Loss of heterozygosity and mutations are the major mechanisms of RB1 gene inactivation in Chinese with sporadic retinoblastoma

We investigated sequence alternation, promoter methylation, and loss of heterozygosity (LOH) of the RB1 gene as possible mechanisms of its inactivation in retinoblastoma. In 42 Chinese patients with sporadic retinoblastoma, the promoter and entire coding region of RB1 were examined for sequence changes. Status of methylation of the CpG-rich island at the 5'end was determined by methylation specific PCR assay. We detected 15 RB1 mutations in 38% (16/42) of the retinoblastoma patients, among them 19% (8/42) were germ-line mutations. A total of nine novel mutations were identified: E54X, S114X, I126S, g73779insG, D718N, IVS2+1G>C, IVS14+1G>C, IVS21+1G>C, and a complex alteration g78177G>T/g78176insTT leading to 543X. Most of them are likely to affect the RB1large pocket domain through the production of truncated gene products. None of the DNA samples showed methylation at the RB1promoter. In 15 cases where both normal and cancerous retinoblastoma tissue specimens were available, allelic loss according to microsatellite markers within or distal to the RB1 locus was analyzed and immunohistological staining for RB1 expression performed. Among them, frequency of LOH at 13q14 was found to be high at 60% (9/15) with no segregation with unilateral tumors. All these nine tumors did not express RB1 protein, showing an association of LOH at the RB1 locus with its loss of expression in retinoblastoma. Our results indicate that the RB1 gene in sporadic retinoblastoma is commonly inactivated because of loss-of-function mutations and loss of heterozygosity but not by the epigenetic phenomenon of promoter hypermethylation.     

Mutation analysis of the BRCA1 and BRCA2 genes results in the identification of novel and recurrent mutations in 6/16 flemish families with breast and/or ovarian cancer but not in 12 sporadic patients with early-onset disease. Mutations in brief no. 224. Online

Since the identification of the BRCA1 and BRCA2 genes (MIM#s 113705 and 600185), more than hundred different mutations throughout both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. We analyzed 12 sporadic female patients with breast cancer before age 35, as well as 16 unrelated families, presenting with either (i) at least 3 first degree relatives with breast and/or ovarian cancer diagnosed at any age, or (ii) at least 2 first and/or second degree relatives with breast and/or ovarian cancer before age 45 years. We performed a protein truncation test for BRCA1 exon 11 and BRCA2 exons 10 and 11 and heteroduplex analysis for all the remaining exons of BRCA1 and 2. Presence of genomic deletions encompassing exons 13 or 22 of BRCA1, known to be Dutch founder mutations, was investigated by PCR. In 6/16 (37.5%) unrelated families the causal mutation in either the BRCA1 or BRCA2 gene was identified. Four different mutations were found in the BRCA1 gene: IVS5+3A>G (intron 5), 1191delC (exon 11), R1443X (exon 13), IVS22+5G>A (intron 22) and two in the BRCA2 gene: 6503delTT (exon 11), 6831delTG (exon 11). 1191delC (BRCA1) and 6831delTG (BRCA2) are novel mutations. IVS5+3A>G in exon 5 of BRCA1 published by Peelen et al. (1997) as a novel Belgian mutation, was identified in one additional family, not fulfilling our inclusion criteria. In the group of 12 sporadic female patients no mutations were found.     

Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF

Constitutional mutations of the RB1 gene are associated with a predisposition to retinoblastoma. It is essential to identify these mutations to provide appropriate genetic counseling in retinoblastoma patients, but this represents an extremely challenging task, as the vast majority of mutations are unique and spread over the entire coding sequence. Since 2001, we have implemented RB1 testing on a routine basis as part of the clinical management of retinoblastoma. As most screening techniques do not meet the requirements for efficient RB1 testing, we have devised a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a quantitative multiplex PCR of short fluorescent fragments (QMPSF) approach to screen for gene rearrangements. We report the results of this comprehensive screening of all exons and promoter of RB1 in 192 unrelated patients, mostly of French origin. Among 102 bilateral and/or familial cases and 90 unilateral sporadic probands, mutations were identified in 83 (81.5%) and 5 (5.5%) cases, respectively. A total of 43 mutations have not been previously reported. The mutational spectrum was found to be significantly different from previous published series, displaying a surprising amount of splice mutations and large deletions. This study demonstrates the reliability of DHPLC for RB1 analysis, but also illustrates the need for a deletion scanning approach. Finally, considering the benefits to retinoblastoma patients, RB1 testing should be widely implemented in routine healthcare because our study clearly illustrates its feasibility.     

