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Biomedical subjects

J C Kaplan

Publications and source records attributed to J C Kaplan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Neurosensory hearing loss in secondary adhalinopathy.

We report mild-to-moderate neurosensory hearing loss and severe childhood autosomal recessive muscular dystrophy with adhalin-deficiency in two siblings from a Bulgarian sibship of Turkish origin. Microsatellite analysis excluded linkage to the adhalin gene, mutations of which cause limb girdle muscular dystrophy (LGMD) 2D, but was compatible with linkage to the gene locus of LGMD 2C on chromosome 13q12. Compound heterozygosity of the affected siblings was detected in this chromosomal region. A severe autosomal recessive form of neurosensory deafness has been linked to the same region (locus NSRD1) which is now contained in a 7 Mb YAC contig. Using polymorphic markers and STS PCR primers mapping in this contig, we did not find evidence for major rearrangements in the suspected region. These preliminary findings are not in favor of, but do not completely exclude a contiguous gene syndrome in these cases. Therefore, we consider a potential role of the putative 13q12 gene product and/or adhalin in neurosensory hearing.

Alleles↗
PubMed1996

A founder mutation in the gamma-sarcoglycan gene of gypsies possibly predating their migration out of India.

We investigated the molecular basis of a severe form of early onset autosomal recessive muscular dystrophy with sarcoglycan (SG) deficiency in seven large Gypsy families living in different parts of Western Europe and apparently not closely related. They were linked to the LGMD2C locus (13q12) suggesting a primary defect in the gamma-SG gene coding for the 35 kDa dystrophin-associated glycoprotein. All of the 18 investigated patients were homozygous for the same G-->A transition in codon 283 producing the replacement of a conserved cysteine of the extra-cellular domain of the protein by a tyrosine. All affected chromosomes in homozygous and heterozygous relatives carried the same allele 5 of the intragenic marker D13S232. Flanking markers were studied to delineate a common ancestral haplotype, the size of which was used to compute the date of the founding mutation. We found evidence that the mutation occurred between 60 and 200 generations ago, therefore possibly predating the commonly accepted date of migration of the Gypsy ancestors out of India.

Biomarkers↗
PubMed1996

Effects of zidovudine-selected human immunodeficiency virus type 1 reverse transcriptase amino acid substitutions on processive DNA synthesis and viral replication.

Certain amino acid substitutions in the reverse transcriptase (RT), including D67N, K70R, T215Y, and K219Q, cause high-level resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine (3'-azidothymidine; AZT) and appear to approximate the template strand of the enzyme-template-primer complex in structural models. We studied whether this set of mutations altered RT-template-primer interaction as well as their effect on virus replication in the absence of inhibitor. When in vitro polymerization was limited to a single association of an RT with an oligodeoxynucleotide-primed heteropolymeric RNA template (a single processive cycle), recombinant-expressed mutant 67/70/215/219 RT synthesized 5- to 10-fold more high-molecular-weight DNA products (>200 nucleotides in length) than wild-type RT. This advantage was maintained as deoxynucleoside triphosphate (dNTP) concentrations were decreased to limiting levels. In contrast, no difference was seen between wild-type and mutant RTs under conditions allowing repeated associations of enzyme with template-primer. Because intracellular dNTP concentrations are low prior to mitogenic stimulation, we compared replication of mutant 67/70/215/219 virus and wild-type virus in peripheral blood mononuclear cells (PBMC) stimulated before and after infection. In the absence of inhibitor, mutant 67/70/215/219 virus had a replication advantage in PBMC stimulated with phytohemagglutinin and interleukin-2 after infection, but virus replication was similar in PBMC stimulated before infection in vitro. The results confirm that RT mutations D67N, K70R, T215Y, and K219Q affect an enzyme-template-primer interaction in vitro and suggest that such substitutions may affect HIV-1 pathogenesis during therapy by increasing viral replication capacity in cells stimulated after infection.

Base Sequence↗
PubMed1996

Analysis of alternative splicing patterns in the cystic fibrosis transmembrane conductance regulator gene using mRNA derived from lymphoblastoid cells of cystic fibrosis patients.

