Identification of WASP mutations in 14 Spanish families with Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations in the WASP gene. The disease is known to be associated with extensive clinical variability, and mutation studies indicate that genotypes are also highly variant among WAS patients. In this study, we performed mutation analysis of the WASP gene in 14 unrelated Spanish families by single strand conformation analysis (SSCA) and sequencing, resulting in the identification of a novel mutation and nine known mutations. No mutation was identified in one family. The ten different mutations include point mutations resulting in amino acid substitutions, stop codons, and small deletions and insertions causing frameshifts. Missense mutations were preferentially located in the amino-terminal part of the protein, exons 2 and 4, whereas stop and frameshift mutations were located in the carboxyl-terminal region, exons 10 and 11. However, in two families, two missense mutations in exon 11 were identified. Our study demonstrates that WASP genotypes have some concordance with the patients' phenotypes, although mutation 1019delC, identified in a family with several affected members, resulted in high intrafamilial clinical variability.     

Two novel mutations identified in the Wiskott-Aldrich syndrome protein gene cause Wiskott-Aldrich syndrome and thrombocytopenia

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are rare X-linked genetic disorders caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene. Both disorders are clinically characterized by chronic thrombocytopenia of small platelets. WAS is a more severe form of the disorder and also courses with eczema, and immune dysfunction. In the present study, we investigated two novel mutations of the WASP gene in two Spanish families with patients clinically diagnosed as having XLT and WAS, respectively. In one of the families a missense mutation in exon 12 (1488A>G), resulting in the highly conserved glutamic residue changing to glycine at position 485 (D485G), was identified in several members. Notably, a female of this family, with clinical signs of XLT, was determined as the carrier of the mutation and showed a skewed pattern of X-inactivation, preferentially inactivating the X-chromosome carrying the wild-type allele. In the case of the second family, we describe a WAS patient with a single base deletion in exon 2 (266-267delA), resulting in a frameshift (at codon 78) that creates a stop codon at amino acid 127. As a consequence, there was no WASP expression.     

WASP gene mutations in Wiskott-Aldrich syndrome and X-linked thrombocytopenia

The WASP gene has been recently cloned from Xp11.23 and shownto be mutated in three patients with the Wiskott-Aldrich syndrome(WAS). We have developed a screening protocol for identifyingWASP gene alterations in genomic DNA and have identified a spectrumof novel mutations In 12 additional unrelated families. Thesemissense, nonsense and frameshift mutations involve eight ofthe 12 exons of the gene. Two mutations creating premature terminationcodons were associated with lack of detectable mRNA on Northernblots. Four amino acid substitutions, Leu27Phe, Thr48lle, Val75Metand Arg477Lys, were found In patients with congenital thrombocytopeniaand no clinically evident immune defect indicating that theWASP gene is the site for mutations in X-linked thrombocytopeniaas well as in WAS. A T-cell line from a WAS patient containedtwo independent DNA alterations, a constitutional frameshiftmutation, also present in peripheral blood leukocytes from thepatient, and a compensatory splice site mutation unique to thecell line. The distribution of eight missense mutations providesvaluable information on amino acids which are essential fornormal protein function, and suggests that sites in the firsttwo exons are hot-spots for mutation.     

Identification of five novel WASP mutations in Chinese families with Wiskott-Aldrich syndrome

The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency caused by mutation in the gene encoding WAS protein (WASP). The disease is characterized by eczema, thrombocytopenia and severe immunodeificency and is associated with extensive clinical heterogeneity. Mutation studies indicated that the mutated genotypes are also highly variable. In this study, we performed PCR-direct sequencing analysis of the WAS gene in six unrelated Chinese families. Five novel mutations identified, included two nonsense mutations (506C-->T, 1388-->T), a small insertion (685-686insCGCA) and two single-base deletions (384delT, 984delC). All of the mutations are predicted to lead to premature translational termination of WASP.     

Molecular diagnosis of Wiskott-Aldrich syndrome in Taiwan.

