Localisation of a gene for mucopolysaccharidosis IIIC to the pericentromeric region of chromosome 8.

Mucopolysaccharidosis type IIIC (MPS IIIC, or Sanfilippo syndrome C) is a rare lysosomal storage disorder caused by a deficiency of acetyl-coenzyme A:alpha-glucosaminide-N-acetyltransferase. Patients develop progressive neuropsychiatric problems, mental retardation, hearing loss, and relatively minor visceral manifestations. The pattern of transmission is consistent with an autosomal recessive mode of inheritance. The aim of this study was to find a locus for MPS IIIC using a homozygosity mapping approach. A genomewide scan was performed on DNA from 27 affected individuals and 17 of their unaffected relatives. Additional patients were recruited, and DNA was obtained from a total of 44 affected individuals and 18 unaffected family members from 31 families from 10 countries. A working candidate interval was defined by looking for excess homozygosity in patients compared with their relatives. Additional markers were genotyped in regions of interest. Linkage analysis was performed to support the informal analysis. Inspection of the genomewide scan data showed apparent excess homozygosity in patients compared with their relatives for markers on chromosome 8. Additional genotyping identified 15 consecutive markers (from D8S1051 to D8S2332) in an 8.3 cM interval for which the genotypes of affected siblings were identical in state. A maximum multipoint lod score of 10.61 was found at marker D8S519. A locus for MPS IIIC maps to an 8.3 cM (16 Mbp) interval in the pericentromeric region of chromosome 8.

Bowen-Conradi syndrome: a clinical and genetic study.

The purpose of the study was to delineate the anomalies and the natural life history of persons with the Bowen-Conradi syndrome [Bowen and Conradi 1976: Birth Defects: Orig Artic Ser XII(6):101-108]. We ascertained 39 cases and personally examined almost all. For those who were not seen, their clinical record were scrutinized. Pedigree analysis of all 39 was done and kinship coefficients computed. The birth prevalence was estimated to be 1/355 live births.

Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.

Haplotype analysis of BRCA2 8765delAG mutation carriers in French Canadian and Yemenite Jewish hereditary breast cancer families.

The BRCA2 8765delAG mutation was previously reported in hereditary breast cancer families of French Canadian and Yemenite Jewish descent. Haplotype analysis, using six microsatellite markers that span BRCA2 and two intragenic polymorphisms, was performed on 8765delAG mutation carriers to determine if there was evidence that the mutations were identical by descent. The alleles of the microsatellite markers most closely flanking BRCA2 (D13S1697 and D13S1701) were found to be identical in state in all the mutation carriers. However, the disease-associated allele of one of the intragenic markers differed between the Yemenite Jews and French Canadian families, indicating that the 8765delAG mutation has independent origins in these two geographically and ethnically distinct populations.

Shwachman-Diamond syndrome with exocrine pancreatic dysfunction and bone marrow failure maps to the centromeric region of chromosome 7.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. A genomewide scan of families with SDS was terminated at approximately 50% completion, with the identification of chromosome 7 markers that showed linkage with the disease. Finer mapping revealed significant linkage across a broad interval that included the centromere. The maximum two-point LOD score was 8.7, with D7S473, at a recombination fraction of 0. The maximum multipoint LOD score was 10, in the interval between D7S499 and D7S482 (5.4 cM on the female map and 0 cM on the male map), a region delimited by recombinant events detected in affected children. Evidence from all 15 of the multiplex families analyzed provided support for the linkage, consistent with a single locus for SDS. However, the presence of several different mutations is suggested by the heterogeneity of disease-associated haplotypes in the candidate region.

Pedigree selection and tests of linkage in a Hutterite asthma pedigree.

We explored methods for kinship and linkage analysis in a Hutterite pedigree comprising 1,544 individuals, 72 of whom were diagnosed with asthma. Subpedigrees were selected by (a) identifying nuclear families containing asthmatics, (b) identifying couples with many asthmatic descendants in an ad hoc manner, and (c) finding the most recent common ancestors of all asthmatics. Markov chain Monte Carlo (MCMC) methods were used to estimate allele sharing in the larger subpedigrees and transmission/disequilibrium tests were performed on nuclear families. On chromosome 5q near the cytokine cluster, modest evidence for linkage to asthma was obtained. Using MCMC, we were able to evaluate the evidence for linkage in complex subpedigrees of several hundred individuals, and hence, incorporate some of the co-ancestry of this founder population.

Linkage of tuberculosis to chromosome 2q35 loci, including NRAMP1, in a large aboriginal Canadian family.

