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PubMed1994

TP53 mutations are frequent in malignant NF1 tumors.

Neurofibromatosis type I (NFI) is a common autosomal dominant disorder with an increased risk for developing benign and malignant tumors. The NFI gene has been cloned and maps to 17q11.2, and the gene product acts as a tumor suppressor gene. Here we analyzed the role of mutations in TP53 in four malignant NFI tumors. Mutations were found in 3 out of 4 tumors. One of these mutations is a common missense mutation in codon 278 in one of the previously identified hot spots for mutations. The two other are hitherto unreported mutations, including a splice mutation of exon 3 and a nonsense mutation in exon 4. In addition, these four tumors also showed loss of heterozygosity (LOH) for markers on chromosome 17 in the region of TP53. Malignant NFI tumors are initiated by a somatic inactivation of the second NFI allele. Tumor progression, however, occurs by accumulation of additional genetic abnormalities, such as homozygous inactivation of TP53, as demonstrated in this paper.

Base Sequence↗
PubMed2002

Molecular analysis in newborns from Texas affected with galactosemia.

The spectrum of mutations in the Galactose-1-phosphate uridyl transferase (GALT) gene is described in 11 cases of classic galactosemia and 38 of Duarte-2 type identified by the Texas Newborn Screening Program. Blinded studies were done by automated DNA sequencing of all the 11 exons and the exon-intron boundaries of the GALT gene using genomic DNA isolated from dry blood spots. Fourteen different mutations (11 missense mutations, 2 nonsense mutations and 1 splicing mutation) were detected in 94 of the 98 mutant alleles (diagnostic efficiency of 96%). The prevalent mutations were N314D (41%), Q188R (37%) and K285N (4%). The other less frequent mutations were IVS2-2A>G and S135L (3% each), T138M (2%) and T23A, H184Q, Y251S, L195P, Q207X, L264X, Q344K, and A345D (1% each). Three novel mutations, T23A, Q207X, and A345D, were identified. Our study supports previous findings that N314D and Q188R are prevalent in Hispanics and Whites and K285N was only observed in Whites. The IVS2-2A->G mutation is probably ethnic specific because it was identified exclusively in Hispanics. S135L, a prevalent mutation in Blacks, was also present in 3 Hispanics. Two unusual genotypes were observed in 2 patients homozygous for the Duarte-2 N314D allele and heterozygous for a novel mutation (Q207X- N314D/N314D in a classic galactosemia and T23A- N314D/N314D in a Duarte-2 case). The detection of GALT gene mutations in newborns from Texas should focus first on N314D, Q188R, K285N, IVS2-2A>G, S135L and T138M. Other exons and exon-intron boundaries would have to be studied if either one or no mutations are found in the primary screening.

DNA Mutational Analysis↗
PubMed1996

Point mutations in Italian patients with classic, non-classic, and cryptic forms of steroid 21-hydroxylase deficiency.

Seventy-three Italian patients affected by steroid 21-hydroxylase deficiency were studied by a PCR-allele-specific oligonucleotide protocol in order to evaluate the presence of eight known point mutations. The majority of chromosomes were found to carry point gene conversions normally present in the pseudogene. Within the classic form, the most common mutations were the splicing mutation A/C-655 to G in intron 2 (34.2%), the nonsense mutation C-1993 to T in exon 8 (10.8%), and the missense mutation T-999 to A in exon 4 (10%). Within the non-classic form, the missense mutation G-1683 to T was the most common (57.7%). Other mutations were either absent, such as the three clustered missense mutations T-1380, T-1383, T-1389 to A in exon 6, or very rare, like the 1761 + T in exon 7 and the C-2108 to T in exon 8. Family genotyping revealed the presence of ten asymptomatic parents carrying mutations in both chromosomes, thus identifying the gene defect in cryptic subjects. Interestingly, the same mutations were found in both symptomatic and asymptomatic forms.

Adrenal Hyperplasia, Congenital↗
PubMed2004

The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: experience of a national referral centre.

