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Biomedical subjects

V C Sheffield

Publications and source records attributed to V C Sheffield.

At least 55 records · Page 3Linked to original sources
PubMed1999

Evaluation and molecular characterization of EHD1, a candidate gene for Bardet-Biedl syndrome 1 (BBS1).

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder characterized by developmental abnormalities including mental retardation, obesity, retinitis pigmentosa, polydactyly, short stature, and hypogenitalism. To date, five BBS loci have been identified. BBS1, located on 11q13, is reported to be the most prevalent form of BBS in the Caucasian population. A positional cloning approach is being used to identify the gene responsible for BBS1. EHD1, a new member of the EH-domain containing proteins, was identified in this study as lying within the BBS1 disease interval. RNA analysis of many tissues revealed that expression of EHD1 is ubiquitous, with elevated levels in the testis. The genomic structure of EHD1 was elucidated by direct BAC sequencing. Following identification of the intron/exon boundaries, mutational analysis was performed by single strand conformation polymorphism and direct sequencing of affected individuals from several large kindreds linked to the BBS1 locus, as well as a cohort of unrelated probands. No disease-causing mutations were identified in this analysis, but several polymorphisms were found.

Amino Acid Sequence↗
PubMed1999

The cloning and developmental expression of unconventional myosin IXA (MYO9A) a gene in the Bardet-Biedl syndrome (BBS4) region at chromosome 15q22-q23.

Bardet-Biedl Syndrome (BBS) is a heterogeneous, autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, syndactyly and/or polydactyly, short stature, and hypogenitalism and is caused by mutations at a number of distinct loci. Using a positional cloning approach for identifying the BBS4 (chromosome 15) gene, we identified and cloned an unconventional myosin gene, myosin IXA (HGMW-approved symbol MYO9A). Since mutations in unconventional myosins are known to cause several human diseases, and since mutations of unconventional myosin VIIa cause retinal degeneration, we evaluated myosin IXA as a candidate for BBS. We exploited PCR-based techniques to clone a 8473-nt cDNA for myosin IXA. A 7644-bp open reading frame predicts a protein with all the hallmarks of class IX unconventional myosins. Human Northern blot analysis and in situ hybridization of mouse embryos reveal that myosin IXA is expressed in many tissues consistent with BBS. Intron/exon boundaries were identified, and myosin IXA DNA and RNA from BBS4 patients were evaluated for mutation.

Amino Acid Sequence↗
PubMed1999

Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness.

CONTEXT: Mutations in the GJB2 gene are the most common known cause of inherited congenital severe-to-profound deafness. The carrier frequency of these mutations is not known. OBJECTIVES: To determine the carrier rate of deafness-causing mutations in GJB2 in the midwestern United States and the prevalence of these mutations in persons with congenital sensorineural hearing loss ranging in severity from moderate to profound, and to derive revised data for counseling purposes. DESIGN: Laboratory analysis, performed in 1998, of samples from probands with hearing loss for mutations in GJB2 using an allele-specific polymerase chain reaction assay, single-strand conformation polymorphism analysis, and direct sequencing. SETTING AND SUBJECTS: Fifty-two subjects younger than 19 years sequentially referred to a midwestern tertiary referral center for hearing loss or cochlear implantation, with moderate-to-profound congenital hearing loss of unknown cause, parental nonconsanguinity, and nonsyndromic deafness with hearing loss limited to a single generation; 560 control neonates were screened for the 35delG mutation. MAIN OUTCOME MEASURE: Prevalence of mutations in the GJB2 gene by congenital deafness status. RESULTS: Of 52 sequential probands referred for congenital sensorineural hearing loss, 22 (42%) were found to have GJB2 mutations. The 35delG mutation was identified in 29 of the 41 mutant alleles. Of probands' sibs, all homozygotes and compound heterozygotes had deafness. Fourteen of 560 controls were 35delG heterozygotes, for a carrier rate expressed as a mean (SE) of 2.5% (0.66%). The carrier rate for all recessive deafness-causing GJB2 mutations was determined to be 3.01% (probable range, 2.54%-3.56%). Calculated sensitivity and specificity for a screening test based on 35delG mutation alone were 96.9% and 97.4%, respectively, and observed values were 94% and 97%, respectively. CONCLUSIONS: Our data suggest that mutations in GJB2 are the leading cause of moderate-to-profound congenital inherited deafness in the midwestern United States. Screening of the GJB2 mutation can be offered to individuals with congenital deafness with high sensitivity and specificity by screening only for the 35delG mutation. A positive finding should establish an etiologic diagnosis and affect genetic counseling.

