一种新的突变引起遗传性凝血因子Ⅴ缺乏症

目的 研究一个遗传性凝血因子Ⅴ(FⅤ)缺乏症家系的基因缺陷。方法 采用比浊法测定凝血酶原时间、凝血酶时间、活化的部分凝血活酶时间；采用凝血因子活性测定法(一步法)和BAELISA法对先证及其家系成员血浆FⅤ活性和抗原进行测定；采用PCR及DNA序列测定技术，对FⅤ基因组DNA中25个外显子和5′非翻译端的序列进行了：PCR扩增，PCR产物回收纯化后直接测序，并经T／A克隆测序对所发现突变进行证实。结果 先证血浆FⅤ严重缺乏，FⅤ活性为1％，FⅤ抗原为1．54％。基因研究显示为复合杂合子，其基因组DNA第4外显子675位核苷酸发生单个碱基A缺失，呈杂合状态，该致病基因来自先证母亲，与来自父方的另外一条等位基因的未知缺陷，共同导致先证血浆FⅤ严重缺乏。结论发现一种新的突变675delA，该缺失引起移码，导致转录提前终止，引起遗传性FⅤ缺乏症。     

A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans

Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional β subunits, expression of which is induced by IFN-γ, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, β type 8 (PSMB8), which encodes one of the β subunits induced by IFN-γ in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patient's tissues. In the patient's skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.     

遗传性蛋白S缺陷症一个新的基因突变

目的 研究一个遗传性蛋白S（PS）缺陷家系的遗传表型及基因型特征。方法 PS活性用凝固法测定,PS抗原用ELISA方法测定。用聚合酶链反应扩增5代家系34个成员中9个PS活性及抗原减低的PS1－15号外显子片段,用单链构象多态性（SSCP）分析cDNA变性后的差异,用测序方法检测突变点。结果 该家系5代9名成员游离PS抗原在10％－45％（正常值为55％－128％）。PS活性在13％－37％（正常值为70％－130％）,较正常参考范围明确降低,二者呈平行下降,但总PS抗原均在正常范围。在这些成员中均检测到10号外显子第163位核苷酸G→T突变,使AGT→ATT,在mRNA转录时,转录为终止密码子,阻断蛋白质的合成。结论 本家系的Ⅲ型PS缺陷症基因分析证明,先证者为杂合子型。在PS10号外显子上第163位核苷酸发生G→突变,在蛋白质合成过程中丝氨酸被终止密码子替代,为目前文献中尚未报道的一个新的基因突变点。     

1例遗传性蛋白S缺陷症患者的基因分析

目的研究一个遗传性蛋白S(PS)缺陷症家系的表型诊断及基因特征。方法PS活性(PS:A)用发色底物法测定,PS抗原(PS:Ag)用ELISA方法测定。用PCR扩增PS基因各个外显子及侧翼序列,用直接测序法检测突变点。结果先证者的PS:Ag和PS:A分别为8·3mg/L和29%,均低于正常。基因检测发现在14号外显子Gln522(CAG)→Stop(TAG)。结论本家系PS基因在14号外显子Gln522(CAG)→Stop(TAG),为国内首次报道的一个新的基因突变。     

A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)

We describe a new congenital disorder of glycosylation, CDG-If. The patient has severe psychomotor retardation, seizures, failure to thrive, dry skin and scaling with erythroderma, and impaired vision. CDG-If is caused by a defect in the gene MPDU1, the human homologue of hamster Lec35, and is the first disorder to affect the use, rather than the biosynthesis, of donor substrates for lipid-linked oligosaccharides. This leads to the synthesis of incomplete and poorly transferred precursor oligosaccharides lacking both mannose and glucose residues. The patient has a homozygous point mutation (221T-->C, L74S) in a semiconserved amino acid of MPDU1. Chinese hamster ovary Lec35 cells lack a functional Lec35 gene and synthesize truncated lipid-linked oligosaccharides similar to the patient's. They lack glucose and mannose residues donated by Glc-P-Dol and Man-P-Dol. Transfection with the normal human MPDU1 allele nearly completely restores normal glycosylation, whereas transfection with the patient's MPDU1 allele only weakly restores normal glycosylation. This work provides a new clinical picture for another CDG that may involve synthesis of multiple types of glycoconjugates.     

