GBA基因复合杂合型突变导致罕见戈谢病Ⅱ型患儿1例

本研究旨在探讨戈谢病Ⅱ型的临床特点及诊断思路.先证者,女, 1岁1个月时因"营养不良,智力运动倒退"来院.患儿生后"嗓子呼噜,痰多",疑诊断"喉喘鸣"; 6个月后体重增长缓慢,被诊断"营养不良"; 9个月后智力运动倒退,接受康复训练无效; 1岁1个月时发现脾肿大、贫血、血小板减少,并出现惊厥发作,来院进行病因分析.外周血白细胞葡萄糖脑苷脂酶(GBA)活性显著减低(0.4 nmol g~(-1)min~(-1),正常对照2.63～25.6 nmol g~(-1)min~(-1)),血浆壳三糖酶活性显著升高(54000.5 nmol L~(-1)min~(-1),正常对照0～25145 nmol L~(-1)min~(-1)).靶向捕获二代测序法及Sanger测序法验证对患儿GBA基因进行分析发现已知致病的复合杂合突变, c.703TC(p.S235P)和c.1205AG(p.Y402C),两个突变分别来自父母,确诊为戈谢病Ⅱ型.患儿1岁4个月在家中"呛奶",窒息死亡.戈谢病是一种常染色体隐性遗传代谢病,Ⅱ型为罕见的类型,预后差.本文患儿隐匿起病,诊断过程曲折,经外周血白细胞溶酶体酶活性分析及基因分析才获得诊断.     

一例急性间歇性血卟啉病病例报道及文献回顾

目的:对患有急性间歇性血卟啉病先证者及其两位直系亲属进行基因突变的分析。方法:采用PCR和一代测序技术分别对患者的HMBS基因的外显子及其旁翼区进行序列分析。结果:检测出先证者HMBS基因11号外显子的旁翼区发生杂合突变c.651+2AG,为剪切突变;从先证者母亲以及女儿的HMBS基因上检测出同样的突变位点。结论:根据先证者的家族史、临床表现及相关代谢检查结果诊断为血卟啉病;基因检测结果提示先证者为急性间歇性血卟啉病;先证者的母亲和女儿存在同样的突变位点,提示先证者母亲及其女儿均患有急性间歇性血卟啉病。     

粘多糖贮积症Ⅱ型患者IDS基因的2个新突变

为了研究粘多糖贮积症II型(MPS Ⅱ)患者发病的分子遗传学机制, 采用PCR扩增艾杜糖-2-硫酸酯酶(IDS)基因突变热点区(外显子2、3、5、7、8和9)、DNA测序分析和限制性内切酶图谱分析的方法, 对2个粘多糖贮积症Ⅱ型家系进行了遗传突变分析。结果表明, 2个家系患者的IDS 基因分别出现IVS 6-1g→a和c.1587~1588 ins T 2个新突变。前者属于单碱基替换, 位于内含子6的3′端剪接位点, 导致跨外显子剪接; 后者属于插入突变, 插入点位于外显子9的cDNA 1,587和1,588碱基之间, 是迄今为止报道的人类IDS基因插入突变中最接近肽链末端的突变, 导致移码突变和转录提前终止。经限制性酶切分析, 证实2个家系中的患者母亲是突变基因的携带者, 符合该病X染色体隐性遗传的规律。另外,在对随机抽取的50名正常人及另外6名不相关的粘多糖病人的测序分析中, 未检测到这2个突变, 说明不是多态性。对于筛查所得的2个新突变是否是患者的致病原因, 尚需进一步证实。     

中国广东Hunter综合征患者的T1140C新突变

应用尿黏多糖含量检测、干血滤纸片直接扩增、PCR产物直接测序法对患者及其父母等的IDS基因的突变热点exons9,3,8进行突变检测。发现患儿IDS基因的exon8发生一新的错义突变,突变部位在第339位密码子（CTA）内,即cDNA第1140bp的T突变为C,导致原339位的“亮氨酸CTA”突变为“脯氨酸CCA”。该患儿为这一突变的半合子,而其母为这一突变的杂合子。该错义突变改变了IDS酶的一级结构和三级空间结构,从而可能引起IDS酶活性大大降低,这可能是该Hunter综合征患者的真正致病原因。     