Adolescent↗
PubMed1999

Expression pattern and in situ localization of the mouse homologue of the human MYOC (GLC1A) gene in adult brain.

The MYOC (GLC1A) gene has recently been associated with both juvenile-onset primary open angle glaucoma (JOAG) and typical late-onset primary open angle glaucoma (POAG). As a result, much scrutiny has been focused on the pathology of these diseases. In order to better understand the pathophysiology of POAG, we have been developing a mouse model of the disease. As a step in this development, we have investigated the expression pattern of Myoc transcripts in embryonic and adult mouse tissue using Northern blot and in situ hybridization analyses. Myoc transcripts were found in high levels in adult eye, heart, brain, skeletal muscle and testis and to a lesser extent in lung and kidney. They were also present, albeit in very low amounts, during mouse embryogenesis. We present new evidence using in situ hybridization analysis that Myoc transcripts were present in widespread regions of the adult brain including the ependymal lining of the third and fourth ventricles, in the choroid plexus, the zonal layer of the junction of the inferior and superior colliculi, the neurons of the habenula, the piriform cortex, the median pre-optic nucleus of the hypothalamus, the olfactory tubercle, and in the inferior olive. In a functional sense, Myoc expression in the ependyma and choroid plexus, two regions of the brain involved in cerebrospinal fluid synthesis and resorption, parallels Myoc expression in the ciliary body and trabecular meshwork of the anterior segment of the eye where aqueous humor synthesis and outflow occur.

Animals↗
PubMed1999

Clinical characterization and linkage analysis of a family with congenital X-linked nystagmus and deuteranomaly.

OBJECTIVES: To identify a congenital nystagmus locus on the X chromosome and to characterize the phenotype of a 4-generation family affected with congenital nystagmus and color deficiency. METHODS: Sixty-five patients underwent an eye examination, including evaluation for the presence of nystagmus and color vision abnormalities. Affected patients and obligate carriers of the congenital nystagmus mutation were genotyped with short tandem repeat polymorphisms located on the X chromosome, and these data were subjected to linkage analysis. RESULTS: Fourteen patients were affected with a horizontal, conjugate, congenital nystagmus. All examined patients had a visual acuity of 20/60 or better. There were no associated ocular or systemic findings except that 18 of the family members had deficient red-green color vision, which was classified as deuteranomaly (the most common form of anomalous trichromacy). Five patients exhibited nystagmus and deuteranomaly. Significant linkage was demonstrated between the nystagmus phenotype and 11 markers from Xq. The maximum lod score was 4.84 (theta = 0) and was obtained with marker DXS8041. Analysis of recombinants defined the disease interval to lie between markers ATA59C05 and DXS1192 (a 5.4-centimorgan region). The proximity of this locus to the red-green opsin gene cluster (11 centimorgans more telomeric) explains the frequent coexistence of nystagmus and color vision deficiency in this family. CONCLUSIONS: We have identified the genetic locus of the X-linked congenital nystagmus gene in this family. The critical interval in this report is less than half the size of the previously described nystagmus locus. These findings will aid in identifying the gene responsible for this condition.

Chromosome Mapping↗
PubMed1999

Expression of the Mf1 gene in developing mouse hearts: implication in the development of human congenital heart defects.

The transcription factor FKHL7 gene has recently been associated with the anterior segment dysgenesis disorder of the eye known as Axenfeld-Rieger anomaly (ARA). A growing body of evidence indicates that mutations in FKHL7 cause not only defects in the anterior segment of the eye but defects in the heart valves and septa as well. In order to evaluate its contribution to normal heart septation and valve formation, expression of the mouse homologue Mf1 in embryonic hearts was analyzed by in situ hybridization. A weak but significant level of Mf1 expression could be detected in the endocardium of mouse embryos as early as day 8.5 post-conception (p.c.). Mf1 expression was undetectable in the hearts of day 9.5 p.c. embryos, but by day 10.5-11 p.c., Mf1 transcripts could be found again in the endocardium of both the atrium and ventricle and a relatively strong signal was observed in the dorsal portion of the septum primum, in what appeared to be the spinal vestibule. At day 13 p.c. when aortic and pulmonary trunks are separated, relatively more Mf1 transcripts were detected in the leaflets of aortic, pulmonary, and venous valves, the ventral portion of the septum primum, as well as in the single layer of cells on the edges of the atrioventricular cushion tissues. Surprisingly, there was no signal detected in the developing interventricular septum. At day 15 p.c., overall Mf1 signals were greatly decreased. However, significant levels of expression could still be observed in the atrial septum, the tricuspid valve, the mitral valve, and in the venous valve but not in the interventricular septum. The temporal and spatial expression patterns of the Mf1 gene in developing mouse hearts suggest that Mf1 may play a critical role in the formation of valves and septa with the exception of the interventricular septum. This is further supported by our studies showing that mutations in the FKHL7 gene were associated with defects in the anterior segment of the eye as well as atrial septal defects or mitral valve defects. Dev Dyn 1999;216:16-27.

