有汗型外胚层发育不良家系基因突变分析及孕早期产前诊断

目的 对1个中国汉族有汗型外胚层发育不良家系进行了基因突变分析,并在此基础上对该家系中已孕11周的胎儿进行了产前诊断,为遗传咨询提供依据.方法 应用PCR扩增和直接双向测序对1个有汗型外胚层发育不良家系2例患者及6名正常成员的GJB6基因(Cx30基因)的全编码区序列进行突变分析.在确定突变后,取胎盘绒毛活检样本进一步行产前诊断.结果 在该家系的2例患者中检测出GJB6基因c.31G＞A的点突变,该突变导致了GJB6蛋白N-末端区域第11位甘氨酸被精氨酸替代(p.G11R),6名正常家系成员均未检测到该突变.产前诊断结果显示胎儿也携带有GJB6基因c.31G＞A突变.在终止妊娠后,流产物突变分析的结果与产前诊断结果一致.结论 GJB6基因c.31G＞A(p.G11R)错义突变是该有汗型外胚层发育不良家系的致病原因,通过基因诊断和产前诊断可以有效阻止致病基因的传递.     

X-连锁无汗性外胚层发育不良一家系产前诊断

目的基因诊断方法及连锁分析对一X连锁无汗性外胚层发育不良（XLHED）家系进行产前诊断。方法提取患儿、患儿母亲外周血以及胎儿脐血中的基因组DNA,PCR扩增该疾病相关基因EDA的五个外显子,并直接测序;选择与该基因连锁的STR位点,位于基因上游的一个（CA）n进行家系内的连锁分析。结果患儿的基因突变未发现,连锁分析提示胎儿为男性未携带风险X染色体,出生后证实胎儿正常。结论基因检测及多态性连锁分析在XLHED家系中进行产前诊断是一种较好的方法。     

眼皮肤白化病Ⅰ型产前基因诊断

目的对已生育过眼皮肤白化病Ⅰ型（oculocutaneous albinism type Ⅰ，OCA1）患儿的两个家系进行酪氨酸酶（tyrosinase，TYR）基因TYR的突变研究和产前基因诊断。方法应用PCR技术扩增TYR基因各外显子、外显子．内含子交界区及启动子区，直接以DNA序列测定技术分析先证者或其父母的基因突变，明确致病性突变后，检测胎儿TYR基因相应位点的DNA序列，获知胎儿的基因型。结果家系1的先证者为R278X和929insC突变复合杂合子；胎儿未获得这2种突变等位基因，基因型和表型均正常。家系2先证者的父母分别为IVS4＋3A→T和G253E突变的杂合子，胎儿只获得了父源性的IVS4＋3A→T突变等位基因，未获得母源性G253E突变等位基因，胎儿为表型正常的致病基因携带者。结论此为中国大陆首次真正意义上的OCA1产前基因诊断；应用上述基因分析方法进行OCA1产前基因诊断是可行的。     

一个眼牙指发育不良家系的基因突变分析

目的 旨在报道一个罕见的中国汉族眼牙指发育不良家系,并对该家系进行基因突变分析.方法 采用PCR及测序方法检测该家系先证者GJA1基因编码区及其侧翼序列的突变,然后在家系成员中验证,并通过Weblogo软件对可疑变异位点进行保守性分析;同时利用PCR、电泳及测序分析方法,检测HOXD13基因突变,排除患者由HOXD13基因突变致病的可能.结果 该家系内患者均携带GJA1基因的杂合突变c.605 C>T,正常个体均无此突变;该突变导致Cx43蛋白第202位氨基酸由精氨酸变成组氨酸(p.R202H);HOXD13基因未见异常.结论GJA1基因c.605 C>T(p.R202H)突变是该中国汉族眼牙指发育不良家系的致病突变,该突变为中国人群中首次报道.     

