Ataxia telangiectasia: more variation at clinical and cellular levels

Ataxia telangiectasia (A‐T) is a rare recessively inherited disorder resulting in a progressive neurological decline. It is caused by biallelic mutation of the ATM gene that encodes a 370 kDa serine/threonine protein kinase responsible for phosphorylating many target proteins. ATM is activated by auto(trans)phosphorylation in response to DNA double strand breaks and leads to the activation of cell cycle checkpoints and either DNA repair or apoptosis as part of the cellular response to DNA damage. The allelic heterogeneity in A‐T is striking. While the majority of mutations are truncating, leading to instability and loss of the ATM protein from the allele, a significant proportion of patients carry one of a small number of mutations that are either missense or leaky splice site mutations resulting in retention of some ATM with activity. The allelic heterogeneity in ATM, therefore, results in an equally striking clinical heterogeneity. There is also locus heterogeneity because mutation of the MRE11 gene can cause an obvious A‐T like disorder both clinically and also at the cellular level and mutation of the RNF168 gene results in a much milder clinical phenotype, neurologically, with the major clinical feature being an immunological defect.     

Mutational analysis of the SH2-kinase linker region of Bruton's tyrosine kinase defines alternative modes of regulation for cytoplasmic tyrosine kinase families

Bruton's tyrosine kinase (Btk) plays critical roles in B cell development and activation. Mutations of Btk cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. An Src homology domain 2-kinase linker region exists in all Src, Abl, ZAP70/Syk and Btk/Tec non-receptor tyrosine kinase families. Missense mutations in the Btk linker region can cause XLA, supporting an essential role for this protein segment. We investigated the regulatory role of the linker region in Btk function by mutational analysis. XLA-causing mutations L369F and R372G abolished Btk-mediated calcium response without affecting Btk protein stability and kinase activity significantly. Although mutation of a well-conserved tryptophan (W260A) in the linker region of the Src family kinase Hck has been shown to cause a hyperactive kinase, an analogous mutation in Btk (W395A) dramatically decreased Btk kinase activity. Tyrosine phosphorylation in the linker region was previously shown to regulate the function of Abl and ZAP70/Syk kinases. Even though tyrosine phosphorylation was detected on tyrosine 375 in the Btk linker region, no significant alteration was observed in Btk-signaling activity and biological function when this tyrosine was mutated in DT-40 cells or in Y375F knock-in mice. Our data and previous studies suggest that each cytoplasmic tyrosine kinase family has evolved a unique strategy to utilize the linker region to regulate the function of the enzyme.     

Large deletions and splicing‐site mutations in the STK11 gene in Peutz‐Jeghers Chilean families

Peutz‐Jeghers syndrome (PJS) is an autosomal dominant disorder characterized by mucocutaneous melanocytic macules, gastrointestinal hamartomatous polyposis and an increased risk of various neoplasms. Germline mutations in the serine/threonine kinase 11 (STK11) gene have been identified as a cause for PJS. The aim of this study was to characterize the genotype of Chilean PJS patients. Mutation screening of 13 patients from eight PJS families was performed using a single strand conformation polymorphism analysis, DNA sequencing and multiplex ligation‐dependent probe amplification assay. The breakpoints of the genomic rearrangements were assessed by a long‐range polymerase chain reaction and sequencing. The results revealed the existence of seven different pathogenic mutations in STK11 gene in seven unrelated families, including three point mutations and four large genomic deletions. Three of these point mutations (43%, 3/7) may be considered as novel. Our results showed that a germline mutation is present in STK11 in 88% of probands fulfilling the diagnostic criteria of PJS. In this study, the combination of two different experimental approaches in the screening of the STK11 in PJS, led to a higher percentage of mutation detection.     

Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.     