Although the majority (65%) of boys with Duchenne muscular dystrophy (DMD) carry a deletion in the dystrophin gene, finding mutations in the remaining families is vital for counselling. We have provided a comprehensive mutation service as a national referral centre for France for over 10 years and we report here our experience. Mutation screening is on mRNA from a muscle biopsy. We have detected 79 mutations in 89 samples referred with a diagnosis of DMD, which is the most comprehensive survey to date of the full range of nondeletion mutations. Although some mutations were nonsense mutations, some frameshift mutations and some splicing mutations, all of them led to the generation of premature stop codons or a shortened product which could be detected using the Protein Truncation Test. We recommend a protocol which is robust and sensitive applied to the entire coding region reverse-transcribed from dystrophin transcripts from muscle biopsy.

Biopsy↗
PubMed2003

Functional analysis of MCCA and MCCB mutations causing methylcrotonylglycinuria.

Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism. This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans. MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized. Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions. We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect. All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones. The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase. The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype.

Amino Acid Sequence↗
PubMed2005

Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric cancer.

We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.

Adult↗
PubMed1997

Novel and characteristic CFTR mutations in Saudi Arab children with severe cystic fibrosis.

More than 600 different CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations have been identified so far that are considered to cause the fatal genetic disorder cystic fibrosis (CF). We have investigated 15 Arab children from 12 families, who were diagnosed as having CF, for mutations in the coding region and in the flanking intron sequences of the CFTR gene. Six different CFTR mutations were identified including two novel mutations, 1548delG in exon 10 and 406-2A-->G in intron 3. Prominent mutations were the splice mutation 3120 + 1G-->A (intron 16) followed by N1303K (exon 21) and 1548delG (exon 10). Most CF children were homozygotes who presented with a severe form of the disease including failure to thrive, recurrent chest infections, particularly with Pseudomonas aeruginosa, and frequent hospital admissions. Identification of the CFTR mutations facilitates molecular investigation of the disease and better understanding of its pathophysiology in Arab children, among whom CF is probably an underdiagnosed disease.

Child↗
PubMed2006

Mutations of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation.

RATIONALE: Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive, genetic disorder characterized by ciliary dysfunction, sino-pulmonary disease, and situs inversus. Disease-causing mutations have been reported in DNAI1 and DNAH5 encoding outer dynein arm (ODA) proteins of cilia. OBJECTIVES: We analyzed DNAI1 to identify disease-causing mutations in PCD and to determine if the previously reported IVS1+2_3insT (219+3insT) mutation represents a "founder" or "hot spot" mutation. METHODS: Patients with PCD from 179 unrelated families were studied. Exclusion mapping showed no linkage to DNAI1 for 13 families; the entire coding region was sequenced in a patient from the remaining 166 families. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on nasal epithelial RNA in 14 families. RESULTS: Mutations in DNAI1 including 12 novel mutations were identified in 16 of 179 (9%) families; 14 harbored biallelic mutations. Deep intronic splice mutations were not identified by reverse transcriptase-polymerase chain reaction. The prevalence of mutations in families with defined ODA defect was 13%; no mutations were found in patients without a defined ODA defect. The previously reported IVS1+2_3insT mutation accounted for 57% (17/30) of mutant alleles, and marker analysis indicates a common founder for this mutation. Seven mutations occurred in three exons (13, 16, and 17); taken together with previous reports, these three exons are emerging as mutation clusters harboring 29% (12/42) of mutant alleles. CONCLUSIONS: A total of 10% of patients with PCD are estimated to harbor mutations in DNAI1; most occur as a common founder IVS1+2_3insT or in exons 13, 16, and 17. This information is useful for establishing a clinical molecular genetic test for PCD.

Adolescent↗
PubMed2000

Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population.

