Characterization of novel Bruton's tyrosine kinase gene mutations in Central European patients with agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency disorder caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). In this study we investigated 10 male patients with XLA-compatible phenotype (agammaglobulinemia and undetectable B cells in peripheral blood) from 9 unrelated Central European families. We identified seven different mutations, six of which were novel. One previously described point mutation caused a premature stop codon (p.C464X), two point mutations resulted in amino acid exchanges (p.W588R; p.G419E), and two point mutations affected splice sites (c.305-1G>A; c.391+1G>A). We further detected one deletion (c.1921_1927del CGTCCCA) and one large duplication. The duplication resulted from Alu element-induced unequal homologous recombination, which was only detectable by extended analysis of cDNA, while direct sequencing of genomic DNA gave a false negative result. Western blot analysis revealed that the patients with the p.W588R and the p.G419E amino acid substitutions, respectively, produced full length BTK, but in clearly diminished amounts. The patient with the 7bp deletion expressed low amounts of protein which might represent truncated BTK. All other genomic alterations resulted in complete loss of BTK protein. In two patients from unrelated families BTK protein expression was normal and no Btk gene mutation was detected. The results of this study further substantiate the importance of using elaborate molecular analysis with different detection techniques to obtain an explicit molecular diagnosis in patients with suspected XLA.     

Analysis of Btk mutations in patients with X-linked agammaglobulinaemia (XLA) and determination of carrier status in normal female relatives: a nationwide study of Btk deficiency in Greece

Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase, critical for B-cell development and function. Mutations that inactivate this kinase were found in families with X-linked agammaglobulinaemia (XLA). In this study the Btk gene was analyzed in 13 registered Greek patients with XLA phenotype originated from 12 unrelated families, in order to provide a definite diagnosis of the XLA. The structure of Btk was analyzed at the cDNA level using the recently developed method, NIRCA (Non-Isotopic-Rnase-Cleavage-Assay). Alterations were detected in all patients and sequencing analysis confirmed the results and defined six novel XLA-associated Btk mutations (three missense mutations: C337G, L346R, L452P; one nonsense mutation: Y392X, and two frameshift alterations: c1211-1212delA, c1306-1307insA). Having defined the genetic alteration in the affected males of these families, the information was used to design polymerase chain reaction (PCR) primers and the Btk segments containing the mutated sequences were amplified from peripheral blood derived genomic DNA of potential female carriers. The PCR products were directly sequenced and carrier status was determined in 12 out of 16 phenotypically normal females analyzed. This protocol can be used once the nature of the Btk mutation has been defined in one of the affected males and provides a convenient, simple and reliable way to determine the carrier status of other female family members. Molecular genetic analysis constitutes a determinative tool for the definitive diagnosis of XLA and may allow accurate carrier and prenatal diagnosis for genetic counselling.     

Molecular analysis of Bruton’s tyrosine kinase gene in Spain

Mutations in Bruton’s tyrosine kinase (BTK) gene result in X linked agammaglobulinemia (XLA). Using Single Strand Conformation Polymorphism (SSCP) followed by direct sequencing 21 mutations were found in 27 patients with an XLA phenotype from 21 unrelated families. We identified 13 novel and 8 known mutations: seven missense (R288W, R544G, P566S, K430E; K374N, L512P, R544S), 5 nonsense (Q196X, Y361X, L249X, Q612X, Q466X), 2 deletions of one nucleotide (A207fsX216, Q612fsX648), 2 deletion‐insertions (V219fsX227, K218fsX228), one insertion of two nucleotides (S572fsX587) and 4 point mutations in donor/acceptor splice sites (g.IVS1+1G>C, g.IVS6+5G>A, g.IVS10+1G>T, g.IVS13‐1GG>CT). Carrier detection was performed in 18 mothers. Only in one case the mutation was found to be de novo. Additionally, BTK mutations were not found in four patients without family history, but with XLA‐compatible phenotype. Hum Mutat 18:84, 2001. © 2001 Wiley‐Liss, Inc.     

