一个掌跖角化病家系的调查

本文报道了一个掌跖角化病家系的调查结果．该家系属于弥漫性掌跖角化病,为常染色体显性遗传．     

人类间隙连接蛋白β-5基因的克隆、表达及突变分析

利用同源筛选与巢式PCR的方法克隆了编码人类间隙连接蛋白b -5的基因GJB5,确定了基因组结构,FISH将其定位于1p33-p35.经RT-PCR分析,GJB5在胎儿皮肤、成人皮肤、胎盘中有表达.采用直接测序的方法对142个感觉神经性听力下降患者及36个其他遗传性疾病家系(5个变异性红皮肤角化病家系,13个Charcot-Marie-Tooth病家系,4个上睑下垂家系和14个视网膜色素变性伴耳聋家系)患者进行了GJB5的突变分析.在两个感觉神经性耳聋患者(M1883,M1589)中发现了两种错义突变(686A→G,H229R;25C→T,L9F);在3个遗传性听力下降的耳聋家系中发现了一种位于内含子剪接受体位点的18 bp的杂合缺失,其中一个家系(娄底家系)中伴有一种错义改变(R265P),18 bp的杂合缺失和错义改变在家系中均不与突变共分离.对缺失携带者进行RT-PCR分析,未能在皮肤组织中发现任何异常的mRNA,mRNA的表达量也未见明显异常.群体分析时发现,199个正常人中有两例此18 bp缺失的携带者.     

一罕见的掌跖角化病和胎儿鱼鳞病家系的遗传学研究

掌跖角化病（Keratoderma palmoplantaris）和胎儿鱼鳞病（Ichthyosis Eetalis）均为人类皮肤系统少见的单基因遗传病。在同一家系中这两种遗传病同时出现,而且掌跖角化病已连续遗传 8代,患者达61人之多,胎儿鱼鳞病亦相继出现3例,实属罕见。     

一个中国Gilbert综合征家系的遗传学分析

对一个中国汉族Gilbert综合征遗传家系致病基因突变位点进行鉴定,以期了解该病的分子遗传学基础。首先提取先证者基因组DNA,PCR扩增尿苷二磷酸葡萄糖醛酸转移酶UGT1A1基因的5个外显子,以琼脂糖电泳鉴定PCR产物,纯化后直接测序鉴定。基因扫描显示,与血清胆红素水平密切相关的UGT1A1基因在第1和第5外显子存在纯合突变,而 UGT1A1基因启动子区域和内含子/外显子剪接边界位点序列未检测到突变。进一步对其他家系成员该基因的相应位点进行突变检测,结果显示他们在第1和第5外显子也存在杂合突变,其中还有两个成员在启动子区域检测到(TA)插入突变。对家系成员未抗凝新鲜血液进行生化检测证实了基因突变分析的结果。综合以上结果发现该家系三种突变并存,致病因素为第1和/或第5外显子突变,为显性遗传,两种突变位点纯合导致先证者出现严重胆红素代谢功能障碍。该家系因此成为Gilbert综合征突变位点及其致病机理研究的一个典型临床病例。     

常染色体显性遗传非综合征性耳聋致病基因定位研究

耳聋具有高度的遗传异质性, 迄今已定位了51个常染色体显性遗传非综合征型耳聋(autosomal dominant non-syndromic sensorineural hearing loss, DFNA)基因位点, 20个DFNA相关基因被克隆。文章收集了一个DFNA巨大家系, 家系中有血缘关系的家族成员共170人, 对73名家族成员进行了详细的病史调查、全身检查和耳科学检查, 提示39人有不同程度的迟发性感音神经性听力下降, 未见前庭及其他系统的异常。应用ABI公司382个常染色体微卫星多态标记进行全基因组扫描连锁分析, 将该家系致聋基因定位于14q12-13处D14S1021-D14S70之间约7.6 cM (3.18 Mb)的区域, 最大LOD值为6.69 (D14S1040), 与已知DFNA9位点有4.7 cM (2.57 Mb)的重叠区, DFNA9致病基因COCH位于重叠区域内。下一步拟进行COCH基因的突变筛查, 以揭示该家系耳聋的分子致病机制。     