The spectrum of Wiskott-Aldrich syndrome (WAS) mutation in Han Chinese residing in Taiwan has not been previously reported. We describe a multidisciplinary approach to the molecular diagnosis of WAS which could be applied to clinical diagnosis, carrier prediction, and prenatal diagnosis. A total of 6 male patients, from 6 independent families, were referred for the molecular diagnosis of WAS. The respective methylation status of the 6 X chromosomes in peripheral blood leukocytes from obligatory female carriers was analyzed initially, followed by analysis of the level of expression of WAS protein (WASP) in peripheral leukocytes from patients, using a Western blotting technique. The analysis of the specific WAS gene mutation was done by direct sequencing. Mutations were identified in the WAS gene of all patients. Mutations identified included p.R13X, p.R41X, p.S82P, IVS1-1 G --> C, p.L342TFsX493, and a large deletion. Four patients had no WASP expression in peripheral leukocytes obtained before bone marrow transplantation. Several female carriers in the families of the 6 patients with such mutations were confirmed. One prenatal diagnosis was made in a fetus and he did not inherit the mutation. The importance of mutations in the first 2 exons of the WAS gene was demonstrated in this study, which represented 5 of the 6 mutations identified in 6 patients. The use of a multidisciplinary approach including DNA and protein analysis is required for molecular diagnosis and genetic counseling of WAS.     

1例Wiskott-Aldrich综合征临床特征和致病基因突变分析

目的:分析1例Wiskott-Aldrich综合征患者的临床特征及分子遗传特征。方法:分析患者的临床特征,收集患者及其父母外周血,提取基因组DNA,针对WASP基因所有编码外显子及外显子和内含子交界处进行PCR扩增测序。结果:我们报道的患者具有典型的WAS表型,临床得分为5分;患儿Coomb’s试验,自身抗体中ANA及类风湿因子均阳性,伴有自身免疫性疾病。患儿为WASP基因第7外显子中第665位核苷酸C突变为T(c.665C>T),导致211位密码子发生无义突变,该位置提前出现终止密码(p.R211X);其母亲为此突变基因携带者。结论:这例男性中国Wiskott-Aldrich综合征患儿由于WASP基因突变致病,此基因型患儿(p.R211X)拥有典型的WAS表型且伴有自身免疫性疾病的临床型为国内首次报道。     

Genetic characteristics of eighty-seven patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. WAS is caused by mutations in the WASP gene which encodes WASP, a 502-amino acid protein. WASP plays a critical role in actin cytoskeleton organization and signalling, and functions of immune cells. We present here the results of genetic analysis of patients with WAS from eleven Eastern and Central European (ECE) countries and Turkey. Clinical and haematological information of 87 affected males and 48 carrier females from 77 WAS families were collected. The WASP gene was sequenced from genomic DNA of patients with WAS, as well as their family members to identify carriers. In this large cohort, we identified 62 unique mutations including 17 novel sequence variants. The mutations were scattered throughout the WASP gene and included single base pair changes (17 missense and 11 nonsense mutations), 7 small insertions, 18 deletions, and 9 splice site defects. Genetic counselling and prenatal diagnosis were applied in four affected families. This study was part of the J Project aimed at identifying genetic basis of primary immunodeficiency disease in ECE countries. This report provides the first comprehensive overview of the molecular genetic and demographic features of WAS in ECE.     

Identification of WASP mutations in 10 Australian families with Wiskott-Aldrich syndrome and X-linked thrombocytopenia

Mutations of the gene encoding the Wiskott-Aldrich syndrome protein (WASP) have been previously shown to be responsible for classical Wiskott-Aldrich syndrome (WAS), isolated X-linked thrombocytopenia (XLT) and severe congenital X-linked neutropenia. AIMS: Identification of WASP mutations in 10 unrelated Australian families presenting with clinical features of WAS/XLT. METHODS: Mutation analysis was performed by PCR and sequence analysis. RESULTS AND CONCLUSIONS: Two novel mutations and seven mutations which have previously been reported were identified. The novel mutations consisted of a missense mutation in exon 2 (C290A) associated with the phenotype of XLT and a mutation in intron 10 (1372+1G>A) in the mother of two boys presenting with a classical WAS phenotype.     

GLA基因的新突变出现在两个典型Fabry家系

目的 报告2个Fabry家系的GLA基因突变特点。方法 2个经临床和病理检查证实的Fabry家系，家系1中连续3代有12人发病，均表现为发作性肢体疼痛；家系2中连续5代有8人发病，多数患者在经末期出现显著的多器官损害表现。对2个家系中的先证者和部分亲属进行PCR扩增其GLA基因的所有7个外显子包括侧翼序列，对PCR产物直接测序。结果 先证者1的GLA基因第1外显子G132T（TGG→TGT）突变，造成W44C替换；先证者2的第6外显子G874C（GCT→CCT），造成A292P替换。两个先证者的母亲都有同其儿子一样的突变，且均为杂合性。结论 经文献检索，两个Fabry家系各存在一个新的GLA基因点突变。同一基因的不同位点的突变导致的临床表现存在很大的差异。由于女性患者和男性一样出现症状，推测这可能是等位基因随机失活导致的显性遗传表现。     