Mutation analysis of the Bruton's tyrosine kinase gene in X-linked agammaglobulinemia: identification of a mutation which affects the same codon as is altered in immunodeficient xid mice

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiencydisease in man, reflecting an arrest In differentiation of pre-Bcells to mature B cell stages. The gene defective in XLA hasbeen identified as a cytoplasmic protein tyrosine kinase, namedbtk (Bruton's tyrosine kinase). Here we report the characterizationof mutations in the btk gene of five unrelated XLA families.Amplified products were generated from cDNA, cloned and sequenced.Three single point mutations and two small insertions were Identified.One of the point mutations and the two Insertions created stopcodons that would lead to truncated btk proteins. In one XLApatient we found a single basepair substitution that alteredthe highly conserved Arg288 within the SH2 domain and wouldtherefore abrogate interactions with substrate phosphotyrosines.In another XLA patient a single basepair substitution was observedthat altered the conserved Arg28 residue in the N-terminal uniqueregion of unknown function. This residue is also mutated inthe xid mouse, which has a different, less severe, B cell deficiency.We conclude that a similar mutation in the btk gene leads Inman to an almost complete arrest at an early stage of B celldifferentiation, but In the mouse to only limited B cell abnormalities.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

Bruton tyrosine kinase gene mutations in Turkish patients with presumed X‐linked agammaglobulinemia

X‐linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B‐cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800‐1821del, 1843‐1847del, 1288‐1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA. Hum Mutat 18:356, 2001. © 2001 Wiley‐Liss, Inc.     

BTK mutations in patients with X-linked agammaglobulinemia: lack of correlation between presence of peripheral B lymphocytes and specific mutations

X-linked agammaglobulinemia (XLA) is a human antibody deficiency that results from mutation of the tyrosine kinase btk. We tested the hypothesis that XLA patients who varied from the classic phenotype of XLA by presence of normal or near normal number of peripheral B lymphocytes would have a set of mutations of BTK that is different from the mutations found in patients without peripheral B lymphocytes. The mutations of BTK we found in two patients with normal numbers of peripheral B lymphocytes have been previously identified in patients without peripheral B lymphocytes. A third patient, without peripheral B cells, was found to express normal levels of wild type btk. Exmination of the mutations of the BTK gene in patients in the BTKbase who were identified as having peripheral B lymphocytes found that these same mutations, or mutations of the same protein domains, were also present in patients identified as lacking peripheral B lymphocytes. Analysis of mutations in BTK has previously led to the conclusion that severity of disease in XLA cannot be predicted from the specific mutation of BTK. The results of this study suggest that whether an XLA patient will develop peripheral B lymphocytes cannot be predicted from the specific mutation of BTK.     

Identification of mutations in the Bruton's tyrosine kinase gene,including a novel genomic rearrangements resulting in large deletion,in Korean X-linked agammaglobulinemia patients

Mutations in the Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). We identified BTK mutations in six patients with presumed XLA from unrelated Korean families. Four out of six mutations were novel: two missense mutations (P565T, C154Y), a point mutation in a splicing donor site (IVS11+1G>A), and a large deletion (a 6.1-kb deletion including BTK exons 11–18). The large deletion, identified by long-distance PCR, revealed Alu-Alu mediated recombination extended from an Alu sequence in intron 10 to another Alu sequence in intron 18, spanning a distance of 6.1 kb. The two known mutations consisted of one missense (G462D) mutation, and a point mutation in a splicing acceptor site (IVS7−9A>G). This study suggests that large genomic rearrangements involving Alu repeats are few but an important component of the spectrum of BTK mutations.     

BRCA1 and BRCA2 mutations in a South American population

A sample of 64 high-risk breast and/or ovarian cancer families from Chile were screened for germline mutations in the coding sequences and exon-intron boundaries of BRCA1 (MIN no. 113705) and BRCA2 (MIN no. 600185) genes using conformation-sensitive gel electrophoresis, and the mutations found were confirmed with direct sequencing. Seven families (10.9%) were found to carry BRCA1 mutations and three families (4.7%) had BRCA2 mutations. Six different pathogenic mutations were detected in BRCA1, four that had been reported previously (c.187_188delAG; c.300T-->G, c.3450_3453delCAAG and IVS17-1G-->A) and two novel mutations (c.2605_2606delTT and c.4185_4188delCAAG). In BRCA2, we found three different pathogenic mutations, two previously described (c.6174delT and c.6503_6504delTT) and one novel mutation (c.5667delT). We also identified nine variants of unknown significance (five in BRCA1 and four in BRCA2). These findings indicate that the Chilean population has a heterogeneous spectrum of prevalent BRCA mutations. Given the results obtained in our study, the screening of the entire BRCA1 and BRCA2 coding regions is necessary for the molecular genetic testing of Chilean high-risk breast/ovarian cancer patients. To our knowledge, this is the first genetic study of BRCA gene mutations conducted in Chile. The Chilean population has a well-known admixed Amerindian-Caucasian ratio and, therefore, our findings are not only important per se, but they constitute the basis for improved and more specific genetic counselling, as well as to support for preventive campaigns geared toward the Chilean population.     