一个蚕豆病遗传家系G6PD基因突变及X染色体失活偏移检测

对一蚕豆病遗传家系的G6PD基因突变进行分析,检测突变后G6PD酶活变化,并对先证者家系进行X染色体失活（XCI）偏移模式检测,从而预测G6PD突变女性携带者患蚕豆病的风险。取家系成员的外周血样,并提取基因组DNA,用聚合酶链式反应（PCR）和DNA测序法进行序列分析,确定先证者突变位点和突变类型及家庭成员遗传情况,若先证者的母亲和姐姐为G6PD突变携带者,则对先证者母亲和姐姐进行X染色体偏移检测以及酶活检测分析,以评估携带者患蚕豆病的风险,同时对研究对象进行随访。结果患者X染色体上G6PD基因发生点突变c.1376G>T;酶活性检测结果显示该突变使G6PD酶活性下降大约25%,导致蚕豆病发生。该家系的两位女性携带者X染色体失活偏移<80%,未来发生蚕豆病的可能性低。     

NOTCH3基因c.520T>G突变致CADASIL一家系报告及文献复习

目的:研究探讨一CADASIL家系的临床特征及基因突变情况。方法:收集同一家系中3例CADASIL患者的临床资料,并对3例患者及先证者之兄进行全外显子测序(Whole Exome Sequencing, WES)。结果:该家系中3例患者临床表现多样,女性患者均有头痛病史,先证者及先证者之姐中年起病,先证者临床表现缺乏特异性,主要表现为头昏,认知功能检查正常,心理评估示轻度焦虑抑郁状态。先证者之姐主要表现为假性球麻痹及锥体束受损,认知功能检查示重度痴呆。先证者之女自4岁起诊断为癫痫-失神发作,认知功能检查示轻度认知功能障碍。影像学显示该家系3例患者均有脑白质病变,且随着年龄增大呈进行性发展,WES显示3例患者均存在NOTCH3基因第4外显子区域杂合突变:c.520T>G,导致氨基酸改变p.Cys174Gly。结论:NOTCH3基因c.520T>G所致该家系的临床表现具有多样性,且该家系中下一代起病较早,临床表现可与父代具有较大异质性,影像学表现可在青少年时期出现,并呈现进行加重的趋势。WES显示该家系中NOTCH3基因突变为第4外显子的杂合突变,该位点突变致CADASIL为国内首次报道。     

脆性X综合征的基因诊断与产前诊断

为了探讨简便、快速、准确、价廉的脆性X综合征的诊断方法,对6个智能低下家系进行了细胞遗传学检查,以及PCR直接扩增FMR1 5^'端(CGG)_{n<\sub>重复序列、RT-PCR扩增FMR1基因的cDNA序列的分子遗传学检查。A家系先证者脆性X染色体高表达（35/273）,分子遗传学检查证实为脆性X综合征全突变患者;B家系先证者及其母亲无脆性X染色体表达,分子遗传学检查证实为非脆性X综合征患者;C家系的男性胎儿脆性X染色体表达（5/93）,先证者及其母亲未发现脆性X染色体,分子遗传学检查证实男性胎儿为脆性X综合征全突变患者,其母亲为前突变携带者,哥哥为嵌合体患者;D家系先证者脆性X染色体高表达17%,其姐姐脆性X染色体5%,分子遗传学检查证实先证者为脆性X综合征全突变患者,其姐姐为嵌合体患者;E家系先证者及其母亲,F家系先证者发现可疑脆性X染色体,分子遗传学检查证实为非脆性X综合征家系。结论： PCR直接扩增FMR1基因(CGG)n<\sub>重复序列联合RT-PCR扩增FMR1基因cDNA 序列简便、快速、价廉。可用于脆性X综合征的筛查、诊断及产前诊断,有推广应用价值。}     

LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症患者的突变分析

为了分析LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT)的突变特点, 文章分别应用PCR结合DNA序列分析方法和PCR-单链构象多态性(PCR-SSCP)结合DNA序列分析方法对6个常染色体显性遗传家系先证者和27个散发病例进行LITAF和RAB7基因突变分析; 应用PCR-SSCP结合DNA序列分析方法对14个常染色体遗传的CMT家系先证者和27个散发患者进行LMNA和MTMR2基因突变分析。结果发现: LITAF基因c.269G→A、c.274A→G序列变异和LMNA基因c.1243G→A、c.1910C→T序列变异, 未发现RAB7和MTMR2基因的序列变异。其中LITAF基因c.269G→A、LMNA基因c.1243G→A和c.1910C→T为新发现的单核苷酸多态; LITAF基因c.274A→G为已知多态。说明LITAF、RAB7、LMNA和MTMR2基因突变在中国人CMT患者中罕见。     

静原鸡ELOVL2基因多态性及其生物信息学分析

极长链脂肪酸延长酶(elongation of very-long-chain fatty acids, ELOVLs)是脂肪酸合成过程中的关键限速酶,在脂类的生物合成及脂肪酸代谢等调控方面作用显著。为研究宁夏地方优良品种静原鸡ELOVL2基因多态性及其编码蛋白质的结构与功能,以原鸡ELOVL2基因序列作为参考序列,针对ELOVL2基因的8个外显子序列设计特异引物并扩增,经DNA混合池测序筛选出存在突变位点的外显子序列,运用PCR-SSCP技术检测其多态性,经序列拼接后对ELOVL2 CDS区序列进行功能生物信息学分析。研究结果表明,静原鸡ELOVL2基因仅exon7和exon8发生单碱基突变(exon7为c.708 G>A, exon8为c.888 T>C),均未引起氨基酸的改变,属同义突变。在exon7和exon8扩增片段中分别检测出3种(AA, AG和GG)、2种(CC和TC)基因型。遗传特性分析显示,E2e7呈中度多态(0.250.05)。生物信息学分析表明,ELOVL2 CDS序列全长为894 bp,共编码297个氨基酸。其原子组成为C1638H2444N394O406S15,分子量为34.63 kD,等电点(pI)为9.40;存在7个跨膜结构,21个磷酸化位点,无信号肽序列,为稳定的水溶性蛋白;ELOVL2基因在进化中高度保守。该研究结果将为进一步研究ELOVL2基因在静原鸡肉质方面的作用及功能提供参考。     

野油菜黄单胞菌中3-羟基脂酰ACP脱水酶功能研究

细菌采用II型脂肪酸系统合成脂肪酸,其中3-羟脂酰ACP脱水酶催化唯一的脱水反应,是细菌生长的关键酶之一.野油菜黄单胞菌(Xcc)引起几乎所有十字花科植物的黑腐病,在全球范围内造成广泛的经济损失.为研究Xcc中3-羟脂酰ACP脱水酶,本研究利用大肠杆菌3-羟脂酰ACP脱水酶FabZ序列同源比对时,发现其与XC₂876 (XcfabZ)编码蛋白具有同源性,序列一致性达到46.1%,同时还具有保守的α螺旋结构和活性位点.将XcfabZ异体遗传互补大肠杆菌fabZ(EcfabZ)条件突变株HW7,结果显示添加IPTG能恢复突变株的生长,初步表明XcFabZ具有3-羟脂酰ACP脱水酶活性.而体外活性分析显示,XcFabZ能在脂肪酸合成的起始反应和延伸反应中发挥3-羟脂酰ACP脱水酶活性作用.本研究不能直接获得XcfabZ基因敲除突变株,但将携带EcfabZ或XcfabZ的表达质粒导入后,获得基因替换突变株,证明XcfabZ是必需基因. EcfabZ替换突变株的脂肪酸组成与野生菌有差异,对逆境条件(高盐、低pH、H₂O₂和SDS)...     

Molecular genetic defect underlying alpha-L-iduronidase pseudodeficiency.