Oncogenic potential is related to activating effect of cancer single and double somatic mutations in receptor tyrosine kinases

Aberrant activation of receptor tyrosine kinases (RTKs) is a common feature of many cancer cells. It was previously suggested that the mechanisms of kinase activation in cancer might be linked to transitions between active and inactive states. Here, we estimate the effects of single and double cancer mutations on the stability of active and inactive states of the kinase domains from different RTKs. We show that singleton cancer mutations destabilize active and inactive states; however, inactive states are destabilized more than the active ones, leading to kinase activation. We show that there exists a relationship between the estimate of oncogenic potential of cancer mutation and kinase activation. Namely, more frequent mutations have a higher activating effect, which might allow us to predict the activating effect of the mutations from the mutation spectra. Independent evolutionary analysis of mutation spectra complements this observation and finds the same frequency threshold defining mutation hotspots. We analyze double mutations and report a positive epistasis and additional advantage of doublets with respect to cancer cell fitness. The activation mechanisms of double mutations differ from those of single mutations and double mutation spectrum is found to be dissimilar to the mutation spectrum of singletons. Hum Mutat 33:1566–1575, 2012. Published 2012 Wiley Periodicals, Inc.*     

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.     

Novel insertions of Bruton tyrosine kinase in patients with X-linked agammaglobulinemia

Mutations in the gene encoding Bruton tyrosine kinase (BTK) result in X-linked agammaglobulinemia (XLA), an immunodeficiency of antibody defect. By using base excision sequence scanning method (BESS) followed by direct sequencing we found in seven unrelated families with a classical XLA phenotype various mutations including six novel mutations (g.64512_64513insC, c.108_109insG, c.1700_1701insACTACAG, g.51375_51376GC>TG, g.63991_63992insGGTAGAAAAAA, c.1956_1957insCA) and a previously known silent polymorphism (c.2031C>T). Except for two mutations, the alterations affect the kinase domain. There was exceptionally high proportion of insertions in the cohort. Frameshift insertion was found altogether in five patients, three of which are on introns, one in upstream region, and one in exon 18 leading to frameshift mutation and truncation of the protein. In the intron 4 there is a substitution of two bases. Carrier detection was performed in four families. In one case the mutation was found to be de novo.     

Mutations in the human LKB1/STK11 gene

The human LKB gene (official HUGO symbol, STK11) encodes a serine/threonine protein kinase that is defective in patients with Peutz-Jeghers syndrome (PJS). PJS is an autosomal dominantly inherited syndrome characterized by hamartomatous polyposis of the gastrointestinal tract and mucocutaneous pigmentation. To date, 145 different germline LKB1 mutations have been reported. The majority of the mutations lead to a truncated protein product. One mutational hotspot has been observed. A 1-bp deletion and a 1-bp insertion at the mononucleotide repeat (C6 repeat, c.837-c.842) between the codons 279-281 have been found in six and seven unrelated PJS families, respectively. However, these mutations account only for approximately 7% of all mutations identified in the PJS families (13/193). A review of the literature provides a total of 40 different somatic LKB1 mutations in 41 sporadic tumors and seven cancer cell lines. Mutations occur particularly in lung and colorectal cancer. Most of the somatic LKB1 mutations result in truncation of the protein. A mutational hotspot seems to be a C6 repeat accounting for 12.5% of all somatic mutations (6/48). These results are concordant with the germline mutation spectrum. However, the proportion of the missense mutations seems to be higher among the somatic mutations (45%) than among the germline mutations (21%), and only seven of the mutations are exactly the same in both of the mutation types.     

The mutation spectrum of the APC gene in FAP patients from southern Italy: detection of known and four novel mutations

Familial adenomatous polyposis (FAP), an autosomal dominantly inherited condition accounting for about 1% of all colorectal cancers, results from mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. The clinical spectrum and severity of FAP varies greatly with the mutation site, and both between and within families. Using the protein truncation test, single strand conformation polymorphism analysis and DNA sequencing, we identified 30 (75%) mutant alleles in 40 unrelated FAP families, for a total of 22 different APC mutations. Of these, 18 are known and 4 are novel: c.1797C>A (C599X), c.893_894delAC, (c.3225T>A; c.3226C>A) and c.4526_4527insT. Of the 30 APC gene mutations, 5 (approximately 17%) are nonsense mutations, 17 (approximately 57%) are small deletions, 5 (approximately 17%) are small insertions and 3 (approximately 10%) are complete deletions. All mutations occurred in single pedigrees, except those at codons 1061 and 1062, each found in two unrelated families, and the mutation at codon 1309 in exon 15, found in five unrelated families. About 40% of mutations, mostly small deletions and insertions, are located at repeated sequences; they promote misalignment-mediated errors in DNA replication and could represent a hot spot mutation region. This study enlarges the spectrum of APC gene mutations and sheds light on the correlation between the site of APC germline mutations and clinical manifestations of FAP.     

