Mutations in myosin VIIA (MYO7A) and usherin (USH2A) in Spanish patients with Usher syndrome types I and II,respectively

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment and retinitis pigmentosa. Three clinical types are known (USH1, USH2 and USH3), and there is an extensive genetic heterogeneity, with at least ten genes implicated. The most frequently mutated genes are MYO7A, which causes USH1B, and usherin, which causes USH2A. We carried out a mutation analysis of these two genes in the Spanish population. Analysis of the MYO7A gene in patients from 30 USH1 families and sporadic cases identified 32% of disease alleles, with mutation Q821X being the most frequent. Most of the remaining variants are private mutations. With regard to USH2, mutation 2299delG was detected in 25% of the Spanish patients. Altogether the mutations detected in USH2A families account for 23% of the disease alleles.     

Map refinement of the Usher syndrome type 1B gene, MYO7A, relative to 11q13.5 microsatellite markers

Usher syndrome (US) is clinically and genetically a heterogeneous group of disorders characterized by the association of deafness with retinitis pigmentosa. So far, eight genes responsible for US have been mapped, of which only the gene responsible for the most common form, USH1B, has been identified. The USH1B is a large gene containing 49 exons and encoding for an unconventional myosin-VIIA (MYO7A). Mutation analysis within the MYO7A gene showed a wide variety of mutations dispersed all over the gene. The present report refines the location of the MYO7A gene relative to microsatellite markers mapped to this region, thereby allowing a reliable and efficient carrier detection by linkage analysis.     

Mutation profile of the MYO7A gene in Spanish patients with Usher syndrome type I

Usher syndrome type I is the most severe form of Usher syndrome. It is an autosomal recessive disorder characterized by profound congenital sensorineural deafness, retinitis pigmentosa, and vestibular abnormalities. Mutations in the myosin VIIA gene (MYO7A) are responsible for Usher syndrome type 1B (USH1B). This gene is thought to bear greatest responsibility for USH1 and, depending on the study, has been reported to account for between 24% and 59% of USH1 cases. In this report a mutation screening of the MYO7A gene was carried out in a series of 48 unrelated USH1 families using single strand conformation polymorphism analysis (SSCP) and direct sequencing of those fragments showed an abnormal electrophoretic pattern. Twenty-five mutations were identified in 23 out of the 48 families studied (47.9%). Twelve of these mutations were novel, including five missense mutations, three premature stop codons, three frameshift, and one putative splice-site mutation. Based on our results we can conclude there is an absence of hot spot mutations in the MYO7A gene and that this gene plays a major role in Usher syndrome.     

一个视网膜色素变性家系的视紫红质基因突变分析

目的　确定常染色体显性遗传视网膜色素变性家系的致病基因及其突变位点,并研究其临床表型。方法　对一个常染色体显性遗传视网膜色素变性(autosomal dominat retinitis pigmentosa,ADRP)家系成员进行了视力、视野及眼底镜检查,并对该家系中先证者进行了视网膜电流图分析。应用聚合酶链反应和直接测序技术,对该家系的所有现存人员的视紫红质基因的外显子进行测序分析。结果　该家系的2 5名成员中12例患者有视紫红质基因(rhodopsin,RH O)的5 12 C>T(P171L)突变,均呈杂合子,该错义突变使密码子171由CCA变成CTA。而未受累者的视紫红质基因表现为野生型。该家系患者的临床表现为5～6岁时出现夜盲,在2 0～30岁逐渐出现视力和视野损害,并先后在4 0～5 0岁前后失明,其中2例患者并发青光眼,先证者的闪烁视网膜电图呈熄灭型。结论　视紫红质基因RH O的一种已知突变5 12 C>T(P171L)是该家系的病因。与国外相同的基因突变类型相比较,该家系发病早、病情进展快、视功能损害较重。     

一个有三例女性X-连锁肾上腺-脑白质营养不良杂合子患者家系基因突变分析

目的 对1个有3例女性杂合子患者的X-连锁肾上腺-脑白质营养不良(X-linked adrenoleukodystrophy,X-ALD)家系进行基因突变分析.方法 提取1例患者的外周血白细胞总RNA,以此为模板,采用长链逆转录-PCR技术,分4个片段扩增ABCD1基因(X-ALD疾病基因)编码序列并对其PCR产物进行直接测序.通过PCR和限制性内切酶分析患者及其家庭成员相应的基因组DNA片段,进一步确证所发现的基因突变.对突变及其对ALD蛋白结构的影响进行生物信息学分析.结果 在患者A月CD1基因第1外显子的第283位密码子发现1个新的错义突变:CAC→CGC(p.H283R),此突变使相应DNA片段的1个Msl Ⅰ酶切位点消失,其子不存在此突变.突变所改变的氨基酸残基在进化上高度保守.p.H283R突变位于ALD蛋白的跨膜结构域,可引起该分子跨膜区整体结构的改变.结论 在国内首次报道了3例杂合子女性X-ALD患者,并在其家系发现了1个新的ABCD1基因突变,即p.H283R突变.     

