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PubMed2006

Characterisation of 96 mutations in 128 unrelated severe haemophilia A patients from France. Description of 62 novel mutations.

Direct sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII<1%) in 128 unrelated haemophiliacs A, negative for intron 22 and intron 1 inversions. A mutation was found in 122/128 patients (95%). Ninety-six distinct mutations were identified in this cohort, 62 of these are novel. They consisted of deletions (7 large and 24 small deletions), insertions (n = 9), associations of insertion/deletion (n = 2), association of deletion/substitution (n = 1), and single nucleotide substitutions (53 point mutations consisting of 31 missense, 20 nonsense, and 2 splicing mutations). Twenty-two patients had developed inhibitors, and among this subgroup 3 large deletions, 6 frameshift, 9 nonsense and 4 missense mutations were detected. For 6 patients, among which one developed an anti-FVIII inhibitor, no mutations were detected in the coding and splicing regions of factor VIII gene. Different approaches of molecular modelling were performed in addition to familial linkage analysis to determine the pathophysiological responsibility of these novel missense mutations.

Blood Coagulation Tests↗

PubMed1997

Novel cystatin B mutation and diagnostic PCR assay in an Unverricht-Lundborg progressive myoclonus epilepsy patient.

Two mutations in the cystatin B gene, a 3' splice mutation and a stop codon mutation, were previously found in patients with progressive myoclonus epilepsy of Unverricht-Lundborg type [Pennacchio et al. (1996): Science 271:1731-1734]. We present here a new mutation 2404deltaTC: a 2-bp deletion within the third exon of the cystatin B gene in an Unverricht-Lundborg patient. This mutation results in a frameshift and consequently premature termination of protein synthesis. Complete sequencing of the coding region and splice junctions of the cystatin B gene showed that neither of the two previously known mutations was present in this patient. The level of cystatin B mRNA in an immortalized cell line was found to be decreased, as had been reported for other Unverricht-Lundborg patients. The new mutation further supports the argument that defects in the cystatin B gene cause the Unverricht-Lundborg form of progressive myoclonus epilepsy. We describe a simple PCR method which can detect the 2404deltaTC deletion. This assay, together with previously described PCR assays for the other two known mutations, should prove useful in confirming clinically difficult diagnoses of Unverricht-Lundborg disease.

Amino Acid Sequence↗

PubMed2005

The splicing machinery is a genetic modifier of disease severity.

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.

Alternative Splicing↗

PubMed2001

Coexpression of normal and mutated CD40 ligand with deletion of a putative RNA lariat branchpoint sequence in X-linked hyper-IgM syndrome.

We describe a novel CD40 ligand (CD40L) splicing mutation in a patient with X-linked hyper-IgM syndrome (X-HIM) associated with alternate splicing of exon 3, resulting in the expression of both full-length and exon-3-skipped CD40L mRNA. The mutation is an 8-bp deletion 25 bp upstream of the intron 2/exon 3 junction which overlaps a putative RNA branchpoint, suggesting that it may impair RNA lariat formation. The exon-3-skipped CD40L transcript encodes a truncated protein (CD40LDeltaE3) encompassing the cytoplasmic, transmembrane, and extracellular stalk domains, but lacking the CD40L receptor binding domain. CD40LDeltaE3 protein expression was readily detectable in transfected Cos cells by immunofluorescence. In cells cotransfected with CD40LDeltaE3 and wild-type CD40L, expression of CD40LDeltaE3 did not inhibit the expression of wild-type CD40L monomers, but strongly inhibited staining by the conformationally sensitive anti-CD40L mAb 5c8, suggesting that CD40LDeltaE3 acts in a dominant negative manner to inhibit the assembly of functional CD40L trimers. This mechanism may contribute to the pathophysiology of CD40L deficiency in X-HIM patients with leaky splice site mutations.

CD40 Ligand↗

PubMed1994

Molecular epidemiology of Tay-Sachs disease in Europe.