Mucopolysaccharidosis type I (i.e., Hurler, Hurler-Scheie, and Scheie syndromes) and type II (i.e., Hunter syndrome) are lysosomal storage disorders resulting from alpha-L-iduronidase (IDUA) deficiency and iduronate-2-sulfatase (IDS) deficiency, respectively. The a priori probability that both disorders would occur in a single individual is approximately 1 in 5 billion. Nevertheless, such a proband was referred for whom clinical findings (i.e., a male with characteristic facies, dysostosis multiplex, and mental retardation) and biochemical tests indicated these concomitant diagnoses. In repeated studies, leukocyte 4 methylumbelliferyl-alpha-L-iduronidase activities in this kindred were as follows: <1.0 nmol/mg protein/h in the proband and proband's clinically normal sister; 45.3 in mother; and 45.7 in father (normal range 65.0-140). Leukocyte L-O-(alpha-iduronate-2-sulfate)-(1->4)-D-O-2,5-anhydro[1-3H]mannitol-6- sulfate activities were as follows: 0.0 U/mg protein/h in the proband; 5.7 in his sister; 4.9 in mother; and 15.0 in father (normal range 11.0-18.4). Multiple techniques, including automated sequencing of the entire IDS and IDUA coding regions, were employed to unravel the molecular genetic basis of these intriguing observations. The common IDS mutation R468W was identified in the proband, his mother, and his sister, thus explaining their biochemical phenotypes. Additionally, the proband, his sister, and his father were found to be heterozygous for a common IDUA mutation, W402X. Notably, a new IDUA mutation A300T was also identified in the proband, his sister, and his mother, accounting for reduced IDUA activity in these individuals; the asymptomatic sister, whose cells demonstrated normal glycosaminoglycan metabolism, is thus a compound heterozygote for W402X and the new allele. This A300T mutation is the first IDUA pseudodeficiency gene to be elucidated at the molecular level.     

Detection of a new mutation (T1140C) in a patient with Hunter syndrome from Guangdong, China

This study identified mutations of the idurnate-2-sulfatase (IDS) gene in a patient with Hunter syndrome, and established a basis for the diagnosis of the prenatal gene of Hunter syndrome. Urine glyeosaminoglycan (GAG) assay was used to make the preliminary diagnosis of mucopolysaccharidosis type II. Polymerase chain reaction (PCR) from dried blood spots and DNA sequencing were applied to analyze hotspot mutations in exons 9,3 and 8 of the IDS gene in the proband and his parents. A new missense mutation (T1140C) in exon 8 of the IDS gene was found by using DNA sequencing. This mutation caused a substitution of codon 339 from CTA (leucine) to CCA (praline). The patient is a hemizygote, and his mother is a heterozygote. The new missense mutation results in a change in the primary and tertiary structure of the IDS protein. It is possible that this mutation severely impairs enzymatic activity and is the underlying basis for the pathology seen in this patient with Hunter syndrome. __________ Translated from Hereditas, 2006, 28(5): 521–524 [译自: 遗传]     

Detection of a new mutation (T1140C) in a patient with Hunter syndrome from Guangdong,China

This study identified mutations of the idumate-2-suffatase (IDS) gene in a patient with Hunter syndrome,and established a basis for the diagnosis of the prenatal gene of Hunter syndrome.Urine glyeosaminoglycan (GAG) assay was used to make the preliminary diagnosis of mucopolysaccharidosis type H.Polymerase chain reaction (PCR) from dried blood spots and DNA sequencing were applied to analyze hotspot mutations in exons 9,3 and 8 of the IDS gene in the proband and his parents.A new missense mutation (T1140C) in exon 8 of the IDS gene was found by using DNA sequencing.This mutation caused a substitution of codon 339 from CTA (leucine) to CCA (praline).The patient is a hemizygote,and his mother is a heterozygote.The new missense mutation results in a change in the primary and tertiary structure of the IDS protein.It is possible that this mutation severely impairs enzymatic activity and is the underlying basis for the pathology seen in this patient with Hunter syndrome.     