四例非综合征型白化病家系的产前基因检测

目的探讨非综合征型白化病产前基因诊断的临床价值和意义。方法本研究应用Sanger测序法分别对四个非综合征型白化病家系进行常见致病基因检测。结果家系1患者在OCA2基因发现致病突变位点c.1441AG和c.727CT,胎儿与患者检测结果一致。家系2患者在SLC45A2基因存在c.478GC纯合突变,胎儿发现该位点杂合突变,为携带者。家系3患者在TYR基因存在致病突变位点c.655GA和c.929ins C,胎儿发现c.655GA杂合突变,为携带者。家系4患者在TYR基因上存在突变位点c.346CT和c.929ins C,但胎儿在TYR基因上未见异常。结论明确患者的致病基因可明显缩短胎儿产前基因诊断的时间,为白化病家庭提供积极且准确的优生指导。     

常染色体显性遗传多囊肾病一个家系的基因检测及产前诊断

目的对一个常染色体显性遗传多囊肾病(autosomal dominant polycystic kidney disease,ADPKD)家系进行基因诊断和产前诊断。方法收集该家系成员的外周血标本及先证者妻子的羊水标本,提取基因组DNA,运用Sanger测序方法对家系成员进行PKD1基因全外显子测序,确定该家系突变位点后,对羊水标本进行产前诊断。结果我们检测出该家系4个病人的PKD1基因均有一个缺失突变C.393-394delTG,该缺失突变为致病突变。进而对先证者妻子的羊水标本进行检测,未发现该缺失突变,结果显示胎儿正常。结论应用Sanger测序技术明确了该家系PKD1基因的致病突变,并对该家系进行了产前诊断。     

应用二代测序对成骨不全家系进行基因突变检测和产前诊断

目的应用全外显子测序技术对一个成骨不全(Osteogenesisimperfect,OI)家系进行分子遗传学检测,确定致病基因,实现该家系的基因诊断及产前诊断。方法对1个成骨不全家系先证者进行全外显子检测,采用生物信息软件对变异位点进行过滤和注释,利用SIFT和Polyphen-2软件对突变位点进行致病分析,预测致病性,结合临床特征,确定患者的致病突变。对先证者及家系其他成员进行Sanger测序验证。抽取脐血,进行产前基因诊断,采用STR法,进行母血污染鉴定,避免因母血污染而导致的误诊。结果通过数据的对比过滤,最终选定COL1A2基因的杂合突变c.4048GA(p.Gly1350Ser)为可疑致病突变,经SIFT和Polyphen-2预测,该突变位点为有害。经Sanger测序证实,该家系的患者均携带该突变位点,家系中正常成员则无此突变。对脐血进行检测,胎儿亦携带该突变位点。结论应用全外显子测序技术对成骨不全家系进行诊断,明确了该家系的基因致病位点,有助于家系的遗传咨询及产前诊断。本研究结果也进一步丰富了COL1A2的突变谱。     

一个X连锁少汗型外胚层发育不良家系的EDA基因c.822G＞T突变分析

目的 对1个少汗型外胚层发育不良家系进行ectodysplasin A(EDA)基因序列分析,为家系成员提供准确病因诊断和遗传咨询.方法 抽取家系中先证者及有血缘关系的家系成员外周静脉血,常规提取基因组DNA,应用聚合酶链式反应、DNA测序技术分析EDA基因编码区序列;选择103名无血缘关系正常人作为对照.结果 在先证者及另1例男性患者的EDA基因第7外显子区域检出c.822G＞T突变,导致第274位的色氨酸变成了半胱氨酸(p.W274C),该突变位点未见文献报道.家系中的5名女性成员中检出c.822G＞T杂合突变,正常男性成员及103名正常对照均不存在此突变.结论 EDA基因c.822 G＞T突变为导致该家系成员少汗型外胚层发育不良的致病突变.     

高分辨熔解曲线技术在Wilson病致病基因突变鉴定中的应用

目的在一个肝豆状核变性(WD)家系中进行致病突变鉴定和产前基因诊断。方法酚-氯仿法提取外周全血或妊娠13周胎儿绒毛组织基因组DNA;利用PCR-Sanger测序技术对先证者ATP7B基因外显子及外显子/内含子衔接区序列进行突变分析;针对先证者携带的突变位点,应用聚合酶链反应(PCR)-高分辨熔解曲线(HRM)方法对先证者家系4名成员进行突变鉴定,并在此基础上为患儿母亲提供产前基因诊断。结果先证者ATP7B基因第8外显子存在纯合致病突变c.2333GT(p.R778L);该家系5名成员和4名群体正常样本分属于3种不同熔解曲线。HRM分析结果与测序结果一致,即:患儿本人为R778L的纯合子,其父母、姐姐和母亲产前诊断的胎儿均为突变杂合子,4名群体正常对照为纯合野生型。结论在一个WD家系中检测到ATP7B基因热点突变c.2333GT(p.R778L),并针对该突变建立了一种基于PCR-HRM技术的快速突变鉴定方法,成功为家系提供产前基因诊断。     