A mutation in the nucleoporin-107 gene causes XX gonadal dysgenesis

Ovarian development and maintenance are poorly understood; however, diseases that affect these processes can offer insights into the underlying mechanisms. XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder that is characterized by underdeveloped, dysfunctional ovaries, with subsequent lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism. Here, we report an extended consanguineous family of Palestinian origin, in which 4 females exhibited XX-GD. Using homozygosity mapping and whole-exome sequencing, we identified a recessive missense mutation in nucleoporin-107 (NUP107, c.1339G>A, p.D447N). This mutation segregated with the XX-GD phenotype and was not present in available databases or in 150 healthy ethnically matched controls. NUP107 is a component of the nuclear pore complex, and the NUP107-associated protein SEH1 is required for oogenesis in Drosophila. In Drosophila, Nup107 knockdown in somatic gonadal cells resulted in female sterility, whereas males were fully fertile. Transgenic rescue of Drosophila females bearing the Nup107^D364N mutation, which corresponds to the human NUP107 (p.D447N), resulted in almost complete sterility, with a marked reduction in progeny, morphologically aberrant eggshells, and disintegrating egg chambers, indicating defective oogenesis. These results indicate a pivotal role for NUP107 in ovarian development and suggest that nucleoporin defects may play a role in milder and more common conditions such as premature ovarian failure.     

抗凝血酶基因G13328A杂合突变导致血栓形成

目的对1个遗传性抗凝血酶（AT）缺陷症家系进行AT抗原（AT：Ag）、活性（AT：A）和基因突变检测并对该突变导致的AT结构和功能的变化进行研究。方法采用免疫比浊法和发色底物法分别检测AT：Ag和AT：A，用PCR法对先证者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。根据基因检测结果，对家系成员相应外显子进行PCR扩增，扩增产物直接测序用大引物法构建突变的AT表达质粒并瞬时转染COS-7细胞，用ELISA法检测细胞培养上清液和细胞内提取物巾AT：Ag。结果先证者的AT：Ag和AT：A分别为179mg／L和42．3％，为Ⅰ型AT缺陷；AT基因测序显示在AT外显子6区存在G13328A杂合突变，导致Ala（GCC）404→Thr（ACC）对家系成员进行的基因测序显示有3人（Ⅱ2、Ⅱ3、Ⅲ2）存在该突变。突变质粒在细胞培养上清液和细胞内的AT：Ag分别为正常人的40％和68％。结论该家系为Ⅰ型遗传性AT缺陷症；G13328A杂合突变是导致先证者血栓形成的原因．     

A novel mutation of the ceruloplasmin gene in a patient with heteroallelic ceruloplasmin gene mutation (HypoCPGM)

We found a novel missense mutation in the ceruloplasmin (Cp) gene in a patient with the heteroallelic Cp gene mutation (HypoCPGM). The patient was a 72-year-old woman who came to our hospital with a 1-year history of postural tremor of the hands. The diagnosis was made based on serum Cp and copper readings which were about half the normal levels, as well as MRI tests of her brain which showed characteristics for hereditary ceruloplasmin deficiency (HCD), known to be caused by the homoallelic Cp gene mutation. Polymerase chain reaction (PCR)-direct sequencing analysis of the Cp gene of the patient revealed a novel point mutation, A to T, at nucleotide position 82 in Exon 1. This mutation changes the Ile28 codon (ATT) to a Phe codon (TTT) (missense mutation). PCR-restriction analysis with restriction enzyme Tsp EI for the mutation revealed that both the patient and her son were heterozygotes for the mutation.     

一例凝血因子Ⅷ B区错义突变导致重型血友病A

目的：检测一例重型血友病Ａ患者（ＳＨ９）的基因突变。方法：用ＰＣＲ、变性梯度凝胶电泳（ＤＧＧＥ）和ＤＮＡ测序检测因子Ⅷ基因突变。先用Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ排除内含子２２倒位，然后应用ＰＣＲ对凝血因子Ⅷ基因进行扩增。扩增范围包括所有外显子及其侧翼内含子序列。结果：片段１４２在ＤＧＧＥ中泳动异常。ＤＮＡ测序证实Ｃ２５３５Ａ导致Ｂ区错义突变８２６Ａｓｐ（ＧＡＣ）→Ｇｌｕ（ＧＡＡ）。结论：该突变可能是导致重型血友病Ａ的原因，但有待进一步研究证实。     

不孕症患者致病基因TUBB8新型突变位点的筛查及分析

不孕症影响着世界范围内8%~12%的育龄人口[1]。40多年来,辅助生殖技术已经取得了巨大进展,但是临床上仍然有部分不孕症病例既没有找到病因也没有找到医治方法。目前,越来越多与不孕相关的基因和易感致病位点被筛选出来,如与透明带形成相关的ZP1、ZP2、ZP3[2]、与卵子死亡相关的PANX1[3]、与卵母细胞成熟相关的PATL2[4]和TUBB8[5]、与受精相关的WEE2[6]、与胚胎发育相关的TLE6[7]和PADI6[8]等。     