A novel termination codon mutation of the WAS gene in a Thai family with Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by microthrombocytopenia, eczema, immunodeficiency, and susceptibility to lymphoid malignancy. Loss-of-function mutations in WAS gene have been identified to cause disorders with platelet defects including WAS and X-linked thrombocytopenia. Mutations anticipated to yield truncated or no protein have been associated with the more severe presentations of WAS. Activating mutations in WAS gene result in an entirely different phenotype, an X-linked severe congenital neutropenia. We describe a Thai family with classic WAS. The proband, a one-year-old boy presented with recurrent mucous bloody diarrhea, recurrent otitis media, chronic eczema, thrombocytopenia, and small platelet sizes. The patient's older brother who also had persistent thrombocytopenia died at the age of seven months from severe pneumonia. Immunoblot analysis demonstrated that the proband's cells lacked WAS protein expression. Mutation analysis of the proband and his mother for the entire coding region of WAS identified a novel type of mutation, a termination codon mutation, X503R. The change is expected to result in an elongated mRNA that would code for a WASP of 581 amino acid residues instead of the normal 502 residues. Because of the absence of WASP expression, we speculate that the termination codon mutation causes reduced mRNA stability. Our findings supported that WAS mutations resulted in no protein are associated with a severe phenotype of WAS.     

Germline mutations in the MEN1 gene: creation of a new splice acceptor site and insertion of 7 intron nucleotides into the mRNA.

The MEN1 tumor predisposition syndrome is caused by mutations in the MEN1 gene on human chromosome 11q13. We screened MEN1 gene exons 1-10 and flanking intron sequences from four different MEN1 families for mutations. In three families, heterozygous germline mutations within the exons were found, two of these representing novel mutations. In another family, all clinically affected members were heterozygous for a point mutation Gright curved arrow A within intron 4. Sequence analysis of cDNA from lymphocytes of the affected patients revealed that the intron mutation created a new acceptor splice site, leading to the inclusion of 7 bp of intronic sequence into the mRNA. The resulting frameshift generates a premature stop in codon 271. Intron borders should thus be screened for mutations in MEN1 diagnostics and cDNA sequence analysis is helpful in identifying pathophysiological consequences of intron mutations.     

Clinical and Molecular Characterization of Thai Patients with Wiskott–Aldrich Syndrome

Wiskott–Aldrich syndrome (WAS) is an X‐linked recessive primary immunodeficiency disorder caused by mutations in the gene encoding the WAS protein (WASP). Classic WAS is characterized by thrombocytopenia with small‐sized platelets, recurrent infections, eczema and increased susceptibility to autoimmune diseases and haematologic malignancies. Here, we reported on seven unrelated Thai individuals with classic WAS. In addition to clinical and immunologic characterization, mutation analysis by PCR‐sequencing the entire coding region of WASP was performed. Recurrent and novel mutations were successfully identified. A nonsense mutation, the c.55C>T (p.Q19X), has not been previously described, expanding the mutational spectrum of WASP. The patient with this newly described mutation developed cow's milk allergy manifesting as angioedema and urticaria and had cytomegalovirus infection that was successfully treated with long‐term ganciclovir. This study reported long‐term follow‐up of seven patients with molecular confirmation of WAS and infrequent features in the patient with classic WAS carrying the novel nonsense mutation.     

A second-site mutation in the initiation codon of WAS (WASP) results in expansion of subsets of lymphocytes in an Wiskott-Aldrich syndrome patient

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the gene encoding WAS protein (WASP ). Recently, somatic mosaicism caused by reversions or second-site mutations has been reported in some inherited disorders including WAS. In this article, we describe somatic mosaicism in a 15-year-old WAS patient due to a second-hit mutation in the initiation codon. The patient originally had a single-base deletion (c.11delG; p.G4fsX40) in the WAS (WASP) gene, which resulted in a frameshift and abrogated protein expression. Subsequently, a fraction of T and natural killer (NK) cells expressed a smaller WASP, which binds to its cellular partner WASP-interacting protein (WIP). The T and NK cells were found to have an additional mutation in the initiation codon (c.1A>T; p.M1_P5del). The results strongly suggest that the smaller WASP is translated from the second ATG downstream of the original mutation, and not only T cells but also NK cells carrying the second mutation acquired a growth advantage over WASP negative counterparts. To our knowledge, this is the first report describing somatic mosaicism due to a second-site mutation in the initiation codon of any inherited disorders.     

