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S H Pearce

Publications and source records attributed to S H Pearce.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mutational analysis of the thyrotropin receptor gene in sporadic and familial feline thyrotoxicosis.

The characterization of a spontaneous animal model equivalent to a human form of thyrotoxicosis would provide a useful resource for the investigation of the human disorder. Feline thyrotoxicosis is the only common form of hyperthyroidism found in domestic or laboratory animals, but its etiopathogenesis remains poorly defined. We have used the polymerase chain reaction (PCR) to amplify codons 480-640 of the previously uncharacterized feline thyrotropin receptor (TSHR) gene, and have determined the DNA sequence in this transmembrane domain region. We have analyzed single stranded conformational polymorphisms in thyroid DNA from 11 sporadic cases of feline thyrotoxicosis and leukocyte DNA from two cases of familial feline thyrotoxicosis. We have also determined the DNA sequence of this region of the TSHR in five of the cases of sporadic feline thyrotoxicosis and the two familial thyrotoxic cats. The normal feline TSHR sequence between codons 480-640 is highly homologous to that of other mammalian TSHRs, with 95%, 92%, and 90% amino acid identity between the feline receptor and canine, human, and bovine TSHRs, respectively. Thyroid gland DNA from 11 cats with sporadic thyrotoxicosis did not have mutations in this region of the TSHR gene. Leukocyte DNA from two littermates with familial feline thyrotoxicosis did not harbor mutations of this region of the TSHR gene. These studies suggest that TSHR gene mutations are not a common cause of feline thyrotoxicosis.

Amino Acid Sequence↗
PubMed1997

Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders.

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.

Adult↗
PubMed1997

Linkage studies of a Missouri kindred with autosomal dominant spondyloepimetaphyseal dysplasia (SEMD) indicate genetic heterogeneity.

A four-generation kindred (14 affected and 10 unaffected members) from Missouri, U.S.A. in which spondyloepimetaphyseal dysplasia (SEMD) had been inherited as an autosomal dominant disorder was investigated for linkage to 13 candidate loci: COL2AI, COL9AI, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, PSACH, FGFR3, decorin, CRTL1, COMP, and PTHRP. Mutations of COL2A1, COL9A2, COL10, and FGFR3 have been reported previously in the Strudwick type of SEMD, multiple epiphyseal dysplasia type 2 (EDM2), the Schmid type of metaphyseal dysplasia, and in achondroplasia, respectively, and the pseudoachondroplasia (PSACH) locus has been mapped to chromosome 19p12. In addition, mutations in COL9 and COL11A are associated with murine forms of degenerative joint disease and chondroplasia, respectively. The family proved informative for 12 of the 13 loci and was uninformative at the decorin locus. Linkage between this form of SEMD, designated the Missouri variant, SEMDMO, and the 12 informative candidate loci was excluded (LOD scores < -2.00 at theta = 0.005 to 0.15), thereby indicating further genetic heterogeneity in these inherited disorders of bone and cartilage development.

Chromosome Aberrations↗
PubMed1997

The calcium-sensing receptor: insights into extracellular calcium homeostasis in health and disease.

The cloning of the BoPCaR1 gene has helped to elucidate many aspects of normal extracellular calcium homeostasis, particularly as applied to the regulation of PTH secretion, calcitonin secretion and renal calcium reabsorption. In addition, loss and gain of function mutations have been found to cause the clinical syndromes of FBH, NSHPT and ADHH respectively. The CaR has also been implicated in the mechanisms leading to primary and uraemic hyperparathyroidism, and autoimmune parathyroid destruction. The recent demonstration that the CaR regulates non-selective cation channel function in rat hippocampal neurones suggests that it may also have a role within the central nervous system that is entirely unrelated to calcium homeostasis (Ye et al. 1996). Finally, the prospect of CaR agonists and antagonists, which may allow PTH secretion to be regulated independently of the serum calcium concentration, also holds much promise for the medical treatment of hyperparathyroidism, renal osteodystrophy and osteoporosis.

Calcium↗
PubMed1996

Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH, FBH, and NSHPT, respectively.

Calcium↗
PubMed1996

A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor.

