Fine mapping of a putative tuberculosis-susceptibility locus on chromosome 15q11-13 in African families

Host genetics plays an important role in individual susceptibility and resistance to infectious diseases, but no genes have yet been identified using genome-wide screens. Twin studies have indicated that tuberculosis susceptibility has a significant host genetic component, and several genes appear to be involved. Recently, a genome-wide linkage analysis of 136 African families identified chromosome 15q11-13 as a region with suggestive evidence of linkage, with a LOD score of 2.0. We tested 10 microsatellite markers and 5 positional candidate genes in this chromosomal region for deviation from random transmission from parents to affected offspring. The polymorphisms, lying in a region of 14 cM, were initially typed in the same 79 Gambian families used in the genome screen. A borderline significant association with a 7 bp deletion in UBE3A (P = 0.01) was found. This polymorphism was then evaluated further in a larger series of families with tuberculosis, including 44 Guinean families and 57 families from South Africa. Testing for association between the deletion and tuberculosis across all the families using the exact symmetry test further supported the association (overall P = 0.002). These fine-mapping data suggest that UBE3A or a closely flanking gene may be a tuberculosis-susceptibility locus.     

Fine structure physical mapping of a 1·9 Mb region of chromosome 13q12

Ann. Hum. Genet. (1997), 61 , 15–24
In this paper we described the physical map of a 1·9 Mb region in chromosome region 13q12. Although in the text we described that the motivation for this effort was to identify the breakpoint in an 8;13 translocation associated with myeloproliferative disease, none of the results presented in fact referred to that localization. However, a reference to the position of a breakpoint was given in Figure 2 and was derived from a figure constructed from early mapping data and submitted by mistake instead of the corrected figure which should not have included this information. We now know that this is not the position of the translocation breakpoint. All of the other mapping details in Figure 2 are correct. The authors regret this error and any inconvenience it may have caused.     

Fine structure physical mapping of a 1·9 Mb region of chromosome 13q12

Through linkage analysis and the identification of structural chromosome rearrangements, a number of disease genes have been mapped to the pericentromeric region of the long arm of chromosome 13. Structural rearrangements, or deletions, of the 13q12 region have been implicated in a range of myeloproliferative neoplasms, and other haematopoietic malignancies. In particular, seven cases of a t(8;13) (p11; q12·1) rearrangement have been noted in patients with an atypical myeloproliferative disorder associated with T-cell leukemia and eosinophilia. We have previously identified a CEPH megaYAC, 943E4, which crosses the translocation breakpoint in archival tumour samples from two patients with this t(8;13) translocation. As an initial step in the characterisation of this translocation breakpoint, we have generated a fine structure physical map of this 1·9 Mb YAC. We have used the method of YAC fragmentation to generate a series of deletion constructs of known size, which provide discreet physical landmarks convenient for mapping genetic markers along the 943E4 YAC. Analysis of these deletion constructs defined the order of ESTs and microsatellite markers in 943E4 as: cen-NIB1257-(ATP1AL1/D13S283)- D13S179E-(D13S504E/D13S505E)-D13S824E-D13S182E-D13S221-tel. These markers have also been assigned to physically defined regions relative to the fragmented YAC endpoints and a derived NotI restriction map.     

Deletion mapping of chromosome region 12q13-24 in colorectal cancer

Colorectal cancer is one of the most common cancers in the world. Colorectal cancer develops after a long and multistep process of carcinogenesis. Inactivation of tumor suppressor genes is among the most important steps in development of colorectal cancer. Analysis of loss of heterozygosity (LOH) is an effective method to determine the localization of tumor suppressor genes. In this study, we used five microsatellite markers to analyze the region 12q13-24 among 47 patients with colorectal cancer. The frequency of LOH and the clinicopathological data were compared using logistic regression and a chi-square test. In 34 of 47 tumor tissues (72%), LOH was detected at least in one marker. The highest LOH frequency was 34%, on the D12S129 locus; the lowest frequency was 23%, on the D12S78 locus. Loss of heterozygosity was detected as 32% on D12S83, 30% on D12S346, and 26% on D12S1660. No statistically significant correlation was found between the frequency of LOH and clinicopathological features (P > 0.05). Chromosome region 12q13-24 contains several known genes that may be candidate tumor suppressor genes, including RASAL1, ITGA7, STAB2, GLIPR1, and SLC5A8. Although the exact roles of these genes in colorectal cancer formation remain to be clarified, the present data point to a tumor suppressor role.     