Using in vitro amplification of cDNA by the polymerase chain reaction, we analyzed alternatively spliced events of cystic fibrosis transmembrane conductance regulator gene in lymphoblastoid cells. Ten alternatively spliced transcripts were identified using analysis of 6 overlapping segments of amplified cDNA, 4 of which have not been described previously. These include transcripts lacking exon 16, 17b, 22 and a transcript resulting from the use of a cryptic acceptor and donor splice sites. Moreover, in 2 cystic fibrosis (CF) patients bearing nonsense mutations E60X or W1282X, we observed that nonsense mutations are associated with an alteration of splice site selection in vivo resulting in exon skipping of constitutive exons or in the use of cryptic splice sites. In addition, even though lymphoblastoid cells are not the relevant tissue to address the question of the relationship between clinical respiratory phenotype and genotype, our results concerning adult CF patients (delta F508/ delta F508) suggest that individual-specific RNA splicing patterns could influence the severity of the CF pulmonary disease. If this phenomenon of alternative splicing events proves to be significant in CF and to be a common feature of disease genes, the study of RNA splicing could become an important tool for the analysis of the genotype-phenotype relationship in many inherited disorders.

Adult↗
PubMed1996

Genotype-phenotype relationships in a cohort of adult cystic fibrosis patients.

In cystic fibrosis (CF), relationships between genotype and phenotype have been shown for pancreatic status but not for pulmonary disease. One hundred and ten adult CF patients were classified according to the expected effect of their mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein: Group 1 (n=48) included deltaF508 homozygotes; Group 2 (n=26), patients with two "severe" mutations and no expected CFTR production; Group 3 (n=17), patients with expected partly functional CFTR corresponding to at least one "mild" mutation; Group 4 (n=19), patients with no mutation identified or only one identified "severe" mutation. As compared to Groups 1 and 2: patients from Groups 3 and 4 had higher arterial oxygen tension (Pa,O2) (9.5+/-1.9 and 9.9+/-1.5 vs 8.8+/-1.5 and 8.3+/-1.7 kPa, respectively p<0.02); and a slower decline in their pulmonary function, estimated by the mean annual loss in forced vital capacity (FVC) (1.2+/-1.0 and 1.5+/-1.1 vs 2.0+/-0.9 and 2.2+/-1.0%, respectively; p<0.01) and in forced expiratory volume in one second (FEV1) (1.7+/-1.1 and 1.9+/-1.3 vs 2.6+/-1.0 and 2.8+/-1.0%, respectively; p<0.005). They had fewer episodes of colonization of the airways by Pseudomonas aeruginosa, and colonization occurred at a more advanced age (median age 25 and 19 vs 15 and 17 yrs, respectively; p<0.01) and required fewer intravenous antibiotic courses (p<0.01). Pancreatic insufficiency was less frequent in Groups 3 (23%) and 4 (63%) than in Groups 1 (100%) and 2 (96%). This study suggests that the phenotype of adult cystic fibrosis patients, including the severity of the lung disease, is related to the severity of the cystic fibrosis transmembrane conductance regulator mutations.

Adolescent↗
PubMed1996

Sequence analysis of the CAG triplet repeats region in the Huntington disease gene (IT15) in several mammalian species.

Huntington's disease (HD) is a progressive neurodegenerative disease linked to abnormally expanded CAG repeats in the first exon of the IT15 gene. Neither HD or other glutamine expansion triplet disease has not yet been described in other species as human. In this study, we sequenced the region containing the CAG repeat expansion region of the corresponding IT15 gene in several mammalian species. In all investigated species, the number of CAG found is equal or inferior to the normal range of human IT15 alleles. The longer uninterrupted CAG stretch was found in the pig and consists of 18 CAG. In the other species, the CAG stretch was shorter and/or interrupted by other triplets. The proline encoding region following the CAG stretch is conserved in the mammalian species studied here and the number of proline is few or not reduced compared to the number of the glutamine codon repeats region.

Amino Acid Sequence↗
PubMed1995

A T to G mutation in the polypyrimidine tract of the second intron of the human beta-globin gene reduces in vitro splicing efficiency: evidence for an increased hnRNP C interaction.