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。}     

两个携带线粒体tRNA~(Met)/tRNA~(Gln)A4401G和tRNA~(Cys)G5821A突变的中国汉族原发性高血压家系的临床及分子遗传学特征

线粒体tRNA基因可能是原发性高血压发病相关的突变热点区域。文章报道了两个具有母系遗传特征的中国汉族原发性高血压家系的临床和分子遗传学特征。家系先证者和其他成员的临床数据表明,家系中母系成员高血压发病的严重程度存在差异,发病年龄也从36~79岁不等。对两个家系先证者使用24对有部分重叠的引物进行线粒体DNA全序列扩增分析,结果发现这两个先证者均携带同质性的tRNAMet/tRNAGlnA4401G和tRNACysG5821A突变,多态性变异位点都属于东亚单体型C。A4401G突变可能通过影响tRNAMet和tRNAGln前体的加工引起线粒体tRNA代谢水平的改变,而tRNACysG5821A突变位于tRNACys氨基酸受体臂,该突变使tRNACys氨基酸受体臂上原有的G6-C67配对消失,可能影响tRNACys空间结构和功能的稳定性,致使线粒体功能障碍。因此,tRNAMet/tRNAGlnA4401G和tRNACysG5821A突变可能是这两个原发性高血压家系的分子致病基础。     

一个I型神经纤维瘤病家系的基因突变分析

目的：I型神经纤维瘤病是一种常见的常染色体显性遗传病,主要累及皮肤和神经系统。其临床表现多样,主要以”咖啡牛奶斑”、皮肤神经纤维瘤、虹膜Lisch结节、腋窝和腹股沟斑点为特征,I型神经纤维瘤病由NF1基因突变所致,神经纤维瘤蛋白是NFI基因编码蛋白,是一种肿瘤抑制蛋白,可抑制细胞的过度生长。NF1基因突变不仅可导致细胞过度生长,还可增加良性及恶性肿瘤的发生风险。本研究中,我们通过基因突变分析,确定中国东北地区一个伴有先天性白内障的I型神经纤维瘤家系NF1基因的突变位点。方法：通过聚合酶链反应（PCR）和NF1基因直接测序分析对家系中的3名患者及2名健康成员进行基因突变检测,以确定其突变位点。结果：此家系呈常染色体显性遗传。通过基因序列分析发现NF1基因第1140密码子第二个碱基呈杂合子点突变C—G,导致一个无义突变S1140X,家系中健康成员和正常对照未检测到此突变存在。结论：通过NF1基因测序分析,我们发现NF1基因的S1140X突变是引起该家系NF1疾病的致病原因,该突变导致NF1基因终止密码提前,神经纤维瘤素蛋白截短。本研究丰富了我国关于I型神经纤维瘤病在眼科的临床表现。     

白边油蝠表皮角质化细胞末端分化的超微结构

蝙蝠是一种唯一能够飞行的哺乳动物,其皮肤的超微结构尚未见报道。在电镜下观察了白边油蝠(Pipistrellus kuhlii)背部和翼膜皮肤的超微结构。表皮的厚度较低(10~12μm),角质层下有1~2层的刺细胞,该刺细胞由相似于鸟类无羽表皮的纤细角化细胞形成。颗粒层不连续且仅有少量小型透明角质颗粒(<0.3μm)。在翼膜的若干区域,表皮简化为一层与角质层相连的基底层。过渡期的角化细胞几乎不存在,提示其角质化过程非常迅速。基底膜上的无数半桥粒在真皮下面形成密集的附着点。大量胶原纤维直接维系在半桥粒和基底膜的致密层上,稀疏的弹性纤维使得蝙蝠表皮在飞行时易于伸展、在飞行后易于迅速折叠而不会受到损伤。与鸟类的表皮相似,蝙蝠角化细胞富有大量的脂质。由于脂质有助于蝙蝠皮肤在飞行中与冷空气流的传热绝缘,大量脂质的存在可能是为补偿蝙蝠翼膜的真皮缺乏厚的脂肪层。研究还表明,毛发较薄(4~7μm),并具有与皮层相似的突状物组成的精细表皮,其表皮细胞形成钩状抓握点使毛发紧紧粘结在一起,通过这种方式毛皮保持紧凑以恒定体温。     

一个遗传性胰腺炎家系中新发现的胰蛋白酶原基因突变

对1个遗传性胰腺炎(hereditary pancreatitis, HP)家系中6例成员和120例无亲缘关系健康人的胰蛋白酶原基因(protease serine 1, PRSS1)进行PCR扩增, 产物纯化后测序, 结合受检者的血清肿瘤标志物、糖尿病相关生化指标以及近亲属的一般临床资料进行分析。结果发现4例家系成员PRSS1基因3号外显子区136位碱基存在C→T杂合性突变, 他们的基因型表现为野生型与突变型杂合现象, 另外在先证者PRSS1基因的3号外显子区171位碱基还存在着一个同义突变点(C→T), 而对照组和家系其他成员中未发现此两种突变, 突变阳性患者表现为乳酸、糖基化血红蛋白和糖类肿瘤标志物(CA19-9、CA125)增高。因此, PRSS1基因3号外显子区136位碱基C→T杂合性突变与该家系遗传性胰腺炎有关, 是该家系中遗传性胰腺炎的遗传易感因素。     