A novel mutation in medium chain acyl-CoA dehydrogenase causes sudden neonatal death.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder of fatty acid oxidation. Most (approximately 80%) cases are homozygous for a single mutation: A to G replacement at nucleotide 985 (A985G). MCAD deficiency typically presents in the second year of life as hypoketotic hypoglycemia associated with fasting and may progress to liver failure, coma, and death. Prompt diagnosis and management may prevent long-term sequelae. MCAD deficiency was verified by analysis of urinary acylglycine and serum acylcarnitine species from two neonates referred for diagnosis. Full-length cDNA and MCAD exon 7 and 11 genomic clones were prepared for sequence analysis. Normal and mutant cDNAs were expressed in bacteria, and enzymatic activity was assayed by the ferricenium hexaflurophosphate method. Four compound heterozygote individuals from two unrelated families with A985G on one allele and a novel G to A mutation at nucleotide 583 (G583A) as the second mutant allele presented with MCAD deficiency in the first week of life. The expressed G583A mutant protein lacks enzymatic activity. This novel mutation, G583A, is associated with severe MCAD deficiency causing hypoglycemia or sudden, unexpected neonatal death. This previously unrecognized phenotype of MCAD deficiency may contribute significantly to preventable infant deaths.     

一种新的抗凝血酶基因突变导致遗传性抗凝血酶缺陷症

目的对1例遗传性抗凝血酶（AT）缺陷症患者及其家系成员AT活性（AT：A）、AT抗原含量（AT：Ag）进行检测及基因分析，探讨该遗传性AT缺陷症发病的分子机制。方法采用发色底物法和免疫比浊法分别检测先证者及其家系成员血浆AT：A和AT：Ag，提取外周血基因组DNA，PCR法扩增AT基因的全部7个外显子及侧翼序列，DNA序列分析AT的基因异常。结果先证者AT：A和AT：Ag分别为45％和97mg／L，为Ⅰ型AT缺陷症。AT基因外显子5区第9833位核苷酸发生杂合性T→A突变，引起Tyr363Stop（Y363X）无义突变。其他家系成员基因测序结果显示有4人（Ⅱ2、Ⅱ6、Ⅲ7、Ⅲ14）存在该突变。结论该家系为I型遗传性AT缺陷症。AT基因外显子5区杂合性9833T—A无义突变引起AT缺陷，导致静脉血栓是该遗传性AT缺陷症的分子致病机制。该突变在国际上尚未见报道。     

A novel 1297-1304delGCCTGCCA mutation in the exon 10 of the thyroid hormone receptor beta gene causes resistance to thyroid hormone.

INTRODUCTION: Resistance to the thyroid hormone (RTH) is an inherited syndrome of reduced tissue responsiveness to hormonal action caused by mutations located in the ligand-binding domain and adjacent hinge region of the thyroid hormone receptor beta (TRbeta) gene. PATIENT: The patient in this study, a 42-year-old Caucasian male, came to medical attention because he experienced atrial fibrillation. Clinical evaluation showed a small and diffuse goiter and biochemical tests revealed markedly elevated concentrations of total T4, total T3, and free T4, normal thyroid-stimulating hormone (TSH) values and slightly increased I131 thyroid uptake at 24 hours. The thyroperoxidase, thyroglobulin, and TSH receptor antibodies were positive. He was treated with cabergoline plus methimazole. This treatment was stopped because of the inconsistent response, monotherapy with tri-iodothyroacetic acid (TRIAC) was then prescribed after molecular diagnosis confirmed RTH syndrome. METHODS: The exons 9 and 10 of the TRbeta gene, including splicing signals and the flanking intronic regions of each intron, were amplified with PCR. DNA sequences from each amplified fragment were performed with the Taq polymerase-based chain terminator method and using the specific TRbeta forward and reverse primers. RESULTS: Direct sequence analysis of the exons 9 and 10 of the TRbeta gene revealed an eight basepair deletion, 1297-1304delGCCTGCCA in exon 10. The mutation produces a frameshift at amino acid 433 and introduces a stop codon TGA at position 461, 85 nucleotides downstream from deletion. This alteration was not detected in either the father or mother of the patient, suggesting a de novo mutation that was confirmed by DNA fingerprint analysis. CONCLUSIONS: In the present study we have identified a novel sporadic mutation corresponding to 1297-1304delGCCTGCCA deletion in the activating function 2 (AF-2) region of TRbeta. To our knowledge, this is the first time that the presence of a partial deletion of eight nucleotides in the TRbeta has been reported.     

一个新的ACTC1基因5’端剪切位点突变可能在先天性心脏病室间隔缺损发病中起重要作用(英文)

背景:ACTC1是先天性心脏病的候选基因,且与人类先天性心脏病房间隔缺损有关。目的:对110个先天性心脏病核心家系中ACTC1基因进行突变筛查。方法:在110个先天性心脏病核心家系与300例无报道有心脏畸形的正常人之间进行对照试验。使用5对引物将ACTC1基因的6个编码区片段进行PCR体外扩增,从PCR产物中筛查基因突变。结果与结论:在ACTC1基因的第五外显子下游5’端剪切位点第3个碱基发现了1个G-A的全新的突变。这个突变存在于1个患有单纯性室间隔缺损的女孩和她30岁的没有报道过心脏畸形的父亲,且该突变没有在300例正常对照组中筛出。提示该突变可能与人类先天性心脏病室间隔缺损有关。