Spectrum of novel mutations in the human PKLR gene in pyruvate kinase-deficient Indian patients with heterogeneous clinical phenotypes

Eighteen unrelated pyruvate kinase (PK)-deficient Indian patients were identified in the past 4 years with varied clinical phenotypes ranging from a mild chronic haemolytic anaemia to a severe transfusion-dependent disorder. We identified 17 different mutations in the PKLR gene among the 36 mutated alleles. Ten novel mutations were identified: 427G>A, 499C>A, 1072G>A, 1180G>T, 1216G>A, 1220A>G, 644delG, IVS5 (+20) C>A, IVS9 (+44) C>T, and IVS9 (+93) A>C. A severe syndrome was commonly associated with some mutations, 992A>G, 1436G>A, 1220A>G, 644delG and IVS9 (+93) A>C, in the PKLR gene. Molecular graphics analysis of human red blood cell PK (RPK), based on the crystal structure of human PK, shows that mutations located near the substrate or fructose 1,6-diphosphate binding site may change the conformation of the active site, resulting in very low PK activity and severe clinical symptoms. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. In particular, the 1216G>A and 1219G>A mutations significantly affect the interdomain interaction because they are located near the catalytic site in the A/B interface domains. The most frequent mutations in the Indian population appear to be 1436G>A (19.44%), followed by 1456C>T (16.66%) and 992A>G (16.66%). This is the first study to correlate the clinical profile with the molecular defects causing PK deficiency from India where 10 novel mutations that produce non-spherocytic haemolytic anaemia were identified.     

Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression

Quantitative multiplex PCR and genomic real-time PCR were used to complete an RB1 mutation analysis in 57 of 433 and 72 of 262 patients with hereditary and isolated unilateral retinoblastoma, respectively. These patients were selected because in previous analyses, which focused mainly on the identification of point mutations, no RB1 mutation was found. We identified gross deletions and insertions in peripheral blood DNA from 26 of 57 patients (46%) with hereditary retinoblastoma, and in six of 72 patients (8.3%) with isolated unilateral disease. In addition, we identified 32 somatic mutations in tumor DNA from 31 of 72 patients (43%) with isolated unilateral retinoblastoma. Together with our previous results, we found that gross RB1 alterations were present in the peripheral blood DNA from 65 of 433 (15%) and 17 of 262 (6.5%) patients with bilateral or familial and isolated unilateral retinoblastoma, respectively. Including reported gross deletions, an analysis of the frequency of breakpoints per intron length shows higher densities in introns 13, 16, 23, and 24. Genotype-phenotype analyses showed that on the whole, carriers of gross deletions develop fewer retinoblastomas compared to patients who are heterozygous for other types of RB1 null mutations. Specifically, carriers of cytogenetic and submicroscopic whole gene deletions often have unilateral tumors only. By contrast, almost all patients with gross deletions with one breakpoint in RB1 have bilateral retinoblastoma.     

Analysis of the PTCH coding region in human rhabdomyosarcoma

Inherited mutations of the human tumor suppressor gene Patched (PTCH) lead to an autosomal dominant disorder known as Nevoid Basal Cell Carcinoma Syndrome (NBCCS). The syndrome is characterized by a combination of developmental abnormalities and a predisposition to tumor formation. Tumors in patients with NBCCS include basal cell carcinoma, medulloblastoma, fibroma and rhabdomyosarcoma (RMS). RMS are also present in 15 % of mice haplodeficient for Ptch. To investigate whether mutations in PTCH are a general feature in rhabdomyosarcomagenesis we sequenced the protein-coding region in sporadic human cases of these tumors. For this purpose we first determined the distribution and frequency of polymorphisms in 23 exons of PTCH in 48 healthy caucasians. Ten new polymorphisms were identified (IVS11 + 15-17del AAA; IVS14 + 25T>C; 2485G>A; IVS15 + 9G>C; IVS17 + 21A>G; 3033T>C; 3149T>C; 3387T>C; 3617G>A; 4080C>T). Next, the PTCH coding region in 14 RMS was sequenced. Whereas one case with LOH at the PTCH locus was detected, none of the cases showed nonsense or missense mutations in the coding region of PTCH. These data do not support the existence of frequent mutations in the protein-coding region of PTCH in RMS.     