Fine deletional mapping of chromosome 4q22-35 region in oral cancer

We analyzed the loss of heterozygosity of the long arm of chromosome 4 in 40 oral cancers, using 16 microsatellite markers based on data from the human genome sequence, and defined the deletional mapping of the region with putative tumor suppressor genes. Our data revealed two distinct commonly deleted regions around the markers, D4S2623 and D4S1644, with an allelic deletion of 44 and 39%, respectively. Additional mapping and use of the markers near one of these hot spots narrowed down the minimally deleted region about 1.5 Mbp around the marker, D4S2623. Caspase 6 is localized 280 kb from the marker, D4S2623. Fine mapping of this region with possible tumor suppressor gene suggests caspase 6 as a putative tumor suppressor gene. Further molecular analysis of caspase 6 should be performed to clarify its role in oral carcinogenesis.     

Fine mapping and positional candidate studies on chromosome 5p13 identify multiple asthma susceptibility loci

BACKGROUND: Genome-wide linkage scans to identify asthma susceptibility loci have revealed many linked regions, including a broad region on chromosome 5p. OBJECTIVE: To identify a 5p-linked asthma or bronchial hyperresponsiveness (BHR) locus. METHODS: We performed fine mapping and positional candidate studies of this region in the Hutterites and an outbred case-control sample from Germany by genotyping 89 single nucleotide polymorphisms (SNPs) in 22 genes. SNP and haplotype analyses were performed. RESULTS: Three genes in a distal region (zinc finger RNA binding protein [ZFR], natriuretic peptide receptor C, and a disintegrin and metalloproteinase domain with thrombospondin type 1 motif [ADAMTS12]) were associated with BHR, whereas 4 genes in a proximal region (prolactin receptor, IL-7 receptor [IL7R], leukemia inhibitory factor receptor [LIFR], and prostaglandin E4 receptor [PTGER4]) were associated with asthma symptoms in the Hutterites. Furthermore, nearly the entire original linkage signal in the Hutterites was generated by individuals who had the risk-associated alleles in ZFR3, natriuretic peptide receptor C, ADAMTS12, LIFR, and PTGER4. Variation in ADAMTS12, IL7R, and PTGER4 were also associated with asthma in the outbred Germans, and the frequencies of long-range haplotypes composed of SNPs at ZFR, ADAMTS12, IL7R, LIFR, and PTGER4 were significantly different between both the German and Hutterite cases and controls. There is little linkage disequilbrium between alleles in these 2 regions in either population. CONCLUSION: These results suggest that a broad region on 5p, separated by >9 Mb, harbors at least 2 and possibly 5 asthma or BHR susceptibility loci. These findings are consistent with the hypothesis that regions providing evidence for linkage in multiple populations may, in fact, house more than 1 susceptibility locus, as appears to be the case for the linked region on 5p. CLINICAL IMPLICATIONS: Identifying asthma or BHR genes could lead to novel therapeutic approaches.     

Fine mapping of a linkage region on chromosome 17p13 reveals that GABARAP and DLG4 are associated with vulnerability to nicotine dependence in European-Americans