Expression studies of mutations underlying Taiwanese Hunter syndrome (mucopolysaccharidosis type II)

Nearly 300 different mutations underlying mucopolysaccharidosis type II (MPS II) have been identified worldwide. To investigate the molecular lesions underlying Taiwanese MPS II, probands and families were identified and screened for iduronate-2-sulfatase (IDS) mutation by single-strand conformation polymorphism and DNA sequencing. Five novel and five previously reported mutations were found. Together with those previously reported, a total of 17 identified missense, small deletion, and nonsense mutations were further characterized by transient expression studies. Transfection of COS-7 cells by the mutated cDNA did not yield active enzyme, demonstrating the deleterious nature of the mutations. A 57% decrease in IDS mRNA level was seen with the 231del6 mutation. Among the 11 missense mutations examined, K347E substitution showed apparent normal maturation and targeting on immunoblot and confocal fluorescence microscopy examination. The other 10 missense mutations showed apparent normal precursor with little or reduced mature forms, indicating normal maturation but incorrect targeting of the mutant enzymes. Among the six deletion and nonsense mutations examined, 1055del12 and E521X showed abnormal maturation. The staining pattern of the truncated W267X and 1184delG proteins suggested retention within early vacuolar compartments. The mutated 231del6 and 1421delAG proteins were unstable and largely degraded. Molecular analysis of the IDS gene will clearly identify the cause of the disease within patients and allow antenatal and family studies. The further characterization of gene mutations may delineate their functional consequences on IDS activity and processing and may enable future studies of genotype–phenotype correlation to estimate a prognosis and to lead to possible therapeutic interventions.Jui-Hung Chang and Shuan-Pei Lin contributed equally to this work     

A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction. The proband was homozygous for this mutation; his mother and a brother are heterozygous. The same mutation was found in a second, apparently unrelated, black GM2-gangliosidosis patient. The second patient was a compound heterozygote, as only one allele carried this mutation. The mother and a brother in this second family are carriers for this mutation, while the father and a noncarrier sister are normal for this region of the gene. The third proband did not have this mutation; nor did the mother of a fourth black proband. Eight other independently ascertained non-black, non-Jewish, GM2-gangliosidosis families did not have this mutation. The observation of the same novel mutation in two unrelated black GM2-gangliosidosis patients indicates that the American black population has segregating within it at least one GM2-gangliosidosis mutation which may be specific to this population and not a result of migration.     

Two sisters with Rett syndrome and non-identical paternally-derived microdeletions in the MECP2 gene

The unique case of two sisters with symptoms of RTT and two quite distinct, novel, and apparently de novo microdeletions of the MECP2 gene is described. One sister possessed an 18 base-pair (bp) deletion (c.1155_1172del18) within the deletion hotspot region of exon 4, whereas the other sister exhibited a 43 bp deletion at a different location in the same exon (c.1448_1461del14+29). Although these lesions occurred on the same paternally-derived X chromosome, this is probably due to chance co-occurrence owing to the relatively high mutation rate of the MECP2 gene rather than to a constitutional mutator phenotype.     

A newly identified lipoprotein lipase (LPL) gene mutation (F270L) in a Japanese patient with familial LPL deficiency

We have systematically investigated the molecular defects resulting in a primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (proband SH) with fasting hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in pre- or postheparin plasma from proband SH. DNA sequence analysis of the LPL gene of proband SH revealed homozygosity for a novel missense mutation of F270L (Phe(270)-->Leu/TTT(1065)-->TTG) in exon 6. The function of the mutant F270L LPL was determined by both biochemical and immunocytochemical studies. In vitro expression experiments on the mutant F270L LPL cDNA in COS-1 cells demonstrated that the mutant LPL protein was synthesized as a catalytically inactive form and its total amount was almost equal to that of the normal LPL. Moreover, the synthesized mutant LPL was non-releasable by heparin because the intracellular transport of the mutant LPL to the cell surface - by which normal LPL becomes heparin-releasable - was impaired due to the abnormal structure of the mutant LPL protein. These findings explain the failure to detect LPL activities and masses in pre- and postheparin plasma of the proband. The mutant F270L allele generated an XcmI restriction enzyme site in exon 6 of the LPL gene. The carrier status of F270L in the proband's family members was examined by digestion with XcmI. The proband was ascertained to be homozygous for the F270L mutation and his parents and sister were all heterozygous. The LPL activities and masses of the parents and the sister (carriers) were half or less than half of the control values. Regarding the phenotype of the carriers, the mother with a sign of hyperinsulinemia manifested hypertriglyceridemia (type IV hyperlipoproteinemia), whereas the healthy father and the sister were normolipidemic. Hyperinsulinemia may be a strong determinant of hypertriglyceridemia in subjects with heterozygous LPL deficiency.