Mapping of a gene for epidermolytic palmoplantar keratoderma to the region of the acidic keratin gene cluster at 17q12–q21

Summary Epidermolytic palmoplantar keratoderma (EPPK) (Vörner-Unna-Thost) is an autosomal dominantly inherited skin disease of unknown etiology characterized by diffuse severe hyperkeratosis of the palms and soles and, histologically, by cellular degeneration. We have mapped a gene for EPPK to chromosome 17q11–q23, with linkage analysis using microsatellite DNA-polymorphisms, in a single large family of 7 generations. A maximum lod score of z=6.66 was obtained with the probe D17S579 at a recombination fraction of =0.00. This locus maps to the same region as the type I (acidic) keratin gene cluster. Keratins, members of the intermediate filament family, the major proteins of the cytoskeleton in epidermis, are differentially expressed in a tissue-specific manner. One acidic keratin, keratin 9 (KRT9), is expressed only in the terminally differentiated epidermis of palms and soles. The KRT9 gene has not yet been cloned; however, since the genes for most acidic keratins are clustered, it is highly probable that it too will map to this region. We therefore propose KRT9 as the candidate gene for EPPK.     

KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.     

Naegeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosa reticularis: two allelic ectodermal dysplasias caused by dominant mutations in KRT14

Naegeli-Franceschetti-Jadassohn syndrome (NFJS) and dermatopathia pigmentosa reticularis (DPR) are two closely related autosomal dominant ectodermal dysplasia syndromes that clinically share complete absence of dermatoglyphics (fingerprint lines), a reticulate pattern of skin hyperpigmentation, thickening of the palms and soles (palmoplantar keratoderma), abnormal sweating, and other subtle developmental anomalies of the teeth, hair, and skin. To decipher the molecular basis of these disorders, we studied one family with DPR and four families with NFJS. We initially reassessed linkage of NFJS/DPR to a previously established locus on 17q11.2-q21. Combined multipoint analysis generated a maximal LOD score of 8.3 at marker D17S800 at a recombination fraction of 0. The disease interval was found to harbor 230 genes, including a large cluster of keratin genes. Heterozygous nonsense or frameshift mutations in KRT14 were found to segregate with the disease trait in all five families. In contrast with KRT14 mutations affecting the central alpha -helical rod domain of keratin 14, which are known to cause epidermolysis bullosa simplex, NFJS/DPR-associated mutations were found in a region of the gene encoding the nonhelical head (E1/V1) domain and are predicted to result in very early termination of translation. These data suggest that KRT14 plays an important role during ontogenesis of dermatoglyphics and sweat glands. Among other functions, the N-terminal part of keratin molecules has been shown to confer protection against proapoptotic signals. Ultrastructural examination of patient skin biopsy specimens provided evidence for increased apoptotic activity in the basal cell layer where KRT14 is expressed, suggesting that apoptosis is an important mechanism in the pathogenesis of NFJS/DPR.     

Directed Expression of a Chimeric Type II Keratin Partially Rescues Keratin 5-null Mice

The crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into cross-linked networks. For IFs comprised of keratins 5 and 14 (K5 and K14), which occur in basal keratinocytes of the epidermis, formation of cross-linked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc) or bundling-deficient wild type KRT8 as a control to the epidermal basal layer of Krt5-null mice to assess the functional importance of keratin IF self-organization in vivo. Such targeted expression of K8bc rescued Krt5-null mice with a 47% frequency, whereas K8 completely failed to do so. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Ex vivo culture of embryonic skin keratinocytes confirmed the ability of K8bc to form IFs in the absence of K5. Additionally, electron microscopy analysis of E18.5 embryonic skin revealed that the striking defects observed in keratin IF bundling, cytoarchitecture, and mitochondria are partially restored by K8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis and underscore the importance of context-dependent regulation for keratin genes and proteins in vivo.     

Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, two unique complex rearrangements consisting of contiguous small insertions and deletions were found in exons 1 and 2 causing L45R/H46S and L120X, respectively. CONCLUSIONS: These studies further define the heterogeneity of mutations causing Fabry disease, permit precise carrier identification and prenatal diagnosis in these families, and facilitate the identification of candidates for enzyme replacement therapy.     

