Identification of multiple quantitative trait loci linked to prion disease incubation period in mice.

Polymorphisms in the prion protein gene are known to affect prion disease incubation times and susceptibility in humans and mice. However, studies with inbred lines of mice show that large differences in incubation times occur even with the same amino acid sequence of the prion protein, suggesting that other genes may contribute to the observed variation. To identify these loci we analyzed 1,009 animals from an F2 intercross between two strains of mice, CAST/Ei and NZW/OlaHSd, with significantly different incubation periods when challenged with RML scrapie prions. Interval mapping identified three highly significantly linked regions on chromosomes 2, 11, and 12; composite interval mapping suggests that each of these regions includes multiple linked quantitative trait loci. Suggestive evidence for linkage was obtained on chromosomes 6 and 7. The sequence conservation between the mouse and human genome suggests that identification of mouse prion susceptibility alleles may have direct relevance to understanding human susceptibility to bovine spongiform encephalopathy (BSE) infection, as well as identifying key factors in the molecular pathways of prion pathogenesis. However, the demonstration of other major genetic effects on incubation period suggests the need for extreme caution in interpreting estimates of variant Creutzfeldt-Jakob disease epidemic size utilizing existing epidemiological models.

Isolated hypercalciuria with mutation in CLCN5: relevance to idiopathic hypercalciuria.

UNLABELLED: Isolated hypercalciuria with mutation in CLCN5: Relevance to idiopathic hypercalciuria. BACKGROUND: Idiopathic hypercalciuria (IH) is the most common risk factor for kidney stones and often has a genetic component. Dent's disease (X-linked nephrolithiasis) is associated with mutations in the CLCN5 chloride channel gene, and low molecular weight (LMW) proteinuria was universally observed in affected males. We sought to identify mutations in CLCN5 or abnormalities in LMW protein excretion in a large group of patients with IH and in a rat model of genetic hypercalciuria. METHODS: One hundred and seven patients with IH (82 adults and 25 children) and one asymptomatic hypercalciuric man with a known inactivating mutation in CLCN5 were studied. Secondary causes of hypercalciuria were excluded in all. The excretion of retinol-binding protein and beta2-microglobulin was measured by immunoassay in 101 patients with IH. Mutation analysis of the CLCN5 gene was performed in 32 patients with IH and in the genetic hypercalciuric stone-forming (GHS) rat strain. RESULTS: LMW protein excretion was normal in 92 patients with IH, and only slight abnormalities were found in the other nine, none of whom had a mutation in CLCN5. One 27-year-old man who had a CLCN5 mutation was found to have isolated hypercalciuria without LMW proteinuria, renal failure, or other evidence of renal disease. Mutation analysis was normal in 32 patients with IH. The CLCN5 sequence was normal in the GHS rat. CONCLUSIONS: Inactivation of CLCN5 can be found in the setting of hypercalciuria without other features of X-linked nephrolithiasis. However, mutations in CLCN5 do not represent a common cause of IH.

A familial syndrome due to Arg648Stop mutation in the X-linked renal chloride channel gene.

We describe a familial syndrome in two brothers who were investigated after the casual discovery of tubular proteinuria in their 1st month of life. During a follow-up of 20 and 11 years, respectively, the two children grew well and were asymptomatic, but developed the same biochemical abnormalities, i.e., tubular proteinuria and hyperphosphaturia, progressive decrease in serum phosphorus below the normal values for age, and an increase in serum 1,25-dihydroxyvitamin D levels over normal values. Moreover, hyperabsorptive hypercalciuria and systemic osteopenia developed and progressively worsened. In both children, at a different age, medullary nephrocalcinosis appeared. The oldest boy suffered a progressive decrease in urinary concentration ability and in glomerular filtration rate. Oral phosphate supplementation led to reversal of all biochemical abnormalities, with the exception of decreased phosphate tubular reabsorption and tubular proteinuria. With long-term phosphate supplementation, a normal bone mass was reached, while progression of nephrocalcinosis was arrested and impairment of renal function was slowed down. In a family study (siblings and parents), the only detectable abnormality was microglobinuria in the mother, thus suggesting a X-linked inheritance of this disorder. In the two probands a mutation within the renal chloride channel gene (CLCN5) was discovered.

Localization of familial benign hypercalcemia, Oklahoma variant (FBHOk), to chromosome 19q13.

Calcium homeostasis by the kidneys and parathyroids is mediated by the calcium-sensing receptor (CaSR), which is located on 3q21-q24 and belongs to family C of the superfamily of G-protein coupled receptors that includes those for metabotropic glutamate, certain pheromones, and gamma-amino butyric acid (GABA-B). Inactivating CaSR mutations result in familial benign hypercalcemia (FBH), or familial hypocalciuric hypercalcemia (FHH), whereas activating mutations result in hypocalcemic hypercalciuria. However, not all FBH patients have CaSR mutations, which, together with the mapping of another FBH locus to 19p13.3, suggests that additional CaSRs or second messengers may be involved. These may be identified by positional cloning, and we therefore performed a genomewide search, using chromosome-specific sets of microsatellite polymorphisms, in an Oklahoma family with an FBH variant (FBHOk), for which linkage to 3q and 19p had been excluded. Linkage was established between FBHOk and eight chromosome 19q13 loci, with the highest LOD score, 6.67 (recombination fraction.00), obtained with D19S606. Recombinants further mapped FBHOk to a <12-cM interval flanked by D19S908 and D19S866. The calmodulin III gene is located within this interval, and DNA sequence analysis of the coding region, the 5' UTR, and part of the promoter region in an individual affected with FBHOk did not detect any abnormalities, thereby indicating that this gene is unlikely to be implicated in the etiology of FBHOk. This mapping of FBHOk to chromosome 19q13 will facilitate the identification of another CaSR or a mediator of calcium homeostasis.

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.

Mutations of CLCN5 in Japanese children with idiopathic low molecular weight proteinuria, hypercalciuria and nephrocalcinosis.

The annual urinary screening of Japanese children above three years of age has identified a progressive renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis. The disorder has been observed in over 60 patients and has a familial predisposition. Mutations of a renal chloride channel gene, CLCN5, have been reported in four such families, and we have undertaken studies in additional patients from 10 unrelated, non-consanguineous Japanese families to further characterize such CLCN5 mutations and to ascertain their prevalence. CLCN5 abnormalities we identified in 7 of the 10 unrelated patients and consisted of 5 mutations (2 nonsense, 1 frameshift and 2 missense), 1 deletion and 1 silent polymorphism. A clustering of these mutations in CLCN5 exons 8 and 10 was observed. Over 80% of the CLCN5 mutations could be readily detected by single stranded conformational polymorphism (SSCP) analysis, thereby providing a useful mutation screening method. Our results, which indicate that over 70% of Japanese patients with this renal tubulopathy have CLCN5 mutations, will help in the genetic and clinical evaluation of children at risk from this disorder.

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.

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Exclusion of ZFM1 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 linkage studies and a 3.8-cM region flanked by PYGM and D11S97 has been defined. The zinc finger in the MEN1 locus (ZFM1) gene, which has also been mapped to this region, represents a candidate gene for MEN1. The ZFM1 gene, which consists of 14 exons, encodes a 623-amino acid protein and exons 2, 8 and 12 encode the putative nuclear localisation signal, a zinc finger motif, and a proline-rich region, respectively. We have investigated these potentially functional regions for germ-line mutations by single-stranded conformational polymorphism (SSCP) analysis in 64 unrelated MEN1 patients. In addition, we performed DNA sequence analysis of all the 14 exons and 13 of the 26 exon-intron boundaries in four unrelated MEN1 patients. A 6-bp deletion that resulted in the loss of two proline residues at codons 479 and 480 in exon 12 was found in one MEN1 patient. However, this did not co-segregate with MEN1 in the family and represented a rare polymorphism. Analysis by SSCP, DNA sequencing, northern blotting, Southern blotting and pulsed field gel electrophoresis revealed no additional genetic abnormalities of ZFM1 in the other MEN1 patients. Thus, our results indicate that ZFM1 is excluded as a candidate gene for MEN1.

Idiopathic low molecular weight proteinuria associated with hypercalciuric nephrocalcinosis in Japanese children is due to mutations of the renal chloride channel (CLCN5).

The annual urinary screening of Japanese children above 3 yr of age has identified a progressive proximal renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrocalcinosis. The disorder, which has a familial predisposition and occurs predominantly in males, has similarities to three X-linked proximal renal tubular disorders that are due to mutations in the renal chloride channel gene, CLCN5. We have investigated four unrelated Japanese kindreds with this tubulopathy and have identified four different CLCN5 mutations (two nonsense, one missense, and one frameshift). These are predicted to lead to a loss of chloride channel function, and heterologous expression of the missense CLCN5 mutation in Xenopus oocytes demonstrated a 70% reduction in channel activity when compared with the wild-type. In addition, single-stranded conformation polymorphism (SSCP) analysis was found to be a sensitive and specific mutational screening method that detected > 75% of CLCN5 mutations. Thus, the results of our study expand the spectrum of clinical phenotypes associated with CLCN5 mutations to include this proximal renal tubular disorder of Japanese children. In addition, the mutational screening of CLCN5 by SSCP will help to supplement the clinical evaluation of the annual urinary screening program for this disorder.

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.

Haemophilia care in Zimbabwe.

OBJECTIVE: To review haemophilia care in Zimbabwe. DESIGN: Retrospective study. SETTING: Cases seen in both central hospitals and health centres in Harare. MAIN OUTCOME MEASURES: Home treatment hospital admission and HIV seroconversion. RESULTS: Of the expected 500 haemophilicacs in a Zimbabwean male population of five million, only 190 had been registered by mid 1993. Home treatment is effective. CONCLUSION: Haemophilia care in Zimbabwe has a good foundation. Home care is effective and needs to be expanded simultaneously with health education for the cases.