Animals↗
PubMed1999

The Pendred syndrome gene encodes a chloride-iodide transport protein.

Pendred syndrome is the most common form of syndromic deafness and characterized by congenital sensorineural hearing loss and goitre. This disorder was mapped to chromosome 7 and the gene causing Pendred syndrome (PDS) was subsequently identified by positional cloning. PDS encodes a putative transmembrane protein designated pendrin. Pendrin is closely related to a family of sulfate transport proteins that includes the rat sulfate-anion transporter (encoded by Sat-1; 29% amino acid sequence identity), the human diastrophic dysplasia sulfate transporter (encoded by DTD; 32%) and the human sulfate transporter 'downregulated in adenoma' (encoded by DRA; 45%). On the basis of this homology and the presence of a slightly modified sulfate-transporter signature sequence comprising its putative second transmembrane domain, pendrin has been proposed to function as a sulfate transporter. We were unable to detect evidence of sulfate transport following the expression of pendrin in Xenopus laevis oocytes by microinjection of PDS cRNA or in Sf9 cells following infection with PDS-recombinant baculovirus. The rates of transport for iodide and chloride were significantly increased following the expression of pendrin in both cell systems. Our results demonstrate that pendrin functions as a transporter of chloride and iodide, but not sulfate, and may provide insight into thyroid physiology and the pathophysiology of Pendred syndrome.

Animals↗
PubMed1999

A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy.

Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) refer to two autosomal dominant diseases characterized by yellow-white deposits known as drusen that accumulate beneath the retinal pigment epithelium (RPE). Both loci were mapped to chromosome 2p16-21 (refs 5,6) and this genetic interval has been subsequently narrowed. The importance of these diseases is due in large part to their close phenotypic similarity to age-related macular degeneration (AMD), a disorder with a strong genetic component that accounts for approximately 50% of registered blindness in the Western world. Just as in ML and DHRD, the early hallmark of AMD is the presence of drusen. Here we use a combination of positional and candidate gene methods to identify a single non-conservative mutation (Arg345Trp) in the gene EFEMP1 (for EGF-containing fibrillin-like extracellular matrix protein 1) in all families studied. This change was not present in 477 control individuals or in 494 patients with age-related macular degeneration. Identification of this mutation may aid in the development of an animal model for drusen, as well as in the identification of other genes involved in human macular degeneration.

Aging↗
PubMed1999

Exclusion of AR-CHED from the chromosome 20 region containing the PPMD and AD-CHED loci.

Congenital hereditary endothelial dystrophy (CHED) is a disorder of the corneal endothelium and has been recognized to segregate in families with both autosomal dominant (AD) and autosomal recessive (AR) modes of transmission. AD-CHED has been previously linked to the pericentric region of chromosome 20. Posterior polymorphous dystrophy (PPMD), a corneal endothelial disorder showing phenotypic overlap with CHED, has also been previously genetically mapped to this region. The genetic interval containing AD-CHED is within the larger genetic interval containing the PPMD locus. This study sought to determine whether AR-CHED segregating in a consanguineous Saudi Arabian pedigree is linked to the previously mapped and overlapping loci for AD-CHED and PPMD on the pericentric region of chromosome 20. Forty members of a consanguineous Saudi Arabian pedigree segregating AR-CHED were ascertained. Short tandem-repeat polymorphic markers from the 20 cM interval on chromosome 20 containing both the PPMD and AD-CHED loci were used to genotype these individuals. LOD score analysis of the genotype data with the MENDEL software package utilizing a model of autosomal recessive inheritance with complete penetrance showed exclusion of CHED from the entire PPMD/AD-CHED interval by utilizing overlapping intervals of LOD scores of at least -2. The results obtained demonstrate that AR-CHED is not allelic to either AD-CHED or PPMD, although it has been proposed that AD-CHED may be allelic to PPMD. Thus, there are at least two genes responsible for CHED and PPMD.

