Geographic distribution of Ala62Thr variant associated to Uric Acid Nephrolithiasis from Sub-Saharan to Mediterranean area

Recently, we identified a susceptibility locus for human Uric Acid Nephrolithiasis (UAN) on 10q21-q22 and showed that a variant (Ala62Thr) of a novel gene (ZNF365) encoding a specific protein (Talanin) is associated with Uric Acid Nephrolithiasis (UAN) (OMIM 605990). We analyzed the frequency of this polymorphism associated with UAN in some ethnic groups from the Mediterranean to the African area. We found that this variant is common in Sardinian (frequency 32%) and Sicilian populations (frequency 23%). Both Mediterranean islands in the Italian peninsula this frequency was 27%. On the contrary, in Burkina Faso and in Benin, both Sub-Saharan countries, a low frequency of this variant predisposing to UAN was found (1.1% and 1.2% respectively). This low frequency of Ala62Thr is associated with a low prevalence of UAN in Burkina Faso and in Benin.     

Genes,diet and uric acid nephrolithiasis

Uric acid represent the final product of purine metabolism: one-third of daily uric acid production is excreted by the gastrointestinal tract and two-thirds by the kidney. A high uric acid excretion with urine, a low urine volume due to dehydration and an acidic urinary pH value have been suggested to be the most important risk factor for uric acid nephrolithiasis (UAN). Recently mutation analysis showed that a variant (Ala62Thr) in a specific protein isoform (Talanin) is associated with UAN. We found that this variant is rather common in the Sardinian (32%) and Sicilian populations (23%), that are Mediterranean islands, as well as in the Italian peninsula (27%). On the contrary, in Burkina Faso and in Benin, both sub-Saharan countries, mesoendemic regions for Plasmodium falciparum malaria and other parasite infections, a low incidence of this variant was found (1.1% and 1.2% respectively). In Burkina Faso and in Benin, the low incidence of Ala62Thr variant is associated with low presence of UAN and the major classes of stones reported are calcium oxalate and calcium phosphate. These low frequencies for Ala62Thr predisposing to UAN in Burkina Faso and in Benin may represent the result of a selective mechanism where the arid conditions of territory and the characteristic alimentary habits of this part of Africa may represent an obstacle to the expansion of mutated allele.     

Evidence of a founder effect and refinement of the hereditary neuralgic amyotrophy (HNA) locus on 17q25 in American families

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder that is associated with episodic recurrent brachial plexus neuropathy. A mutation for HNA maps to chromosome 17q25. To refine the HNA locus further, we carried out genetic linkage studies in seven pedigrees with a high density set of DNA markers from chromosome 17q25. All pedigrees demonstrated linkage to chromosome 17q25, and an analysis of recombinant events placed the HNA locus within an interval of approximately 1 Mb flanked by markers D17S722 and D17S802. In order to test the power of linkage disequilibrium mapping, we compared genotypes of 12 markers from seven pedigrees that were from the United States and that showed linkage to chromosome 17q25. The haplotypes identified a founder effect in six of the seven pedigrees with a minimal shared haplotype that further refines the HNA locus to an interval of approximately 500 kb. These findings suggest that, for the pedigrees from the United States, there are at least two different mutations in the HNA gene.     

Mapping of a milk production quantitative trait locus to a 420-kb region on bovine chromosome 6

A QTL affecting milk production traits was previously mapped to an interval of 7.5 cM on chromosome 6 in Norwegian dairy cattle. This article aimed to refine this position by increasing the map density in the region by a set of single-nucleotide polymorphisms and analyzing the data with a combined linkage and linkage disequilibrium approach. Through a series of single- and multitrait and single- and multipoint analyses, the QTL was positioned to an interval surrounded by the genes ABCG2 and LAP3. As no recombinations were detected in this interval, physical mapping was required for further refining. By using radiation hybrid mapping as well as BAC clones, the bovine and human comparative maps in the region are resolved, and the QTL is mapped within a distance of 420 kb.     

Closing in on the Rieger syndrome gene on 4q25: mapping translocation breakpoints within a 50-kb region.

Rieger syndrome (RGS) is an autosomal dominant disorder of morphogenesis affecting mainly the formation of the anterior eye chamber and of the teeth. RGS has been localized to human chromosome 4q25 by linkage to epidermal growth factor (EGF). We have constructed a detailed physical map and a YAC contig of the genomic region encompassing the EGF locus. Using FISH, several YACs could be shown to cross the breakpoint in two independent RGS patients with balanced 4q translocations. Alu- and LINE-fragmentation of a 2.4-Mb YAC generated a panel of shorter YACs ranging in size from 2.4 Mb to 75 kb. Several fragmentation YACs were subcloned in cosmids, which were mapped to specific subregions of the original YAC by hybridization to the fragmentation panel to further refine the localization of the translocation breakpoints, allowing mapping of the breakpoints to within the most-telomeric 200 kb of the original 2.4-Mb YAC. FiberFISH of cosmids located in this 200-kb region mapped the two translocation breakpoints within a 50-kb region approximately 100-150 kb centromeric to D4S193, significantly narrowing down the candidate region for RGS. The mapping data and resources reported here should facilitate the identification of a gene implicated in Rieger syndrome.     

