Toward localization of the Werner syndrome gene by linkage disequilibrium and ancestral haplotyping: lessons learned from analysis of 35 chromosome 8p11.1-21.1 markers

Werner syndrome (WS) is an autosomal recessive disorder characterized by premature onset of a number of age-related diseases. The gene for WS, WRN, has been mapped to the 8p 11.1-21.1 region with further localization through linkage disequilibrium mapping. Here we present the results of linkage disequilibrium and ancestral haplotype analyses of 35 markers to further refine the location of WRN. We identified an interval in this region in which 14 of 18 markers tested show significant evidence of linkage disequilibrium in at least one of the two populations tested. Analysis of extended and partial haplotypes covering 21 of the markers studied supports the existence of both obligate and probable ancestral recombinant events which localize WRN almost certainly to the interval between D8S2196 and D8S2186, and most likely to the narrower interval between D8S2168 and D8S2186. These haplotype analyses also suggest that there are multiple WRN mutations in each of the two populations under study. We also present a comparison of approaches to performing disequilibrium tests with multiallelic markers, and show that some commonly used approximations for such tests perform poorly in comparison to exact probability tests. Finally, we discuss some of the difficulties introduced by the high mutation rate at microsatellite markers which influence our ability to use ancestral haplotype analysis to localize disease genes.     

Mapping of the spinal muscular atrophy (SMA) gene to a 750-kb interval flanked by two new microsatellites

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557. We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA. The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557. We tested all our key recombinant families with these markers. In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823. Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA. Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval.     

Allelic loss on chromosome 7q in ovarian adenocarcinomas: two critical regions and a rearrangement of the PLANH1 locus

The presence of a tumour suppressor gene on chromosome 7q is indicated by cytogenetic, loss of heterozygosity (LOH) and chromosome transfer studies. One candidate gene in this region is Plasminogen Activator Inhibitor-1 (PAI-1). The PAI-1 gene product is involved in proteolysis and may therefore influence tumour spread and invasion. We have analysed a series of 139 ovarian epithelial tumours at four loci in the region 7q21-q31 which includes the PAI-1 gene. The highest rates of loss were found in malignant tumours (FIGO stages I-IV) at markers D7S471 (38%, 20/52 informative cases) and D7S522 (34%, 15/44). No loss was seen in benign tumours and only one out of 27 (4%) informative LMP tumours demonstrated LOH. The smallest region of overlap (SRO) lies between D7S471 and PAI-1. We also identified a rearrangement in one tumour in the PAI-1 gene, suggesting that this may be the inactivated gene in this region. In addition LOH at the more distal marker, D7S522, which lies outside the SRO, shows significant association with stage (P=0.0343) and with LOH on chromosome 13 (P=0.0024). This is in contrast to all other markers examined. These data suggest the presence of two critical regions on 7q which may be important in subsets of epithelial ovarian tumours.     

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Detailed deletion mapping of chromosome 6q has shown that the highest percentage of loss of heterozygosity (LOH) is located at 6q25-q27 and suggested that an ovarian cancer associated tumor suppressor gene may reside in this region. To further define the smallest region of common loss, we used 12 tandem repeat markers spanning a region no more than 18 cM, located between 6q25.1 and 6q26, to examine allelic loss in 54 fresh and paraffin embedded invasive ovarian epithelial tumor tissues. Loss of heterozygosity was observed more frequently at the loci defined by marker D6S473 (14 of 32 informative cases, 44%) and marker D6S448 (17 of 40 informative cases, 43%). Detailed mapping of chromosome 6q25-q26 in these tumor samples identified a 4 cM minimal region of LOH between markers D6S473 and D6S448 (6q25.1-q25.2). Loss of heterozygosity at D6S473 correlated significantly both with serous versus non-serous ovarian tumors (P=0.040) and with high grade versus low grade specimens (P=0.023). The results suggest that a 4 cM deletion unit located at 6q25.1-q25.2 may contain the putative tumor suppressor gene which may play a role in the development and progression of human invasive epithelial ovarian carcinomas (IEOC).     

Benign recurrent intrahepatic cholestasis (BRIC): evidence of genetic heterogeneity and delimitation of the BRIC locus to a 7-cM interval between D18S69 and D18S64

Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. The gene was previously assigned to chromosome 18q21, using a shared segment analysis in three families from the Netherlands. In the present study we report the linkage analysis of an expanded sample of 14 BRIC families, using 15 microsatellite markers from the 18q21 region. Obligate recombinants in two families place the gene in a 7-cM interval, between markers D18S69 and D18S64. All intervening markers had significant LOD scores in two-point linkage analysis. Moreover, we identified one family in which the BRIC gene seems to be unlinked to the 18q21 region, or that represents incomplete penetrance of the BRIC genotype.     