Identification of mutations of Bruton's tyrosine kinase gene (BTK) in Brazilian patients with X-linked agammaglobulinemia

Mutations in the Bruton tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in the peripheral blood. We evaluated 17 male Brazilian patients from 13 unrelated families who showed markedly reduced numbers of blood B cells and hypogammaglobulinemia. BTK gene analysis detected mutations in 10 of the 13 presumed XLA families. Seven mutations (Q196X, G613D, R28L, 251-273del, Q234X, H364P, and R13X) had been reported previously, whereas the remaining three mutations (M501T, IVS15+1G>C, and IVS14+1G>A) were novel. Mutation IVS15+1G>C occurred in a splice donor site and caused exons 15 and 16 to be skipped, and IVS14+1G>A might cause exon 14 to be skipped. Flow cytometry revealed deficient expression of BTK protein in 10 of the 13 families. This is the first report of the diagnosis of XLA by analysis of mutations of the BTK gene in Brazilian patients.     

Splice-site contribution in alternative splicing of PLP1 and DM20: molecular studies in oligodendrocytes

Mutations in the proteolipid protein 1 (PLP1) gene cause the X-linked dysmyelinating diseases Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia 2 (SPG2). We examined the severity of the following mutations that were suspected of affecting levels of PLP1 and DM20 RNA, the alternatively spliced products of PLP1: c.453G>A, c.453G>T, c.453G>C, c.453+2T>C, c.453+4A>G, c.347C>A, and c.453+28_+46del (the old nomenclature did not include the methionine codon: G450A, G450T, G450C, IVS3+2T>C, IVS3+4A>G, C344A, and IVS3+28-+46del). These mutations were evaluated by information theory-based analysis and compared with mRNA expression of the alternatively spliced products. The results are discussed relative to the clinical severity of disease. We conclude that the observed PLP1 and DM20 splicing patterns correlated well with predictions of information theory-based analysis, and that the relative strength of the PLP1 and DM20 donor splice sites plays an important role in PLP1 alternative splicing.     

Molecular analysis in Fabry disease in Spain: fifteen novel GLA mutations and identification of a homozygous female

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A gene (GLA). Here we report molecular studies in 22 unrelated Spanish patients with Fabry disease ( 20 males and two females). Fifteen novel mutations were identified. In addition 7 previously described mutations and two previously reported polymorphisms were detected. The 15 novel mutations comprise: eight missense E48K (c.142G>A), W81S (c.242G>C), D170H (c.508G>C), W226C (c.678G>T), Q279R (c.836A>G), C382Y (c.1145G>A), I407K (c.1220T>A), L414S (c.1241T>C); one nonsense W95X (c.284G>A); one insertion Y216fsX15 (c.646_647insT); two small deletions G346fsX1 (c.1037delG), K426fsX23 (c.1277_1278delAA); one gross deletion comprising exons 5, 6, 7; one complex mutation (insertion and deletion) A368fsX24 (c.1102delGinsTTATAC), and one splice-site mutation IVS4+1G>A (c.639+1G>A). One of the females was found homozygous for Q279R mutation and she presented with the classic phenotype since the age of 8 years, this case extending into women the severe phenotype observed in classically affected males. Mutation analysis provided precise identification for 30 heterozygotes among female relatives and detection of a de novo mutation. The molecular studies on Spanish Fabry patients here reported further contribute to the identification of new mutations in this disease, and allow reliable detection of heterozygotes which has consequences for genetic counselling and for treatment.     

Co-existence of two functional mutations on the same allele of the human ferrochelatase gene in erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is an autosomal dominant disease with incomplete penetrance due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the final step of the heme biosynthetic pathway. The clinical phenotype of EPP results from co-inheritance of a mutated allele and a wild-type low-expressed allele of the FECH gene. To date, more than 88 different mutations have been identified in the FECH gene of patients with EPP. There are evidences suggesting that an entire haplotype (-251G, IVS1-23T and IVS3-48C) reduces allele expression. In this study, we searched for the -251A/G, IVS1-23C/T and IVS3-48T/C polymorphisms in two unrelated Italian families with EPP. In all the patients, carrying the -250G>C mutation in the promoter region, the IVS3-48C on the other allele showed apparent homozygosity and absence of Mendelian segregation. By RNA and long polymerase chain reaction analysis, we identified a deletion of 5576 bp (g12490_18067), including exons 3 and 4, in cis with the -250G>C mutation in the promoter.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

