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Biomedical subjects

M Goossens

Publications and source records attributed to M Goossens.

At least 109 records · Page 6Linked to original sources
PubMed1994

Hb Taybe (alpha 38 or 39 THR deleted): an alpha-globin defect, silent in the heterozygous state and producing severe hemolytic anemia in the homozygous.

Several alpha-chain hemoglobin variants have been described as responsible, in homozygous or compound heterozygous patients, for a chronic hemolytic disease that overlaps thalassemia and Heinz bodies hemolytic anemia phenotypes. These variants are present in trace amounts together with some Hb H in the lysate of the patients. In the asymptomatic heterozygous carriers, they are usually not detected by electrophoretic methods. Hb Taybe is an example of such an unstable and thalassemic alpha-hemoglobin variant. This hemoglobin was observed in a young Israeli Arab woman having suffered since birth from a severe and highly regenerative hemolytic anemia for which she was splenectomized at age sixteen. The structural abnormality was characterized by protein chemistry as the deletion of a threonine residue at position alpha 38 or 39 and assigned to the alpha 1 gene by selective DNA sequencing. This structural modification is localized in helix C, which is a highly conserved 3(10) helix participating in the alpha 1 beta 2 contact and close to the alpha 1 beta 1 interface. The propositus and two siblings, who were also anemic, were found to be homozygous for the molecular defect, although the abnormal Hb was not detected in the latters. Consanguinity in this family demonstrated the threshold effect in the clinical manifestations of such alpha-gene disorders since heterozygotes were clinically and biologically normal.

Adult↗
PubMed1994

Recurrent nonsense mutations within the type VII collagen gene in patients with severe recessive dystrophic epidermolysis bullosa.

The generalized mutilating form of recessive dystrophic epidermolysis bullosa (i.e., the Hallopeau-Siemens type; HS-RDEB) is a life-threatening disease characterized by extreme mucocutaneous fragility associated with absent or markedly altered anchoring fibrils (AF). Recently, we reported linkage between HS-RDEB and the type VII collagen gene (COL7A1), which encodes the major component of AF. In this study, we investigated 52 unrelated HS-RDEB patients and 2 patients with RDEB inversa for the presence, at CpG dinucleotides, of mutations changing CGA arginine codons to premature stop codons TGA within the COL7A1 gene. Eight exons containing 10 CGA codons located in the amino-terminal domain of the COL7A1 gene were studied. Mutation analysis was performed using denaturing gradient gel electrophoresis of PCR-amplified genomic fragments. Direct sequencing of PCR-amplified products with altered electrophoretic mobility led to the characterization of three premature stop codons, each in a single COL7A1 allele, in four patients. Two patients (one affected with HS-RDEB and the other with RDEB inversa) have the same C-to-T transition at arginine codon 109. Two other HS-RDEB patients have a C-to-T transition at arginine 1213 and 1216, respectively. These nonsense mutations predict the truncation of approximately 56%-92% of the polypeptide, including the collagenous and the noncollagenous NC-2 domains. On the basis of linkage analysis, which showed no evidence for locus heterogeneity in RDEB, it is expected that these patients are compound heterozygotes and have additional mutations on the other COL7A1 allele, leading to impaired AF formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Expression and binding properties of two isoforms of the human growth hormone receptor.

Two isoforms of the human growth hormone receptor mRNA, one containing exon 3 (encoding an extracellular domain of the receptor), hGHR, and one excluding exon 3, hGHRd3, have been described. To study the cellular distribution of the two types of messengers we have analysed a panel of tissues. Both isoforms were expressed independently or simultaneously depending on the tissue studied. To investigate the binding properties of hGHRd3 we have cloned its cDNA in a eukaryotic expression vector; transient expression in COS-7 cells showed that the receptor without exon 3 was expressed on the plasma membrane and was able to bind human growth hormone (hGH) with the same high affinity as hGHR. Human lactogen (hCS) removed 125I-hGH bound to the full-length and exon 3-excluding receptors to the same extent. These results show that hGHR and hGHRd3 have tissue-specific expression and share identical binding properties for hGH and hCS and leave open the possibility that exon 3 might influence receptor signalling.

Amino Acid Sequence↗
PubMed1993

A rapid, efficient, and sensitive assay for simultaneous detection of multiple cystic fibrosis mutations.