Chromosome Mapping↗
PubMed1999

Complement factor H gene mutation associated with autosomal recessive atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (HUS) presents with the clinical features of hypertension, microangiopathic hemolytic anemia, and acute renal failure. Both dominant and recessive modes of inheritance have been reported. This study describes the genetic and functional analysis of a large Bedouin kindred with autosomal recessive HUS. The kindred consists of several related nuclear families in which all parent unions of affected children are consanguineous. A previous report demonstrated that a dominant form of HUS maps to chromosome 1q and that complement factor H (CFH), a regulatory component of the complement system, lies within the region and is involved in the dominant disorder. Early-onset and persistent hypocomplementemia in this Bedouin kindred prompted us to evaluate the CFH gene. Linkage analysis was performed, demonstrating linkage between the disorder and the markers near the CFH gene. Mutation analysis of the CFH coding region revealed a single missense mutation. Functional analyses demonstrate that the mutant CFH is properly expressed and synthesized but that it is not transported normally from the cell. This is the first study reporting that a recessive, atypical, early-onset, and relapsing HUS is associated with the CFH protein and that a CFH mutation affects intracellular trafficking and secretion.

Age of Onset↗
PubMed1999

Analysis of myocilin mutations in 1703 glaucoma patients from five different populations.

A glaucoma locus, GLC1A, was identified previously on chromosome 1q. A gene within this locus (encoding the protein myocilin) subsequently was shown to harbor mutations in 2-4% of primary open angle glaucoma patients. A total of 1703 patients was screened from five different populations representing three racial groups. There were 1284 patients from primarily Caucasian populations in Iowa (727), Australia (390) and Canada (167). A group of 312 African American patients was from New York City and 107 Asian patients from Japan. Overall, 61 different myocilin sequence variations were identified. Of the 61 variations, 21 were judged to be probable disease-causing mutations. The number of probands found to harbor such mutations in each population was: Iowa 31/727 (4.3%), African Americans from New York City 8/312 (2.6%), Japan 3/107 (2.8%), Canada 5/167 (3.0%), Australia 11/390 (2.8%) and overall 58/1703 (3. 4%). Overall, 16 (76%) of 21 mutations were found in only one population. The most common mutation observed, Gln368Stop, was found in 27/1703 (1.6%) glaucoma probands and was found at least once in all groups except the Japanese. Studies of genetic markers flanking the myocilin gene suggest that most cases of the Gln368Stop mutations are descended from a common founder. Although the specific mutations found in each of the five populations were different, the overall frequency of myocilin mutations was similar ( approximately 2-4%) in all populations, suggesting that the increased rate of glaucoma in African Americans is not due to a higher prevalence of myocilin mutations.

Asian People↗
PubMed1999

Two different mutations in the thyroid peroxidase gene of a large inbred Amish kindred: power and limits of homozygosity mapping.

Approximately 10% of newborns with congenital hypothyroidism are unable to convert iodide into organic iodine. This iodide organification defect has a prevalence of 1 in 40,000 newborns and may be caused by defects in the thyroid peroxidase enzyme (TPO), the hydrogen peroxide-generating system, the TPO substrate thyroglobulin, or inhibitors of TPO. We identified a high incidence of severe hypothyroidism due to a complete iodide organification defect in the youngest generation of five nuclear families belonging to an inbred Amish kindred. Genealogical records permitted us to trace their origin to an ancestral couple 7-8 generations back and to identify an autosomal recessive pattern of inheritance. Initial studies of homozygosity by descent using two polymorphic markers within the TPO gene showed no linkage to the phenotype. In fact, 4 of 15 affected siblings from 2 of the nuclear families were heterozygous, resulting in homozygosity values of 73% and 53% in affected and unaffected family members, respectively. A genome-wide homozygosity screen using DNA pools from affected and unaffected family members localized the defect to a locus close to the TPO gene. Linkage analysis using 4 additional polymorphic markers within the TPO gene reduced the number of homozygous unaffected siblings to zero without altering the percent homozygosity initially found in the affected. Sequencing of the TPO gene revealed 2 missense mutations, E799K and R648Q. TPO 779K was found in both alleles of the 11 affected homozygotes, both mutations were present in each of the 3 affected compound heterozygotes, and there were no TPO mutations in 1 subject with hypothyroidism of different etiology. These results demonstrate the power of the DNA pooling strategy in the localization of a defective gene and the pitfalls of linkage analysis when 2 relatively rare mutations coexist in an inbred population.

