests a Type 3 Gaucher phenotype. The only previous report of this mutation was also from a member of the Cree Nation, who has had a rather similar clinical course. A protocol is described for the isolation of genomic DNA from formalin-fixed bone marrow aspirate archival specimens obtained from the deceased for subsequent PCR-based sequence analysis and mutation detection. This technique will be applicable to the screening of this and other populations for the frequency of known Gaucher mutations where traditional DNA sources are unavailable.     

卵清白蛋白特异性T细胞免疫诱导BALB/c小鼠的调节性免疫应答

目的：研究T细胞免疫后正常小鼠的调节性免疫应答，方法：应用体外扩增的卵清白蛋白（OVA）特异的T细胞克隆免疫BALB／c小鼠，3H－TdR掺入法分析细胞增殖，3H－TdR标记靶细胞检测杀伤T细胞的杀伤效应，间接免疫荧光法分析血清中抗T细胞抗体水平。结果：T细胞免疫后能诱导BALB/c小鼠产生调节性T细胞的增殖反应，对靶细胞的杀伤效应以及针对于活化的T细胞的体液免疫应答，并进一步降低机体对OVA抗原的应答，结论：T细胞免疫能诱导正常机体的调节性免疫应答。     

Functional relationship between T15 and J558 idiotypes in BALB/c mice.

In inbred strains of mice, antiphosphorylcholine (PC) and anti-alpha 1,3 dextran (DEX) antibodies are structurally distinct from each other and have been shown to exhibit noncross-reactive antigen binding and idiotypic specificities. However, the prototype anti-PC and anti-DEX antibodies, TEPC15 and J558, respectively, were shown to be connected via a common autoantiidiotypic monoclonal antibody isolated from newborn BALB/c mice. The capacity of various monoclonal anti-PC and anti-DEX antibodies as well as the antigens PC and DEX to modulate T15 and J558 idiotypes in BALB/c mice was tested by their administration to newborn mice. Anti-PC antibodies of the T15 idiotype injected into 2-4-day-old mice, at a time when T15+ anti-PC precursors develop in BALB/c mice, suppressed the anti-PC response of these mice at 6 weeks of age. Similarly, J558 antibodies injected into 8-12-day-old mice, at a time when J558 precursors normally develop, suppressed the response to DEX. As a further demonstration of this connectivity, the injection of J558 into 4-day-old mice led to a down modulation of T15 idiotype, whereas both T15 and a minor idiotype-expressing antibody M167 when injected into 8-12-day-old mice caused a reduction in expression of the J558 idiotype. As predicted from in vitro analysis, injection of anti-PC antibodies of the M167 idiotype 2 to 4 days after birth enhanced the subsequent response to PC. However, anti-PC antibodies expressing another minor M603 idiotype did not affect the PC response. The results parallel the in vitro enhancement of M167 antibodies but not M603 on T15 binding to antiidiotype in vitro. Similarly, anti-DEX antibodies expressing the M104E idiotype had no detectable effects on the capacity to respond to PC or DEX or on the expression of T15 and J558 idiotypes as adults. Exposure of newborn mice to PC led to a dramatic reduction in the response to DEX as adults, whereas exposure to DEX at this stage of development had no effect on response to PC as adults. Collectively, these observations provide evidence for a complex functional connectivity between T15 and J558 idiotype-bearing B cells during ontogeny and extend our previous observations that development of these idiotypes is regulated by idiotype-directed interactions between B cells or their immunoglobulin products.