We describe the use of DGGE multiplex systems for rapid analysis of 15 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which about half of the known CF molecular defects are clustered. We have previously determined the spectrum of mutations affecting the CFTR gene in the French population using a strategy based on denaturing gradient gel electrophoresis (DGGE) of amplified gene segments. Analysis of CF patients' DNA with five DGGE multiplex systems permitted us to characterize nearly 35% of non-delta F508 CF alleles and increased the CF allele detection rate to almost 82% in this population. This simple and rapid multiplex analysis strategy, which allows a significant proportion of the most frequent CF mutations in Caucasians to be detected, will be helpful in the implementation of genetic screening programs.

Alleles↗
PubMed1993

Detection of sequence variations in the human insulin-receptor gene by parallel denaturing gradient gel electrophoresis.

We developed a parallel denaturing gradient gel electrophoresis (DGGE) protocol to maximize the detection of nucleotide variants in the DNA sequence coding for the mature insulin receptor and in splice site junctions. The melting behaviours of exons 2 to 22 and flanking intronic sequences were computer-simulated using two programs, MELT87 and SQHTX. The data obtained from computer analysis were used to select primers for amplification by polymerase chain reaction and optimal electrophoretic conditions. The ability of this protocol to detect nucleotide changes at the insulin-receptor locus was assessed by studying amplified DNA of a patient with leprechaunism whose insulin-receptor mutations were known and by screening the insulin-receptor gene for polymorphisms in a population of unrelated caucasian individuals. Our results demonstrate that this DGGE protocol is sensitive since it detected (1) sequence variants reported to be undetectable by means of parallel DGGE, (2) previously characterized insulin-receptor nucleotide variants, and (3) unreported polymorphisms at the insulin-receptor locus of caucasian individuals. It is also simple as perpendicular denaturing gradient gels are not required. Application of this protocol will facilitate the search for molecular defects underlying the pathogenesis of insulin resistance observed in genetic syndromes of severe insulin resistance as well as in other metabolic disorders. In addition, its ability to detect several regions of the insulin-receptor gene displaying a number of common polymorphic sites and being multiallelic will contribute to linkage studies in families with diabetic and/or insulin-resistant subjects.

Base Sequence↗
PubMed1993

Screening for cystic fibrosis in dried blood spots of newborns.

We propose a newborn cystic fibrosis (CF) screening test based on the analysis of dried blood spot DNA by a strategy involving simple or multiplex denaturing gradient gel electrophoresis (DGGE) of PCR products of CFTR gene fragments, in conjunction with the immunoreactive-trypsin (IRT) assay. From May 1988 to May 1992 we have performed a neonatal screening programme of 42,000 newborns in Brittany. We identified 450 infants with an elevated IRT level. From this cohort, to evaluate the feasibility of measuring IRT in conjunction with mutation analysis in Guthrie cards, a pilot study was initially conducted on 200 individuals with normal IRT and 150 with raised IRT levels. Furthermore, a retrospective study was performed on 189 IRT positive cards, involving mutation scanning of exons 10 and 11 of the CFTR gene, which contains a number of frequent mutations including the deletion delta F508. We show that this approach has several implications for neonatal CF screening especially in decreasing the recall rate and detecting CF carriers.

Algorithms↗
PubMed1993

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling.

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0 U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G(-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5' region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patient's mother and a grandmother. The different phenotypes associated with the G(-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.

Adult↗
PubMed1993

A missense mutation in the rod domain of keratin 14 associated with recessive epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a group of epidermal blistering diseases almost invariably transmitted as a dominant trait, which has recently been shown to arise from mutations in keratins 14 and 5 (K14 and K5). We describe a family with recessive EBS in which the disease is tightly linked to the substitution of the highly conserved glutamic acid-144 to alanine in the first helical segment of the rod domain of keratin 14. In contrast, linkage with keratin 5 was excluded. The loss of an ionic interaction with keratin 5 is likely to affect K14-K5 heterodimer formation. Our data suggest that this mutation underlies EBS in our family, and that mutations in keratin genes may impair the mechanical integrity of basal keratinocytes in a recessive as well as dominant fashion.

Alanine↗
PubMed1993

A homozygous insertion-deletion in the type VII collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa.

The Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a life-threatening autosomal disease characterized by loss of dermal-epidermal adherence with abnormal anchoring fibrils (AF). We recently linked HS-RDEB to the type VII collagen gene (COL7A1) which encodes the major component of AF. We describe a patient who is homozygous for an insertion-deletion in the FN-4A domain of the COL7A1 gene. This defect causes a frameshift mutation which leads to a premature stop codon in the FN-5A domain, resulting in a marked diminution in mutated mRNA levels, with no detectable type VII collagen polypeptide in the patient. Our data suggest strongly that this null allele prevents normal anchoring fibril formation in homozygotes and is the underlying cause of HS-RDEB in this patient.

Base Sequence↗
PubMed1993

Spectrum of growth hormone receptor mutations and associated haplotypes in Laron syndrome.

Laron syndrome is a rare autosomal recessive disorder characterized by resistance to growth hormone (GH). In 10 patients of different ethnic origins, we have analyzed all the GH receptor (GHR)-coding exons along with their splice junctions and 6 intragenic polymorphic sites defining several GHR gene haplotypes. This allowed us to identify the mutations in the 20 chromosomes studied and to describe a new GHR haplotype. Eleven different mutations associated with various GHR haplotypes were observed; they included 3 nonsense mutations, 3 splice defects and 5 missense mutations. Of the 11 mutations, 8 were novel. All the mutations involved the exoplasmic domain of the receptor and all the missense mutations were clustered in a short polypeptide segment. Most of the missense mutations affected residues conserved among GHRs from different species and the related molecules that belong to the cytokine receptor superfamily. Adding to the 5 mutations so far described, these findings illustrate the allelic heterogeneity of this syndrome and document the independent origin of the molecular defects, all features of clinical relevance for genetic counselling.

Alleles↗
PubMed1993

Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.

Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross-linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.

Cross-Linking Reagents↗
PubMed1993

Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing.

Germline mutations in the RB1 gene confer hereditary predisposition to retinoblastoma. The majority of these mutations occur de novo and differ from one patient to another. Cytogenetics and Southern blotting were shown to detect less than 15% of constitutional rearrangements. In this study we used the polymerase chain reaction (PCR) combined with denaturant gradient gel electrophoresis (DGGE) to detect point mutations or small deletions and insertions in a pool of 120 unrelated retinoblastoma patients. Partial DGGE analysis of the RB1 gene enabled us to identify sequence alterations generating stop codons, leading to amino acid substitution or affecting splice sites as well as several polymorphisms. Most of the nucleotide changes detected are flanked by direct repeats. The approach described here has proved to be a useful method for the detection of germline mutations in the RB1 gene.

Base Sequence↗
PubMed1993

Hepatitis C virus infection can mimic type 1 (antinuclear antibody positive) autoimmune chronic active hepatitis.

Hepatitis C virus (HCV) has been shown to induce anti-liver-kidney microsomal-1 (LKM1) antibody positive chronic active hepatitis, simulating type 2 autoimmune chronic active hepatitis. The cases of five patients presenting with features of type 1 (antinuclear antibody positive) autoimmune chronic active hepatitis and extrahepatic autoimmune manifestations, in whom immunosuppressive treatment had no effect on liver disease are presented. In these patients, HCV infection could be shown by the presence in serum of anti-HCV antibodies and HCV-RNA detected by polymerase chain reaction. These cases suggest the following: (a) chronic HCV infection can mimic type 1, as well as type 2, autoimmune chronic active hepatitis; (b) HCV infection might be systematically sought in patients presenting with features of type 1 autoimmune chronic active hepatitis, with special care in patients who are unresponsive to immunosuppressive treatment.

Aged↗
PubMed1993

Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene.

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Base Sequence↗
PubMed1993

Twenty-four novel hemophilia B mutations revealed by rapid scanning of the whole factor IX gene in a French population sample.

Full scanning of the factor IX gene by means of denaturing gradient gel electrophoresis enabled us to determine the molecular defects in 48 out of 49 hemophiliacs and to evaluate the spectrum of factor IX mutations in the French population. Our results further document the high molecular heterogeneity of the disease and the efficiency of this rapid screening method for disease-causing mutations. This direct approach, which is based on computer-aided analysis of the whole coding, promoter and exon-flanking factor IX gene sequences, proved to be helpful for carrier detection and prenatal diagnosis in most hemophilia B families, including sporadic cases. Moreover, we were able to identify 24 novel molecular defects of various natures in the factor IX gene.

Base Sequence↗