Death-associated protein kinase (DAP kinase) alteration in soft tissue leiomyosarcoma: Promoter methylation or homozygous deletion is associated with a loss of DAP kinase expression

The death-associated protein kinase (DAP kinase) was initially identified as a positive mediator of programmed cell death induced by interferon-gamma. To investigate the potential role and the alteration of the DAP kinase gene in soft tissue leiomyosarcoma (LMS), we first searched for homozygous deletion and promoter hypermethylation in 45 LMSs for which genomic DNA was available, using differential PCR and methylation-specific PCR, respectively. Promoter methylation was recognized in 10 of 45 cases (22%), and homozygous deletion was detected in 3 of 45 cases (7%). p53 mutation was detected in 11 of 45 LMS cases (24%). Cases with DAP kinase alteration or p53 mutation showed a close correlation with high French Federation of Cancer Centers grade or with poor prognosis (P = 0.0244, P = 0.0491, respectively). Next, to determine that DAP kinase promoter methylation or homozygous deletion is involved in the down-regulation of DAP kinase expression, we examined the expression of DAP kinase protein by immunohistochemistry. Decreased expression of DAP kinase protein was recognized in 13 of 45 LMS cases (29%). Seven of 13 cases (54%) with decreased expression of DAP kinase protein revealed promoter methylation or homozygous deletion of DAP kinase, and the methylation status or homozygous deletion of its gene showed a close correlation with decreased DAP kinase expression (P = 0.0300). In conclusion, although DAP kinase alteration was relatively rare, DAP kinase alteration and/or p53 mutation may associate with tumor progression in soft-tissue LMSs. Furthermore, although further detailed analyses are necessary, promoter methylation or homozygous deletion status of DAP kinase may present a major alternative mechanism of a loss of or decrease in DAP kinase expression.     

中国人遗传性非息肉病性结直肠癌MSH6基因胚系突变的测序研究

目的探讨中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系中MSH6基因胚系突变。方法采用PCR-直接测序的方法检测39个无胚系MSH2及MLH1基因突变、符合不同临床标准的中国人HNPCC家系先证者MSH6基因各外显子胚系突变;对137名正常人胚系基因组DNA进行错义突变相应外显子的测序分析。应用Envision二步法检测有突变的先证者肿瘤组织MSH6蛋白表达。结果在39个HNPCC先证者中共发现6个MSH6基因的胚系突变,分别位于第4、6、9和第10外显子;突变类型为4个错义突变、1个无义突变、1个剪接区的插入突变;对4个错义突变的相应外显子的测序分析显示:137名正常人胚系基因组DNA5例具有第6外显子1163密码子处的c.3488A>T的错义突变,约占3.65%(5/137),为单核苷酸多态性(single nucleotide polymorphism,SNP);其余错义突变在正常人群中均未发现。在6例有MSH6基因胚系突变家系的肿瘤组织中免疫组化染色除1例为SNP的肿瘤组织MSH6蛋白阳性表达外,其余均为阴性表达。经过查询国际HNPCC突变数据库及SNP数据库证实上述突变中5个为国际上尚未报道的病理性突变,1个为新发现的SNP。结论MSH6基因胚系突变在符合不同临床标准的中国人HNPCC中均起一定作用,对无MSH2及MLH1基因胚系突变的先证者行MSH6基因胚系突变的测序分析对确诊HNPCC家系是必要的。     

Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.     

