3个遗传性抗凝血酶缺陷症家系的表型和基因型关系的研究

目的探讨遗传性抗凝血酶缺陷症家系表型和和基因型的关系。方法对3个遗传性抗凝血酶缺陷症家系(家系1~3)作表型和基因型诊断:常规检测活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(Fg)、凝血酶时间(TT)以筛查凝血功能,发色底物法检测蛋白C、蛋白S和抗凝血酶活性(PC∶A,PS∶A及AT∶A),免疫比浊法检测抗凝血酶抗原(AT∶Ag),Western blot检测血浆中抗凝血酶的分子量和含量;PCR扩增AT基因所有外显子及侧翼序列,DNA测序并进行基因分析。针对新基因突变,在100例正常人中检测相应突变以排除多态性,用TA克隆PCR产物测序鉴定杂合碱基缺失突变。结果 3名先证者均为反复发作的静脉血栓患者,凝血指标及PC∶A和PS∶A都正常,AT∶A分别为正常人(100%)的60%、52%和60%,AT∶Ag分别为16.9、14.1和11.4 mg/dl,Western blot显示3位先证者的血浆AT蛋白分子量正常(58 kD)而含量低于正常;基因分析发现3名先证者各携带1个杂合突变,分别为g.8263 C>T(Leu340Phe)、g.5894-6 del TTC(Phe122del)和g.5898 T>G(Phe123Cys)。3个家系中与先证者表型相似的成员,则携带有相同的AT基因突变;但除家系2先证者的父亲有静脉血栓外,家系1和3中含有相应AT基因突变的家庭成员均无血栓发生。结论 3名先证者遗传性抗凝血酶缺陷症分别由Leu340Phe、Phe122del和Phe123Cys突变所致,其中Leu340Phe和Phe123Cys突变为国际上首次报道。     

新生儿筛查发现4例MCCD患儿的临床和基因分析

目的对4例经由新生儿筛查发现的不同临床类型的3-甲基巴豆酰辅酶A羧化酶缺乏症(3-methylcrotonyl-coenzyme A carboxylase deficiency,MCCD)患儿,用尿气相色谱质谱和基因分析证实其诊断。方法对新生儿筛查中C4DC+C5OH0.6μmol/L的新生儿召回复查,同时检测其母亲的C4DC+C5OH浓度。用尿气相色谱质谱分析对MCCD疑诊病例进行临床诊断,再通过基因分析进一步证实。结果通过基因诊断确诊MCCD 3例,包括MCCD患儿1例,父源性MCCD 1例,母源性MCCD 1例。另有1例临床诊断的MCCD患儿经基因检测仅找到1个致病位点。结论对新生儿筛查中发现的C4DC+C5OH增高的新生儿家系(包括母亲和父亲)应行MS/MS检测。疑似MCCD患者基因检测仅发现1个致病位点时不要轻易否定MCCD的诊断,建议定期随访。     

15例遗传性凝血因子V缺陷症先证者的临床特征与基因突变分析

摘要:目的︰分析15个遗传性凝血因子V(FV)缺陷症先证者的临床特征与基因突变类型,初步探讨其可能的分子致病机制。方法采用一期凝固法和ELISA法分别检测FV活性(FV:;C)和FV抗原(FV:Ag)。用PCR扩增患者F5基因的25个外显子及其侧翼序列,并直接测序。利用蛋白质模型分析其可能的分子机制。结果在5例FV;C大于10%的先证者中,仅有1例出现轻微出血症状;在10例FV:C小于10%的先证者中,7例表现出各种出血症状。15例先证者共检出12个基因突变位点(其中8个为新的突变,1个为致病的多态性)。蛋白质模型分析表明,所有6种错义突变都会导致FV蛋白的构象改变,其中2种(p.Ser1781Arg和p.Asp96Hlis)会减少氢键数量,从而导致局部蛋白质结构不稳定。结论这些遗传性FV缺陷症先证者的FV水平与各自的F5基因突变有关,其FV水平与出血症状具有较强的相关性。     

Molecular analysis of the CYP21A2 gene in Chinese patients with steroid 21-hydroxylase deficiency

Objective

21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), a family of autosomal recessive disorders involving impaired cortisol synthesis. This study aimed to design a reliable and rational approach for identifying mutations in the CYP21A2 gene and to characterize the molecular basis of 21-OHD in 30 Chinese patients.