BACKGROUND: The calcium-sensing receptor regulates the secretion of parathyroid hormone in response to changes in extracellular calcium concentrations, and mutations that result in a loss of function of the receptor are associated with familial hypocalciuric hypercalcemia. Mutations involving a gain of function have been associated with hypocalcemia in two kindreds. We examined the possibility that the latter type of mutation may result in a phenotype of familial hypocalcemia with hypercalciuria. METHODS: We studied six kindreds given a diagnosis of autosomal dominant hypoparathyroidism on the basis of their hypocalcemia and normal serum parathyroid hormone concentrations, a combination that suggested a defect of the calcium-sensing receptor. The hypocalcemia was associated with hypercalciuria, and treatment with vitamin D resulted in increased hypercalciuria, nephrocalcinosis, and renal impairment. Mutations in the calcium-sensing-receptor gene were identified by DNA-sequence analysis and expressed in human embryonic kidney cells (HEK-293). RESULTS: Five heterozygous missense mutations (Asn118Lys, Phe128Leu, Thr151Met, Glu191Lys, and Phe612Ser) were detected in the extracellular domain of the calcium-sensing-receptor gene and shown to cosegregate with the disease. Analysis of the functional expression of three of the mutant receptors in HEK-293 cells demonstrated shifts in the dose-response curves so that the extracellular calcium concentrations needed to produce half-maximal increases in total inositol phosphate in the cells were significantly (P=0.02 to P<0.001) lower than those required for the wild-type receptor. CONCLUSIONS: Gain-of-function mutations in the calcium-sensing receptor are associated with a familial syndrome of hypocalcemia with hypercalciuria that needs to be distinguished from hypoparathyroidism.

Adolescent↗
PubMed1996

Expression and characterization of inactivating and activating mutations in the human Ca2+o-sensing receptor.

Nearly 30 mutations have been identified to date in the coding region of the extracellular calcium-sensing receptor (CaR) that are associated with inherited human hypo- and hypercalcemic disorders. To understand the mechanisms by which the mutations alter the function of the receptor may help to discern the structure-function relationships in terms of ligand-binding and G protein coupling. In the present studies, we transiently expressed eight known CaR mutations in HEK293 cells. The effects of the mutations on extracellular calcium- and gadolinium-elicited increases in the cytosolic calcium concentration were then examined. Seven inactivating mutations, which cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, show a reduced functional activity of the receptor because they may 1) reduce its affinity for agonists; 2) prevent conversion of the receptor from a putatively immature, high mannose form into the fully glycosylated and biologically active form of the CaR, in addition to lowering its affinity for agonists; or 3) fail to couple the receptor to and/or activate its respective G protein(s). Conversely, one activating mutation, which causes a form of autosomal dominant hypocalcemia, appears to increase the affinity of the receptor for its agonists.

Blotting, Western↗
PubMed1996

A common molecular basis for three inherited kidney stone diseases.

Kidney stones (nephrolithiasis), which affect 12% of males and 5% of females in the western world, are familial in 45% of patients and are most commonly associated with hypercalciuria. Three disorders of hypercalciuric nephrolithiasis (Dent's disease, X-linked recessive nephrolithiasis (XRN), and X-linked recessive hypophosphataemic rickets (XLRH)) have been mapped to Xp11.22 (refs 5-7). A microdeletion in one Dent's disease kindred allowed the identification of a candidate gene, CLCN5 (refs 8,9) which encodes a putative renal chloride channel. Here we report the investigation of 11 kindreds with these renal tubular disorders for CLCN5 abnormalities; this identified three nonsense, four missense and two donor splice site mutations, together with one intragenic deletion and one microdeletion encompassing the entire gene. Heterologous expression of wild-type CLCN5 in Xenopus oocytes yielded outwardly rectifying chloride currents, which were either abolished or markedly reduced by the mutations. The common aetiology for Dent's disease, XRN and XLRH indicates that CLCN5 may be involved in other renal tubular disorders associated with kidney stones.

Amino Acid Sequence↗
PubMed1996

Calcium-sensing receptor mutations in familial hypocalciuric hypercalcaemia with recurrent pancreatitis.

OBJECTIVE: Pancreatitis is an unusual complication of the benign disorder familial hypocalciuric hypercalcaemia (FHH) such that it could represent a distinct subgroup of FHH. In order to study this, we investigated three FHH kindreds with recurrent pancreatitis for mutations of the extracellular calcium-sensing receptor (CaR) to identify a possible common genetic aetiology for typical FHH and that associated with pancreatitis. PATIENTS AND METHODS: Three FHH kindreds (18 affected, 14 unaffected members) in which the proband had presented with recurrent pancreatitis were identified. The entire 3234bp coding region of the CaR gene was examined by direct DNA sequencing using fluorochrome labelled dideoxy-terminators. Mutations were confirmed and demonstrated to co-segregate with FHH by restriction enzyme analysis. RESULTS: Three novel heterozygous missense mutations (Asn178Asp, Arg220Gln and Pro221Ser) in the extracellular domain of the CaR were identified in each of the probands. These mutations, which co-segregated with the hypercalcaemia, were not detected as common polymorphisms in 55 unrelated normocalcaemic controls. CONCLUSIONS: Familial hypocalciuric hypercalcaemia with recurrent pancreatitis is associated with calcium-sensing receptor mutations, and thus this variant has the same genetic aetiology as typical familial hypocalciuric hypercalcaemia.