Genetic analysis of 14 families with Schnyder crystalline corneal dystrophy reveals clues to UBIAD1 protein function

Schnyder crystalline corneal dystrophy (SCCD) is a rare autosomal dominant disease characterized by progressive corneal opacification resulting from abnormal deposition of cholesterol and phospholipids. Recently, six different mutations on the UBIAD1 gene on chromosome 1p36 were found to result in SCCD. The purpose of this article is to further characterize the mutation spectrum of SCCD and identify structural and functional consequences for UBIAD1 protein activity. DNA sequencing was performed on samples from 36 individuals from 14 SCCD families. One affected individual was African American and SCCD has not been previously reported in this ethnic group. We identified UBIAD1 mutations in all 14 families which had 30 affected and 6 unaffected individuals. Eight different UBIAD1 mutations, 5 novel (L121F, D118G, and S171P in exon 1, G186R and D236E in exon 2) were identified. In four families with DNA samples from both affected and unaffected individuals, the D118G, G186R, T175I, and G177R mutations cosegregated with SCCD. In combination with our previous report, we have identified the genetic mutation in UBIAD1 in 20 unrelated families with 10 (including 5 reported here), having the N102S mutation. The results suggest that N102S may be a mutation hot spot because the affected families were unrelated including Caucasian and Asian individuals. There was no genotype phenotype correlation except for the T175I mutation which demonstrated prominent diffuse corneal haze, typically without corneal crystals. Protein analysis revealed structural and functional implications of SCCD mutations which may affect UBIAD1 function, ligand binding and interaction with binding partners, like apo E.     

帕金森病致病基因LRRK2的研究进展

富含亮氨酸重复序列激酶2基因(1eucine-rich repeat kinase 2 gene,LRRK2)是家族性常染色体显性遗传性帕金森病8型的致病基因,其突变所致帕金森病具有与原发性帕金森病极为相似的临床表现,但神经病理改变却呈现出多样性.且突变类型、发生频率在不同人种间存在显著性差异.IRRK2在体内分布广泛,功能复杂,具有多个功能结构域,序列比对后推测,LRRK2可能作为一种对底物蛋白进行磷酸化修饰的多功能激酶或支架蛋白来发挥功能.对LRPK2正常生理功能及致病机制的研究,有助于深入探索帕金森病的发病机制以及寻求有效的防治措施.     

Successful valganciclovir treatment of post‐transplant cytomegalovirus infection in the presence of UL97 mutation N597D

Mutations in the human cytomegalovirus (CMV) UL97 protein kinase are the most common mechanism of ganciclovir (GCV) resistance in the clinical setting. A CMV strain with a previously unrecognized UL97 mutation N597D was identified in the blood of a heart transplant recipient who experienced a persistent CMV infection with high viral loads accompanying pain and fever while receiving valganciclovir (valGCV) therapy. The N597D mutation was transferred by mutagenesis to an antiviral sensitive CMV strain for analysis of antiviral susceptibility by standardized phenotypic assay. Recombinant phenotyping showed N597D conferred a less than twofold increase in GCV IC₅₀ compared to the sensitive control strain. Despite the presence of this mutation, valGCV eventually resolved the infection after 6 weeks of therapy. A subsequent CMV reactivation was also responsive to valganciclovir. This case illustrates the diversity of UL97 mutations in the codon segment 590–607 usually associated with GCV resistance, with some mutations producing minimal levels of resistance that do not preclude a therapeutic response to the drug. Accurate interpretation of genotypic test results ultimately requires experimental determination of the level of resistance conferred by newly discovered UL97 mutations. J. Med. Virol. 81:507–510, 2009. © 2009 Wiley‐Liss, Inc.     

