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K Stephens

Publications and source records attributed to K Stephens.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification of NF1 mutations in both alleles of a dermal neurofibroma.

A hallmark clinical feature of neurofibromatosis 1 (NF1) is multiple dermal neurofibromas, benign tumours that typically appear in early adolescence and increase in numbers throughout life. The pathogenesis of these tumours is not known. One domain of the NF1 gene product, neurofibromin, stimulates the intrinsic GTPase of Ras, and inactivation of both NF1 alleles has been demonstrated in specific malignancies. These observations support the contention that the NF1 gene product is a tumour suppressor that is involved in the Ras signal transduction pathway. Even though accumulating evidence demonstrates that NF1 acts as a tumour suppressor in some cells, mutations have not been identified in both NF1 alleles in dermal neurofibromas. Using standard techniques to analyse DNA extracted from benign neurofibromas, numerous investigators failed to identify loss of heterozygosity (LOH) in multiple tumours. In contrast to these reports, Colman et al. demonstrated NF1 LOH of dermal neurofibromas derived from 2 of 5 NF1 patients, yet the constitutional NF1 mutations in these patients were not identified, and the extent of the somatic deletions beyond the NF1 locus were not established. In this study, we show that a dermal neurofibroma from an NF1 individual who has a constitutional deletion of the entire NF1 locus harbours a 4-bp deletion of NF1 exon 4b in the other allele. This is the first definitive identification of a somatic mutation which is limited to the NF1 locus in a benign neurofibroma from an NF1 individual in whom the constitutional NF1 mutation is known.

Alleles↗
PubMed1996

The detection of contiguous gene deletions at the neurofibromatosis 1 locus with fluorescence in situ hybridization.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized primarily by the development of multiple neurofibromas and pigmentary changes. The recent identification of contiguous gene deletions in NF1, a previously unrecognized molecular basis for this disorder, raises important questions regarding deletion frequency in the patient population and the role that contiguous genes may play in the physical manifestations of NF1 patients. To facilitate the identification of patients with large NF1 deletions, we have isolated clones carrying large genomic segments from the NF1 locus and tested their efficacy as probes for fluorescence in situ hybridization (FISH). Clone P1-9 spans approximately 65 kb of the NF1 gene, including exons 2-11, and clone P1-12 carries approximately 55 kb of NF1 intron 27B. FISH studies performed with P1-9, P1-12, and a set of overlapping 1F10 cosmid clones mapping telomeric to the NF1 locus identified large deletions in two new neurofibromatosis type 1 patients who, like previously characterized deletion patients, had mildly dysmorphic facial features and large numbers of cutaneous neurofibromas.

Chromosome Aberrations↗
PubMed1995

Methods for rapid detection of a recurrent nonsense mutation and documentation of phenotypic features in neurofibromatosis type 1 patients.

We have developed a rapid screening method to detect a recurrent mutation in the neurofibromatosis type 1 gene. Using gene amplification and hybridization with allele-specific oligonucleotides, we screened 97 unrelated affected individuals for the recurrent C-->T substitution in codon 1947. The mutation was detected in 1 patient and found to cosegregate with the disease phenotype in the patient's family. Although the estimated prevalence of this mutation is low, rapid screening of different patient cohorts would identify multiple individuals carrying the same mutation. Such data would provide the first opportunity for examining correlations between phenotypic characteristics and molecular genotype and would allow clinicians to offer early diagnosis and prenatal screening to affected families. A format for the comparison of phenotypic features in other settings is presented.

Base Sequence↗
PubMed1995

A common keratin 5 gene mutation in epidermolysis bullosa simplex--Weber-Cockayne.

The Weber-Cockayne subtype of epidermolysis bullosa simplex is an inherited skin-fragility disorder characterized by basal keratinocyte lysis and epidermal blistering confined primarily to the hands and feet. The disorder results from a mutation in either the keratin 5 or keratin 14 gene, which encode the peptide components of the obligate heterodimeric keratin intermediate filaments of the basal cell. We have determined that a T-->G substitution mutation in keratin 5, which results in a Ile-->Ser change at codon 161, is common among patients with the Weber-Cockayne disease variant, accounting for six of 13 cases tested. The observed high frequency of this mutation may result from either a mutational hot spot or a founder effect. The potential utility of this common mutation in confirming disease status in some at-risk individual is discussed.

Base Sequence↗
PubMed1995

Epidermolysis bullosa simplex: a keratin 5 mutation is a fully dominant allele in epidermal cytoskeleton function.