In a patient with a beta-thalassemia intermedia, a mutation was identified in the second intron of the human beta-globin gene. The U-->G mutation is located within the polypyrimidine tract at position -8 upstream of the 3' splice site. In vivo, this mutation leads to decreased levels of the hemoglobin protein. Because of the location of the mutation and the role of the polypyrimidine tract in the splicing process, we performed in vitro splicing assays on the pre-messenger RNA (pre-mRNA). We found that the splicing efficiency of the mutant pre-mRNA is reduced compared to the wild type and that no cryptic splice sites are activated. Analysis of splicing complex formation shows that the U-->G mutation affects predominantly the progression of the H complex towards the pre-spliceosome complex. By cross-linking and immunoprecipitation assays, we show that the hnRNP C protein interacts more efficiently with the mutant precursor than with the wild-type. This stronger interaction could play a role, directly or indirectly, in the decreased splicing efficiency.

Base Sequence↗
PubMed1995

Four new mutations in the NADH-cytochrome b5 reductase gene from patients with recessive congenital methemoglobinemia type II.

Recessive congenital methemoglobinemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases. In the type I form, cyanosis is the only symptom, and the soluble enzyme is defective in red blood cells. In the type II form, cyanosis is associated with severe mental retardation and neurologic impairment; the enzymatic defect is systemic, involving both soluble and membrane-bound isoforms. We characterized mutations responsible for cytb5r deficiency in three unrelated patients with severe RCM type II. The first patient presented a homozygous exon 5 skipping. The only mutation detected was a homozygous G to C transversion at position +8, downstream from the 5' splice site of exon 5. We suggest that this unusual mutation might be responsible for the abnormal splicing of the primary transcripts, resulting in frameshift with premature STOP codon. The second mutation found corresponds to a homozygous C to T transition changing the Arg-218 codon to a premature STOP codon in exon 8. The third case was a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation with replacement of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele carried a 3-bp deletion (TGA) of nucleotides 815 to 817, modifying two contiguous codons in exon 9 of the cDNA with loss of Met-272. These results confirm the genetic polymorphism of cytb5r gene mutations identified in RCM type II, as observed for the mutations described in the RCM type I, and shed light on the molecular bases of the two different diseases associated with cytb5r deficiency.

Amino Acid Sequence↗
PubMed1995

Mutation heterogeneity of cystic fibrosis in France: screening by denaturing gradient gel electrophoresis using psoralen-modified oligonucleotide.

In order to establish counseling guidelines and aid carrier risk assessment, we sought to establish the frequencies of cystic fibrosis (CF) mutations that are present in CF families living in the Ile de France region, a region notable for its ethnic heterogeneity. We studied 470 CF chromosomes in which we identified more than 90% of the CF mutations. We systematically screened 21 exons and the adjacent sequences of the CF transmembrane conductance regulator (CFTR) gene by denaturing gradient gel electrophoresis using chemical clamps. We detected 41 different CF mutations located in 14 exons. One of these mutations had not been previously described. Given the heterogeneity of these mutations, population screening does not appear to be readily feasible in our population.

Base Sequence↗
PubMed1995

Three novel sequence variations in the 5' upstream region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: two polymorphisms and one putative molecular defect.

More than 400 sequence alterations have been identified in the whole coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene corresponding to the 27 exons and their exon-intron boundaries. However, in some CF chromosomes, no mutation has yet been detected. In such cases, we have explored the promoter and the sequence up to position -1000 from the cap site, by using denaturing gradient gel electrophoresis. This study concerning 35 CF chromosomes has allowed us to identify three novel sequence variations located in the 5' upstream region of the gene. The T to G substitution located at position -895 from the cap site could be considered as a polymorphic variation. The second substitution (C to T at position -816) has been detected on only one CF chromosome, but does not concern a regulatory DNA element previously described. Conversely, the third substitution (a T to G substitution at position -741 from the cap site) is located at a potential AP-1 binding site. We have investigated, by electrophoretic mobility shift assay, the ability of this region to bind nuclear factors. We have found that the normal sequence between -740/-745 does not bind either the AP-1 transcription factor or AP-1 related proteins, and that the T to G-741 mutated sequence exhibits an abnormal binding pattern suggesting the possible deleterious effect of still unknown negative trans-acting factors.