The abnormalities in the gene coding for the beta-hexosaminidase alpha subunit were analysed from fibroblast's RNAs of 42 Tay-Sachs patients (seven with adult or late onset of Tay-Sachs disease and 35 with infantile Tay-Sachs disease). After first strand synthesis by random priming, PCR was used to amplify in two overlapping fragments (868 and 949 bp) the entire coding region. These amplified products were first studied for changes in size by agarose gel electrophoresis to screen for splicing mutations leading to exon skipping or cryptic splice site activation. For each patient, the two overlapping cDNA fragments were subjected to chemical mismatch cleavage analysis using hydroxylamine to modify C-containing mismatches and osmium tetroxide to modify T-containing mismatches. DGGE was used to screen for mutations in the coding region spanning exon 2 to exon 6, a region putatively encompassing the active site and therefore a potential hot spot of mutations associated with Tay-Sachs disease. To increase the sensitivity of the technique, a 30 bp GC-clamp has been added at the 5' end of the sense oligonucleotide to amplify a fragment of 629 bp. The computerized analysis found that single base changes in domain spanning from nt 313 to nt 693 can be distinguished. Fragments displaying an altered melting behavior or a cleaved product were further analysed by direct sequencing of the amplified material. These methods as a whole allowed us to identify 30/38 alleles studied (79%) with 15 point mutations and one 4 bp insertion detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1998

Search for mutations near the alternatively spliced 8-amino-acid exon in the GABAA receptor gamma 2 subunit gene and lack of allelic association with alcoholism among four aboriginal groups and Han Chinese in Taiwan.

The alternatively spliced 8-amino-acid exon for the GABAA receptor gamma2 subunit gene (GABRC2) has been postulated to mediate behavioral actions of alcohol. A rapid search for splice-junction mutations near the 8-amino-acid exon using restriction enzymes which normally recognize sequences near or in the exon gave negative results among 217 alcoholics in four aboriginal groups (Ami, Atayal, Bunun and Paiwan) and Han Chinese in Taiwan. The role of the GABRC2 gene in alcoholism was further assessed by a comparison of allelic frequencies revealed by a NciI RFLP between case and control groups. No significant association of alcohol dependence with GABRC2 alleles was observed. These results suggest that the GABRC2 gene probably does not play an essential role in predisposition to alcoholism in the sample population.

Alcoholism↗

PubMed1998

Constant denaturant gel electrophoresis (CDGE) in BRCA1 mutation screening.

Screening for mutations in the breast and ovarian cancer susceptibility gene, BRCA1, is complicated by the wide spectrum of mutations found in this large gene. In the present study a constant denaturant gel electrophoresis (CDGE) mutation screening strategy was established for approximately 80% of the genomic coding sequence (exons 2, 11, 13-16, 20, 24). This strategy was applied to screen genomic DNA from 50 familial breast and/or ovarian cancer patients who had previously been examined for BRCA1 mutations by SSCP. A total of 14 carriers of 12 distinct disease-associated mutations and 7 carriers of 6 distinct rare substitutions leading to amino acid substitutions were identified. The SSCP failed to detect 40% of the different deletions/insertions (4/10) and 75% (6/8) of the different base substitutions leading to terminating codons or rare amino acid changes. SSCP did, however, identify one rare base substitution that could not be detected in the CDGE screening. To evaluate the CDGE mutation screening strategy further, 25 unrelated patients from Norwegian breast and/or ovarian cancer families were examined for BRCA1 mutations using a combined genomic DNA/cDNA approach covering the entire coding sequence of the gene. A total of six mutation carriers were detected, all of whom had cases of ovarian cancer in their families. Three patients from independent families carried an 1135insA mutation in exon 11, two others had a Gly484ter and an 1675delA mutation, respectively, and the sixth carried a splice mutation (5194-2 a-->c) causing deletion of exon 18. CDGE may become an efficient tool in diagnostic and population based screening for BRCA1 mutations.