To explore the relationship between abnormal keratin molecules, 10-nm intermediate filament (IF) organization, and epidermal fragility and blistering, we sought to determine the functional consequences of homozygosity for a dominant keratin defect. We describe a family with an autosomal dominant skin-blistering disorder, epidermolysis bullosa simplex, Koebner subtype (EBS-K), that has a novel point mutation, occurring in the keratin 5 gene (KRT5), that predicts the substitution of an evolutionarily conserved lysine by an asparagine residue (K173N). Unlike previous heterozygous mutations located within the initial segment of domain 1A of keratin molecules, K173N heterozygosity did not result in severe disease or clumping of keratin filaments. One family member was found to be homozygous for the K173N allele, having inherited it from each of her affected first-cousin parents. Despite a lack of normal keratin 5 molecules, and an effective doubling of abnormal molecules, available for heterodimerization with keratin 14 during IF formation, there were no significant differences in the clinical severity or the ultrastructural organization of the keratin IF cytoskeleton of the homozygous individual. These data demonstrate that the K173N mutation behaves as a fully dominant allele and indicate that a limited number of abnormal keratin molecules are sufficient to impair cytoskeletal function and elicit epidermal fragility and blistering.

Alleles↗
PubMed1994

Autosomal dominant cyclic hematopoiesis: exclusion of linkage to the major hematopoietic regulatory gene cluster on chromosome 5.

Autosomal dominant cyclic hematopoiesis (ADCH), or cyclic neutropenia, is a genetic disorder characterized by cyclic oscillations of neutrophils and other blood cells. To determine if the gene for ADCH mapped within the major hematopoietic regulatory gene cluster at 5q23.3-q33.3, we tested five families with ADCH for genetic linkage between the disorder and loci on chromosome 5q. Two-point analyses gave significant evidence in favor of excluding linkage between ADCH and the hematopoietic genes granulocyte-macrophage colony-stimulating factor (CSF), interleukins 3, 4, 5, and 9, and the receptor of macrophage-CSF. Furthermore, the exclusion data provide evidence for rejecting the hypothesis that ADCH may be encoded by a new gene mapping within this cluster.

Chromosome Aberrations↗
PubMed1994

Evidence for locus heterogeneity in human autosomal dominant split hand/split foot malformation.

Split hand/split foot (SHSF; also known as ectrodactyly) is a human developmental disorder characterized by missing central digits and other distal limb malformations. An association between SHSF and cytogenetically visible rearrangements of chromosome 7 at bands q21-q22 provides compelling evidence for the location of a causative gene at this location, and the locus has been designated SHFD1. In the present study, marker loci were localized to the SHFD1 critical region through the analysis of somatic cell hybrids derived from individuals with SHSF and cytogenetic abnormalities involving the 7q21-q22 region. Combined genetic and physical data suggest that the order of markers in the SHFD1 critical region is cen-D7S492-D7S527-(D7S479-D7S491)-SHFD1-++ +D7S554-D7S518-qter. Dinucleotide repeat polymorphisms at three of these loci were used to test for linkage of SHSF to this region in a large pedigree that demonstrates autosomal dominant SHSF. Evidence against linkage of the SHSF gene to 7q21-q22 was obtained in this pedigree. Therefore, combined molecular and genetic data provide evidence for locus heterogeneity in autosomal dominant SHSF. We propose the name SHSF2 for this second locus.

Adult↗
PubMed1994

Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3-q22.1.

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHfD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON-D7S812-SHFD1-D7S811-ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electrophoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.

Adult↗
PubMed1994

Deletions spanning the neurofibromatosis 1 gene: identification and phenotype of five patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by marked variation in clinical severity. To investigate the contribution to variability by genes either contiguous to or contained within the NF1 gene, we screened six NF1 patients with mild facial dysmorphology, mental retardation, and/or learning disabilities, for DNA rearrangement of the NF1 region. Five of the six patients had NF1 gene deletions on the basis of quantitative densitometry, locus hemizygosity, and analysis of somatic cell hybrid lines. Analyses of hybrid lines carrying each of the patient's chromosomes 17, with 15 regional DNA markers, demonstrated that each of the five patients carried a deletion > 700 kb in size. Minimally, each of the deletions involved the entire 350-kb NF1 gene; the three genes--EVI2A, EVI2B, and OMG--that are contained within an NF1 intron; and considerable flanking DNA. For four of the patients, the deletions mapped to the same interval; the deletion in the fifth patient was larger, extending farther in both directions. The remaining NF1 allele presumably produced functional neurofibromin; no gene rearrangements were detected, and RNA-PCR demonstrated that it was transcribed. These data provide compelling evidence that the NF1 disorder results from haploid insufficiency of neurofibromin. Of the three documented de novo deletion cases, two involved the paternal NF1 allele and one the maternal allele. The parental origin of the single remaining expressed NF1 allele had no dramatic effect on patient phenotype. The deletion patients exhibited a variable number of physical anomalies that were not correlated with the extent of their deletion. All five patients with deletions were remarkable for exhibiting a large number of neurofibromas for their age, suggesting that deletion of an unknown gene in the NF1 region may affect tumor initiation or development.