High frequency of OPA1 mutations causing high ADOA prevalence in south-eastern Sicily, Italy

Optic atrophy type 1 (OPA1) gene mutation causes autosomal dominant optic atrophy (ADOA, MIM #165500). Prevalence of ADOA ranges from 1:50,000 in most populations to 1:12,000 in Denmark. Seventy members of nine families were analysed for the presence of OPA1 gene mutations by polymerase chain reaction (PCR) and direct sequencing. We identified three OPA1 gene mutations in 48 patients with variable signs of optic atrophy. Two mutations, c.784-21_784-22insAluYb8 and c.876_878delTGT, were found in two different families. The third mutation, c.869G>A, was found in 28 patients from seven families. The haplotype analysis data suggested that the c.869G>A mutation is a founder mutation. Our main result suggests a higher ADOA prevalence in south-eastern Sicily than previously found in Denmark. This is because of not only the founder effect but also to the presence of three different mutations in the geographical area of the study. Our hypothesis is that a combination of social pressure because of blindness and migration factors is involved. In fact, in Siracusa, a provincial capital in south-eastern Sicily, St. Lucy, the patron saint of the blind was born and died.     

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.     

Mutation of the <Emphasis Type="Italic">BTK</Emphasis> Gene and Clinical Feature of X-Linked Agammaglobulinemia in Mainland China

Introduction  X-Linked agammaglobulinemia is a prototypical humoral immunodeficiency with the mutation of the Bruton’s tyrosine kinase gene. Methods  We investigated the gene mutation and clinical features of 30 Chinese X-linked agammaglobulinemia (XLA) patients from 27 families. There were 26 mutations, including 11 novel and 15 recurrent mutations, distributing over the entire gene. The nucleotide and amino acid aberration, 1129C>T(H333Y) and 1196T>A(I355N), in SH2 have not been reported before. Five (I355N, W124R, R520X, I590F, G594E) of the 24 mutations not detected in the mothers receiving gene analysis were determined to be de novo. Two mutations occurred within intronic splice-site sequences (intron5(−2)A>G, intron17(−2)A>T). Results and Discussion  There are eight mutations in the PH domain, two mutations in the SH3 domain, three mutations in the SH2 domain, one mutation in the TH domain, and other 16 mutations in the TK domain. The mutations of protein domain is most common in TK (53%) domain and then in PH(8%) domain. Missense and nonsense mutations were found equal in 46% of the detected mutations. All of the patients are alive, but one died of liver cancer. Clinical features and serum Igs levels range variedly and were not correlated with genotypes. Our results demonstrated molecular genetic characteristics of XLA in mainland China.     

The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 80 patients from 72 families by means of a PCR-based mutation screening strategy, consisting of heteroduplex analysis, restriction enzyme digest, DNA single strand electrophoresis, and direct sequencing. For a subset of patients mutation screening with DHPLC was established which turned out to be as fast and as sensitive as the more conventional methods. Fifteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in smaller previous studies, p.A150P (65%), p.A175D (11%) and p.N335K (8%) were the most common mutated alleles, followed by c.360_363delCAAA, p.R60X, p.Y204X, and c.865delC. Eight novel mutations were identified in eight families with HFI: a small indel mutation (c.1044_1049delTTCTGGinsACACT), two small deletions (c.345_372del28; c.841_842delAC), two splice site mutations (c.113-1G>A, c.799+2T>A), one nonsense mutation (c.612T>G (p.Y204X)), and two missense mutations (c.532T>C (p.C178R), c.851T>C (p.L284P)). By mutation screening for the three most common ALDOB mutations by DHPLC in 2,000 randomly selected newborns we detected 21 heterozygotes. Based on these data and after correction for less common and private ALDOB mutations, HFI prevalence in central Europe is estimated to be 1:26,100 (95% confidence interval 1: 12,600-79,000).     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families

Hereditary non-polyposis colorectal cancer (HNPCC), the most common hereditary colon cancer syndrome, is a dominant disorder caused by germline defects in mismatch repair (MMR) genes. Identification of MMR gene mutations can have direct clinical implications in counseling and management of HNPCC families. We screened 44 HNPCC and 97 suspected HNPCC Korean families for germline mutations in three MMR genes: MLH1, MSH2 and MSH6. We identified twelve novel mutations: nine in MLH1(c.632_633insT, c.808_811delACTT, c.845C>G, c.1625A>C, c.1730+1delG, c.1907T>C, c.1918C>T, c.2104-2A>G and c.2170T>A), two in MSH2 (c.1886A>G, c.1316_1318delCCT) and one in MSH6 (c.3488A>T). In addition, two statically significant cSNPs in MLH1: c.1128T>C ( p=0.008 in HNPCC and p=0.037 in early-onset CRC) and c.2168C>A ( p<0.001 in HNPCC). Interestingly, the most frequent mutation, c.1757_1758insC in MLH1, was a founder mutation inherited from a common Korean ancestor.     