An ancestral core haplotype defines the critical region harbouring the North Carolina macular dystrophy gene (MCDR1)

Autosomal dominant North Carolina macular dystrophy (NCMD) or central areolar pigment epithelial dystrophy (CAPED) is an allelic disorder that maps to an approximately 7.2 cM interval between DNA markers at D6S424 and D6S1671 on 6q14-q16.2. The further refinement of the disease locus has been hindered by the lack of additional recombination events involving the critical region. In this study, we have identified three multigeneration families of German descent who express the NCMD phenotype. Genotyping was carried out with a series of markers spanning approximately 53 cM around the NCMD locus, MCDR1. Genetic linkage between the markers and the disease phenotype in each of the families could be shown. Disease associated haplotypes were constructed and provide evidence for an ancestral founder for the German NCMD families. This haplotype analysis suggests that a 4.0 cM interval flanked by markers at D6S249 and D6S475 harbours the gene causing NCMD, facilitating further positional cloning approaches.     

Loss of heterozygosity in tuberous sclerosis hamartomas

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.     

Search for a founder mutation in idiopathic focal dystonia from Northern Germany

Both the discovery of the DYT1 gene on chromosome 9q34 in autosomal dominant early-onset torsion dystonia and the detection of linkage for one form of adult-onset focal dystonia to chromosome 18p (DYT7) in a family from northern Germany provide the opportunity to further investigate genetic factors in the focal dystonias. Additionally, reports of linkage disequilibrium between several chromosome 18 markers and focal dystonia, both in sporadic patients from northern Germany and in members of affected families from central Europe suggest the existence of a founder mutation underlying focal dystonia in this population. To evaluate the role of these loci in focal dystonia, we tested 85 patients from northern Germany who had primary focal dystonia, both for the GAG deletion in the DYT1 gene on chromosome 9q34 and for linkage disequilibrium at the chromosome 18p markers D18S1105, D18S1098, D18S481, and D18S54. None of these patients had the GAG deletion in the DYT1 gene. Furthermore, Hardy-Weinberg analysis of markers on 18p in our patient population and in 85 control subjects from the same region did not support linkage disequilibrium. Taken together, these results suggest that most cases of focal dystonia in patients of northern German or central European origin are due neither to the GAG deletion in DYT1 nor to a proposed founder mutation on chromosome 18p but must be caused by other genetic or environmental factors.     

Human bladder cancers and normal bladder mucosa present the same hot spot of heterozygous chromosome-9 deletion

Loss of heterozygosity (LOH) on chromosome 9 is the most frequent genetic alteration in bladder cancer identified to date, suggesting the presence of key gene(s) for this pathology. In this study, we examined 44 bladder tumors and 21 normal bladder samples for LOH on both arms of chromosome 9. Sixteen microsatellite markers, 12 on the short arm (encompassing 9p21-22) and 4 on the long arm (encompassing 9q33-34), were chosen for their highly frequent alterations in bladder cancer. LOH for at least one marker was identified in 42 tumor samples (95.5%), and 14 tumors (32%) displayed LOH for all informative tested markers. Detailed analysis showed that 2 markers on chromosome 9p (D9S157 and D9S156) had the highest frequencies of allelic loss (about 70%), independent of tumor grade and stage. The same study was performed on the 21 normal bladder mucosa samples: 50% of informative cases presented a single specific LOH at the D9S156 locus. Normal samples showing LOH at this locus were therefore screened with 3 novel microsatellite markers in the 810-kb region incorporating D9S156. Using this marker, we found no further heterozygous loss in this region. This result allows different interpretations of the D9S156 loss in normal bladder mucosa, and suggests that D9S156 may be more an indicator of bladder epithelium impairment than a tumor-initiation marker. Similarly, this unexpected result calls in question the interpretation of LOH studies.     

Reiter''s disease in an 11-year-old boy

The breast basic conserved gene (D16S444E) is a candidate tumor suppressor gene previously mapped to human chromosome 16. We determined the map position of D16S444E more precisely using a somatic mouse x human hybrid panel and fluorescence in situ hybridization on metaphase spreads and interphase nuclei. We show that the D16S444E gene is localized on band 16q24.3 and is located between APRT and D16S44.     