蛋白激酶C对高迁移率蛋白I的磷酸化及其位点分析

纯化的重组人HMGI可被蛋白激酶C（PKC）迅速磷酸化,其化学计量达到2.0～2．5mol／mol,经过蛋白酶水解得到3个磷酸化肽段。磷酸氨基酸分析和氨基酸测序结果表明,该蛋白分子的44和64位的丝氨酸（Ser）是PKC主要的磷酸化位点,其中Ser－64在其第二个DNA结合结构域的C端近旁,而Ser-44则位于第一和第二个DNA结合结构域之间。另外,在75～78位的4个苏氨酸中存在低水平的磷酸化。该结果提示HMGI上PKC的磷酸化位点和CDC2激酶的位点显著不同。后者的位点主要为53和78位的苏氨酸。     

Protein S gene analysis reveals the presence of a cosegregating mutation in most pedigrees with type I but not type III PS deficiency

DNA sequence analysis of the protein S gene (PROS1) in 22 Spanish probands with type I or III PS deficiency, has allowed the identification of 10 different mutations and 2 new sequence variants in 15 probands. Nine of the mutations, 8 of which are novel, cosegregate with type I or quantitative PS deficiency in 12 of the 13 pedigrees analyzed. One of these mutations (Q238X) also cosegregates with both type I and III PS‐deficient phenotypes coexisting in a type I/III pedigree. Another mutation identified in a pedigree with these two PS phenotypes is the missense mutation R520G, present in the homozygous form in the type I propositus and in the heterozygous form in his type III relatives. By contrast, no cosegregating PROS1 mutation has been found in any of the six families with only type III phenotypes. Three of these families, as well as the two families with type I and I/III phenotypes where no other PROS1 mutation has been identified, segregate the P allele of the S460P variant, although this allele does not always cosegregate with the deficient phenotype. From these results we conclude that while mutations in PROS1 are the main cause of type I PS deficiency, the molecular basis of the type III phenotype is probably more complex, with many cases not being explained by a PROS1 mutation. Hum Mutat 14:30–39, 1999. © 1999 Wiley‐Liss, Inc.     

KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs)

Mutually exclusive KIT and PDGFRA mutations are central events in GIST pathogenesis, and their understanding is becoming increasingly important, because specific treatment targeting oncogenic KIT and PDGFRA activation (especially imatinib mesylate) has become available. KIT mutations in GIST are clustered in four exons. Most common are exon 11 (juxtamembrane domain) mutations that include deletions, point mutations (affecting a few codons), and duplications (mostly in the 3' region). The latter mutations most often occur in gastric GISTs. Among gastric GISTs, tumors with deletions are more aggressive than those with point mutations; this does not seem to hold true in small intestinal GISTs. Exon 9 mutations (5-10%) usually are 2-codon 502-503 duplications, and these occur predominantly in intestinal versus gastric GISTs. Lesser imatinib sensitivity of these tumors has been noted. Kinase domain mutations are very rare; GISTs with such mutations are variably sensitive to imatinib. PDGFRA mutations usually occur in gastric GISTs, especially in the epithelioid variants; their overall frequency is approximately 30% to 40% of KIT mutation negative GISTs. Most common is exon 18 mutation leading Asp842Val at the protein level. This mutation causes imatinib resistance. Exon 12 and 14 mutations are rare. Most mutations are somatic (in tumor tissue only), but patients with familial GIST syndrome have consitutitonal KIT/PDGFRA mutations; >10 families have been reported worldwide with mutations generally similar to those in sporadic GISTs. GISTs in neurofibromatosis 1 patients, children, and Carney triad seem to lack GIST-specific KIT and PDGFRA mutations and may have a different disease mechanism. Secondary mutations usually occur in KIT kinase domains in patients after imatinib treatment resulting in resistance to this drug. Mutation genotyping is a tool in GIST diagnosis and in assessment of sensitivity to kinase inhibitors. This is a US government work. There are no restrictions on its use.     

On the sequence-directed nature of human gene mutation: the role of genomic architecture and the local DNA sequence environment in mediating gene mutations underlying human inherited disease

Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher order features of the genomic architecture. The human genome is now recognized to contain "pervasive architectural flaws" in that certain DNA sequences are inherently mutation prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here, we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of noncanonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair and may serve to increase mutation frequencies in generalized fashion (i.e., both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease.