一个先天性肾性尿崩症家系精氨酸血管升压素受体2基因的突变检测

采集临床诊断为X-连锁肾性尿崩症的一家系3例患者及其12名亲属的血液样本，抽提基因组DNA，通过PCR扩增精氨酸血管升压素2型受体（arginine vasopressin receptor 2，AVPR2）基因的全部编码区，并直接测序。在3例患者中发现AVPR2基因的突变：g1236T→C（L292P），他们的母亲在该位点均为杂合突变。     

TRβ基因突变致甲状腺激素抵抗综合征

目的 研究一甲状腺激素抵抗综合征家系的甲状腺激素受体β( thyroid hormone reecptorβ)的基因突变情况.方法 提取患者及其家系5名成员的外周血基因组DNA,PGR分段扩增TRβ基因1-10号外显子,产物直接进行DNA测序检测突变位点.结果 测序结果显示,该家系中有2名成员TRβ基因第10外显子1642位核苷酸发生C转换为A的错义突变,使该位点所编码的氨基酸由脯氨酸变为苏氨酸(P453T),此种突变为杂合子突变.结论 经基因测序检测诊断证实患者TRβ基因第10外显子存在P453T突变,该突变可能导致了甲状腺激素抵抗综合征发生.     

FGFR3基因G1138A突变导致软骨发育不全

目的对一例临床诊断为软骨发育不全(ACH)的患者及其父母的成纤维细胞生长因子受体3(FGFR3)进行基因突变检测。方法提取患者及其家庭成员外周血DNA,对FGFR3基因的全部编码氨基酸外显子及相邻内含子区设计引物,进行PCR扩增,并对扩增产物进行测序。结果 FGFR3基因第10外显子发生了cDNA第1138位G到A的转换,使得其所编码蛋白FGR3的第380位氨基酸由甘氨酸变为精氨酸。结论 FGFR3基因的G1138A杂合突变为该患者发病的原因,进一步说明了该突变为热点突变。     

高胆固醇血症低密度脂蛋白受体基因点突变的研究

目的：建立低密度脂蛋白－受体（ＬＤＬ－Ｒ）基因点突变监测方法,从基因水平对３０例原发性高胆固醇血症患者进行筛选、明确诊断。 方法：将聚合酶链式反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）与银染技术相结合,用１９对引物对ＬＤＬ－Ｒ基因的全部１８个外显子进行检测,３０例患者中,对筛查出的突变用ＰＣＲ产物直接测序或克隆测序的方法明确突变的性质和位置。 结果：确立的ＰＣＲ－ＳＳＣＰ条件稳定,银染方法灵敏度高、重复性好,且避免了同位素污染。３０例患者中,发现１例定位于外显子１４的杂合子点突变患者,ＰＣＲ产物直接测序证实第６７１位密码子发生错义突变：ＣＣＣ→ＣＧＣ,导致脯氨酸→精氨酸;１例纯合子家族性高胆固醇血症患者外显子７发生点突变,克隆测序证实密码子３０８位发生错义突变：ＴＡＣ→ＴＧＣ,导致半胱氨酸→酪氨酸;２例杂合子患者外显子４的３’部分ＳＳＣＰ出现相同异常带型。查阅文献,测序证实的突变皆是新的突变。 结论：建立的ＰＣＲ－ＳＳＣＰ方法可用于高胆固醇血症患者ＬＤＬＲ基因点突变的筛检。研究结果从基因水平证实中国家族性高胆固醇血症患者ＬＤＬ－Ｒ基因突变具有多样性的特点。     

雄激素受体基因新突变致雄激素不敏感综合征

目的 分析2例雄激素不敏感综合征患者及其家系的临床及分子遗传学.方法 收集2例雄激素小敏感综合征患者的临床资料,从患者及其家系成员的外周血单个核细胞抽提基因组DNA,应用PCR扩增雄激素受体基因并直接测序,明确患者及其父母基因有无突变.结果 患者1表现为女性外生殖器、单侧乳房发育、原发性闭经、阴毛腋毛缺如.患者2表现为男性化不全,体毛稀少、双侧乳房发育、尿道下裂.基因检测证实患者1雄激素受体基因第2号外显子第579位密码子点突变(S579N),并证实为一新突变.患者2第5号外显子第747位密码子点突变(V747M).结论 该2例雄激素受体不敏感综合征系分别由雄激素受体基因S579N及V747M所致,其中S579N突变尚未见文献报道.     

罕见的CYP17A1基因外显子剪接突变导致17α-羟化酶缺陷的分子病因学研究

目的 对一例46XY性发育异常的患者进行CYP17A1致病基因分析,并探讨新的突变对患者表型的影响.方法 对患者及其父母的CYP17A1基因的8个外显子进行PCR扩增,扩增产物直接测序.将野生型和含有新突变位点的突变型PCR片段连入表达载体,构建Mini-gene系统,转染HEK-293T细胞,RT-PCR观察新突变位点对剪切的影响.进一步构建野生型和剪切异常的CYP17A1 cDNA全长表达质粒,转染HEK-293T细胞,体外检测CYP17A1酶活性.结果 基因分析显示患者的CYP17A1基因为复合杂合突变,其中一个等位基因为外显子6中c.985_987delinsAA突变,另一等位基因含有新的同义突变(c.1263 G＞A:GCG＞GCA).体外研究表明,这种同义突变会产生新的剪切位点,导致CYP17A1基因mRNA异常剪切,剪切产物中CYP17A1的415位氨基酸残基后缺失6或7个氨基酸.体外转染和酶活性检测证实异常剪切产物使酶活性丧失;但此突变对剪切的影响并不是完全的,在患者体内还应存在部分正常的剪切产物,发挥残余酶活性,与患者的表型相一致.结论 本研究首次报道了由于CYP17A1基因外显子剪切突变导致的17α-羟化酶缺乏的患者,并且启动对异常剪切体的功能研究.     

中国人低血钾性周期性麻痹家系:SCN4A新突变位点

目的　通过检测相关基因的突变位点来研究低钾性周期性麻痹 (HOKPP)这一常染色体显性遗传疾病的遗传学病因。方法　运用PCR扩增及反应产物直接测序的方法 ,对一个中国人低钾周麻家系 3 2名成员进行基因筛查 ,并运用亚克隆方法进行证实。结果　在编码Na通道的基因SCN 4A上第 2 0 14位核苷酸存在一个新点突变 (CGT TGT) ,为杂合突变 ,并引起相应编码氨基酸的改变 ,由原来的精氨酸变为半胱氨酸 (R672C) ,此突变为HOKPP一个新的突变类型 ,并且经亚克隆的方法进一步证实了此杂合突变。结论　在一中国人HOKPP家系的SCN4A 12号外显子上发现了新的点突变 (C2 0 14T ) ,它只在HOKPP患者中存在。家系中各患者的表现各不相同 ,提示了R672C突变型可能具有不完全外显的特性。     

心脏肌球蛋白结合蛋白C基因S236G新突变致肥厚型心肌病表型研究

目的 研究中国人肥厚型心肌病的致病基因突变位点,寻找国人特有的热点突变并分析基因型与临床表型的相互关系.方法 在100例肥厚型心肌病患者以及120名健康对照者中进行心脏型肌球蛋白结合蛋白C(MYBPC3)基因突变筛查,聚合酶链式反应(PCR)扩增基因功能区外显子片段并对PCR产物进行测序分析.结果 在3例肥厚型心肌病患者中发现MYBPC3基因第6号外显子第706位碱基由T转换为C,结果导致第236位的丝氨酸(Ser,s)转变为甘氨酸(Gly,G),正常对照组相同位置未发现异常.该突变在西方人中未见报道,携带该突变的肥厚型心肌病患者呈现不同的临床表型.结论 首次在中国人肥厚型心肌病患者中发现MYBPC3基因S236G突变,其在中国人肥厚型心肌病患者中占有一定的比例,是热点突变之一.     

甲状腺激素受体β基因H435Y突变所致甲状腺激素抵抗综合征:一家系研究

[提要]对一个包括先证者在内共28位成员的甲状腺激素抵抗综合征家系进行临床调查,并提取先证者及其家系14位成员(其他13位成员拒绝抽血)外周血白细胞基因组DNA,对甲状腺激素受体(thyroid hormone receptor,TR)β基因的第1～ 10外显子进行PCR扩增,PCR产物进行DNA测序检测突变位点.测序结果显示,该家系中有3名成员TRβ第10外显子1 303位核苷酸发生C转换为T的错义突变,使该位点编码的氨基酸由组氨酸变为酪氨酸(H435Y),此突变为杂合子突变,可能导致了甲状腺激素抵抗综合征的发生.     

感染性心内膜炎链球菌耐药基因检测与分析

目的通过分析感染性心内膜炎链球菌的耐药基因,以便有效控制链球菌耐药性的进一步发展,从而为感染性心内膜炎的治疗提供依据。方法从感染性心内膜炎患者血培养标本中分离链球菌,提取DNA,采用PCR方法检测耐药基因,并作测序分析。结果 PCR检测链球菌9种耐药基因,其中gyrA、parC、tetM基因扩增均阳性,且同源性较高。所有菌株的tetM基因同源性均为100%。有3株发生突变,突变位点分别为81位、87位和79位,且突变株均对氟喹诺酮类抗菌药物耐药。链球菌耐药基因编码氨基酸序列比对图显示,gyrA第81位由丝氨酸(Ser)突变为精氨酸(Arg),parC第87位由精氨酸(Arg)变异为亮氨酸(Leu),parC第79位由丝氨酸(Ser)突变为苯丙氨酸(Phe),因而可能导致耐药性的产生。结论分离自感染性心内膜炎患者的链球菌含有gyrA、parC、tetM基因,这3种基因可能与链球菌的耐药性有关。     

Identification and characterization of a novel X-linked AVPR2 mutation causing partial nephrogenic diabetes insipidus: a case report and review of the literature

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease characterized by a malfunctioning renal response to the antidiuretic hormone arginine vasopressin (AVP) due to mutations in the AVPR2 gene. A limited number of mutations in the AVPR2 gene resulting in partial phenotype have been described so far. In this mini-review the retrospective analysis of 13 known AVPR2 mutations that have been previously shown in vitro to partially abolish AVPR2 function is described, along with a novel mutation diagnosed in a kindred with partial NDI. In the present study, a 14 year old male and his 73 year old maternal grandfather were diagnosed with partial NDI based on the clinical phenotype, the water deprivation test and the inadequate response to 1-desamino-8-d-arginine vasopressin (DDAVP) administration. Sequencing analysis of the AVPR2 gene revealed the novel missense mutation p.N317S (g.1417A > G) in both patients. This mutation was re-created by site directed mutagenesis in an AVPR2 cDNA expression vector and was functionally characterized, in terms of arginine vasopressin (AVP) and DDAVP response. AVPR2 activity of the p.N317S mutant receptor after the AVP and DDAVP administration, as assessed by cAMP production was reduced and impaired when compared to cells that expressed the wild type AVPR2 gene. In conclusion, the affected members of this family have X-linked NDI with partial resistance to AVP, due to a missense mutation in the AVPR2 gene.     

Partial nephrogenic diabetes insipidus caused by a novel mutation in the AVPR2 gene

Objective To identify the molecular basis and clinical characteristics of X‐linked congenital nephrogenic diabetes insipidus (CNDI) presenting with an unusual phenotype characterized by partial resistance to AVP. Subjects The proband was admitted at the age of 4 years with a history of polydipsia and polyuria since infancy. Initial clinical testing confirmed a diagnosis of diabetes insipidus (DI). Urine osmolarity rose during fluid deprivation and after 20 µg of intranasal desmopressin [1‐deamino‐8‐d ‐arginine‐vasopressin (dDAVP)]. A similar DI phenotype was found in his brother. Methods The coding regions of the AVP gene and the AVP receptor 2 (AVPR2) genes were sequenced in two affected and three unaffected family members. Clinical studies included a fluid deprivation test, intranasal dDAVP challenge, infusion of graded doses of dDAVP and AVP, and measurements of 24‐h urine output before and at the end of a 7‐day therapeutic trial of intranasal dDAVP. Results A novel missense mutation (1454C > A) in exon 3 of the AVPR2 gene predicting a Ser329Arg substitution was identified in the X‐chromosome of the two affected brothers and in one of the X‐chromosomes in the mother. The AVPR2 gene was normal in two unaffected siblings. Under basal conditions, the 24‐h urine volumes of the two affected boys were 5·5 l (229 ml/kg) and 3·5 l (192 ml/kg), the urine osmolalities were 78 and 90 mosm/kg, and plasma AVP 13·5 and 19·0 pg/ml. Urine osmolalities increased to 573 and 720 mosm/kg while plasma AVP levels were practically unchanged, 13·6 and 8·8 pg/ml, during fluid deprivation. Infusion of AVP resulted in urine osmolalities of 523 and 623 mosm/kg at plasma AVP levels of 58 and 42 pg/ml. Infusion of dDAVP had a similar effect, while treatment with standard doses of intranasal dDAVP had no effect on urine output. Discussion The affected members of this Belgian kindred have CNDI with partial resistance to AVP caused by a mutation in the AVPR2 gene that differs from any of the six mutations reported previously to produce this phenotype. Because the resistance to AVP is partial, this form of CNDI can be difficult to distinguish by indirect diagnostic tests from partial pituitary and dipsogenic DI.     