一个Wiskott-Aldrich综合征家系融S基因的突变分析CSCD

目的探讨1个汉族Wiskott-Aldrich综合征（Wiskott-Aldrich syndrome）家系WAS基因的突变情况。 方法PCR扩增WAS基因的外显子及外显子与内含子的连接区域,对PCR产物直接进行正、反向测序并与数据库进行比对,确定有无WAS基因突变以及突变的位点。测定家系成员B细胞活化因子（B-cell activating factor, BAFF）的水平。结果家系中2例患者均携带WAS基因第2外显子c.257G〉A的半合子突变,先证者的母亲为c.257G〉A的杂合突变携带者,其他表型正常家系成员均未检测到该突变,经查阅人类基因突变数据库证实该突变为已知致病突变。家系中患者血清BAFF水平明显高于表型正常的成员。结论WAS基因c.257G〉A的突变可能是该Wiskott-Aldrich综合征家系的致病原因。     

Seven novel mutations in the APC gene of Portuguese families with familial adenomatous polyposis: correlation with phenotype.

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). In the present study, we have used the protein truncation test to screen for mutations in exon 15 and exons 1-14 of the APC gene and denaturing gradient gel electrophoresis to analyze exons 1-14. We have studied nine unrelated FAP kindreds, eight with the classical phenotype and one with an atypical phenotype, with several family members exhibiting fewer than 50 colonic polyps. The combined use of these two methodologies allowed the identification of seven novel mutations, with two unrelated families sharing the same mutation. All mutations were chain terminating: six resulted from small deletions, one from a small insertion, and one was a point mutation, resulting in a premature stop codon. Seven mutations were located in exon 15 of the APC gene, one was in exon 10, and the remaining one, which corresponded to the kindred with an atypical phenotype, was located in exon 4.     

Deep intronic KRIT1 mutation in a family with clinically silent multiple cerebral cavernous malformations

Loss‐of‐function mutations in CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10 genes are identified in the vast majority of familial cases with multiple cerebral cavernous malformations (CCMs). However, genomic DNA sequencing combined to large rearrangement screening fails to detect a mutation in 5% of those cases. We report a family in which CCM lesions were discovered fortuitously because of the investigation of a developmental delay in a boy. Three members of the family on three generations had typical multiple CCM lesions and no clinical signs related to CCM. No mutation was detected using genomic DNA sequencing and quantitative multiplex PCR of short fluorescent fragments (QMPSF). cDNA sequencing showed a 99‐nucleotide insertion between exons 5 and 6 of CCM1, resulting from a mutation located deep into intron 5 (c.262+132_262+133del) that activates a cryptic splice site. This pseudoexon leads to a premature stop codon. These data highly suggest that deep intronic mutations explain part of the incomplete mutation detection rate in CCM patients and underline the importance of analyzing the cDNA to provide comprehensive CCM diagnostic tests. This kind of mutation may be responsible for apparent sporadic presentations due to a reduced penetrance.     

PALB2 mutations in European familial pancreatic cancer families

Slater EP, Langer P, Niemczyk E, Strauch K, Butler J, Habbe N, Neoptolemos JP, Greenhalf W, Bartsch DK. PALB2 mutations in European pancreatic cancer families. Recently, PALB2 was reported to be a new pancreatic cancer susceptibility gene as determined by exomic sequencing, as truncating PALB2 mutations were identified in 3 of 96 American patients with familial pancreatic cancer (FPC). Representing the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC) and the German National Case Collection for Familial Pancreatic Cancer (FaPaCa), we evaluated whether truncating mutations could also be detected in European FPC families. We have directly sequenced the 13 exons of the PALB2 gene in affected index patients of 81 FPC families. An index patient was defined as the first medically identified patient, stimulating investigation of other members of the family to discover a possible genetic factor. None of these patients carried a BRCA2 mutation. We identified three (3.7%) truncating PALB2 mutations, each producing different stop codons: R414X, 508‐9delAG and 3116delA. Interestingly, each of these three families also had a history of breast cancer. Therefore, PALB2 mutations might be causative for FPC in a small subset of European families, especially in those with an additional occurrence of breast cancer.     

Molecular biology of the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency associated with thrombocytopenia, bloody diarrhea, eczema, recurrent infections, and a high incidence of malignancies. X-linked thrombocytopenia (XLT) is a milder form with predominant platelet abnormalities. Both are caused by mutations of the cytoplasmic WAS protein (WASP). To date, mutations of WASP have been identified in over 340 families and consist of missense and nonsense mutations, deletions and insertions, and splice site mutations. There is a striking correlation between phenotype and genotype. The complex gene product of WASP has multiple functional domains that contribute to actin polymerization, cell motility, intracellular signaling, and apoptosis. Understanding the molecular basis of WAS/XLT not only explains the highly variable clinical phenotype, but also affects the medical management of this serious congenital disorder.