A two-stage association study was conducted targeting a genomic region on chromosome 17p13 that we reported likely to harbor susceptibility gene(s) for nicotine dependence (ND). Participants were 2037 subjects from 602 nuclear families of either African-American (AA) or European-American (EA) origin from our Mid-South Tobacco Family (MSTF) cohort. We first examined 10 single nucleotide polymorphisms (SNPs) in six genes within the targeted region of about 90 kb to determine which SNP/gene was associated with ND, assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerstr?m Test for ND (FTND). Individual SNP analysis revealed that SNPs rs17710 and rs222843 in GABA(A) receptor-associated protein (GABARAP) exhibited a significant association with at least one age- and gender-adjusted ND measure in the EA sample and rs222843 remained significant with the FTND after correction for multiple testing (P = 0.009). Although no SNP in DLG4 was significantly associated with ND, we found a G-G haplotype with a frequency of 14.2% formed by SNPs rs2242449 and rs507506 within the gene that showed significant inverse associations with all three ND measures [P = 0.003, 0.015 and 0.024, for SQ (defined as the number of cigarettes smoked per day), HSI and FTND, respectively]. We also found an A-A haplotype with a frequency of 8.8% formed by SNPs rs17710 and rs222843 in GABARAP, which revealed significant associations with all three ND measures (P = 0.006, 0.019 and 0.024, for SQ, HSI and FTND, respectively). To confirm these findings with a better coverage of GABARAP and DLG4, we conducted a second-stage association analysis by genotyping four more SNPs for GABARAP and nine more for DLG4 on the same set of samples. Our results from the second stage of individual SNP- and/or haplotype-based association analysis supported our finding of significant association of the DLG4 gene with ND. No significant association of GABARAP or DLG4 with ND was detected in the AA sample. Further, by comparing the linkage signal before and after adjustment for the SNPs of GABARAP and DLG4, we found that inclusion of the SNPs of the two genes as covariates largely reduced the linkage signal in the EA sample, but kept nearly unchanged in the AA sample. Taken together, our two-stage association analysis and linkage analysis results indicate that the GABARAP and DLG4 genes are involved in the etiology of ND in EA smokers. Further investigation of neurobiological mechanisms of the two genes in the etiology of ND is thus warranted.     

Identification of a prostate cancer susceptibility gene on chromosome 5p13q12 associated with risk of both familial and sporadic disease

Genetic heterogeneity is a difficulty frequently encountered in the search for genes conferring susceptibility to prostate cancer. To circumvent this issue, we selected a large prostate cancer pedigree for genome-wide linkage analysis from a population that is genetically homogeneous. Selected cases and first-degree relatives were genotyped with Affymetrix 10K SNP arrays, identifying a 14 Mb haplotype on chromosome 5 (5p13-q12) inherited identical-by-descent (IBD) by multiple cases. Microsatellite genotyping of additional deceased case samples confirmed that a total of eight cases inherited the common haplotype (P=0.0017). Re-sequencing of eight prioritised candidate genes in the region in six selected individuals identified 15 SNPs segregating with the IBD haplotype, located within the ITGA2 gene. Three of these polymorphisms were selected for genotyping in an independent Tasmanian data set comprising 127 cases with familial prostate cancer, 412 sporadic cases and 319 unaffected controls. Two were associated with prostate cancer risk: rs3212649 (OR=1.67 (1.07-2.6), P=0.0009) and rs1126643 (OR=1.52 (1.01-2.28), P=0.0088). Significant association was observed in both familial and sporadic prostate cancer. Although the functional SNP remains to be identified, considerable circumstantial evidence, provided by in vivo and in vitro studies, supports a role for ITGA2 in tumour development.     

Identification of a I-cM region of common deletion on 13q14 associated with human prostate cancer.

Frequent allelic losses on chromosome arm 13q are observed in carcinomas of the head and neck, breast, ovary, and pituitary gland. We analyzed 59 primary prostate tumors (stage B, 18 patients; C, 12 patients; D1, 4 patients; and endocrine therapy-resistant cancer death, 25 patients), as well as 18 metastatic tissues from 14 of the 25 cancer death patients for loss of heterozygosity (LOH) using 35 microsatellite markers on chromosome arm 13q. Of the 59 primary tumors, 31 (53%) showed LOH involving at least one locus. Detailed deletion mapping identified a distinct commonly deleted region in the I-cM interval flanked by D13S153 and D13S273 on 13q14 and this region overlapped a part of the RB1 gene. Paired DNAs were available from both primary and metastatic tumors in the 14 cases of cancer death; among those pairs, we detected LOH on 13q in seven (50%) primary tumors, and in all metastatic foci (P = 0.0029). Moreover, the regions lost in metastatic tissues were more extensive than those seen in the corresponding primary tumors. These results suggest that inactivation of a putative tumor suppressor gene(s) including the RB1 gene on 13q14 plays an important role in human prostate cancer.     