1例神经纤维瘤病家系致病基因鉴定及产前诊断分析

目的 探讨Ⅰ型神经纤维瘤病家系进行基因检测与产前诊断的临床价值。方法 应用高通量测序对先证者行基因检测，进一步通过Sanger测序验证突变位点，然后检测其家系NF1基因突变位点，明确致病突变后通过绒毛活检术取胎儿样本进行产前基因诊断。结果 高通量测序结果提示先证者样本在NF1基因编码区发现两处杂合变异，分别是c.702G>A同义突变和c.3044T>G(p.Leu1015Arg)错义突变，其父亲，母亲，大姐，二姐，三姐及妻子并未检测出NF1错义突变位点。产前诊断结果表明胎儿样本在NF1基因未发现异常突变。结论 通过高通量测序联合Sanger测序技术可快速筛查出先证者致病突变，从而指导产前诊断，降低异常儿的出生。     

Connexin30 mutations responsible for hidrotic ectodermal dysplasia cause abnormal hemichannel activity

Clouston syndrome or hidrotic ectodermal dysplasia (HED) is a rare dominant genodermatosis characterized by palmoplantar hyperkeratosis, generalized alopecia and nail defects. The disease is caused by mutations in the human GJB6 gene which encodes the gap junction protein connexin30 (Cx30). To gain insight into the molecular mechanisms underlying HED, we have analyzed the consequences of two of these mutations (G11R Cx30 and A88V Cx30) on the functional properties of the connexons they form. Here, we show that the distribution of Cx30 is similar in affected palmoplantar skin and in normal epidermis. We further demonstrate that the presence of the wild-type protein (wt Cx30) improves the trafficking of mutated Cx30 to the plasma membrane where both G11R and A88V Cx30 co-localize with wt Cx30 and form functional intercellular channels. The electrophysiological properties of channels made of G11R and A88V Cx30 differ slightly from those of wt Cx30 but allow for dye transfer between transfected HeLa cells. Finally, we document a gain of function of G11R and A88V Cx30, which form functional hemichannels at the cell surface and, when expressed in HeLa cells, generate a leakage of ATP into the extracellular medium. Such increased ATP levels might act as a paracrine messenger that, by altering the epidermal factors which control the proliferation and differentiation of keratinocytes, may play an important role in the pathophysiological processes leading to the HED phenotype.     

Frequencies of gap- and tight-junction mutations in Turkish families with autosomal-recessive non-syndromic hearing loss

Mutations in genes encoding gap- and tight-junction proteins have been shown to cause distinct forms of hearing loss. We have now determined the GJB2[connexin 26 (Cx26)] mutation spectrum in 60 index patients from mostly large Turkish families with autosomal-recessive inherited non-syndromic sensorineural hearing loss (NSSHL). GJB2 mutations were found in 31.7% of the families, and the GJB2-35delG mutation accounted for 73.6% of all GJB2 mutations. The carrier frequency of GJB2-35delG in the normal Turkish population was found to be 1.17% (five in 429). In addition to the described W24X, 233delC, 120delE and R127H mutations, we also identified a novel mutation, Q80R, in the GJB2 gene. Interestingly, the Q80R allele was inherited on the same haplotype as V27I and E114G polymorphisms. As little is known about the mutation frequencies of most other recently identified gap- and tight-junction genes as a cause for hearing loss, we further screened our patients for mutations in GJB3 (Cx31), GJA1 (Cx43), DeltaGJB6-D13S1830 (Cx30) and the gene encoding the tight-junction protein, claudin 14 (CLDN14). Several novel polymorphisms, but no disease-associated mutations, were identified in the CLND14 and GJA1 genes, and we were unable to detect the DeltaGJB6-D13S1830 deletion. A novel putative mutation, P223T, was found in the GJB3 gene in heterozygous form in a family with two affected children. Our data shows that the frequency of GJB2 mutations in Turkish patients with autosomal-recessive NSSHL and the carrier rate of the GJB2-35delG mutation in the Turkish population, is much lower than described for other Mediterranean countries. Furthermore, mutations in other gap- and tight-junction proteins are not a frequent cause of hearing loss in Turkey.     

Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment

Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing loss cases. Approximately half result from mutations in the connexin 26 (Cx26) gene, GJB2, in Caucasian populations. Heterozygous mutations in GJB2 occasionally co-occur with a deletion of part of GJB6 (connexin 30; Cx30). It is estimated that approximately 1% of deafness is maternally inherited, due to mutations in mitochondrial DNA (mtDNA). Few studies have focused on the frequency of mutations in connexins or mtDNA in African American (AA) and Caribbean Hispanic (CH) admixture populations. In this study, we performed bidirectional sequencing of the GJB2 gene and polymerase chain reaction (PCR) screening for the common GJB6 deletion, as well as PCR/RFLP analysis for three mutations in mtDNA (A1555G, A3243G, A7445G), in 109 predominantly simplex AA and CH individuals. Variations found were a 101T > C (M34T; 1/101 cases), 109G > A (V37I; 1/101), 35delG (mutation; 4/101, (3/4) of non-AA/CH ethnicity), 167delT (mutation; 1/101), 139G > T (mutation; E47X; 1/101 homozygote, consanguineous), -15C > T (1/101), 79G > A (V27I; 9/101), 380G > A (R127H; 4/101; Guyana, India, Pakistan ethnicity), 670A > C (Indeterminate; K224Q; 1/101), 503A > G (novel; K168R; 3/101) and 684C > A (novel; 1/101). All but one of the AA and CH patients had monoallelic variations. There were no hemizygous GJB6 deletions in those with monoallelic GJB2 variations. We also did not identify any patients with the three mutations in mtDNA. Bidirectional sequencing of the GJB2 gene was performed in 187 AA and Hispanic healthy individuals. Our results reveal that GJB2 mutations, GJB6 deletions, and mtDNA mutations may not be significant in these minority admixture populations.     

High prevalence of V37I genetic variant in the connexin-26 (GJB2) gene among non-syndromic hearing-impaired and control Thai individuals

Hearing loss is highly prevalent with a worldwide incidence of 1-2 per 1000 newborns. Several previous studies have demonstrated that mutations of connexin 26 (Cx26 or GJB2) are responsible for most cases of the recessive non-syndromic sensorineural hearing loss (NSSHL). Certain mutations have been described frequently among various populations, which include 35delG, 167delT, and 235delC. Recently, a missense mutation, V37I, was reported as a pathogenic change in East Asian affected individuals. To identify genetic variants associated with NSSHL in Thai population, we performed mutation analysis of Cx26 in 166 unrelated probands with NSSHL and 205 controls. We identified seven novel genetic variants in Cx26. We also identified a high prevalence of the V37I mutation among both affected probands (11.1%) and control subjects (8.5%), which suggests that the pathologic role of V37I may be modified by other genes. Our data support previous studies that show heterogeneity in the frequencies and types of mutations in Cx26 within populations and among ethnicities and that before clinical significance and causality can be attributed to a genetic variant, functional characterization is necessary.     

GJB2: the spectrum of deafness-causing allele variants and their phenotype

Genetic testing was completed on 1,294 persons with deafness referred to the Molecular Otolaryngology Research Laboratories to establish a diagnosis of DFNB1. Exon 2 of GJB2 was screened for coding sequence allele variants by denaturing high-performance liquid chromatography (DHPLC) complemented by bidirectional sequencing. If two deafness-causing mutations of GJB2 (encoding Connexin 26) were identified, further screening was not performed. If only a single deafness-causing mutation was identified, we screened for the g.1777179_2085947del (hereafter called del(GJB6-D13S1830); GenBank NT_024524.13) and mutations in the noncoding region of GJB2. Phenotype-genotype correlations were evaluated by categorizing mutations as either protein truncating or nontruncating. A total of 205 persons carried two GJB2 exon 2 mutations and were diagnosed as having DFNB1; 100 persons carried only a single deafness-causing allele variant of exon 2. A total of 37 of these persons were c.35delG carriers, and 51 carried other allele variants of GJB2. Persons diagnosed with DFNB1 segregating two truncating/nonsense mutations had a more severe phenotype than persons carrying two missense mutations, with mean hearing impairments being 88 and 37%, respectively (P < 0.05). The number of deaf c.35delG carriers was greater than expected when compared to the c.35delG carrier frequency in normal-hearing controls (P < 0.05), suggesting the existence of at least one other mutation outside the GJB2 coding region that does not complement GJB2 deafness-causing allele variants.     