Hereditary spherocytosis (HS) is the most common hemolytic anemia of congenital origin in the Japanese population. Among 844 cases of 520 kindred with congenital red cell membrane disorders studied at the Kawasaki Medical School in the last 25 years (1975-1999), 407 cases (48.2%) of 215 kindred had HS. Among the recent 60 kindred with HS, autosomal dominant (AD) transmission was proven in 19. The remaining 41 non-AD HS included 1) homozygous patients with autosomal recessive inheritance, 2) HS patients with de novo gene mutations, and 3) mild HS with AD inheritance. The extent of clinical severity in the non-AD HS cases was nearly identical to that in the AD cases. The incidence of membrane protein abnormalities in our 60 Japanese HS kindred was unique: there were lower ankyrin deficiencies (7%), moderate band 3 deficiencies (20%), and much higher protein 4.2 deficiencies (45%), with 28% of unknown etiology. The incidence of membrane protein deficiencies corresponded to that determined by gene analyses; i.e., mutations mostly in band 3 and/or in protein 4.2 genes and fewer ankyrin gene mutations. In the band 3 gene, 11 mutations pathognomonic for HS were identified (3 frameshift and 8 missense mutations). There were 5 mutations of the protein 4.2 gene (3 missense mutations, 1 nonsense mutation, and 1 splicing mutation) pathognomonic for HS. On the other hand, 2 missense mutations were detected in the ankyrin gene in this study. The genetic abnormalities in our HS patients correlated well with the phenotypic ultrastructural abnormalities of red cell membranes in situ. Ankyrin mutations (ankyrin Marburg and ankyrin Stuttgart with frameshift mutations) were associated mostly with a disrupted cytoskeletal network, and band 3 mutations (band 3 Kagoshima with frameshift mutation) typically demonstrated anomalies of intramembrane particles (IMPs). Protein 4.2 mutations (homozygotes of protein 4.2 Nippon) with complete protein 4.2 deficiency showed abnormalities of both the cytoskeletal network and IMPs.

Animals↗
PubMed2006

[Analysis of MYH7, MYBPC3 and TNNT2 gene mutations in 10 Chinese pedigrees with familial hypertrophic cardiomyopathy and the correlation between genotype and phenotype].

OBJECTIVE: The aim of this study was to screen the disease-causing gene mutations and investigate the genotype-phenotype correlation in 10 Chinese pedigrees with familial hypertrophic cardiomyopathy (HCM). METHODS: There are 91 family members from these 10 pedigrees and 5 members were normal mutated carriers, 23 members were HCM patients (14 male) aged from 1.5 to 73 years old. The functional regions of myosin heavy chain gene (MYH7), cardiac myosin-binding protein C (MYBPC3) and cardiac troponin T gene (TNNT2) were screened with PCR and direct sequencing technique. Clinical information from all patients was also evaluated in regard to the genotype. RESULTS: Mutations were found in 5 out of 10 pedigrees. Mutations in MYH7 (Arg663His, Glu924Lys and Ile736Thr) were found in 3 pedigrees and 3 patients from these pedigrees suffered sudden death at age 20-48 years old during sport. Mutations in MYBPC3 were found in 2 pedigrees, 1 with complex mutation (Arg502Trp and splicing mutation IVS27 + 12C > T) and 1 with novel frame shift mutation (Gly347fs) and the latter pedigree has sudden death history. No mutation was identified in TNNT2. CONCLUSIONS: Although the Han Chinese is a relatively homogeneous ethnic group, different HCM gene mutations were responsible for familiar HCM suggesting the heterogeneity nature of the disease-causing genes and HCM MYH7 mutations are associated with a higher risk of sudden death in this cohort. Furthermore, identical mutation might result in different phenotypes suggesting that multiple factors might be involved in the pathogenesis of familiar HCM.

Adolescent↗
PubMed1995

Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome.

Germ-line mutations of the tumor-suppressor gene p53 have been observed in some families with the Li-Fraumeni syndrome (LFS), a familial cancer syndrome in which affected relatives develop a diverse set of early-onset malignancies including breast carcinoma, sarcomas, and brain tumors. The analysis of the p53 gene in LFS families has been limited, in most studies to date, to the region between exon 5 and exon 9. In order to determine the frequency and distribution of germ-line p53 mutations in LFS, we sequenced the 10 coding exons of the p53 gene in lymphocytes and fibroblast cell lines derived from 15 families with the syndrome. Germ-line mutations were observed in eight families. Six mutations were missense mutations located between exons 5 and 8. One mutation was a nonsense mutation in exon 6, and one mutation was a splicing mutation in intron 4, generating aberrant shorter p53 RNA(s). In three families, a mutation of the p53 gene was observed in the fibroblast cell line derived from the proband. However, the mutation was not found in affected relatives in two families and in the blood from the one individual, indicating that the mutation probably occurred during cell culture in vitro. In four families, no mutation was observed. This study indicates that germ-line p53 mutations in LFS are mostly located between exons 5 and 8 and that approximately 50% of patients with LFS have no germ-line mutations in the coding region of the p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1999

A common splice site mutation is shared by two families with different type 2N von Willebrand disease mutations.