PIP: A retrospective study was conducted to evaluate the status of hemophilia care in Zimbabwe. Parirenyatwa Hospital in Harare has the only hemophilia clinic in Zimbabwe. This monthly clinic facilitates diagnosis, registration, and long-term management of hemophilia. In mid 1993, there were 190 registered hemophilia cases in Zimbabwe. During 1991-1993, only 70 patients were seen more than once in the clinic. The National Blood Transfusion Service (NTSB) supplies blood products for hemophiliacs. Solvent-detergent treated Factor VIII and IX (FVIII and FIX, respectively) concentrates are imported from South Africa. They are the most common blood products used in Harare. Laboratory staff screen fresh frozen plasma and cryoprecipitate for HIV antibody and hepatitis B surface antigen. Five NTSB branches also distribute blood products. Blood products are expensive. Most hemophiliacs are covered by a social welfare program. 45 hemophiliac cases had been receiving home care since 1987. 67% of 24 home care patients receiving FVIII did not store FVIII packs in a refrigerator. Most home care patients injected blood products 0-6 hours from onset of symptoms (e.g., nosebleed). About 33% did not know how to calculate the dose required. All home care patients were satisfied with treatment. In 1992, Parirenyatwa Hospital registered 3 deaths of hemophiliacs. When considering only the 70 regular clinic attenders, the mortality rate for 1992 was 5.7%. Of the 73 hemophiliac cases tested for HIV infection, 32% tested positive. All HIV-positive hemophiliac cases began treatment for hemophilia before 1986, the year before HIV testing of hemophiliacs started. So far, about 33% of hemophiliacs tested positive for hepatitis C. The only social support system for hemophiliacs is the Zimbabwe Hemophilia Association. None of the 38 hemophiliacs screened for coagulation factor inhibitors had any inhibitors. Hemophilia care in Zimbabwe has a good start and can be used as a model for other developing countries. Expansion and close supervision of the effective home treatment program is advised.

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.

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.

Isolation and partial characterization of a chloride channel gene which is expressed in kidney and is a candidate for Dent's disease (an X-linked hereditary nephrolithiasis).

Dent's disease, an X-linked renal tubular disorder, is a form of Fanconi syndrome which is characterized by proteinuria, hypercalciuria, nephrocalcinosis, kidney stones and renal failure. Previous studies localised the gene responsible to Xp11.22, within a microdeletion involving the hypervariable locus DXS255. Further analysis using new probes which flank this locus indicate that the deletion is less than 515 kb. A 185 kb YAC containing DXS255 was used to screen a cDNA library from adult kidney in order to isolate coding sequences falling within the deleted region which may be implicated in the disease aetiology. We identified two clones which are evolutionarily conserved, and detect a 9.5 kb transcript which is expressed predominantly in the kidney. Sequence analysis of 780 bp of ORF from the clones suggests that the identified gene, termed hCIC-K2, encodes a new member of the CIC family of voltage-gated chloride channels. Genomic fragments detected by the cDNA clones are completely absent in patients who have an associated microdeletion. On the basis of the expression pattern, proposed function and deletion mapping, hCIC-K2 is a strong candidate for Dent's disease.

Haemophilia in Zimbabwe.

Of the 120 haemophiliacs diagnosed in Zimbabwe in 8 years, haemophilia A and Christmas disease accounted for 90% and 10% respectively (i.e. a ratio of 9:1). Although the clinical and laboratory parameters were essentially similar to those previously described in Caucasian, African and other populations in the World, sub-haemophiliac cases are probably still being missed particularly in very busy health centres where the index of suspicion is low and malnutrition and infectious disease predominate and therefore readily attract the attention of most health workers. However, with the steadily improving socio-economic status and decentralization of health care facilities, more of these cases are likely to be diagnosed. Major constraints in the diagnosis and management of haemophilia in an African setting are succinctly discussed; including home therapy; and the implications of recent findings of HIV sero-positivity. The study serves as evidence that haemophilia is common in Zimbabwe contrary to earlier published literature.

Infant search strategies with containers that move but do not alter the location at which contents can be found.

A number of hypotheses about infants' delayed search accuracy have been based upon the notion that a location associated with repeated retrieval of the object attains privileged status. Infants may need strong cues to search at a new location. However, a test is reported in which performance of 12- and 15-month-old infants was shown to be indifferent to the location. The results were reliable at an individual level. The data accord with previous research upon the canonicality effect in infant spatial search, an effect which is taken to index an experiential constraint on spatial discrimination. The experimental design thus serves as a discriminative test between three approaches: the original privileged location hypothesis, a newer spatial-contrast hypothesis, and a wider approach which focuses on experiential constraints on learning, including the canonicality effect.