Child, Preschool↗
PubMed1999

The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.

Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes.

Bacteriophage P1↗
PubMed1998

Identification and mutation analysis of a cochlear-expressed, zinc finger protein gene at the DFNB7/11 and dn hearing-loss loci on human chromosome 9q and mouse chromosome 19.

The DFNB7/11 locus for autosomal recessive non-syndromic hearing loss (ARNSHL) has been mapped to an approx. 1.5 Mb interval on human chromosome 9q13-q21. We have determined the cDNA sequence and genomic structure of a novel cochlear-expressed gene, ZNF216, that maps to the DFNB7/11 interval. The mouse orthologue of this gene maps to the murine dn (deafness) locus on mouse chromosome 19. The ZNF216 gene is highly conserved between human and mouse, and contains two regions that show homology to the putative zinc linger domains of other proteins. To determine it mutations in ZNF216 might be the cause of hearing loss at the DFNB7/11 locus, we screened the coding region of this gene in DFNB7/11 families by direct sequencing. No potential disease-causing mutations were found. In addition, Northern blot analysis showed no difference in ZNF216 transcript size or abundance between dn and control mice. These data Suggest that the ZNF216 gene is unlikely to be responsible for hearing loss at the DFNB7/11 and dn loci.

Algorithms↗
PubMed1998

Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A)

BACKGROUND: A substantial proportion of cases of glaucoma have a genetic basis. Mutations causing glaucoma have been identified in the chromosome 1 open-angle glaucoma gene (GLC1A), which encodes a 57-kd protein known as myocilin. The normal role of this protein and the mechanism by which mutations cause glaucoma are not known. METHODS: We screened 716 patients with primary open-angle glaucoma and 596 control subjects for sequence changes in the GLC1A gene. RESULTS: We identified 16 sequence variations that met the criteria for a probable disease-causing mutation because they altered the predicted amino acid sequence and they were found in one or more patients with glaucoma, in less than 1 percent of the control subjects. These 16 mutations were found in 33 patients (4.6 percent). Six of the mutations were found in more than 1 subject (total, 99). Clinical features associated with these six mutations included an age at diagnosis ranging from 8 to 77 years and maximal recorded intraocular pressures ranging from 12 to 77 mm Hg. CONCLUSIONS: A variety of mutations in the GLC1A gene are associated with glaucoma. The spectrum of disease can range from juvenile glaucoma to typical late-onset primary open-angle glaucoma.

Age of Onset↗
PubMed1998

Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss.

Mutations in the Cx26 gene have been shown to cause autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNB1 locus on chromosome 13q12. Using direct sequencing, we screened the Cx26 coding region of affected and nonaffected members from seven ARNSHL families either linked to the DFNB1 locus or in which the ARNSHL phenotype cosegregated with markers from chromosome 13q12. Cx26 mutations were found in six of the seven families and included two previously described mutations (W24X and W77X) and two novel Cx26 mutations: a single base pair deletion of nucleotide 35 resulting in a frameshift and a C-to-T substitution at nucleotide 370 resulting in a premature stop codon (Q124X). We have developed and optimized allele-specific PCR primers for each of the four mutations to rapidly determine carrier and noncarrier status within families. We also have developed a single stranded conformational polymorphism (SSCP) assay which covers the entire Cx26 coding region. This assay can be used to screen individuals with nonsyndromic hearing loss for mutations in the CX26 gene.

Alleles↗
PubMed1998

Short tandem repeat polymorphic markers for the rat genome from marker-selected libraries.

In an effort to generate a genome-wide set of high-quality polymorphic markers for the rat, we used the marker-selection method, which has already been proven useful for the development of markers, especially for the human genome. Small-insert (300-900 bp) rat genomic libraries were constructed with an estimated complexity of three genome equivalents and enriched for short tandem repeat sequences (STRs). The enriched libraries were found to contain 45% (CA)n and 27% (GATA)n, representing at least a 50-fold enrichment over unselected small insert genomic libraries. A subset of 2160 STR-containing clones, primarily of the (GATA)n class of repeats, were sequenced. PCR primers flanking the repeats were synthesized from some of the sequences from the (CA)n and (GATA)n classes of STRs and tested for polymorphism in a panel of eight inbred rat strains. This strategy yielded 147 polymorphic markers, which mapped with high odds to all chromosomes by linkage in three F2 populations. The integration of these STR markers with other rat genetic markers and mapping reagents will facilitate the mapping of disease genes in the rat and the identification of loci associated with complex mammalian phenotypes.

Animals↗