BRCA1 Protein↗

PubMed2001

Tay-Sachs screening in the Jewish Ashkenazi population: DNA testing is the preferred procedure.

A unique screening program for the identification of Tay-Sachs Disease (TSD) heterozygotes has been performed in the tradi- tional Orthodox Ashkenazi Jewish (AJ) community since 1983. In recent years the program has utilized the biochemical assay for the determination of hexosaminidase A levels by the heat inactivation technique as well as by direct DNA analysis. The three mutations which were analyzed were those that have been shown to be prevalent among AJ TSD patients and carriers, namely the four nucleotide insertion mutation in exon 11 (1278+TATC), the splice mutation at the 5' end of intron 12 (1421+1g-->c), and the adult mutation, a Gly(269)-->Ser substitution in exon 5 (G269S). A total of 103,133 individuals were tested by biochemical analysis, and 38,197 of them were also assayed by DNA testing. Furthermore, 151 chromosomes from TSD patients or obligate heterozygotes were subjected to DNA analysis for one of the three mutations. DNA testing of the latter identified one of the three AJ mutations in every case, predicting a very high detection rate of heterozygotes in this community by this method. By contrast, the sensitivity of the enzyme assay ranged from 93.1% to 99.1% depending on the exclusion (inclusion) of inconclusive results as positive, while the specificity ranged from 88.1% to 98.8% depending on the inclusion (exclusion) of inconclusive results as positive. Our results strongly support the use of DNA testing alone as the most cost-effective and efficient approach to carrier screening for TSD in individuals of confirmed Ashkenazi Jewish ancestry.

DNA↗

PubMed1991

Polymorphic DNA haplotypes at the phenylalanine hydroxylase locus and their relation to phenotype in Swedish phenylketonuria families.

The genetic heterogeneity at the phenylalanine hydroxylase (PAH) locus was studied in 88 families including 93 of the 105 children with phenylketonuria (PKU) or hyperphenylalaninemia (HPA) detected through the Swedish neonatal screening program from 1966 to the end of 1986. Haplotypes based on eight restriction fragment length polymorphisms (RFLPs) at the PAH locus could be constructed for 132 normal and 136 mutant alleles. The normal alleles were of 27 different RFLP haplotypes, 9 of which have not been described previously, but there was a dominance of a few haplotypes common to many European populations. The distribution of mutant alleles was significantly different from that in neighboring countries, even though over 90% of all mutant alleles were confined to six RFLP haplotypes, also prevalent in other European populations. Allele-specific oligonucleotide hybridization analysis for the Arg408 to Trp408 mutation and for the G to A splicing mutation in intron 12 showed exceptions to the previously reported linkage of these mutations to mutant haplotypes 2 and 3, respectively. Correlation of mutant alleles with clinical phenotypes pointed to the presence of at least two different mutations associated with each of six haplotypes. We argue that PKU/HPA in the Swedish population may be caused by at least 13 different mutations in addition to the 4 already identified. The theoretical informativity of RFLP analysis in heterozygote detection and prenatal diagnosis in PKU/HPA families was estimated at approximately 85%. Carrier detection could, in effect, be accomplished for 88% of the 56 healthy siblings in the families studied.

Alleles↗

PubMed1994

Characterization of mutations on the rare duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency.

We have defined the mutations causing congenital adrenal hyperplasia in three Swedish patients carrying a rare haplotype containing two mutated steroid 21-hydroxylase genes (CYP21) in addition to one pseudogene (CYP21P). The presence of such haplotypes complicates genetic diagnosis and screening of mutations in 21-hydroxylase deficiency, and we show how these genotypes can be resolved by amplification and analysis of each gene separately. In all cases, the rare haplotype carried the same combination of disease-causing mutations; one of the genes had the splice mutation at base 659 in intron 2, and the other had the nonsense mutation at base 1999 in exon 8 (CAG to TAG). We have thus characterized the most common haplotype containing duplicated CYP21 genes. The frequency of this haplotype is low, and if additional such haplotypes are present, they are rare in this population.