Analysis of DNA elements that modulate myosin VIIA expression in humans

Usher syndrome�Ib (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE‐19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE‐19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A ‐2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, ‐2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B. Hum Mutat 14:354, 1999. © 1999 Wiley‐Liss, Inc.     

A novel missense mutation (402C → T) in exon 1 in the EDA gene in a family with X-linked hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (EDA), or Christ-Siemens-Touraine syndrome, is clinically characterized by hypohidrosis, hypoodontia and hypotrichosis. The X-linked form of the disease has been mapped to Xq12-q13.1, and a gene from this region has recently been cloned. This gene encodes a predicted transmembrane protein of 135 amino acids, which was found to be expressed in keratinocytes, hair follicles, and sweat glands. A variety of rearrangements in this gene have been found in patients with hypohidrotic ectodermal dysplasia.
We have screened the probands from nine unrelated Danish families with hypohidrotic ectodermal dysplasia for mutation in exon 1 of the EDA-gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). In one large kindred we identified a novel missense mutation (402C → T), which changes a histidine to tyrosine at position 54 in the protein. This mutation cosegregates with the disease in the family and is the first mutation described which affects the predicted transmembrane, hydrophobic domain of the protein.     

A novel nonsense mutation at Glu-631 in a Spanish family with complement component 7 deficiency

Deficiency of the seventh component of complement (C7D) is frequently associated with recurrent neisserial infections. We report in the present study the genetic basis for C7D in a Spanish family. We used exon-specific polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis as a screening step for mutations, followed by direct sequencing of the target exon. The mutation in the proband was a homozygous G-to-T transversion at nucleotide 1957, the first nucleotide of the codon GAG for Glu-631, leading to a stop codon TAG (E631X). Our result provides further evidence that the molecular pathogenesis of C7D is heterogeneous. Received: October 20, 1998 / Accepted: December 25, 1998     

良性家族性新生儿惊厥 KCNQ2 基因新突变

目的 对一个中国良性家族性新生儿惊厥(benign familial neonatal convulsions，BFNC)家系进行基因诊断，并探讨其分子发病机理。方法 对该家系进行详细的临床检查及疾病基因的连锁分析。应用聚合酶链反应(polymerase chain reaction，PCR)-DNA直接测序，并用PCR-单链构象多态(single strand conformation polymorphism，SSCP)对先证者、家系内16人及家系外72名无血缘关系的正常入进行KCNQ2基因突变分析。结果 连锁分析提示该家系与KCNQ2基因连锁，并排除与KCNQ3基因连锁。PCR—DNA直接测序在先证者发现．KCNQ2基因突变193ldelG，PCR—SSCP发现家系内其他患者均出现与先证者相同的异常SSCP条带，而72名正常人未出现此异常条带。结论 KCNQ2基因突变是中国人BFNC的发病原因之一，193ldelG是国内外未曾报道过的新突变，连锁分析结合基因突变分析可对BFNC患者进行基因诊断。     

Detection of a novel missense mutation in the mevalonate kinase gene in one Chinese family with DSAP

Disseminated superficial actinic porokeratosis (DSAP) is the most common form of porokeratosis and a severe chronic autosomal dominant cutaneous disorder with high genetic heterogeneity. Recently, the mevalonate kinase (MVK) gene has been identified as a candidate gene responsible for DSAP and multiple mutations have been reported. Here, we report identification of a novel missense mutation in the MVK gene in a Chinese family with DSAP. A 50-year-old male was diagnosed as proband of DSAP based on the clinical and histological findings, which show numerous hyperpigmented macules by physical examination and cornoid lamella by skin biopsy. Similar skin symptoms were also observed in his father, who died many years ago. We prepared genomic DNA from the proband, unaffected individuals from his family members, as well as 100 unrelated healthy controls. PCR was then conducted using the above genomic DNA as template and the MVK gene-specific primers. The PCR product was subjected to direct sequencing and the sequence was compared to that of MVK gene within the NCBI database. We detected a heterozygous C to G transition at nucleotide 643 in exon 7 of MVK gene of the proband. This will result in an amino acid change at codon 215 (P.Arg215Gly.), which is from an arginine codon (CGA) to a Glycine codon (GGA). We did not detect any mutation in the unaffected family members or the 100 unrelated healthy controls, demonstrating that this is a novel missense mutation in MVK gene and therefore, contributes to the molecular diagnosis of DSAP.     