Mutation spectrum of phenylketonuria in Iranian population

Identification of molecular basis of phenylketonuria (PKU) in Iran has been accomplished through the analysis of 248 unrelated chromosomes from 124 Iranian classic PKU subjects. Phenylalanine hydroxylase (PAH) gene mutations were analyzed through a combined approach in which p.S67P, p.R252W, p.R261Q, p.R261X, p.L333F, IVS10-11G>A, IVS11+1G>C, p.L364del, p.R408Q and p.R408W mutations were first screened by PCR of PAH gene exons 3, 7, 10, 11 and 12, followed by digestion with the appropriate digestion enzymes. Subsequently SSCP analysis for exons 2, 6, 7 and 11 of the PAH gene and finally, sequencing of 13 PAH gene exons have been used to study uncharacterized PKU chromosomes. 26 different mutations were found. The predominant mutation in this population sample was IVS10-11G>A, with a frequency of 24.6%. Nine mutations (IVS10-11G>A, p.R261Q, p.P281L, IVS11+1G>C, p.K363>NFS, p.R243X, IVS2+5G>C, p.R261X and p.R252W) represent almost 84% of all PKU chromosomes studied. IVS10-11G>A mutation is the major PKU-causing mutation throughout the Mediterranean region. The finding of the high prevalence of this mutation in Iranian population is consistent with the historical and geographical links between Iranian and Mediterranean populations.     

中国北方地区苯丙氨酸羟化酶基因的突变构成

目的了解中国人苯丙氨酸羟化酶（phenylalanine hydroxylase,PAH）基因的突变构成。方法应用PCR单链构象多态结合序列分析检测230例苯丙酮尿症患儿PAH基因全部外显子及其两侧内含子。结果（1）在460个PAH等位基因中检测出75种不同的突变基因,总检测率达94.6%（435/460）,其中3种突变基因（S251-R252〉SfsX89、Y387D和A389G）尚未见到报道。常见的突变基因为：R243Q（21.7%）、EX6-96A〉G（10.2%）、R111X（8.3%）、R413P（6.5%）和Y356X（6.1%）。较常见的突变基因为：V399V（4.1%）、IVS4-1G〉A（3.5%）、IVS7＋2T〉A（2.2%）和R241C（2.2%）。大部分突变集中在第3、5、6、7、11和12外显子及其两侧内含子区域。（2）检测出10种多态性位点,突变率高的4个位点IVS3-22C〉T（56.7%）、IVS10＋97G〉A（75.9%）、Q232Q（89.0%）和V245V（81.9%）,提示PAH基因cDNA序列存在人种差异。结论中国人PAH基因的突变构成与欧洲人群完全不同,与亚洲其它人群有频率的差异。     

经典型苯丙酮尿症苯丙氨酸羟化酶基因的新突变鉴定

目的研究经典型苯丙酮尿症(phenylketonuria, PKU)基因突变.方法应用聚合酶链反应,单链构象多态分析和DNA直接测序等技术,对内蒙古地区32个PKU家系苯丙氨酸羟化酶(phenylalanine hydroxylase, PAH)基因第3～12外显子进行了鉴定分析. 结果检出14种PAH基因点突变R243Q (12/64)、Y356X(6/64)、Y204C(5/64)、R261Q(2/64)、Y161S(2/64)、R252Q(1/64)、R111X(2/64)、D282G(1/64)、S303P(1/64)、G239D(1/64)、R413P(1/64)、IVS7nt+2(2/64)、IVS4nt+3(1/64)、IVS9nt+34(2/64),经检索国际PAH基因突变数据统计库(截至到2004年7月),确认IVS4nt+3(G>C)、IVS9nt+34(G>A)为国际首次发现的新突变,S303P(T>C) 、D282G(A>G)为国内首次报道的新突变.结论内蒙古人群苯丙氨酸羟化酶基因存在突变的多样性,R243Q、Y356X、Y204C是PAH基因的突变热点.     

Mutation detection in the duplicated region of the polycystic kidney disease 1 (PKD1) gene in PKD1-linked Australian families

Screening for disease-causing mutations in the duplicated region of the PKD1 gene was performed in 17 unrelated Australian individuals with PKD1-linked autosomal dominant polycystic kidney disease. Exons 2-21 and 23-34 were assayed using PKD1-specific PCR amplification and direct sequencing. We have identified 12 novel probably pathogenic DNA variants, including five truncating mutations (Q563X, c.5105delAT, c.5159delG, S2269X, c.9847delC), two in-frame deletions (c.7472del3, c.9292del39), and two splice-site mutations (IVS14+1G>C, IVS16+1G>T). Three of the mutations (G381C, Y2185D, G2785D) were predicted to lead to the replacement of conserved amino acid residues, with ensuing changes in protein conformation. Defects in the duplicated region of PKD1 thus account for 63% of our patients. Together with the previously detected mutations (Q4041X, R4227P) in the 3 region of the gene, the study has achieved an overall mutation detection rate of 74%. In addition, we have detected 31 variants (nine novel and 22 previously published) that did not segregate with the disease and were considered to be neutral polymorphisms. Three of the nine novel polymorphisms were missense mutations with a predicted effect on protein conformation, emphasizing the problems of interpretation in PKD1 mutation screening.     