Design and methods

Copy number variations were investigated by multiplex ligation-dependent probe amplification (MLPA). Locus-specific polymerase chain reaction (PCR)/restriction endonuclease analysis was then used to verify CYP21A2 rearrangement products and prevent allele dropout. Direct sequencing of rearrangement products was performed to further refine recombination breakpoint locations. Direct sequencing of the entire CYP21A2 gene was used to detect microconversions.

Results

We successfully characterized 60 CYP21A2 alleles from 30 patients with genetic defects. The most common one was intron 2 splice mutation (38.3%). Eighteen alleles with large gene deletions/conversions were identified, which accounted for nearly one-third (30.0%) of the genetic defects. Among these, three types of CYP21A1P/CYP21A2 chimeric genes (CH-1, CH-2, and CH-4) were characterized. Two novel CYP21A2 rearrangement genes were revealed and further demonstrated to be located downstream of the TNXB gene.

Conclusions

Our results indicate that the stepwise diagnostic procedure involving MLPA analysis, locus-specific PCR/restriction endonuclease analysis, and direct DNA sequencing can provide detailed genetic information about Chinese 21-OHD patients, which is helpful for characterizing structural rearrangements of CYP21A2.     

17 alpha‐hydroxylase deficiency: A case report of young Chinese woman with a rare gene mutation

We report a young adult woman with 17 alpha‐hydroxylase deficiency (17α‐OHD) in Shandong province of China. The patient carried compound heterozygous mutations in the CYP17A1 gene: c.985–987 delinsAA (p.Tyr329LysfsX90) and c.1486C > T (p.Arg496Cys). The patient''s hypertension and hypokalemia were resolved after taking medications of glucocorticoid, aldactone, and calcium antagonists.     

Novel mutations in ETFDH gene in Chinese patients with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency

Background

Multiple acyl-CoA dehydrogenase deficiency (MADD, OMIM 231680) or glutaric aciduria type II (GAII) is an inherited autosomal recessive disease affecting fatty acid, amino acid and choline metabolism, due to mutations in one of three genes namely, electron transfer flavoprotein alpha-subunit, ETFA (OMIM 608053), electron transfer flavoprotein β-subunit, ETFB (OMIM 130410) and electron transfer flavoprotein dehydrogenase, ETFDH (OMIM 231675). Some MADD patients are responsive to riboflavin treatment with an excellent prognosis. Recently, mutations in ETFDH were found to be responsible for all riboflavin-responsive MADD patients. In this study, we present the clinical features and molecular studies of 2 Chinese families with riboflavin-responsive MADD.

Methods

Genomic DNA was extracted from peripheral blood samples or skin fibroblast cultures from the patients and normal controls. The thirteen exons of ETFDH were amplified by PCR. PCR products were sequenced in both forward and reverse directions. To rule out mutations in other genes, phenotype segregation was studied in the families by microsatellite markers in the proximity of the 3 genes, ETFA, ETFB and ETFDH.

Results

Four novel mutations in ETFDH were detected in the 2 families. In family 1, a frame shift mutation, c.1355delG which introduced a premature-termination codon (PTC), I454X in exon 11 of ETFDH was found. Another mutation was a c.250G>A transition in exon 3 of ETFDH, A84T. In family 2, two novel missense mutations were identified, P137S, in exon 4 and G467R in exon 11. No carrier of these four mutations was identified from about 150 alleles of healthy Chinese control subjects.

Conclusions

Four novel mutations (3 missenses and 1 deletion) in ETFDH were found in Chinese families that presented with riboflavin-responsive MADD, which further expands the list of mutations found in patients with riboflavin-responsive MADD. Furthermore, we illustrated the utility of phenotype-genotype segregation in MADD families to prioritize genes for sequencing or to rule out the presence of disease causing mutation in other genes in MADD and other diseases caused by multiple genes.     

深圳盐田地区G6PD缺乏症发病率与基因突变调查

目的了解深圳盐田区G6PD缺乏症的流行现状、基因突变谱,探讨其诊断方法及流程。方法采用G6PD／6PGD定量比值法检测G6PD酶活性,反向点杂交及DNA测序检测G6PD基因突变。结果该区G6PD基因突变人群携带率为4．50％,基因突变以C．1388G〉A、c．1376G〉T和c．95A〉G为主。结论该区为包含其他罕见或少见突变类型的复杂G6PD基因突变谱,基于G6PD酶活性的表型筛查有明显的漏检率,基因检测结合表型筛查可大大提高诊断准确性与可靠性。     

Child with adenylosuccinate lyase deficiency caused by a novel complex heterozygous mutation in the ADSL gene: A case report

BACKGROUNDAdenylosuccinate lyase (ADSL) deficiency is a rare autosomal-recessive defect of purine metabolism caused by mutation of the ADSL gene. It can cause severe neurological impairment and diverse clinical manifestations, including epilepsy.CASE SUMMARYHere, we describe a 3-year-old Chinese boy who had both psychomotor retardation and refractory epilepsy. Magnetic resonance imaging showed myelin hypoplasia. Electroencephalography findings supported a diagnosis of epilepsy. Whole-exon sequencing revealed the presence of a novel complex heterozygous mutation in the ADSL gene: The splicing mutation c.154-3C>G and the missense mutation c.71C>T (p. Pro24Leu). Considering the patient’s clinical presentation and genetic test results, the complex heterozygous mutation was predicted to prevent both ADSL alleles from producing normal ADSL, which may have led to ADSL deficiency. Finally, the child was diagnosed with ADSL deficiency.CONCLUSIONWe identified a novel complex heterozygous mutation in the ADSL gene associated with ADSL deficiency, thus expanding the known spectrum of pathogenic mutations that cause ADSL deficiency. Additionally, we describe epilepsy that occurs in patients with ADSL deficiency.     

六味地黄颗粒对晚期肝肾阴虚型结直肠癌患者基因表达谱的差异分析

目的：筛选肝肾阴虚型晚期结直肠癌（CRC）患者使用六味地黄颗粒前后的基因表达谱差异,探索六味地黄颗粒药效的基因组学机制。方法选取晚期肝肾阴虚型CRC患者以六味地黄颗粒进行干预,21 d为1个疗程。干预期间患者不接受其他西医及中医药治疗。21 d后再次提取外周血,进行基因芯片检测,并与干预前芯片结果进行自身对照分析。结果与干预前相比,干预后基因芯片检测发现129个差异基因条,其中128个上调,1个下调。基因功能（GO）富集分析结果显示,干预前后共发现254个基因GO存在显著差异（P＜0．05或P＜0．01）。在生物过程中,凝血功能相关的基因占41．5％;在细胞组成中,45．5％的差异基因与细胞质膜有关;在分子功能方面,64．9％的差异基因与结合有关。结论晚期CRC肝肾阴虚型患者应用六味地黄颗粒干预前后存在差异表达基因,功能分析显示六味地黄颗粒可增强患者凝血功能,增加钙离子结合。     

儿童期获诊的CYP17A1变异的临床及遗传学特征

【摘要】目的：细胞色素P450家族17亚家族A成员1（CYP17A1）基因变异临床罕见,多在青春期后由于严重高血压或青春期发育延迟等获诊,生存质量受到影响。该文拟探讨儿童期CYP17A1基因变异患儿的临床及遗传学特征,实现早诊断早治疗的目的。方法：回顾分析2017年～2022年在河南省儿童医院郑州儿童医院确诊的4例CYP17A1变异所致的17α-羟化酶/17,20碳链裂解酶缺陷症（17-OHD）患儿的临床表现、实验室及影像学检查结果、基因突变特点并进行文献复习。结果：4例诊断时年龄分别为1岁 - 10岁6月,均无阳性家族史,染色体核型均为46,XY。社会性别：例1为男性,余3例为女性;主诉分别为阴茎短小、腹股沟肿块、体检发现血压增高。例1外生殖器为男性,表现为阴茎短小、尿道下裂,余3例均为幼女外阴。例3、例4均为2级高血压。例1、例2、例4经彩超检查分别在阴囊、腹股沟、腹股沟内环口发现睾丸。4例患儿8点皮质醇（COR）、睾酮（T）、雄烯二酮（AD）均降低,促肾上腺皮质激素（ACTH）、孕酮（P）、促黄体生成素（LH）、卵泡刺激素（FSH）均升高,17羟孕酮（17-OHP）均正常。血钾轻度减低（3.44～3.48mmol/L）。CYP17A1基因1例纯合突变,3例复合杂合突变,其中 c.563A＞G p.(Tyr188Cys)和c.436+1G>T为既往未报道过的新突变位点,3例均存在 c.985_987delinsAA变异。均给予口服氢化可的松治疗。检索文献获得我国有8例儿童期获诊的染色体核型为46,XY的17-OHD患儿,年龄1.5岁-10岁,主诉为外生殖器异常3例、发现血压高3例、发现血钾低1例、阴唇包块1例;其中6例携带c.985_987delinsAA变异。结论：外生殖器异常、腹股沟/阴唇包块及无意中发现的高血压是46,XY 17-OHD儿童期主要表现,早期规范糖皮质激素替代治疗可有效防治17-OHD的并发症;CYP17A1 c.985_987delinsAA变异推测为我国17-OHD患儿的热点变异。     

抗凝血酶基因A9850G突变导致一个新的遗传性抗凝血酶缺陷症家系

目的对一个遗传性抗凝血酶(AT)缺陷症家系进行临床表型诊断和基因突变检测。方法用AT活性(AT:A)和抗原含量(AT:Ag)作实验诊断;用聚合酶链反应(PCR)对先证者AT基因的7个外显子及其侧翼内含子序列进行扩增,PCR产物纯化后直接测序,检测其基因突变(H is369Arg),为国际首次报道。结果先证者表现为AT基因外显子5区的第9850位有A→G的杂合突变。结论该突变是遗传性AT缺陷症的一个新的突变位点,可以导致血栓形成。     

A novel missense mutation (Asn5→Ile) in lecithin: cholesterol acyltransferase (LCAT) gene in a Japanese patient with LCAT deficiency

We identified a novel missense mutation in the lecithin: cholesterol acyltransferase gene in a new case of lecithin:cholesterol acyltransferase (LCAT) deficiency. The patient was a 64-year-old diabetic Japanese male who showed an extremely low level of serum high-density lipoprotein-cholesterol, corneal opacities, anemia, and proteinuria. Both the patient's LCAT activity and mass were markedly low. DNA sequence analysis of the LCAT gene showed an A-to-T transition at base 97 in exon 1, and predicted a change in asparagine to isoleucine at the 5th amino acid of the protein. Restriction analysis of polymerase chain reaction-amplified DNA usingAse I showed that the patient was homozygous for this mutation. Our results suggested that asparagine 5 was an important amino acid and substitution with isoleucine caused marked reduction of LCAT activity and mass, resulting in LCAT deficiency.     

女性原发性骨质疏松肾虚三证与性激素变化的关系

目的探讨女性原发性骨质疏松(POP)肾虚三证与性激素变化的关系为临床辨证施治提供客观诊断依据。方法选择116例有肾虚三证的老年女性为研究对象,其中肾气虚52例,肾阴虚36例,肾阳虚28例,另选正常对照组50例,同步检测血清雌二醇(E2)、睾酮(T)、T/E2并进行统计学分析。结果与对照组相比,肾虚三证T、T/E2显著上升(P<0.05或0.01),肾阳虚T值较肾气虚亦有显著差异(P<0.05),肾虚三证E2较对照组显著下降(P<0.01)但组间无显著差异(P>0.05),逐步判别分析结果表明:性激素水平可作为POP肾虚三证的客观诊断指标。结论(1)女性POP患者性激素T、T/E2的变化按肾气虚、肾阴虚、肾阳虚逐渐升高,E2则逐渐降低;(2)女性POP患者性激素水平变化与肾虚三证关系密切,其变化依肾气虚、肾阴虚、肾阳虚逐渐明显;(3)究提示性激素水平变化可作为判断女性POP肾虚三型的客观指标。     

云南省葡萄糖-6-磷酸脱氢酶基因突变型的初步研究

目的 分析云南人群中葡萄糖－６－磷酸脱氢酶（Ｃ６ＰＤ）缺乏症患的基因突变类型及分布特点。应用自然或错配引物介导的聚合酶链反应／限制性酶切分析方法。检测６０例云南籍Ｇ６ＰＤ缺乏症患中Ｇ１３８８Ａ、Ｇ１３７６Ｔ、Ｃ１０２４Ｔ、Ｇ３９２Ｔ、Ｃ５９２Ｔ、Ａ９５Ｇ６种Ｇ６ＰＤ基因突变类型。结果 ６０例Ｇ６ＰＤ缺乏症患中,共检出６种Ｇ６ＰＤ基因突变类型,其中Ｇ１３８８Ａ突变７例（２８．３％）、Ｇ１３７６     

一种新的IDUA基因复合杂合突变致姐弟同患Ⅰ型黏多糖贮积症

摘要：目的：分析一家系中2例黏多糖贮积症Ⅰ型（MPSⅠ）患儿α-L-艾杜糖醛酸酶（IDUA）基因突变。 方法：用PCR结合直接测序技术分析患儿IDUA基因14个外显子及侧翼序列的突变,对107例健康人行限制性酶切分析以排除新发现的突变为多态变异。 结果：2例患儿均携带IDUA基因复合杂合突变p.M1T/p.T179R,其父母分别为p.M1T突变和p.T179R突变的携带者。107例健康人未见p.T179R突变。 结论：p.M1T/p.T179R突变可能是该家系2例患儿的致病原因。