Characterization of germline mutations of the gene encoding Bruton's tyrosine kinase in families with X-linked agammaglobulinemia

Bruton's tyrosine kinase (Btk) has been identified as the protein responsible for the primary immunodeficiency X-linked agammaglobulinemia (XLA) and has been described as a new member of Srcrelated cytoplasmic protein tyrosine kinases. We have recently characterized the structure of the entire gene encoding Btk and developed a polymerase chain reaction (PCR)-based assay to detect germline mutations within it. In this report we describe six mutations, five of which are novel, of the Btk gene in patients with XLA and demonstrate the inheritance pattern of the defect within the families of the affected individuals. The mutations found include two nonsense and two missense mutations, a single base deletion at an intron acceptor splice site, and a 16-bp insertion. A single Strand conformation polymorphism was also found in the 5′ end of intron 8 with the same assay. This technique has provided a powerful tool for direct analysis of the Btk gene for the diagnosis of XLA and carrier detection. The identification of new mutations may eventually reveal the role of Btk in the signaling pathways involved in B-cell development. © 1995 Wiley-Liss, Inc.     

Genomic organization of the Btk gene and exon scanning for mutations in patients with X-linked agammaglobulinemia

The defective gene responsible for the recessively inheritedimmunodeficiency X-linked agammaglobulinemia (XLA) has beenshown to encode a cytoplasmic protein tyrosine kinase of theSrc family designated Btk (Bruton's tyrosine kinase). To facilitatethe search for germline mutations of the Btk gene, we have characterizedits genomic structure. Eighteen introns were positioned withinthe approximately 37 kb gene. Each of the exon/intron boundarieswere defined and sequenced, and all but two conform to consensussequences. We have utilized the genomic organization of Btkand the intervening sequence data to design an assay for amplifyingeach of the 19 exons from XLA patient DNA for single strandconformation polymorphism (SSCP) analysis. By using this methodwe have identified mutations in 12 of 14 unrelated affectedmales: seven different base substitutions and two small deletions.Two of the mutations described in exon 15 of the kinase domainwere found in more than one patient and may represent a mutationhot spot. Exon scanning has proven to be a valuable method foridentifying the patient mutations in genomic DNA without theuse of cDNA. The mutations are easily confirmed with directsequencing of the amplified exons. This approach will greatlybenefit XLA family studies involving carrier detection and prenataldiagnosis. In addition, the mutations identified may revealresidues involved in the specific protein interactions necessaryin the B-cell developmental pathway, of which Btk is an integralcomponent.     

Mutation analysis of the parkin and PINK1 genes in American Caucasian early-onset Parkinson disease families

Mutations in the parkin gene and the PTEN-induced putative kinase 1 gene (PINK1) have been identified as the most common causes of autosomal recessive early-onset Parkinson disease (EOPD). To investigate the presence of the parkin and PINK1 gene mutation(s) and to explore genotype-phenotype correlations in American Caucasian families with EOPD from North American, we screened these two genes in probands of six families by direct sequencing, semi-quantitative PCR and RT-PCR. No PINK1 gene mutation was found in any of the probands, but compound heterozygous mutations (EX 3 del and EX 3_4 del) in the parkin gene were identified in one family. Extended analysis of the parkin-positive family showed the phenotype of patients was that of classic autosomal recessive EOPD, characterized by early age at onset, slow progression, beneficial response to levodopa, and levodopa-related motor complications. Three heterozygous mutation carriers (EX 3 del or EX 3_4 del) were free of any neurological symptoms. None of 62 healthy controls harbored EX 3 del or EX 3_4 del mutation. Our data suggest that compound heterozygous mutations (EX 3 and EX 3_4 del) in the parkin gene were the cause of EOPD in one of six Caucasian families; heterozygous EX 3 del and heterozygous EX 3_4 del forms were insufficient to cause this disorder, consistent with a loss-of-function mechanism of the parkin mutations. The results may provide new insights into the cause and diagnosis of PD and have implications for genetic counseling.