Adrenal Hyperplasia, Congenital↗

PubMed1999

Structure of the gene for congenital nephrotic syndrome of the finnish type (NPHS1) and characterization of mutations.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin.

Amino Acid Sequence↗

PubMedDate not supplied

Novel mutation identified in the PAH gene.

The investigation of a DNA-amplified fragment of a phenylketonuria (PKU) patient by sequencing reveals a novel mutation in the PAH gene. This mutation represents the deletion of a single base (guanine) localized at the intron 11/exon 12 junction. This newly described mutation may be a frameshift or a splicing mutation. The identified mutation expresses phenotypically as the severe form of PKU.

Amino Acid Sequence↗

PubMed2000

In a novel form of IFN-gamma receptor 1 deficiency, cell surface receptors fail to bind IFN-gamma.

Complete IFN-gamma receptor ligand-binding chain (IFNgammaR1) deficiency is a life-threatening autosomal recessive immune disorder. Affected children invariably die of mycobacterial infection, unless bone marrow transplantation is undertaken. Pathogenic IFNGR1 mutations identified to date include nonsense and splice mutations and frameshift deletions and insertions. All result in a premature stop codon upstream from the segment encoding the transmembrane domain, precluding cell surface expression of the receptors. We report herein two sporadic and two familial cases of a novel form of complete IFNgammaR1 deficiency in which normal numbers of receptors are detected at the cell surface. Two in-frame deletions and two missense IFNGR1 mutations were identified in the segment encoding the extracellular ligand-binding domain of the receptor. Eight independent IFNgammaR1-specific mAb's, including seven blocking antibodies, gave recognition patterns that differed between patients, suggesting that different epitopes were altered by the mutations. No specific binding of (125)I-IFN-gamma to cells was observed in any patient, however, and the cells failed to respond to IFN-gamma. The mutations therefore cause complete IFNgammaR1 deficiency by disrupting the IFN-gamma-binding site without affecting surface expression. The detection of surface IFNgammaR1 molecules by specific antibodies, including blocking antibodies, does not exclude a diagnosis of complete IFNgammaR1 deficiency.

Adolescent↗

PubMed2003

Follow-up of 68 children with congenital adrenal hyperplasia due to 21-hydroxylase deficiency: relevance of genotype for management.

The phenotype of congenital adrenal hyperplasia (CAH) varies greatly. The purpose of this study was to optimize diagnosis and follow-up by comparing phenotype with genotype. Sixty-eight patients with CAH due to 21-hydroxylase deficiency were studied by clinical, hormonal, and molecular genetic methods. Patients were classified according to predicted mutation severity: group 0, null mutation (17.6%); group A, homozygous for IVS2 splice mutation or compound heterozygous for IVS2 and null mutations (33.8%); group B, homozygous or compound heterozygous for I172N mutation (14.7%); group C, homozygous or compound heterozygous for V281L or P30L mutations (26.5%); and group D, mutations with unknown enzyme activity (7.4%). All group 0 and A patients had the salt-wasting form, and group C had nonclassical forms. Group B included five salt-wasting and five simple virilizing forms. Groups 0 and A were younger at diagnosis (P < 0.02), and females were more virilized than those in group B. Group B had higher basal plasma 17-hydroxyprogesterone (564 +/- 162 nmol/liter) and testosterone (11 +/- 3 nmol/liter) levels than group C [59 +/- 13 nmol/liter (P < 0.001) and 1.4 +/- 0.2 nmol/liter (P < 0.005), respectively]. Hydrocortisone doses given to groups 0, A, and B were similar at all ages, but lower in group C (P < 0.01). Final height was below target height in classical (n = 16; -2 +/- 0.2 SD score; P < 0.02) and nonclassical (n = 11; -1.2 +/- 0.4 SD score; P < 0.03) forms. The severity of the genetic defects and the clinical-laboratory features are well correlated. Genotyping, combined with neonatal screening and optimal medical and surgical treatment, can help in the management of CAH.