McArdle disease: the mutation spectrum of PYGM in a large Italian cohort

Deficiency of the muscle isozyme of glycogen phosphorylase is causative of McArdle disease or Glycogen storage disease type V (GSD-V), the most common autosomal recessive disorder of glycogen metabolism. The typical clinical presentation is characterized by exercise intolerance with cramps, and recurrent myoglobinuria. To date, 46 mutations in the PYGM gene have been detected in GSD-V patients. We report the mutational spectrum in 68 Italian patients. We identified 30 different mutations in the PYGM gene, including 19 mutations that have not been reported previously. The novel mutations include: eight missense mutations (c.475G>A, p.G159R; c.689C>G, p.P230R; c.1094C>T, p.A365E; c.1151C>A, p.A384D; c.1182C>T, p.R428C; c.1471C>T, p.R491C; c.2444A>C, p.D815A; c.2477G>C, p.W826S), two nonsense mutations (c.1475G>A, p.W492X; c.1627A>T, p.K543X), five splice site mutations (c.855 +1G>C; c.1092 +1G>A; c. 1093-1G>T; c.1239 +1G>A; c.2380 +1G>A), and four deletions (c.715_717delGTC, p.V239del; c.304delA, p.N102DfsX4; c.1970_2177del, p.V657_G726; c.2113_2114delGG, p.G705RfsX16). Whereas we confirmed lack of direct correlation between the clinical phenotype and the genotype, we also found that the so-called 'common mutation' (p.R50X) accounted for about 43% of alleles in our cohort and that no population-related mutations are clearly identified in Italian patients.     

New RB1 oncogenic mutations and intronic polymorphisms in Serbian retinoblastoma patients: genetic counseling implications

The purpose of this work was to identify germ line RB1 mutations in 16 Serbian retinoblastoma patients for genetic counselling. Mutation analysis was carried out by PCR directed sequencing of the 27 exons. Loss of heterozygosity for two RB1 intragenic markers was also analyzed in 14 tumour samples. Five new RB1 oncogenic mutations (g.2078 del C, g.77047_48 del GC, g.78117_8 del TT, g.160797 del T, and g.64439+2 T>C) and two recurrences (R445X and Q383X) have been found in this study. In addition, four intronic variants were observed germ line in some unilateral patients. Two of these variants (g.44668-15T/G, and g.166204-8T/A) are discussed as potential oncogenic mutation candidates. The results show the relevance of studies aimed to investigate the role of intronic variants in exon splicing regulation. Such studies will help to disclose hidden retinoblastoma susceptibilities, important for accurate genetic counselling.     

Identification of four novel RB1 germline mutations in Korean retinoblastoma patients

To elucidate RB1 germline mutations in Korean retinoblastoma patients, DNA samples from 14 children with bilateral (including three familial cases) and 19 children with unilateral retinoblastoma were analyzed. We found germline mutations in three out of 14 bilateral cases and one out of 19 unilateral cases. There were no germline mutations in the three familial cases. PCR-SSCP from each exon showed bandshifts in four patients which, upon sequencing, were shown to be K616E in exon 19 (c.1846A>G), an AA insertion in exon 7 (c.684-685insAA), R500G in exon 16 (c.1498A>G), and an A insertion in exon 23 (c.2391-2392insA), respectively. Hum Mutat 18:252, 2001.     

A molecular study of first and second RB1 mutational hits in retinoblastoma patients

RB1 mutations accountable for biallelic inactivation are crucial events in the development of retinoblastoma because a first mutation (M1) predisposes to retinoblastoma while a second mutation (M2) is required for tumor development. Mutational analyses of this gene showed a wide spectrum of genetic alterations (single base substitutions, insertions, or deletions, as well as small and large deletions). The most frequent second hit in retinoblastoma patients is loss of heterozygosity (LOH) followed by promoter methylation. Molecular analyses of RB1 mutations were conducted in 36 patients (20 unilateral and 16 bilateral) using polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP) analysis, sequencing, and LOH analysis. Sixty-four amplified fragments showing abnormal SSCP patterns were sequenced, and mutations were confirmed in five patients (13.89%). Four mutations were located at coding regions, and a fifth one was found at an exon-intron junction. Two mutations were C-->T transitions, two were small-length deletions, and one was a G-->A transition. A total of 47.05% patients showed LOH. In one patient, the parental origin of the mutated allele was detected: the allele retained in the tumor was the paternal one. This work helps to characterize the spectrum of mutations in the Brazilian population, and to confirm that formaldehyde-fixed paraffin tissue can provide valuable information on the RB1 status in retinoblastoma patients.