The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the von Hippel Lindau disease gene

Von Hippel Lindau disease (VHL) is a rare autosomal dominant disease associated with tumors and cysts in multiple organ systems. The VHL disease gene is tightly linked to the polymorphic DNA marker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomeric and RAF1 on its centromeric side. Two additional markers, D3S1038 and D3S601, have also been identified, and these markers, like D3S720, are very tightly linked to VHL. Previously 93 cosmid clones were mapped to the larger region, 3p24.2-pter, surrounding the VHL disease gene. Using a Southern-based screening strategy on pools of YAC clones we have isolated a contig of overlapping YAC clones that extends about 0.7 megabase centromeric, and about 1.3 megabases telomeric of D3S720 and contains all three tightly linked VHL markers. Individual YACs in this contig were hybridized to grids containing cosmids localized between 3p24.2-pter and to several cosmids localized by fluorescent in situ hybridization (FISH) to 3p25. A total of 28 cosmids were positioned on this contig of overlapping YAC clones. We have also identified homologous YAC clones to many additional cosmid clones localized between 3p24.2-p25, although these have not yet been precisely localized relative to the contig of YAC clones. This contig of YAC clones probably contains the VHL disease gene and should facilitate the isolation and characterization of this gene.     

MALT lymphoma of labial minor salivary gland in an immunocompetent child with a gastric Helicobacter pylori infection

Autosomal recessive nonsyndromic sensorineural deafness segregating in a large consanguineous Indian family was mapped to chromosome 11p14-p15.1 defining a new locus, DFNB18. A maximum lod score of 4.4 at theta = 0 was obtained for the polymorphic micro-satellite marker D11S1888. Haplotype analysis localizes this gene between markers D11S1307 and D11S2368, which is approximately 1.6 cM and encompasses the region of Usher syndrome type 1C (USH1C). We postulate that DFNB18 and USH1C are allelic variants of the same gene.     

Mapping of a breast cancer tumor suppressor gene locus to a 4-cM interval on chromosome 18q21

DPC4 and DCC, putative tumor suppressor genes implicated in the genesis of several types of human cancer, lie on the long arm of human chromosome 18. We examined 200 primary breast cancers for allelic losses on chromosome 18, using 15 microsatellite markers distributed along the long arm. Allelic loss was detected most frequently (29-30%) at loci mapped to 18q21. Deletion mapping of the 34 tumors showing partial or interstitial deletions identified a commonly deleted region within the 4-cM interval flanked by D18S474 and D18S487 at 18q21.1-q21.3. Although this interval included the DPC4 and DCC genes, we excluded DPC4 from candidacy when polymerase chain reaction-single-strand conformation polymorphism analysis of each exon failed to detect abnormalities in any of the 54 breast cancers that exhibited loss of heterozygosity involving 18q. Allelic loss on 18q was found more frequently in tumors of the solid tubular histological type (24 of 55, 44%) than in other types (24 of 113, 21%) (P = 0.0049). The results suggest that a tumor suppressor gene located within the 4-cM region at 18q21, either DCC or another gene not yet identified, may play a role in the development of some sporadic breast cancers, particularly those of the solid tubular type.     

Tn10 transposition via a DNA hairpin intermediate

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88.     

Localization of the hemochromatosis gene close to D6S105

The hemochromatosis (HC) gene is known to be linked to HLA-A (6p21.3); however, its precise location has been difficult to determine because of a lack of additional highly polymorphic markers for this region. The recent identification of short tandem repeat sequences (microsatellites) has now provided this area with a number of markers with similar polymorphic index to the HLA serological polymorphisms. Using four microsatellites--D6S105, D6S109, D6S89, and F13A--together with the HLA class I loci HLA-A and HLA-B in 13 large pedigrees clearly segregating for HC, we have been able to refine the location of the HC gene. We identified no recombination between HC and HLA-A or D6S105, and two-point analyses placed the HC gene within one centimorgan (cM) of HLA-A and D6S105 (HLA-A maximum of the lod score [Zmax] of 9.90 at recombination fraction [theta] of 0.0, and D6S105 Zmax of 8.26 at theta of 0.0). The markers HLA-B, D6S109, D6S89, and F13A were separated from the HC locus by recombination, defining the centromeric and telomeric limits for the HC gene as HLA-B and D6S109, respectively. A multipoint map constructed using HLA-B, HLA-A, and D6S109 indicates that the HC gene is located in a region less than 1 cM proximal to HLA-A and less than 1 cM telomeric of HLA-A. These pedigree data indicate an association between HC and specific alleles at HLA-A and D6S105 (i.e., HLA-A3 and D6S105 allele 8).(ABSTRACT TRUNCATED AT 250 WORDS)     