Polyuria and polydipsia in a young child: diagnostic considerations and identification of novel mutation causing familial neurohypophyseal diabetes insipidus

A 3-year 5-month-old boy was seen for second opinion regarding polydipsia and polyuria. Previously, a diagnosis of primary polydipsia was made after normal urine concentration after overnight water deprivation testing. The boy’s father, paternal grandfather, and paternal aunt had diabetes insipidus treated with desmopressin acetate. Based on this young boy’s symptoms, ability to concentrate urine after informal overnight water deprivation, and family history of diabetes insipidus, we performed AVP gene mutation testing. Analysis of the AVP gene revealed a novel mutation G54E that changes a normal glycine to glutamic acid, caused by a guanine to adenine change at nucleotide g.1537 (exon 2) of the AVP gene. Commonly, patients with familial neurohypophyseal diabetes insipidus (FNHDI) present within the first 6 years of life with progressively worsening polyuria and compensatory polydipsia. Since these patients have progressive loss of arginine vasopressin (AVP), they may initially respond normally to water deprivation testing and have normal pituitary findings on brain MRI. Genetic testing may be helpful in these patients, as well as preemptively diagnosing those with a mutation, thereby avoiding unnecessary surveillance of those unaffected.     

An alternative strategy for identification of type IIA VWD mutations

Summary. A 306 base pair segment of the von Willebrands factor (VWF) gene between nucleotides 4627 and 4932 was amplified by PCR using DNA from a type IIA VWD patient. The amplified DNA was cloned in the plasmid pCRII. Clones of the VWF gene and pseudogene were distinguished by their KpnI restriction patterns. A series of six VWF gene clones was sequenced. A single C to T point mutation at nucleotide 4789 resulting in the substitution of arginine 834 by tryptophan was identified in four clones. This mutation, which destroys a BstEII restriction site, was also detected in amplified DNA of affected relatives of the patient. The method described avoids difficulties associated with strategies employed previously for identification of VWD mutations.     

A new mutation of the arginine vasopressin-neurophysin II gene in a family with autosomal dominant neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomally dominant inherited disorder with a typical onset at one to six years of age. The genetic locus of FNDI is the arginine vasopressin-neurophysin II (AVP-NPII) gene. The gene encoding the precursor hormone (prepro-AVP-neurophysin II) is located in the chromosomal region 20p13 and contains three exons. Mutations that cause FNDI have been found to occur within the signal peptide of the prepro-AVP-neurophysin II precursor, within the coding sequence for neurophysin II and the vasopressin-coding sequence. A family (four members with FNDI, two without FNDI) in three consecutive generations was investigated. Index case was a now 22-year old man with a history of severe polyuria (18 L/day) and polydipsia first recognized at about 4-5 months of age. The arginine vasopressin-neurophysin II gene was investigated by direct sequencing of PCR products amplified from each exon. Subsequently, a restriction analysis was performed to verify the sequencing results. The affected individuals were found to have a missense mutation in exon 2 at nucleotide position 1887 (G to C) of the AVP-NPII gene. Using both restriction enzyme digestion and sequence analysis, the mutation was found in all affected family members, but not in the unaffected members studied. This mutation (1887 G to C) represents a novel mutation of the AVP-NPII gene.     