Adolescent↗

PubMed1998

Apparent congenital athyreosis contrasting with normal plasma thyroglobulin levels and associated with inactivating mutations in the thyrotropin receptor gene: are athyreosis and ectopic thyroid distinct entities?

Loss-of-function mutations in the TSH receptor gene (TSH-R), usually leading to asymptomatic hyperthyrotropinemia, have been reported since 1995 in a total of eight pedigrees, with a pattern of transmission suggesting autosomal recessive inheritance. Although normal TSH secretion and action are not necessary for normal migration of the thyroid analage, they are essential for normal thyroid growth and function. In keeping with this concept, we report a severely hypothyroid boy with a normally located but very hypoplastic and hypofunctional thyroid caused by TSH-R loss-of-function mutations. The propositus' maternal great aunt also had apparent athyreosis. The propositus had undetectable uptake on 99mpertechnetate scintigraphy but normal plasma thyroglobulin at 15 days of age. He was found to be a compound heterozygote for TSH-R mutations, with the maternal allele carrying a splicing mutation (G to C transversion at position +3 of the donor site of intron 6) and the other allele a deletion of two nucleotides (2 bases of codon 655 in exon 10). The great aunt's TSH-R was normal. We also report the sex ratio of hypothyroid newborns referred to our center since 1989 with apparent athyreosis (5 girls, 7 boys) and with ectopic thyroid tissue (41 girls, 15 boys). We conclude that different genetic and nongenetic mechanisms for athyreosis and ectopic thyroid are likely, and that these two distinct entities are themselves heterogeneous. Our results further show that inactivating mutations in TSH-R may account for some cases of apparent congenital athyreosis and should be suspected, especially if plasma thyroglobulin levels are normal.

Amino Acid Sequence↗

PubMed2002

Serum GH response to pharmacological stimuli and physical exercise in two siblings with two new inactivating mutations in the GH-releasing hormone receptor gene.

OBJECTIVE: Inactivating mutations of the GH-releasing hormone receptor (GHRHR) gene (GHRHR) cause familial isolated GH deficiency (IGHD) type IB. The GH response to physical exercise (PE) in patients lacking GHRHR has never been studied. We hypothesized that subjects lacking functional GHRHR may be a model to study GH response to PE. DESIGN: We have analyzed peripheral genomic DNA of a family with two sibs affected by IGHD IB for mutations in the GHRHR, studied the patients' GH response to different GH secretagogues and to PE, and examined the morphology of their pituitary gland by magnetic resonance imaging (MRI). METHODS: The GHRHR was analyzed by direct sequencing of the 13 exons, intron-exon boundaries, and of the proximal 327 bp of the promoter region in the index case. The patients' GH response to GHRH and standardized PE was studied twice, using a GH ultrasensitive assay in the second round of testing. RESULTS: Both subjects were compound heterozygotes for two previously undescribed mutations in the GHRHR that are predicted to cause complete lack of functional GHRHR protein: a nonsense mutation in codon 43 (Q43X), and a splice mutation at the beginning of intron 3 (IVS3+1G-->A). MRI showed hypoplasia of their anterior pituitaries. Both subjects had a small but detectable increase in serum GH after maximal PE. CONCLUSIONS: GHRHR mutations need to be considered in IGHD IB patients even in the absence of parental consanguinity, and patients lacking GHRHR may provide a model to study the mechanism by which PE influences GH secretion.

Base Sequence↗

PubMed2002

Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes.