Refined genetic mapping of autosomal dominant retinitis pigmentosa locus RP18 reduces the critical region to 2 cM between D1S442 and D1S2858 on chromosome 1q

Linkage analysis was performed on a large Danish family to refine the position of RP18, the locus for autosomal dominant retinitis pigmentosa, mapped previously between D1S534 and D1S305 in chromosome 1p13-q21. We genotyped the family members for five microsatellite-type DNA polymorphisms and mapped RP18 between D1S422 and D1S2858 to a region of less than 2 cM. No obvious candidate gene has yet been assigned to the chromosomal interval defined here.     

Autosomal dominant hypophosphatemic rickets is linked to chromosome 12p13

Autosomal dominant hypophosphatemic rickets (ADHR) is an inherited disorder of isolated renal phosphate wasting, the pathogenesis of which is unknown. We performed a genome-wide linkage study in a large kindred to determine the chromosome location of the ADHR gene. Two-point LOD scores indicate that the gene is linked to the markers D12S314 [Z(theta) = 3.15 at theta = 0.0], vWf [Z(theta) = 5.32 at theta = 0.0], and CD4 [Z(theta) = 3.53 at theta = 0.0]. Moreover, multilocus analysis indicates that the ADHR gene locus is located on chromosome 12p13 in the 18-cM interval between the flanking markers D12S100 and D12S397. These data are the first to establish a chromosomal location for the ADHR locus and to provide a framework map to further localize the gene. Such studies will permit ultimate identification of the ADHR gene and provide further insight into phosphate homeostasis.     

New polymorphic microsatellite markers place the haemochromatosis gene telomeric to D6S105

The haemochromatosis gene (HFE) is linked to both HLA-A and D6S105 on the short arm of chromosome 6 but these markers are separated by approximately 2 Mb of DNA. Most chromosomes carrying HFE have a common haplotype which extends from HLA-A to D6S105 and includes HLA-F. To localise the gene more precisely we have examined 10 microsatellite markers extending over a genetic distance of approximately 5 cM from D6S265 (within 100 kb of HLA-A on the centromeric side) to D6S299 (telomeric). The order of markers is D6S265, HLA-F, D6S258, D6S306, CS3, D6S105, D6S464, CS5, D6S461 and D6S299. We confirm that haemochromatosis appears to originate from a founder mutation which has multiplied in the population through successive generations. This mutation is associated with the haplotype D6S306-5, CS3-3, D6S105-8, D6S464-9 and CS5-4 which is found on approximately 70% of HFE chromosomes. We have applied a new and powerful, likelihood analysis for linkage disequilibrium. The maximum value of lambda (proportion of total possible association between a marker and disease) is 0.74 for marker CS5 (allele 4). A multipoint analysis also gives a maximum likelihood near marker CS5. We conclude that the HFE gene is likely to be located telomeric of D6S105 and close to CS5.     

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.     

Homozygosity and linkage-disequilibrium mapping of the urofacial (Ochoa) syndrome gene to a 1-cM interval on chromosome 10q23-q24

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disease characterized by congenital obstructive uropathy and abnormal facial expression. The patients present with enuresis, urinary-tract infection, hydronephrosis, and voiding dysfunctions as a result of neurogenic bladders. To map the UFS gene, a genome screen using a combination of homozygosity-mapping and DNA-pooling strategies was performed in 20 selected patients, one patient pool, and three control pools (unaffected relatives). After analyses of 36 randomly chosen markers, D10S677 was identified as being linked to and associated with UFS, as suggested by a significant excess of homozygosity in patients compared with that in unaffected relatives (P < 10(-6)), as well as by the allelic-frequency differences between the patient pool and control pools. Ten additional markers flanking D10S677 and covering a 22-cM region then were analyzed to fine-map the UFS gene by use of haplotype (linkage disequilibrium) analysis. All 31 patients were found to be homozygous for two closely linked markers (D10S1726 and D10S198) located approximately 5 cM telomeric to D10S677, whereas only 12% of the unaffected relatives were homozygous for both markers (P < 10(-19)). Several patients are heterozygous at two markers immediately flanking D10S1726/D10S198, one on the centromeric side (D10S1433) and the other on the telomeric side (D10S603). These recombinational events place the UFS gene near D10S1726/D10S198 and within a 1-cM interval defined by D10S1433 and D10S603 on chromosome 10q23-q24.