Adult↗
PubMed1996

Loss of heterozygosity studies at the retinoblastoma and breast cancer susceptibility (BRCA2) loci in pituitary, parathyroid, pancreatic and carcinoid tumours.

OBJECTIVE: Allelic deletion of the retinoblastoma (Rb) gene on chromosome 13 has been reported in both pituitary and parathyroid tumours. We have investigated the roles of the Rb and the hereditary breast cancer susceptibility gene (BRCA2), which lie within 25 cM of each other on chromosome 13q12-14, in the multi-step aetiology of endocrine tumours. PATIENTS AND MEASUREMENTS: Seventy-seven endocrine tumours (43 anterior pituitary, 22 parathyroid, 7 carcinoid, and 5 pancreatic islet cell tumours) with paired leucocytes have been examined for loss of heterozygosity (LOH) at the Rb and BRCA2 loci by using specific oligonucleotide primers for the PCR amplification of microsatellite polymorphisms at three intragenic Rb markers, Rb1.20, Rbi4 and D13S153, and D13S260 which is linked to the BRCA2 locus. RESULTS: Seventy-five of the 77 tumour-leucocyte pairs were informative and LOH was detected in 1 of 16 non-functioning pituitary tumours, 1 of 8 prolactinomas, 3 of 19 parathyroid adenomas and 1 of 1 parathyroid carcinoma. All the 3 parathyroid adenomas with LOH were associated with aggressive clinical and histopathological features. Allele loss was not detected in any of the 16 somatotrophinomas, 2 corticotrophinomas, 1 gonadotrophinoma, 7 carcinoid tumours (6 bronchial, 1 metastatic intestinal) or 5 pancreatic islet cell tumours that were informative. CONCLUSIONS: These results demonstrate that allelic deletions of the 13q12-14 region occur in some pituitary adenomas and 16% of parathyroid adenomas. The extensive loss, which involves both the Rb gene and the BRCA2 locus, suggests that tumour suppressor genes in this region other than Rb or BRCA2 may be involved in the development and progression of some endocrine tumours.

BRCA2 Protein↗
PubMed1995

Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis).

Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus.

Adult↗
PubMed1995

Linkage studies in a kindred from Oklahoma, with familial benign (hypocalciuric) hypercalcaemia (FBH) and developmental elevations in serum parathyroid hormone levels, indicate a third locus for FBH.

A five-generation kindred (19 affected, two obligate carriers and 20 unaffected) from Oklahoma USA, in which familial benign (hypocalciuric) hypercalcaemia (FBH) was associated with a developmental elevation in serum parathyroid hormone (PTH) levels, has been investigated for linkage to the candidate chromosomal regions 3q21-q24 and 19p13.3, 11q13, and 11p15, to which the genes for FBH, multiple endocrine neoplasia type 1 (MEN1) and PTH have been mapped respectively. By means of 17 polymorphic markers from these regions, linkage was excluded [LOD scores < -2.00 at (theta) = 0.05-0.25]. In addition, an analysis of multipoint crossovers and use of the LINKMAP program confirmed the exclusion from these regions. Thus, this form of FBH, designated the Oklahoma variant FBH(Ok), is not linked to markers that segregate with FBH, MEN1 and PTH; our results indicate further genetic heterogeneity and the presence of a third locus for FBH.

Chromosome Mapping↗
PubMed1995

Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism.

Familial benign hypercalcemia (FBH) and neonatal hyperparathyroidism (NHPT) are disorders of calcium homeostasis that are associated with missense mutations of the calcium-sensing receptor (CaR). We have undertaken studies to characterize such CaR mutations in FBH and NHPT and to explore methods for their more rapid detection. Nine unrelated kindreds (39 affected, 32 unaffected members) with FBH and three unrelated children with sporadic NHPT were investigated for mutations in the 3,234-bp coding region of the CaR gene by DNA sequencing. Six novel heterozygous (one nonsense and five missense) mutations were identified in six of the nine FBH kindreds, and two de novo heterozygous missense mutations and one homozygous frame-shift mutation were identified in the three children with NHPT. Our results expand the phenotypes associated with CaR mutations to include sporadic NHPT. Single-stranded conformational polymorphism analysis was found to be a sensitive and specific mutational screening method that detected > 85% of these CaR gene mutations. The single-stranded conformational polymorphism identification of CaR mutations may help in the distinction of FBH from mild primary hyperparathyroidism which can be clinically difficult. Thus, the results of our study will help to supplement the clinical evaluation of some hypercalcemic patients and to elucidate further the structure-function relationships of the CaR.

Amino Acid Sequence↗