Adolescent↗
PubMed1993

A keratin 14 mutational hot spot for epidermolysis bullosa simplex, Dowling-Meara: implications for diagnosis.

Recently, two patients with the Dowling-Meara subtype of epidermolysis bullosa simplex (EBS-DM) were reported with different mutations in codon 125 of the keratin 14 gene. To determine whether these are common mutations, we screened ten EBS-DM patients and their families using single nucleotide primer extension. Four of ten unrelated EBS-DM patients had a G-->A substitution at base pair 434 of codon 125, whereas one case out of ten had a C-->T substitution at position 433 of the same codon. The G434A alteration cosegregated with the disorder in two multigenerational families; no recombination events were detected. In these two families, linkage analysis provided significant evidence in favor of linkage between G434A and the EBS-DM phenotype, with a LOD score of 3.29 at a recombination rate of 0%. Codon 125 substitutions identified in three unrelated sporadic EBS-DM patients were not found in their clinically unaffected parents. Together, these data provide compelling genetic evidence that the codon 125 substitutions are causal for EBS-DM. The high frequency of mutation at this site in individuals with EBS-DM now makes DNA-based diagnosis of this disorder feasible.

Amino Acid Sequence↗
PubMed1993

Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.

Agrobacterium tumefaciens↗
PubMed1993

From the clinic to the research laboratory. The role of the clinician in molecular genetic studies.

BACKGROUND: The first physician to examine a patient with a genetic disorder or birth defect is usually a specialist in a field other than genetics. The presentation of certain categories of patients of particular interest to molecular genetics research may be distinct. The recognition of these patients by clinicians is fundamental to the study of genetic disorders at the DNA level. OBSERVATIONS: Neurofibromatosis type 1 is a paradigm for how the study of a single genetic disease and its multiple molecular features has been facilitated by the use of various categories of patients. Other examples of interest to dermatologists, surgeons, and other specialists are discussed to demonstrate how the identification of key patients was instrumental in studies of gene localization and subsequent cloning, gene clusters or contiguous gene deletion syndromes, or mutation phenomena such as imprinting, uniparental disomy, and gonadal mosaicism. The molecular researcher has limited access to surgical specimens, and the donation of skin, tumor, and other tissues may lead to increased knowledge of new mutations in somatic mosaicism, or loss of heterozygosity of tumor suppression genes in cancer. CONCLUSIONS: Guidelines are suggested to alert the physician to each of these categories of individuals with unusual presentation, as well as to recognize that the study of families with rare disorders may enable scientists to locate the responsible genes. The teamwork of clinician and molecular researcher is essential for the advancement of our understanding of DNA mechanisms in genetic disease. The ethics involved in referral of patients to molecular genetic research studies are discussed.

Chromosome Aberrations↗
PubMed1992

Preferential mutation of the neurofibromatosis type 1 gene in paternally derived chromosomes.

An interesting feature of neurofibromatosis type 1 (NF1) is its high mutation rate of 1 x 10(-4) per gamete per generation. The molecular basis for frequent NF1 mutation in unknown; the gene is not deletion prone. We have found that in all ten families examined, the apparent new NF1 mutation occurred on the paternally-derived chromosome. The probability of observing this result by chance is less than 0.001 assuming an equal frequency of mutation of paternal and maternal NF1 genes. We hypothesize a role for genomic imprinting that may either enhance mutation of the paternal NF1 gene or confer protection from mutation to the maternal NF1 gene.

Chromosomes, Human↗
PubMed1992

The gene for a novel epidermal antigen maps near the neurofibromatosis 1 gene.

Recently the M17S1 gene, encoding an epidermal antigen thought to play a role in cell adhesion, was mapped to chromosome bands 17q11-q12, placing it in the vicinity of the gene for the genetic disorder neurofibromatosis 1 (NF1). The pleomorphic cutaneous lesions of NF1 and the precedent for other genes being embedded within the NF1 gene prompted us to investigate whether the M17S1 gene mapped near, or within, the NF1 gene. Genetic linkage analyses revealed that M17S1 was tightly linked to NF1 and mapped within the interval bounded by D17S58 and D17S54. Physical mapping of an M17S1 cDNA on somatic cell hybrids, yeast artificial chromosomes, and an NF1 patient with a deletion involving an entire NF1 allele demonstrated that M17S1 is located at least 180 kb centromeric to the NF1 gene. The distance between the genes suggests that M17S1 is unlikely to contribute to the NF1 phenotype since a gross chromosomal rearrangement would be required to disrupt expression of both genes.

Animals↗