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.)

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.     

Physical mapping of the cystic fibrosis region by pulsed-field gel electrophoresis

The gene for cystic fibrosis (CF) is known to be flanked by the closely linked DNA markers met and J3.11 on chromosome 7. Using the technique of pulsed-field gel electrophoresis, we have constructed a complete overlapping restriction map of approximately 3000 kb of DNA in this region. The met and J3.11 probes are found to be between 1300 and 1800 kb apart, which compares well with their genetic distance of 1-2 cM. The CF gene must be located within this interval, and the availability of this physical map should be of considerable utility in mapping additional clones as the search for the gene proceeds.     

Localization of psoriasis-susceptibility locus PSORS1 to a 60-kb interval telomeric to HLA-C

Recent genome scans have established the presence of a major psoriasis-susceptibility locus in the human leukocyte antigen (HLA) complex on chromosome 6p21.3. To narrow the interval for candidate gene testing, we performed a linkage-disequilibrium analysis of 339 families, with the use of 62 physically mapped microsatellite markers spanning the major histocompatibility complex (MHC). As detected by use of the transmission/disequilibrium test (TDT), individual markers yielded significant linkage disequilibrium across most of the MHC. However, the strongest evidence for marker-trait disequilibrium was found in an approximately 300-kb region extending from the MICA gene to the corneodesmosin gene. Maximum-likelihood haplotypes were constructed across the entire MHC in the original sample and across a 1.2-Mb region of the central MHC in an expanded sample containing 139 additional families. Short (two- to five-marker) haplotypes were subjected to the TDT using a "moving-window" strategy that reduced the variability of TDT P values relative to the single-locus results. Furthermore, the expanded sample yielded a sharp peak of evidence for linkage disequilibrium that spanned approximately 170 kb and that was centered 100 kb telomeric to HLA-C. The 1.2-Mb interval was further dissected by means of recombinant ancestral haplotype analysis. This analysis identified risk haplotype 1 (RH1), which is a 60-kb fragment of ancestral haplotype 57.1, on all identifiable HLA risk haplotypes. One of these haplotypes exhibits significant linkage disequilibrium with psoriasis but does not carry Cw6, which is the HLA allele most strongly associated with the disease. These results demonstrate that RH1 is highly likely to carry the disease allele at PSORS1, and they exclude HLA-C and corneodesmosin with a high degree of confidence.     

Chromosome landing at the tomato Bs4 locus

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.     

Unraveling Genomic Complexity at a Quantitative Disease Resistance Locus in Maize

Multiple disease resistance has important implications for plant fitness, given the selection pressure that many pathogens exert directly on natural plant populations and indirectly via crop improvement programs. Evidence of a locus conditioning resistance to multiple pathogens was found in bin 1.06 of the maize genome with the allele from inbred line “Tx303” conditioning quantitative resistance to northern leaf blight (NLB) and qualitative resistance to Stewart’s wilt. To dissect the genetic basis of resistance in this region and to refine candidate gene hypotheses, we mapped resistance to the two diseases. Both resistance phenotypes were localized to overlapping regions, with the Stewart’s wilt interval refined to a 95.9-kb segment containing three genes and the NLB interval to a 3.60-Mb segment containing 117 genes. Regions of the introgression showed little to no recombination, suggesting structural differences between the inbred lines Tx303 and “B73,” the parents of the fine-mapping population. We examined copy number variation across the region using next-generation sequencing data, and found large variation in read depth in Tx303 across the region relative to the reference genome of B73. In the fine-mapping region, association mapping for NLB implicated candidate genes, including a putative zinc finger and pan1. We tested mutant alleles and found that pan1 is a susceptibility gene for NLB and Stewart’s wilt. Our data strongly suggest that structural variation plays an important role in resistance conditioned by this region, and pan1, a gene conditioning susceptibility for NLB, may underlie the QTL.     