Functional characterization of the molecular defects causing nephrogenic diabetes insipidus in eight families

X-Linked nephrogenic diabetes insipidus (NDI) is a rare inherited disorder characterized by the excretion of abnormal large volumes of diluted urine mainly caused by mutations in the V2 vasopressin receptor (AVPR2) gene. By screening NDI patients for mutations within the AVPR2 gene we have identified three novel (I46K, F105V, I130F) and four recurrent (D85N, R106C, R113W, Q225X) mutations. In addition, a recurrent missense mutation (A147T) within the aquaporin-2 gene was identified in a female patient with autosomal recessive NDI associated with sensorineural deafness. Selected clinical data of the NDI patients were compared with the results from the in vitro studies. Functional analysis of I46K and I130F revealed reduced maximum agonist-induced cAMP responses as a result of an improper cell surface targeting. In contrast, the F105V mutation is delivered to the cell surface and displayed an unchanged maximum cAMP response, but impaired ligand binding abilities of F105V were reflected in a shifted concentration-response curve toward higher vasopressin concentrations. As the extracellularly located F105 is highly conserved among the vasopressin/oxytocin receptor family, functional analysis of this residue implicates an important role in high affinity agonist binding.     

Germline FAS gene mutation in a case of ALPS and NLP Hodgkin lymphoma

FAS germline mutations have been associated with the development of autoimmune lymphoproliferative syndrome (ALPS). Occurrence of Hodgkin lymphoma (HL) has been reported in 2 families with ALPS. In both families an uncle of the index patient developed HL. A 15-year-old boy with autoimmune thrombopenia, lymphadenopathy, and splenomegaly for 6 years was studied. In an axillary lymph node biopsy nodular lymphocyte predominant (NLP) HL was diagnosed; in the areas between the nodules a proliferation of double-negative blastic T cells were present, suggestive of ALPS. Analysis for the presence of a FAS gene mutation using the denaturing gradient gel electrophoresis technique indicated a mutation in exon 9. Direct sequence analysis revealed a mutation causing a substitution of arginine with glutamine at codon 234. Because ALPS and NLP HL are both highly infrequent conditions, the occurrence in at least 3 families suggests a causative relationship between germline FAS gene mutations and NLP HL.     

Two unrelated patients with familial hyperproinsulinemia due to a mutation substituting histidine for arginine at position 65 in the proinsulin molecule: identification of the mutation by direct sequencing of genomic deoxyribonucleic acid amplified by polymerase chain reaction

Mutations in the insulin gene can impair the bioactivity of the insulin molecule. Previously, two classes of mutations have been identified: 1) those that impair posttranslational processing of proinsulin to insulin, and 2) those that alter the structure of the insulin molecule, thereby reducing the affinity of the molecule for the insulin receptor. We have investigated two apparently unrelated patients, both of which have mutations that inhibit the conversion of proinsulin to insulin. By directly sequencing genomic DNA amplified by polymerase chain reaction, we have demonstrated that both patients are heterozygous for the same point mutation converting codon 65 from an arginine (CGT) to a histidine (CAT) codon. Because Arg65 is one of the two dibasic amino acids at the site of proteolytic cleavage between the insulin A-chain and C-peptide, this mutation explains the impairment in the cleavage of proinsulin to insulin. Interestingly, the same His65 mutation has been identified in the insulin gene of a Japanese kindred with familial hyperproinsulinemia. Thus, this mutation has occurred in three apparently unrelated kindreds from two different racial groups. This observation is consistent with the hypothesis that the dinucleotide sequence CpG, the first two nucleotides in the arginine (CGT) codon, is a "hot spot" for mutations.     

X-连锁遗传性肾性尿崩症研究进展

X-连锁遗传性肾性尿崩症较为少见,临床表现各异,精氨酸加压素受体2(AVPPR2)基因突变检测是其主要确诊手段.因常规对症治疗仅可部分缓解患者多饮、多尿的临床症状,个体差异显著.基于基因功能及相关药物的研究进展,药物伴侣如AVPR2拮抗剂或激动剂成为目前的研究热点,该类药物在一定程度上可恢复部分突变受体的结构和(或)生理活性,为该病的治疗提供潜在可能性,但仍需更多的研究来进一步证实其临床疗效和安全性.     

Aminoglycoside pretreatment partially restores the function of truncated V(2) vasopressin receptors found in patients with nephrogenic diabetes insipidus

By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V(2) vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.