Hermansky-Pudlak syndrome (HPS) has evolved into a group of genetically distinct disorders characterized by oculocutaneous albinism, a storage pool deficiency, and impaired formation or trafficking of intracellular vesicles. HPS-1 results from mutations in the HPS1 gene and affects approximately 400 individuals in northwest Puerto Rico due to a 16-bp duplication in exon 15. Another 13 mutations have been reported in non-Puerto Ricans. HPS1 codes for a 79.3 kDa cytoplasmic protein of unknown function. HPS-1 patients typically develop fatal pulmonary fibrosis in their fourth decade. HPS-2 is caused by mutations in ADTB3A, which codes for the beta3A subunit of the adaptor protein-3 complex, AP3. This coat protein complex has been localized to the TGN as well as to a peripheral endosomal compartment. Evidence indicates that AP3 plays a role in the stepwise process of vesicular trafficking which leads to formation of the melanosomal, platelet dense body and lysosomal compartments. All three known HPS-2 patients had childhood neutropenia and infections. HPS-3 results from mutations in HPS3 and affects central Puerto Ricans homozygous for a 3904-bp deletion removing exon 1. At least 8 non-Puerto Rican patients have other HPS3 mutations, including an IVS5+1G->A splicing mutation in five Ashkenazi Jewish patients. HPS3 codes for a 113.7 kDa protein of unknown function. HPS-3 manifests with mild hypopigmentation and bleeding. All types of HPS are diagnosed by whole mount electron microscopic demonstration of absent platelet dense bodies, and molecular diagnoses are available for the Puerto Rican HPS1 and HPS3 founder mutations. Mouse and Drosophila models provide candidates for new genes causing HPS in humans. These genes will reveal the pathways by which specialized vesicles of lysosomal lineage arise within cells.

Animals↗

PubMed2003

Molecular epidemiological study of alpha- and beta-thalassemia in Sihui city.

OBJECTIVE: To investigate alpha- and beta-thalassemia (alpha- and beta-thal) gene frequencies and gene mutation spectrum in the population of Sihui City. METHODS: The umbilical cord blood samples from 1 007 neonates and peripheral blood samples from 1 524 apparently healthy adults for pre-marriage health check in Sihui city were collected for molecular epidemiologic study of alpha- and beta-thal respectively. The diagnostic standard for alpha-thal was the presence of Hb Bart's, and that for beta-thal was both the decrease of mean corpuscular volume (MCV<80 fl) and the increase of Hb A(2) level (> or = 3.5%). The samples of identified subjects with positive thal genotypes were further examined with PCR-based DNA analysis for determining the alpha- or beta-globin gene genotype, while those from subjects with positive genotypes but without mutations known to Chinese subjects were subjected to DNA sequence analysis of beta-globin gene. In addition, the alpha-thal alleles, -alpha(3.7) and -alpha(4.2) were examined in all umbilical cord blood samples. RESULTS AND CONCLUSION: Of all the 1 007 umbilical cord blood samples, 110 were identified as from alpha-thal gene carriers, 3 from patients Hb H disease and 1 from patients with hydrops fetalis, which meant an alpha-thal gene frequency of 11.72% (118/1 007). Three types of alpha-gene deletion were identified in this cohort, with the frequency of 53.4% (--SEA)), 34.7% (-alpha(3.7)) and 11.9% (-alpha(4.2)) respectively. By examining the peripheral venous blood samples from the 1,524 healthy adult subjects, 59 subjects were found to be beta-thal gene carriers with a rate of 3.87% (59/1,524), whose genotypes were determined and from whom 7 beta-thal mutations were identified. Of these 59 beta-thal gene carriers, 11 were diagnosed as having heterozygotes compound for beta- and alpha-thal genes with the deletion of the --(SEA) in 7 cases and -alpha(3.7) in 4 cases respectively, showing an incidence of 0.72% (11/1,524). The three commonest point mutations, beta CD41-42 (-CTTT) frameshift mutation, beta IVS2-654(C-->T) aberrant splicing mutation and beta-28 (A-->G) transcription mutation occurred with a total frequency of 84.75% among subjects with beta-thal allele mutations. In addition, a novel mutation, beta-globin gene promoter -90 (C-->T) allele was detected for the first time in Chinese subjects.

Adult↗