Hitchhiking mapping reveals a candidate genomic region for natural selection in three-spined stickleback chromosome VIII

Identification of genes and genomic regions under directional natural selection has become one of the major goals in evolutionary genetics, but relatively little work to this end has been done by applying hitchhiking mapping to wild populations. Hitchhiking mapping starts from a genome scan using a randomly spaced set of molecular markers followed by a fine-scale analysis in the flanking regions of the candidate regions under selection. We used the hitchhiking mapping approach to narrow down a selective sweep in the genomic region flanking a candidate locus (Stn90) in chromosome VIII in the three-spined stickleback (Gasterosteus aculeatus). Twenty-four microsatellite markers were screened in an approximately 800-kb region around the candidate locus in three marine and four freshwater populations. The patterns of genetic diversity and differentiation in the candidate region were compared to those of a putatively neutral set of markers. The Bayesian FST-test indicated an elevated genetic differentiation, deviating significantly from neutral expectations, at a continuous region of approximately 20 kb upstream from the candidate locus. Furthermore, a method developed for an array of microsatellite markers rejected neutrality in a region of approximately 90 kb flanking the candidate locus supporting the selective sweep hypothesis. Likewise, the genomewide pattern of genetic diversity differed from the candidate region in a bottleneck analysis suggesting that selection, rather than demography, explains the reduced genetic diversity at the candidate interval. The neutrality tests suggest that the selective sweep had occurred mainly in the Lake Pulmanki population, but the results from bottleneck analyses indicate that selection might have operated in other populations as well. These results suggest that the narrow interval around locus Stn90 has likely been under directional selection, but the region contains several predicted genes, each of which can be the actual targets of selection. Understanding of the functional significance of this genomic region in an ecological context will require a more detailed sequence analysis.     

Allelic loss mapping and physical delineation of a region harboring a thymic lymphoma suppressor gene on mouse chromosome 16

Our previous mapping of allelic loss in gamma-ray induced thymic lymphomas in F(1) hybrid and backcross mice between BALB/c and MSM strains identified three regions with high frequencies of allelic loss which probably harbor a tumor suppressor gene. One region, Tlsr7, exists near the D16 Mit122 locus on chromosome 16. This study has further localized Tlsr7 by constructing a physical map and scanning a total of 587 thymic lymphomas. The map consists of 13 overlapping BAC clones and isolation of BAC-derived polymorphic probes leads to fine mapping of allelic losses. Eleven lymphomas show informative breakpoints of allelic loss regions relative to the flanking markers on the map. Pulsed-field gel electrophoresis of NotI digests of the clones shows that the commonly lost region is localized within an approximately 300 kb interval near D16Mit192. This map is invaluable to facilitate the identification of genes in the Tlsr7 region.     

Fine mapping of the schizophrenia susceptibility locus on chromosome 1q22

Schizophrenia is a serious neuropsychiatric illness estimated to affect approximately 1% of the general population. As part of a genome scan for schizophrenia susceptibility loci, we have previously reported a maximum heterogeneity four-point lod score of 6.50 on chromosome 1q21-22 in a group of 22 medium-sized Canadian families, selected for study because multiple relatives were clinically diagnosed with schizophrenia or schizoaffective disorder. We have now conducted fine mapping of this locus in the same set of individuals using 15 genetic markers spanning an approximately 15-cM interval. Parametric linkage analysis with GENEHUNTER v2.1 and VITESSE v2.0 produced a maximum multipoint heterogeneity lod score of 6.50, with a Zmax-1 support interval of <3 cM, corresponding to approximately 1 Mb. Physical mapping and sequence analysis from this region confirmed the presence of an approximately 81-kb tandem duplication, containing low-affinity IgG receptor genes and heat shock protein genes. The sequences of the two copies of this duplication are approximately 97% identical, which has led to the collapse of the two copies into one in the June 2002 NCBI Build 30 of the Human Genome. This duplication may be involved in genomic instability, leading to gene deletion, and so presents an intriguing candidate locus for schizophrenia susceptibility.     

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.     

Linkage-disequilibrium mapping narrows the Fukuyama-type congenital muscular dystrophy (FCMD) candidate region to <100 kb.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with brain anomalies. After our initial mapping of the FCMD locus to chromosome 9q31-33, we have further defined the locus within a approximately 5-cM region between D9S127 and D9S2111 and have found linkage disequilibrium between FCMD and D9S306 in this candidate region on 9q31. The high prevalence of FCMD among the Japanese, who are a relatively isolated population, provides an opportunity to utilize linkage-disequilibrium mapping. We developed three new microsatellites, near D9S306, from the FCMD YAC contig, determined their positions on YACs, and performed linkage-disequilibrium mapping with these markers and other newly published loci. The maximum value of p(excess), which represents the strength of linkage disequilibrium, was obtained at D9S2107; and this value showed a relatively steady rise and fall across the region that is likely to contain FCMD. Distances between FCMD and each marker were presumed to be approximately 1 Mb, approximately 350 kb, approximately 140 kb, approximately 20 kb, approximately 280 kb, approximately 450 kb, and approximately 740 kb for D9S306, A107XF9, D9S2105, D9S2107, D9S172, D9S299, and D9S2109, respectively. Haplotype analysis using the three closest markers D9S2105, D9S2107, and D9S172 indicated that most FCMD-bearing chromosomes are derived from a single ancestral founder and suggested that these markers can be used for the diagnosis of sporadic FCMD. Thus, the FCMD gene is most likely to lie within a region of <100 kb containing D9S2107.