Interstitial duplications of chromosome region 15q11q13: Clinical and molecular characterization

Duplications of chromosome region 15q11q13 often occur as a supernumerary chromosome 15. Less frequently they occur as interstitial duplications [dup(15)]. We describe the clinical and molecular characteristics of three patients with de novo dup(15). The patients, two males and one female (ages 3–21 years), had nonspecific findings that included autistic behavior, hypotonia, and variable degrees of mental retardation. The extent, orientation, and parental origin of the duplications were assessed by fluorescent in situ hybridization, microsatellite analyses, and methylation status at D15S63. Two patients had large direct duplications of 15q11q13 [dir dup(15)(q11q13)] that extended through the entire Angelman syndrome/Prader-Willi syndrome (AS/PWS) chromosomal region. Their proximal and distal breaks, at D15S541 or D15S9 and between D15S12 and D15S24, respectively, were comparable to those found in the common AS/PWS deletions. This suggests that duplications and deletions may be the reciprocal product of an unequal recombination event. These two duplications were maternally derived, but the origin of the chromatids involved in the unequal crossing over in meiosis differs. In one patient, the duplication originated from two different maternal chromosomes, while in the other patient it arose from the same maternal chromosome. The third patient had a much smaller duplication that involved only D15S11 and parental origin could not be determined. There was no obvious correlation between phenotype and extent of the duplication in these patients. Am. J. Med. Genet. 79:82–89, 1998. © 1998 Wiley-Liss, Inc.     

Linkage and association studies of atopy and the chromosome 11q13 region

The clinical syndrome atopy is largely determined by genetic factors. In 1989, the first linkage of markers within and flanking the chromosomal region 11q13 and atopy was reported. In the following years, the gene coding for the beta chain of the high affinity IgE receptor was localised to this region and two polymorphisms in this gene have been shown to be associated with the atopic phenotype. We investigated two independent populations (population based and outpatient department) with different degrees of clinical symptoms. Using highly polymorphic markers we could find no evidence for linkage or allelic association of this particular genomic region to the atopic phenotype defined by enhanced IgE responsiveness (p>0.05). Neither did we succeed in finding either of the two polymorphisms described, nor could we identify any other polymorphisms within the gene. However, we found weak evidence for linkage in asthmatic sib pairs regarding maternal alleles (p=0.03). We conclude from our data that in our populations the gene for the beta chain of the high affinity IgE receptor is of minor importance for enhanced IgE responsiveness, and that it might influence atopy with clinical signs like asthma through maternally derived alleles.     

Fine mapping of a multiple sclerosis locus to 2.5 Mb on chromosome 17q22-q24

Genome-wide linkage analyses performed in a Finnish study sample have identified four potential predisposing loci for multiple sclerosis (MS). Here we made an effort to restrict the wide linkage region on chromosome 17 with a dense set of 31 markers using multipoint linkage analyses and monitoring for shared marker alleles in MS chromosomes. We carried out the linkage analyses in 22 Finnish multiplex MS families originating from a regional subisolate that shows an exceptionally high prevalence of MS in order to minimize the genetic and environmental heterogeneity of the study sample. Thirty markers on the 23 cM initial interval gave positive pairwise LOD scores. We monitored for shared haplotypes among affected family members within a family, and identified an approximately 4 cM region flanked by the markers D17S1792 and ATA43A10 in 17 out of the 22 families (77.3%). The multipoint linkage analyses using Genehunter and SIMWALK 2.40 provided further evidence for the same 4 cM region, for example a maximal multipoint NPL score of 5.98 (P<0.0002). We observed nominal evidence for association to MS, with one marker flanking the shared region, and this association was replicated in the additional set of families. Using the combined power of linkage, association and shared haplotype analyses, we were thus able to restrict the MS locus on chromosome 17q from 23 cM to a 4 cM region covering a physical interval of approximately 2.5 Mb. Thus, this study describes the restriction of an MS locus outside the HLA region into a segment approachable by molecular tools.     

