The von Willebrand factor predicted unpaired cysteines are essential for secretion.

BACKGROUND: von Willebrand factor (VWF) contains free thiols that mass spectroscopy has located to nine cysteines: two in the D3 domain (Cys889 and Cys898) and seven in the C domains (Cys2448, Cys2451, Cys2453, Cys2490, Cys2491, Cys2528, and Cys2533) (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). It has been suggested that these free thiols function to regulate the self-association of VWF through thiol-disulfide exchange (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). However, recent structural modeling has predicted that these cysteines are, in fact, disulfide-bonded (Blood, 118, 5312; Blood, 120, 449). OBJECTIVES: To use mutation and expression analyses to investigate how these conflicting reports might be compatible with the synthesis and expression of VWF. METHODS AND RESULTS: Both full-length VWF and VWF fragments with cysteine to alanine mutations of the nine cysteines and two predicted binding partners (Cys2431 and Cys2468) failed to secrete. Mutation of a cysteine pair, C2431A/C2453A, similarly resulted in a failure to secrete, indicating that this is not secondary to creation of an unpaired thiol. Deletion mutants containing seven of these cysteines, conforming to hypothesized domain boundaries, also failed to secrete: ∆C1C6 (2255-2720), ∆C3C4 (2429-2577), ∆C3 (2429-2496), and ∆C4 (2497-2577). Analysis of cell lysates and immunofluorescence confirmed that the mutants were retained within the endoplasmic reticulum (ER). Coexpression with wild-type VWF rescued secretion of some mutants to a limited extent. CONCLUSIONS: These data suggest: first, that pairing of cysteines implicated in free thiol exchange is essential for correct folding of the VWF molecule, and unpairing must occur following exit from the ER or secretion from the cell; and second, that intact C domains are essential for efficient VWF secretion and must interact in the ER.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

Renal chloride channel, CLCN5, mutations in Dent's disease.

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. Patients with Dent's disease may also suffer from rickets and other features of the renal Fanconi Syndrome. Patients may have mutations in the X-linked renal chloride channel gene, CLCN5, which encodes a 746-amino-acid protein with 12-13 transmembrane domains. We have investigated the 11 coding exons of CLCN5 for mutations in eight unrelated patients with Dent's disease. Leukocyte DNA was used for the polymerase chain reaction amplification of CLCN5 and the products analyzed for single-stranded conformational polymorphisms (SSCPs). Abnormal SSCPs were sequenced and revealed eight mutations. These consisted of three nonsense mutations (Arg34Stop, Arg648Stop, Arg704Stop), four deletions involving codons 40, 86, 157, and 241, and one acceptor splice consensus sequence mutation tgcag --> tgaag. The mutations were confirmed either by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis. In addition, an analysis of 110 alleles from 74 unrelated normal individuals demonstrated that the DNA sequence changes were not common polymorphisms. All of the mutations predict truncated chloride channels that are likely to result in a functional loss. Thus, our findings expand the spectrum of CLCN5 mutations associated with Dent's disease and the results will help to elucidate further the functional domains of this novel chloride channel.

Localisation of X linked recessive idiopathic hypoparathyroidism to a 1.5 Mb region on Xq26-q27.

X linked recessive idiopathic hypoparathyroidism (HPT) has been observed in two kindreds from Missouri, USA. Affected subjects, who are males, suffer from infantile onset of epilepsy and hypocalcaemia, which appears to be the result of an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcaemic. The gene causing HPT has been previously mapped to a 7 cM interval, flanked centromerically by F9 and telomerically by DXS98, in Xq26-q27, and an analysis of mitochondrial DNA has established a common ancestry for these two kindreds. In order to define further the map location of HPT and thereby facilitate its isolation, we have undertaken linkage studies using polymorphic loci whose order has been established as Xcen - DXS1001 - DXS294 - DXS102 - F9 - DXS1232 - DXS984 - CDR1 - DXS105 - DXS1205 - DXS1227 - DXS98 - DXS52 - Xqter, within this region. Our results established linkage (lod score > 3) between HPT and eight of these 12 loci and indicated that the most likely location of HPT was within a 1.5 Mb interval flanked centromerically by F9 and telomerically by DXS984. Thus, the results of this study have helped to refine the map location of HPT, and this will facilitate the identification of this putative developmental gene and its role in the embryological formation of the parathyroids.

Mutational analysis of PHEX gene in X-linked hypophosphatemia.

Hypophosphatemic rickets is commonly an X-linked dominant disorder (XLH or HYP) associated with a renal tubular defect in phosphate transport and bone deformities. The XLH gene, referred to as PHEX, or formerly as PEX (phosphate regulating gene with homologies to endopeptidases on the X-chromosome), encodes a 749-amino acid protein that putatively consists of an intracellular, transmembrane, and extracellular domain. PHEX mutations have been observed in XLH patients, and we have undertaken studies to characterize such mutations in 46 unrelated XLH kindreds and 22 unrelated patients with nonfamilial XLH by single stranded conformational polymorphism and DNA sequence analysis. We identified 31 mutations (7 nonsense, 6 deletions, 2 deletional insertions, 1 duplication, 2 insertions, 4 splice site, 8 missense, and 1 within the 5' untranslated region), of which 30 were scattered throughout the putative extracellular domain, together with 6 polymorphisms that had heterozygosity frequencies ranging from less than 1% to 43%. Single stranded conformational polymorphism was found to detect more than 60% of these mutations. Over 20% of the mutations were observed in nonfamilial XLH patients, who represented de novo occurrences of PHEX mutations. The unique point mutation (a-->g) of the 5'untranslated region together with the other mutations indicates that the dominant XLH phenotype is unlikely to be explained by haplo-insufficiency or a dominant negative effect.

Characterization of mutations in patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.

Mapping of the gene encoding the B56 beta subunit of protein phosphatase 2A (PPP2R5B) to a 0.5-Mb region of chromosome 11q13 and its exclusion as a candidate gene for multiple endocrine neoplasia type 1 (MEN1).

The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined. In the course of constructing a conting, we have identified the location of the gene encoding the B56 beta subunit of protein phosphatase 2A (PP2A), which is involved in cell signal transduction pathways and thus represents a candidate gene for MEN1. We have searched for mutations in the PP2A-B56 beta coding region, together with the 5' and 3' untranslated regions in six MEN1 patients. DNA sequence abnormalities were not identified and thus the PP2A-B56 beta gene is excluded as the candidate gene for MEN1. However, our precise localisation of PP2A-B56 beta to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this generich region.

Localisation of a gene causing endocrine neoplasia to a 4 cM region on chromosome 1p35-p36.

The development of some endocrine tumours, such as medullary thyroid carcinomas, phaeochromocytomas, anterior pituitary adenomas, and parathyroid adenomas involve a putative tumour suppressor gene located on chromosome 1p32-pter, a region that represents 111 cM. In order to refine the location of this gene, 93 endocrine tumours (39 parathyroid adenomas, 40 anterior pituitary adenomas, seven pancreatic islet cell adenomas, and seven carcinoids) were investigated for loss of tumour heterozygosity (LOH) using the seven polymorphic loci 1pter-D1S228-D1S507-D1S234-D1S476-D1S22 0-D1S207-D1S206-1cen. LOH was detected in 27% of the parathyroid tumours and in 7.5% of the pituitary tumours, but in none of the pancreatic islet cell or carcinoid tumours. In addition, seven of the 10 parathyroid tumours that showed LOH of chromosome 1p facilitated a more precise mapping of this putative tumour suppressor gene; five tumours involved a loss only of the telomeric locus D1S228, whereas two other tumours showed LOH at the centromeric loci D1S507, D1S234, D1S476, and D1S220, but not D1S228. Thus, our results have mapped this tumour suppressor gene implicated in endocrine tumours to a 4 cM region flanked by D1S228 and D1S507 on chromosome 1p35-p36.

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.

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.

Clinical studies of multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid, pancreatic islet and anterior pituitary tumours. To facilitate a screening programme for MEN1, we investigated 709 people (364 males and 345 females, age range 1-84 years) from 62 MEN1 families, and 36 non-familial MEN1 patients. Of those investigated, 220 (95 males and 125 females, age range 8-79 years) suffered from MEN1. Parathyroid, pancreatic and pituitary tumours occurred in 95%, 41% and 30% of the patients, respectively. Parathyroid tumours were the first manifestation of MEN1 in 87% of patients, and amongst the pituitary and pancreatic tumours, somatotrophinomas and gastrinomas were more common in patients above the age of 40 years, whilst insulinomas occurred more frequently in patients below the age of 40 years. Biochemical screening indicated that the penetrance of MEN1 by the ages of 20, 35 and 50 years was 43%, 85% and 94%, respectively, and that the development of MEN1 was confined to first-degree relatives in 91% of patients and to second-degree relatives in 9% of patients. These findings have helped to define a proposed screening programme for MEN1.

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.

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of mu-calpain as the multiple endocrine neoplasia type 1 gene.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM- SEA-D11S913-D11S970-D11S97- D11S146-INT2-D11S971-D11S533-11qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.

Construction of a YAC contig and an STS map spanning 3.6 megabase pairs in Xp22.1.

We have constructed a 3.6 Mb sequence tagged sites (STS)-based yeast artificial chromosome (YAC) contig, consisting of 58 individual YAC clones, spanning the region PDHA1 and DXS451 on Xp22.1. In addition to establishing the order of PDHA1, ISPK-1, DXS2504, DXS1528 and the 13 known polymorphic loci as Xpter-PDHA1-DXS443-DXS3424-ISPK-1-DXS12 29-DXS2504-DXS1528-DXS365-DXS7101- DXS1683-DXS1052-DXS274-DXS92-DXS1226-DX S41-DXS989-DXS451-Xcen, we have also developed 35 novel STSs from YAC end clones. These results provide a high density of STS markers (approximately 1 per 70 kb). Furthermore, a detailed long-range restriction map of the contig has been constructed with rare-cutter enzymes and this has refined and verified the physical distances between markers inferred from YAC sizes and their STS content. The integration of the physical mapping data with previous genetic mapping data and the use of STSs and non-chimeric YAC clones reported here should facilitate the construction of a transcript map of this region and the positional cloning of disease genes in this portion of Xp22.1.

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.

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.

Dent's disease, a renal Fanconi syndrome with nephrocalcinosis and kidney stones, is associated with a microdeletion involving DXS255 and maps to Xp11.22.

Dent's disease is a familial proximal renal tubular disorder which is associated with low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, kidney stones and renal failure. The mode of inheritance and the primary defect for this disorder are unknown. An analysis of 5 unrelated British families revealed a greater disease severity in males and an absence of male to male transmission. This suggested an X-linked inheritance and we investigated this further by linkage studies in 33 members (12 affected, 21 unaffected) from two 3-generation families. Twenty X-linked polymorphic markers were used and linkage was established with the Xp11 loci ARAFI, DXS426, DXS255 and DXS988 with peak LOD scores and recombination fractions (theta) of 5.42 (theta = 0.000), 3.61 (theta = 0.000), 5.48 (theta = 0.000) and 4.25 (theta = 0.045) respectively. In addition, DXS255 revealed a microdeletion in the affected members of one family, thereby further localising Dent's disease to Xp11.22. Combined multilocus linkage analysis and deletion mapping studies defined the locus order Xpter-MAOB-(ARAFI, DXS426)-SYP-TFE3-(DXS255, DENT'S)-DXS988-Xcen, thereby mapping the microdeletion associated with Dent's disease to a 4 centiMorgan interval flanked by TFE3 and DXS988. Thus, Dent's disease is an X-linked disorder which is associated with a microdeletion of Xp11.22, and a further characterisation of this gene will help to elucidate the factors controlling proximal renal tubular function and the development of kidney stones.

Association of somatotrophinomas with loss of alleles on chromosome 11 and with gsp mutations.

The molecular pathology of somatotrophinomas has been investigated by a combined search for dominant mutations of the gene encoding the Gs alpha protein and for recessive mutations involving chromosome 11q13, which contains the gene causing multiple endocrine neoplasia type 1 (MEN1). Somatotrophinomas and peripheral leukocytes were obtained from thirteen patients with acromegaly; one patient also suffered from MEN1. Five DNA probes identifying restriction fragment length polymorphisms from 11q revealed allele loss in pituitary tumors from five (four non-MEN1 and one MEN1) patients. Deletion mapping revealed that the region of allele loss common to the somatotrophinomas involved 11q13. An analysis for similar allelic deletions at 12 other loci from chromosomes 1-5, 7-9, 12-14, and 17 did not reveal generalized allele loss in the somatotrophinomas. These results, which represent the first report of chromosome 11 allele loss occurring in non-MEN1 somatotrophinomas, indicate that a recessive oncogene on 11q13 is specifically involved in the monoclonal development of somatotrophinomas. In addition Gs alpha mutations were detected in two non-MEN1 somatotrophinomas, one of which also revealed allele loss of chromosome 11. Thus, our results reveal that the development of somatotrophinomas is associated with alterations in both dominant and recessive oncogenes and further characterization of these genetic abnormalities will help to elucidate the multistep etiology and progression of somatotrophinomas.

Mapping the gene causing X-linked recessive nephrolithiasis to Xp11.22 by linkage studies.

X-linked recessive nephrolithiasis is associated with kidney stones and renal tubular dysfunction in childhood progressing to renal failure in adulthood. The primary defect causing this renal tubular disorder is unknown and determining the chromosomal location of the mutant gene would represent an important step toward defining the biochemical basis. We have performed linkage studies in 102 members (10 affected males, 47 unaffected males, 15 obligate heterozygote females, and 30 unaffected females) from five generations of one family. As genetic markers we used 10 cloned human X chromosome fragments identifying restriction fragment length polymorphisms and seven pairs of oligonucleotide primers identifying microsatellite polymorphisms. Linkage with the locus DXS255 was established with a peak LOD score = 5.91 at 3.6% recombination, thereby localizing the X-linked recessive nephrolithiasis gene to the pericentromeric region of the short arm of the X chromosome (Xp11.22). Multilocus analysis indicated that the mutant gene was distal to DXS255 but proximal to the Duchenne muscular dystrophy locus on Xp. Thus, the gene that causes X-linked recessive nephrolithiasis maps to the pericentromeric region of the short arm of the X chromosome (Xp11.22), and further characterization of this gene will help to elucidate the factors controlling renal tubular function and mineral homeostasis.