An epidemic of tuberculosis occurred in a community of Aboriginal Canadians during the period 1987-89. Genetic and epidemiologic data were collected on an extended family from this community, and the evidence for linkage to NRAMP1, a candidate gene for susceptibility to mycobacterial diseases, was assessed. Individuals were grouped into risk (liability) classes based on vaccination, age, previous disease, and tuberculin skin-test results. Under the assumption of a dominant mode of inheritance and a relative risk of 10, which is associated with the high-risk genotypes, a maximum LOD score of 3.81 was observed for linkage between a tuberculosis-susceptibility locus and D2S424, which is located just distal to NRAMP1, in chromosome region 2q35. Significant linkage was also observed between a tuberculosis-susceptibility locus and a haplotype of 10 NRAMP1 intragenic variants. No linkage to the major histocompatibility-complex region on chromosome 6p was observed, despite distortion of transmission from one member of the oldest couple to their affected offspring. The ability to assign individuals to risk classes was crucial to the success of this study.

Low prevalence of psychoses among the Hutterites, an isolated religious community.

OBJECTIVE: The authors estimated the prevalence of psychoses among the Hutterites in Manitoba, Canada, who lived in 102 communal farms or colonies. The study stemmed from an earlier epidemiological survey of North American Hutterite colonies (1950-1953), in which a low prevalence of psychoses was documented. METHOD: Psychiatrically ill individuals identified during the previous survey were rediagnosed with DSM-IV criteria. A current provincial health insurance claims database was queried anonymously for the period June 1992-May 1997, and the prevalence rate of disease among Hutterites, identified by distinctive surnames and unique postal addresses, was compared with the rate in the entire population of the province of Manitoba and in a comparison group of persons with Hutterite surnames but with addresses outside the Hutterite colonies. RESULTS: The annual prevalence of schizophrenia among the communal Hutterites, estimated from the database search by using ICD-9 criteria, was consistent with the prevalence found in the prior epidemiological survey (annual mean of 1.2/1,000 population, compared with 1.3/1,000 in the prior survey). The database search yielded a significantly lower prevalence for schizophrenia and other functional psychoses among communal Hutterites as well as among the comparison group, compared to the total Manitoba population. There was also lower prevalence for affective psychoses and adjustment reaction disorders among the communal Hutterites, compared to the total Manitoba population. Rates for neurotic disorders were elevated both among the communal Hutterites and the comparison group. CONCLUSIONS: The prevalence of specific psychoses was reduced among the Hutterites, although neurotic disorders were more prevalent. These findings suggest some specificity, although possible artifacts such as ascertainment bias must be considered. Further research is needed to examine genetic and environmental factors that may contribute to reduced prevalence of specific psychoses among the Hutterites.

Localization of a recessive gene for North American Indian childhood cirrhosis to chromosome region 16q22-and identification of a shared haplotype.

North American Indian childhood cirrhosis (NAIC, or CIRH1A) is an isolated nonsyndromic form of familial cholestasis reported in Ojibway-Cree children and young adults in northwestern Quebec. The pattern of transmission is consistent with an autosomal recessive mode of inheritance. To map the NAIC locus, we performed a genomewide scan on three DNA pools of samples from 13 patients, 16 unaffected siblings, and 22 parents from five families. Analysis of 333 highly polymorphic markers revealed 3 markers with apparent excess allele sharing among affected individuals. Additional mapping identified a chromosome 16q segment shared by all affected individuals. When the program FASTLINK/LINKAGE was used and a completely penetrant autosomal recessive mode of inheritance was assumed, a maximum LOD score of 4.44 was observed for a recombination fraction of 0, with marker D16S3067. A five-marker haplotype (D16S3067, D16S752, D16S2624, D16S3025, and D16S3106) spanning 4.9 cM was shared by all patients. These results provide significant evidence of linkage for a candidate gene on chromosome 16q22.

Exclusion of linkage of Shwachman-Diamond syndrome to chromosome regions 6q and 12q implicated by a de novo translocation.

Shwachman-Diamond syndrome is a rare genetic disorder of unknown pathogenesis involving exocrine pancreatic insufficiency and hematological and skeletal abnormalities. There is broad clinical variability; the extent of heterogeneity is unknown but comparisons within a large cohort of patients show no striking differences between patients of families with single or multiple affected offspring. Segregation analysis of a cohort of 69 families has suggested an autosomal recessive mode of inheritance. A single constitutional de novo chromosome rearrangement was reported in a Japanese patient involving a balanced translocation, t(6;12)(q16.2;q21.2), thereby suggesting possible loci for a genetic defect. Evenly spaced microsatellite markers spanning 26-32 cM intervals from D6S1056 to D6S304 and D12S375 to D12S346 were analyzed for linkage in members of 13 Shwachman-Diamond syndrome families with two or three affected children. Two-point lod scores were calculated for each marker under assumptions of recessive inheritance and complete penetrance. Negative lod scores indicated exclusion of both chromosome regions. Further, affected sibs were discordant for inheritance of chromosomes in most families based on constructed haplotypes. The cytogenetic abnormality is not associated with most cases of Shwachman-Diamond syndrome.