Base Sequence↗
PubMed1995

HIV-1 DNA in fibroblast cultures infected with urine from HIV-seropositive cytomegalovirus (CMV) excretors.

Interactions between HIV-1 and CMV may be important in the pathogenesis of AIDS. We have studied whether active CMV infection alters the cell tropism of HIV-1 in dually-infected individuals. Urines from HIV-seropositive individuals excreting CMV were compared to urines from CMV non-excretors. Sixty-six urines from HIV-seropositive individuals were tested. Infectious HIV-1 was not detected in any of the concentrated urines tested. The urines were filtered, concentrated, DNase-treated and cultured on HIV-1 non-permissive human forestin fibroblasts. HIV-1 DNA was detected by PCR with pol gene primers in 5 of 39 MRHF cell cultures inoculated with CMV culture positive urine (p = 0.037). HIV-1 DNA was not detected by PCR in uninfected fibroblasts, in fibroblasts inoculated with CMV uninfected urine from 27 HIV-seropositive patients or in fibroblasts cultured with 9 CMV culture positive urines from 16 HIV-seronegative renal transplant recipients. Supernatant fluid from an HIV-1 PCR-positive culture was passaged onto another fibroblast monolayer, and these cells were negative for HIV-1 DNA. Direct inoculation of fibroblasts with HIV-1 did not yield evidence of infection by PCR. CMV infection may facilitate HIV-1 DNA entry into ordinarily non-permissive cells.

Cells, Cultured↗
PubMed1995

Diagnosis of "sporadic" Huntington's disease.

The diagnosis of Huntington's disease (HD) in patients with progressive chorea and mental impairment, but without similarly affected relatives, remains uncertain and impedes genetic counseling. Twenty patients with suspected HD, but with no family history of the disease underwent molecular analysis of the CAG repeat in the IT15 gene for HD. Eighteen patients displayed the HD expanded allele and two had CAG repeats in the normal range. Neuropsychological tests could be performed in 12 of the 20 patients. Of these 10 with the expanded allele presented the deficits typical of HD, but not the two patients without the HD mutation. This study shows that a neuropsychological pattern is specific to patients with the expanded CAG and that most isolated patients with suspected HD are in fact affected.

Adult↗
PubMed1995

Sequence analysis of the CCG polymorphic region adjacent to the CAG triplet repeat of the HD gene in normal and HD chromosomes.

The CAG expansion responsible for Huntington's disease (HD) is followed by an adjacent polymorphic CCG repeat region which may interfere with a PCR based diagnosis. We have sequenced this region in 52 unrelated HD patients, from both normal and HD chromosomes. Fifty percent of the normal alleles were (CCG)7(CCT)2, 48% (CCG)10(CCT)2, and 2% (CCG)7(CCT)3. In contrast (CCG)7(CCT)2 was found in 85% of the HD alleles which represents significant linkage disequilibrium with the HD mutation.

Alleles↗
PubMed1995

Diagnostic usefulness of the polymorphism of the GT dinucleotide and the polythymidine tract in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

15-20% of the CF mutation are expected to be rare and escape detection by systems designed to screen for common mutations. The highly polymorphic simple repeats would be particularly useful for genetic diagnosis in CF families where the mutations have not been identified. In this study, we used denaturing gradient gel electrophoresis with psoralen-modified oligonucleotide primers to study the GTnTm polymorphism previously identified at the intron 8 - exon 9 junction of the CFTR gene. Twelve characteristic patterns were identified. The most frequent genotype in CF alleles was GT10T9 and in non-CF alleles GT11T7. In this study, the heterozygous incidence is 70% in unrelated CF carriers. This polymorphism is full informative in 45% and half-informative in 50%. We conclude, that this polymorphism, easy to study by a relatively simple, rapid and cheap procedure, would be particularly useful in genetic counselling for CF and prenatal diagnosis in CF families in which mutations have not been yet identified.

Cystic Fibrosis↗