视网膜色素变性中GUCA1B、GNGT1和RGS9基因突变筛查

为寻找视网膜色素变性的致病基因,从120个家系收集视网膜色素变性先证者,制备基因组DNA。应用PCR―异源双链-SSCP法,分析GUCA1B基因4个外显子、GNGT1基因编码区和RGS9基因视网膜特异性转录区,寻找基因变异。序列分析确定突变。结果表明,31人的GUCA1B基因外显子1存在T/C多态。所有先证者中均未检测到GUCA1B、GNGT1和RGS9基因突变。认为本组病例未发现GUCA1B、GNGT1和RGS9基因的突变。 Abstract:To screen possible disease-causing mutations in the GUCA1B gene,GNGT1 gene,and the alternative-splicing region of RGS9 gene in 120 probands with retinitis pigmentosa,genomic DNA was collected from 120 probands with retinitis pigmentosa out of 120 families.The coding sequences of the GUCA1B and GNGT1 genes and the alternative splicing region of the RGS9 gene were analyzed by using PCR-heteroduplex-SSCP method.Mutation was confirmed by DNA sequencing.A T/C polymorphism was identified in exon 1 of the GUCA1B gene in 31 of the 120 probands.Heteroduplex-SSCP analysis of the GUCA1B and GNGT1 coding regions and RGS9 alternative splicing region showed no mutations in 120 patients with retinitis pigmentosa.We found no evidence that mutation in GUCA1B,GNGT1,or RGS9 gene is a cause of retinitis pigmentosa.     

Arginine‐ but not alanine‐rich carboxy‐termini trigger nuclear translocation of mutant keratin 10 in ichthyosis with confetti

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant‐negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)‐terminus, from poly‐glycine to either a poly‐arginine or ‐alanine tail. Previous studies on the type of C‐terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C‐termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C‐terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine‐rich C‐terminus. However, this was not observed with K10 featuring an alanine‐rich C‐terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild‐type and truncated forms of K10. This study demonstrates that, of the various 3′ frameshift variants of KRT10, exclusively arginine‐rich C‐termini lead to nuclear localization of K10.     

Epidermolysis bullosa simplex: a keratin 5 mutation is a fully dominant allele in epidermal cytoskeleton function.

To explore the relationship between abnormal keratin molecules, 10-nm intermediate filament (IF) organization, and epidermal fragility and blistering, we sought to determine the functional consequences of homozygosity for a dominant keratin defect. We describe a family with an autosomal dominant skin-blistering disorder, epidermolysis bullosa simplex, Koebner subtype (EBS-K), that has a novel point mutation, occurring in the keratin 5 gene (KRT5), that predicts the substitution of an evolutionarily conserved lysine by an asparagine residue (K173N). Unlike previous heterozygous mutations located within the initial segment of domain 1A of keratin molecules, K173N heterozygosity did not result in severe disease or clumping of keratin filaments. One family member was found to be homozygous for the K173N allele, having inherited it from each of her affected first-cousin parents. Despite a lack of normal keratin 5 molecules, and an effective doubling of abnormal molecules, available for heterodimerization with keratin 14 during IF formation, there were no significant differences in the clinical severity or the ultrastructural organization of the keratin IF cytoskeleton of the homozygous individual. These data demonstrate that the K173N mutation behaves as a fully dominant allele and indicate that a limited number of abnormal keratin molecules are sufficient to impair cytoskeletal function and elicit epidermal fragility and blistering.     

肌萎缩侧索硬化患者SOD1基因突变检测及突变与临床表型的关系

应用PCR技术结合DNA直接测序方法对8例临床确诊为家族性肌萎缩侧索硬化(Familiar amyotrophic lateral sclerosis,FALS)家系的先证者进行铜锌超氧化物歧化酶基因(SOD1)的突变筛查,在3例先证者中检出2种SOD1基因突变,其中,2例携带了位于4号外显子的错义突变Cys111Tyr(c.332G>A),另1例携带了位于5号外显子的错义突变Gly147Asp(c.440G>A),这2种突变在中国ALS患者中属首次报道。该结果扩大了中国FALS患者的SOD1基因突变谱,对研究中国FALS患者SOD1基因突变特点和分布规律有一定帮助。分析携带这2个突变患者的临床特点,提示Cys111Tyr突变导致的临床表型相对温和,而Gly147Asp突变可导致病情进展较快。该结果有待在更多的病例中进行证实。