A gene for autosomal recessive limb-girdle muscular dystrophy in Manitoba Hutterites maps to chromosome region 9q31-q33: evidence for another limb-girdle muscular dystrophy locus.

Characterized by proximal muscle weakness and wasting, limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of clinical disorders. Previous reports have documented either autosomal dominant or autosomal recessive modes of inheritance, with genetic linkage studies providing evidence for the existence of at least 12 distinct loci. Gene products have been identified for five genes responsible for autosomal recessive forms of the disorder. We performed a genome scan using pooled DNA from a large Hutterite kindred in which the affected members display a mild form of autosomal recessive LGMD. A total of 200 markers were used to screen pools of DNA from patients and their siblings. Linkage between the LGMD locus and D9S302 (maximum LOD score 5.99 at recombination fraction .03) was established. Since this marker resides within the chromosomal region known to harbor the gene causing Fukuyama congenital muscular dystrophy (FCMD), we expanded our investigations, to include additional markers in chromosome region 9q31-q34.1. Haplotype analysis revealed five recombinations that place the LGMD locus distal to the FCMD locus. The LGMD locus maps close to D9S934 (maximum multipoint LOD score 7.61) in a region that is estimated to be approximately 4.4 Mb (Genetic Location Database composite map). On the basis of an inferred ancestral recombination, the gene may lie in a 300-kb region between D9S302 and D9S934. Our results provide compelling evidence that yet another gene is involved in LGMD; we suggest that it be named "LGMD2H."

Limb girdle muscular dystrophy in Manitoba Hutterites does not map to any of the known LGMD loci.

Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of disorders affecting primarily the shoulder and pelvic girdles. Autosomal dominant and recessive forms have been identified; 8 have been mapped and 1 more has been postulated on the basis of exclusion of linkage. An autosomal recessive muscular dystrophy was first described in 1976 in the Hutterite Brethren, a North American genetic and religious isolate [Shokeir and Kobrinsky, 1976; Clin Genet 9:197-202]. In this report, we discuss the results of linkage analysis in 4 related Manitoba Hutterite sibships with 21 patients affected with a mild autosomal recessive form of LGMD. Because of the difficulties in assigning a phenotype in some asymptomatic individuals, stringent criteria for the affected phenotype were employed. As a result, 7 asymptomatic relatives with only mildly elevated CK levels were assigned an unknown phenotype to prevent their possible misclassification. Two-point linkage analysis of the disease locus against markers linked to 7 of the known LGMD loci and 3 other candidate genes yielded lod scores of < or = -2 at theta = 0.01 in all cases and in most cases at theta = 0.05. This suggests that there is at least 1 additional locus for LGMD.

Evolution of a repeat sequence in the parathyroid hormone-related peptide gene in primates.

A polymorphism of the variable number of tandem repeat (VNTR) type is located 97 bp downstream of exon VI of the parathyroid hormone-related peptide (PTHrP) gene in humans. The repeat unit has the general sequence G(TA)nC, where n equals 4-11. In order to characterize the evolutionary history of this VNTR, we initially tested for its presence in 13 different species representing four main groups of living primates. The sequence is present in the human, great apes, and Old World monkeys, but not in New World monkeys; and this region failed to PCR amplify in the Loris group. Thus, the evolution of the sequence as part of the PTHrP gene started at least 25-35 millions years ago, after divergence of the Old World and New World monkeys, but before divergence of Old World monkeys and great apes and humans. The structural changes occurring during evolution are characterized by a relatively high degree of sequence divergence. In general, the tandem repeat region tends to be longer and more complex in higher primates with the repeat unit motifs all being based on a TA-dinucleotide repeat sequence. Intra-species variability of the locus was demonstrated only in humans and gorilla. The divergence of the TA-dinucleotide repeat sequence and the variable mutation rates observed in different primate species are in contrast to the relative conservation of the flanking sequences during primate evolution. This suggests that the nature of the TA-dinucleotide repeat sequence, rather than its flanking sequences, is responsible for generating variability.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of polymorphisms and sequence variants in the human homologue of the mouse natural resistance-associated macrophage protein gene.

The most common mycobacterial disease in humans is tuberculosis, and there is evidence for genetic factors in susceptibility to tuberculosis. In the mouse, the Bcg gene controls macrophage priming for activation and is a major gene for susceptibility to infection with mycobacteria. A candidate gene for Bcg was identified by positional cloning and was designated "natural resistance-associated macrophage protein gene" (Nramp1), and the human homologue (NRAMP1) has recently been cloned. Here we report on (1) the physical mapping of NRAMP1 close to VIL in chromosome region 2q35 by PCR analysis of somatic cell hybrids and YAC cloning and (2) the identification of nine sequence variants in NRAMP1. Of the four variants in the coding region, there were two missense mutations and two silent substitutions. The missense mutations were a conservative alanine-to-valine substitution at codon 318 in exon 9 and an aspartic acid-to-asparagine substitution at codon 543 in the predicted cytoplasmic tail of the NRAMP1 protein. A microsatellite was located in the immediate 5' region of the gene, three variants were in introns, and one variant was located in the 3' UTR. The allele frequencies of each of the nine variants were determined in DNA samples of 60 Caucasians and 20 Asians. In addition, we have physically linked two highly polymorphic microsatellite markers, D2S104 and D2S173, to NRAMP1 on a 1.5-Mb YAC contig. These molecular markers will be useful to assess the role of NRAMP1 is susceptibility to tuberculosis and other macrophage-mediated diseases.

Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach.

We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome.

Association of apolipoprotein epsilon 4 allele and neuropathologic findings in patients with dementia.

Apolipoprotein E (APOE) is a lipoprotein expressed in liver and brain as one of three isoforms (APOE 2, APOE 3 and APOE 4). Recent findings suggest that the presence of APOE 4 is associated with an increased risk for both familial Alzheimer's disease and late-onset Alzheimer's disease. We extended these observations by determining the frequency of APOE alleles in patients with pathologically confirmed Alzheimer's Disease (AD), Parkinson's disease (PD), diffuse Lewy Body disease (DLBD), AD with concomitant PD pathology, demented PD patients without or with concomitant AD pathology and in schizophrenics with a progressive dementia (SCHIZ+DEM). The APOE genotype was determined by restriction digestion of polymerase chain reaction-amplified DNA isolated from frozen brain samples. The frequency of the APOE epsilon 4 allele was highest among sporadic AD and DLBD patients (0.30 and 0.38, respectively) and lowest in the SCHIZ+DEM and non-demented PD patients (0.06 and 0.1, respectively). Thus, the APOE epsilon 4 allele is over-represented selectively in patients with dementias associated with plaques and tangles and/or cortical Lewy bodies, but not in demented schizophrenics or non-demented PD patients.

Molecular biology of diabetes insipidus.

The identification, characterization, and mutational analysis of three different genes, namely the prepro-arginine-vasopressin-neurophysin II gene (prepro-AVP-NPII), the arginine-vasopressin receptor 2 gene (AVPR2), and the vasopressin-sensitive water channel gene (aquaporin-2, AQP2), provide the basis for our understanding of three different hereditary forms of diabetes insipidus: autosomal dominant neurogenic diabetes insipidus, X-linked nephrogenic diabetes insipidus, and autosomal recessive nephrogenic diabetes insipidus, respectively. These advances provide diagnostic tools for physicians caring for these patients.

X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis.

In X-linked nephrogenic diabetes insipidus (NDI) the urine of male patients is not concentrated after the administration of the antidiuretic hormone arginine-vasopressin. This disease is due to mutations in the V2 receptor gene that maps to chromosome region Xq28. In 1969, Bode and Crawford suggested that most NDI patients in North America shared common ancestors of Ulster Scot immigrants who arrived in Halifax in 1761 on the ship Hopewell. A link between this family and a large Utah kindred was also suggested. DNA was obtained from 17 affected male patients from the "Hopewell" kindred and from four additional families from Nova Scotia and New Brunswick who shared the same Xq28 NDI haplotype. The Utah kindred and two families (Q2, Q3) from Quebec were also studied. The "Hopewell" mutation, W71X, is a single base substitution (G-->A) that changes codon 71 from TGG (tryptophan) to TGA (stop). The W71X mutation was found in affected members of the Hopewell and of the four satellite families. The W71X mutation is the cause of X-linked NDI for the largest number of related male patients living in North America. Other families (Utah, Q2 and Q3) that are historically and ethnically unrelated bear other mutations in the V2 receptor gene.

Molecular characterization of an intragenic minisatellite (VNTR) polymorphism in the human parathyroid hormone-related peptide gene in chromosome region 12p12.1-p11.2.

The human parathyroid hormone-related peptide (hPTHrP) gene in chromosome region 12p12.1-p11.2 plays an important role in mammalian development and specifically in skeletogenesis. We have characterized a VNTR polymorphism in the hPTHrP gene that is located in an intron 100-bp downstream of exon VI that encodes a 3' untranslated region. By PCR analysis eight different alleles were identified in a group of 112 unrelated individuals. All eight alleles were sequenced and the repeat unit was identified as the general sequence [G(TA)nC]N, where n = 4 to 11 and N = 3 to 17. This polymorphic sequence-tagged site will be useful for mapping chromosome 12p and will aid in testing for linkage of genetic diseases to the hPTHrP gene.