Use of a multiplex PCR/sequencing strategy to detect both connexin 30 (GJB6) 342 kb deletion and connexin 26 (GJB2) mutations in cases of childhood deafness

Hearing loss is a common congenital disorder that is frequently associated with mutations in the Cx26 gene (GJB2). Three recent reports that found a large deletion in another DFNB1 gene, Cx30 (GJB6), suggest that this defect may cause nonsyndromic recessive hearing loss through either a homozygous deletion of Cx30, or digenic inheritance of a Cx30 deletion and a Cx26 mutation in trans. We designed a simple diagnostic strategy with multiplex PCR followed by direct sequencing to allow for the simultaneous detection of Cx26 mutations and Cx30 deletions, and evaluated its effectiveness as a clinical genetic test by examining 200 DNA samples. In the 108 samples from deaf subjects, two digenic mutations were identified in Cx26 and Cx30 (E47X/342 kb deletion and 167delT/342 kb deletion); 69 had only Cx26 mutations (29 biallelic, 40 singleton), including two novel frameshift mutations 511-512insAACG and 358-360delAG; and 37 had no detectable mutation in either Cx26 or Cx30. Our deletion mapping suggested that the proximal breakpoint of all reported Cx30 large deletions are between the nucleotide 444 and 627 at the Cx30 coding region within a maximal interval of 78 or 184 bp. This simultaneous examination of Cx26 and Cx30 is a practical and efficient diagnostic approach for patients with nonsyndromic congenital deafness.     

Clinical features of patients with GJB2 (connexin 26) mutations: severity of hearing loss is correlated with genotypes and protein expression patterns

Mutations in the GJB2 (connexin 26, Cx26) gene are the major cause of nonsyndromic hearing impairment in many populations. Genetic testing offers opportunities to determine the cause of deafness and predict the course of hearing, enabling the prognostication of language development. In the current study, we compared severity of hearing impairment in 60 patients associated with biallelic GJB2 mutations and assessed the correlation of genotypes and phenotypes. Within a spectrum of GJB2 mutations found in the Japanese population, the phenotype of the most prevalent mutation, 235delC, was found to show more severe hearing impairment than that of V37I, which is the second most frequent mutation. The results of the present study, taken together with phenotypes caused by other types of mutations, support the general rule that phenotypes caused by the truncating GJB2 mutations are more severe than those caused by missense mutations. The present in vitro study further confirmed that differences in phenotypes could be explained by the protein expression pattern.     

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.     

Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness.

Mutations in four members of the connexin gene family have been shown to underlie distinct genetic forms of deafness, including GJB2 [connexin 26 (Cx26)], GJB3 (Cx31), GJB6 (Cx30) and GJB1 (Cx32). We have found that alterations in a fifth member of this family, GJA1 (Cx43), appear to cause a common form of deafness in African Americans. We identified two different GJA1 mutations in four of 26 African American probands. Three were homozygous for a Leu-->Phe substitution in the absolutely conserved codon 11, whereas the other was homozygous for a Val-->Ala transversion at the highly conserved codon 24. Neither mutation was detected in DNA from 100 control subjects without deafness. Cx43 is expressed in the cochlea, as is demonstrated by PCR amplification from human fetal cochlear cDNA and by RT-PCR of mouse cochlear tissues. Immunohistochemical staining of mouse cochlear preparations showed immunostaining for Cx43 in non-sensory epithelial cells and in fibrocytes of the spiral ligament and the spiral limbus. To our knowledge this is the first alpha connexin gene to be associated with non-syndromic deafness. Cx43 must also play a critical role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph.     

High prevalence of the W24X mutation in the gene encoding connexin-26 (GJB2) in Spanish Romani (gypsies) with autosomal recessive non-syndromic hearing loss

Molecular testing for mutations in the gene encoding connexin-26 (GJB2) at the DFNB1 locus has become the standard of care for genetic diagnosis and counseling of autosomal recessive non-syndromic hearing impairment (ARNSHI). The spectrum of mutations in GJB2 varies considerably among the populations, different alleles predominating in different ethnic groups. A cohort of 34 families of Spanish Romani (gypsies) with ARNSHI was screened for mutations in GJB2. We found that DFNB1 deafness accounts for 50% of all ARNSHI in Spanish gypsies. The predominating allele is W24X (79% of the DFNB1 alleles), and 35delG is the second most common allele (17%). An allele-specific PCR test was developed for the detection of the W24X mutation. By using this test, carrier frequencies were determined in two sample groups of gypsies from different Spanish regions (Andalusia and Catalonia), being 4% and 0%, respectively. Haplotype analysis for microsatellite markers closely flanking the GJB2 gene revealed five different haplotypes associated with the W24X mutation, all sharing the same allele from marker D13S141, suggesting that a founder effect for this mutation is responsible for its high prevalence among Spanish gypsies.