中国人遗传性B型短指(趾)家系中 ROR2基因突变的鉴定

目的　确定一个中国人 B型短指 (趾 )家系中是否存在 ROR2基因突变。方法　从一个中国人B型短指 (趾 )家系的患者外周血中提取基因组 DNA,PCR扩增 ROR2基因外显子 8和部分外显子 9,PCR产物直接测序 ;同时进行 PCR产物 TA克隆和插入片段测序。结果　在患者中发现 ROR2基因第 9外显子1398～ 1399ins A杂合突变 ,与国外发现的致病性突变一致。结论　本文首次报告中国人 B型短指 (趾 )中的 ROR2基因突变。     

A novel missense mutation of the EDA gene in a Mongolian family with congenital hypodontia

X-linked hypohidrotic ectodermal dysplasia (HED) is a rare disease characterized by the hypoplasia or absence of eccrine glands, dry skin, scant hair, and dental abnormalities. Here, we report a Mongolian family with congenital absence of teeth inherited in an X-linked fashion. The affected members of the family did not show other HED characteristics, except hypodontia. We successfully mapped the affected locus to chromosome Xq12-q13.1, and then found a novel missense mutation, c.193C>G, in the ectodysplasin A (EDA) gene in all affected males and carrier females. The mutation causes arginine to be replaced by glycine in codon 65 (R65G) in the juxtamembrane region of EDA. In addition, 33% (3/9) of female carriers have a skewed X-chromosome inactivation pattern. Our result strongly suggests that the c.193C>G mutation is the disease-causing mutation in this family.Ran Tao, Buhe Jin, Shen Zheng Guo, and Wei Qing contributed equally to this work.     

一个近亲婚配家系中的一种P基因新突变

目的在DNA水平上对1个有2例患者的姨表兄妹近亲婚配家系中的眼皮肤白化病患者进行分型诊断。方法用PCR扩增先证者P基因及TYR基因各外显子、外显子-内含子交界区及3＇端和5＇端非翻译区，以DNA序列测定技术检测基因突变，以DNA测序技术和限制性内切酶酶切法检测该家系其他成员及群体中正常人的相应基因位点。结果先证者和其白化病妹妹为P基因A787T突变纯合子，其父母和表型正常的弟弟均为A787T突变杂合子。先证者TYR基因未见突变。群体中102名表型正常者中无A787T突变等位基因。结论在基因水平确定我国存在眼皮肤白化病Ⅱ型，同时发现了一种P基因病理性新突变。     

A new mutation in the proteolipid protein (PLP) gene in a German family with pelizaeus-merzbacher disease

A C-to-T transition in exon 4 of the PLP gene was found in 2 affected males and two obligate carriers in a German family with Pelizaeus–Merzbacher disease. The mutation, which causes loss of an HphI site and changes amino acid 155 from threonine to isoleucine, was absent from 108 normal chromosomes. There are 5 concordances and 1 discrepancy between these results and those obtained by magnetic resonance imaging in this family.     

遗传性多发性骨软骨瘤的基因诊断及产前诊断

目的 研究家族遗传性骨软骨瘤病(hereditary multiple exostoses,HME)的致病基因及产前诊断.方法 应用连锁分析方法对一个HME家系EXT1、EXT2和EXT3基因进行分析.致病基因定位后,用PCR-测序法进行了突变分析.结果 在该家系中EXT2基因第6外显子发生1个新的无义突变(c.1006C>T),该突变导致第336位编码谷氨酰胺的密码子CAA变为终止密码子TAA(Gln336X).根据上述结果配合遗传咨询进行了产前诊断,结果显示胎儿正常.结论 在家族遗传性骨软骨瘤家系中发现一新的EXT2基因突变,并应用于产前诊断.     

Hereditary angioedema in a Jordanian family with a novel missense mutation in the C1-inhibitor N-terminal domain

Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) is an autosomal dominant disease caused by mutations in the SERPING1 gene. A Jordanian family, including 14 individuals with C1-INH-HAE clinical symptoms, was studied. In the propositus and his parents, SERPING1 had four mutations leading to amino acid substitutions. Two are known polymorphic variants (c.167T>C; p.Val34Ala and c.1438G>A; p.Val458Met), the others are newly described. One (c.203C>T; p.Thr46Ile) is located in the N-terminal domain of the C1-inhibitor protein and segregates with angioedema symptoms in the family. The other (c.800C>T; p.Ala245Val) belongs to the serpin domain, and derives from the unaffected father. DNA from additional 24 family members were screened for c.203C>T mutation in the target gene. All individuals heterozygous for the c.203C>T mutation had antigenic and functional plasma levels of C1-inhibitor below 50% of normal, confirming the diagnosis of type I C1-INH-HAE. Angioedema symptoms were present in 14 of 16 subjects carrier for the c.203T allele. Among these subjects, those carrying the c.800T variation had more severe and frequent symptoms than subjects without this mutation. This family-based study provides the first evidence that multiple amino acid substitutions in SERPING1 could influence C1-INH-HAE phenotype.