Physical mapping of the human chromosome 11q23 region containing the ataxia-telangiectasia locus

Two breakpoints within chromosome 11q23 were characterized with 29 DNA probes to establish a physical map of the region. This region is notable in that it contains at least 14 functional genes which are also syntenic in the mouse (chromosome 9). Chromosome 11q23 includes these markers: STMY, CLG, NCAM, DRD2, APOA1, APOC3, APOA4, CD3E, CD3D, CD3G, PBGD, THY1, ets-1, and cbl-2. The two breakpoints, herein called "X;11" and "4;11," defined a region of approximately 8 cM containing the APO and CD3 complexes as well as the polymorphic marker D11S29. DRD2 localized centromeric to the X;11 breakpoint despite evidence for close genetic linkage to D11S29, suggesting that DRD2 lies close to the X;11 breakpoint. THY1, PBGD, and cbl-2 localized telomeric to the 4;11 breakpoint and thus to the [D11S29--APO--CD3] grouping as well. The physical map helps to correlate the cytogenetic and linkage maps of this region. It also suggests that the human 11q23 syntenic grouping is inverted with respect to its murine counterpart. Based on this physical map and on our primary linkage map of the 11q23 region, we are able to confirm a preliminary localization of the gene for ataxia-telangiectasia group A (ATA) to a region centromeric to the interval defined by D11S144 (pYNB3.12) and THY1.     

Deletion mapping of endocrine tumors localizes a second tumor suppressor gene on chromosome band 11q13

Multiple endocrine neoplasia type 1 syndrome (MEN1, MIM 131100), an autosomal dominant disease, is characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. These tumors also occur sporadically. Both the familial (MEN1) and the sporadic tumors reveal loss of heterozygosity (LOH) for chromosome band 11q13 sequences. Based on prior linkage and LOH analyses, the MEN1 gene was localized between PYGM and D11S460. Recently, the MEN1 gene (menin) has been cloned from sequences 30-kb distal to PYGM. We performed deletion mapping on 25 endocrine tumors (5 MEN1 and 20 sporadic) by using 21 polymorphic markers on chromosome band 11q13. Of these, two (137C7A, 137C7B) were derived from PYGM-containing BAC (bacterial artificial chromosome-137C7) sequences, one from INT2-containing cosmid sequences and the marker D11S4748, a (CA)₂₀ repeat marker that was developed by us. The LOH analysis shows that the markers close to the MEN1 (menin) gene were not deleted in three of the tumors. These tumors, however, showed LOH for distal markers. Thus, the data suggest the existence of a second tumor suppressor gene on chromosome band 11q13. Genes Chromosomes Cancer 22:130–137, 1998. © 1998 Wiley-Liss, Inc.     

Fine mapping of a region on chromosome 21q21.11-q22.3 showing linkage to type 1 diabetes

Background: Results of a Scandinavian genome scan in type 1 diabetes mellitus (T1D) have recently been reported. Among the novel, not previously reported chromosomal regions showing linkage to T1D was a region on chromosome 21.

Objective: To fine map this region on chromosome 21.

Methods and results: The linked region was initially narrowed by linkage analysis typing microsatellite markers. Linkage was significantly increased, with a peak NPL score of 3.61 (p = 0.0002), suggesting the presence of one or several T1D linked genes in the region. The support interval for linkage of 6.3 Mb was then studied by linkage disequilibrium (LD) mapping with gene based single nucleotide polymorphisms (SNPs). Thirty two candidate genes were identified in this narrowed region, and LD mapping was carried out with SNPs in coding regions (cSNPs) of all these genes. However, none of the SNPs showed association to T1D in the complete material, whereas some evidence for association to T1D of variants of the TTC3, OLIG2, KCNE1, and CBR1 genes was observed in conditioned analyses. The disease related LD was further assessed by a haplotype based association study, in which several haplotypes showed distorted transmission to diabetic offspring, substantiating a possible T1D association of the region.