Using an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.

Alleles↗
PubMed1996

Spectrum of spontaneously occurring mutations in the HPRT gene of the Chinese hamster V79 cell mutant V-H4, which is homologous to Fanconi anemia group A.

The mitomycin C (MMC)-hypersensitive Chinese hamster V79 cell mutant V-H4 has a cellular phenotype similar to Fanconi anemia (FA), and has been shown to be homologous to FA group A. To examine consequences of the defect in V-H4 cells on spontaneous mutagenesis, we studied the frequency and nature of spontaneous mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus in this mutant and the parental V79 cells. The mutation rates expressed as the number of mutations per cell per generation were 8.7 x 10(-7) and 3.7 x 10(-7) for V-H4 and V79 cells respectively. The molecular spectrum of 42 spontaneous hprt mutants of V-H4 cells was determined and compared with the previously described spectrum of spontaneous mutations at the HPRT locus of Chinese hamster V79 cells. The spectra of spontaneous mutations in the hprt gene of both cell lines are predominated by base pair substitutions and splice mutations. Among the base changes, V-H4 shows a larger frequency of transitions (13/42; 31%) than transversions (3/42; 7%), whereas in V79 transversions are observed more often than transitions (P < 0.001; Wilcoxon test). The frequency of splice mutations in V-H4 (17/42; 40%), which affects exon 4 almost exclusively, is not significantly different from V79. The fraction of deletions in V-H4 is low (6/42; 14%), and comparable to the level in V79. This is in contrast with the published molecular spectrum of spontaneous hprt mutants in FA (group D) cells, which consists predominantly of deletions.

Animals↗
PubMed1998

Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency.

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5' splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor-like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the triangle upEGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.

Animals↗
PubMed1990

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing.

We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.

Base Sequence↗
PubMed1998

Molecular basis of an apolipoprotein[a] null allele: a splice site mutation is associated with deletion of a single exon.

Apolipoprotein[a] (apo[a]), a unique component of atherogenic lipoprotein[a], is highly polymorphic in human and nonhuman primates. Null alleles, producing no detectable circulating Lp[a] or apo[a] isoforms, are found at high frequencies. The molecular basis of null alleles is not yet known. In baboons, approximately two-thirds of null alleles do not produce detectable hepatic transcripts (transcript negative nulls), and one-third of null alleles produce normal amounts of apo[a] transcripts (transcript positive nulls). We have cloned apo[a] cDNA from a baboon carrying a transcript positive null allele defective in secretion from primary hepatocytes. Compared with wild-type cDNA, the null allele contained an in-frame 47 amino acid deletion in the protease domain corresponding to one exon of the apo[a] gene. The null allele contains an A-->T substitution in the third nucleotide position of the intron downstream of the deleted exon which alters the donor splice site consensus sequence. Thus, this null is likely due to a mutation that prevents normal mRNA splicing, yielding a shortened protein that may be defective in intramolecular interactions required for normal processing and secretion of apo[a]. This is the first report of a molecular basis for apo[a] null alleles.

Alleles↗
PubMed2000

Two novel mutations in splice donor sites of CYP11B1 in congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency.

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Genetic analysis showed two new base substitutions of CYP11B1, a conservative transition at the last base of exon 5, and a IVS8+4A-->G transition in intron 8. Difficulties with suppressive therapy resulted in severe hypertension. A laparoscopic adrenalectomy was decided which lead to normalization of blood pressure. In vitro, steroidogenesis by adrenal cells showed no measurable 11beta-hydroxylase activity. Analysis of CYP11B1 mRNA by RT-PCR and sequencing showed expression of a mRNA which lacked exon 8, presumably resulting from the intron 8 mutation. In addition a highly truncated mRNA was detected corresponding to exons 1, 2, 8, 9, with the loss of exons 3-7, presumably related to the exon 5 mutation. Western blot analysis showed a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus adrenalectomy in this patient allowed effective treatment of severe hypertension and helped to understand the mechanisms of two novel mutations responsible for aberrant splicing of CYP11B1.

Adrenal Cortex↗