Conclusions: Although a single gene variant responsible for the observed linkage could not be identified, there was evidence for several combinations of markers, and for association of markers in conditioned analyses, supporting the existence of T1D susceptibility genes in the region.

     

FISH characterization of head and neck carcinomas reveals that amplification of band 11q13 is associated with deletion of distal 11q

In order to characterize homogeneously staining regions (HSR) and other 11q13 rearrangements identified cytogenetically, we performed fluorescence in situ hybridization (FISH) using a CCND1cosmid and five YAC clones spanning chromosomal bands 11q13–14 on metaphase cells from 14 primary and one metastatic head and neck carcinomas. At the cytogenetic level, a total of 17 HSR were detected in ten cases: five were in derivative chromosomes 11 in band 11q13, and 12 were located in other derivative chromosomes. Other forms of 11q13 rearrangements were observed in five cases, whereas two cases had normal chromosomes 11. FISH analysis demonstrated that all HSR but two were derived from the 11q13 band. The size of the amplicon varied from case to case, but the amplification always included the region covered by YAC 55G7, which contains the CCND1 locus. The amplification of CCND1was confirmed by use of a CCND1cosmid. We also showed that most of the cases (9 of 11) with 11q13 amplification had lost material from distal 11q. The breakpoints were mapped by FISH and were shown to cluster to the region between YACs 55G7 and 749G2. We conclude that loss of gene(s) in distal 11q may be as important as amplification of genes in 11q13 for the biological aggressiveness of head and neck carcinomas. Genes Chromosomes Cancer 22:312–320, 1998. © 1998 Wiley-Liss, Inc.     

Linkage analysis of a candidate region in Scandinavian sib pairs with multiple sclerosis reveals linkage to chromosome 17q

To date, four genome screens have been completed in the demyelinating autoimmune disease multiple sclerosis (MS). Although these screens failed to identify any loci with major effects on susceptibility, several novel regions of potential linkage were suggested, including the long arm of chromosome 17. In order to further pursue this promising region we have investigated six highly polymorphic microsatellite markers in 115 Scandinavian families with MS affected sib pairs. Multipoint linkage analysis revealed a peak maximum likelihood score (MLS) of 0.9 in the region of marker D17S787. Stratifying the results on the basis of HLA-DR2 status showed that the linkage was not limited to families segregating for the HLA-DR2 allele as has previously been suggested. In conclusion, our results further support the proposal that a multiple sclerosis susceptibility locus is contained on chromosome 17q.     

Mapping of nasopharyngeal carcinoma tumor-suppressive activity to a 1.8-megabase region of chromosome band 11q13

Nasopharyngeal carcinoma (NPC) is a malignancy that is particularly prevalent among populations from Southern China and Southeast Asian countries. Evidence for a genetic contribution to the disease has been documented, although the genetic basis for NPC development is not yet fully understood. Previous functional evidence of tumor-suppressive activity on chromosome band 11q13 in NPC was obtained using a microcell-mediated chromosome-transfer approach with HONE1 NPC cells. In the present study, this region was subjected to a detailed investigation of microcell hybrids and their tumor segregants using microsatellite analysis to narrow down the region of tumor-suppressive activity. Fluorescence in situ hybridization was also performed with BAC and cosmid probes to confirm the microsatellite data. The critical region responsible for tumor suppression was narrowed down to a 1.8-Mb interval, which does not tolerate an additional normal allele by chromosome transfer. One or two alleles from either endogenous or exogenous chromosomes at 11q13 were consistently eliminated during tumor growth. Results of this study suggest that a candidate tumor-suppressor gene, not the MEN1 gene, maps between D11S4907 and GSTP1 in NPC.