Linkage of a candidate gene locus to familial combined hyperlipidemia: lecithin:cholesterol acyltransferase on 16q

Familial combined hyperlipidemia (FCHL) is a common lipid disorder characterized by elevated levels of plasma cholesterol and triglycerides that is present in 10% to 20% of patients with premature coronary artery disease. To study the pathophysiological basis and genetics of FCHL, we previously reported recruitment of 18 large families. We now report linkage studies of 14 candidate genes selected for their potential involvement in the aspects of lipid and lipoprotein metabolism that are altered in FCHL. We used highly polymorphic markers linked to the candidate genes, and these markers were analyzed using several complementary, nonparametric statistical allele-sharing linkage methodologies. This current sample has been extended over the one in which we identified an association with the apolipoprotein (apo) AI-CIII-AIV gene cluster. We observed evidence for linkage of this region and FCHL (P<0.001), providing additional support for its involvement in FCHL. We also identified a new locus showing significant evidence of linkage to the disorder: the lecithin:cholesterol acyltransferase (LCAT) locus (P<0.0006) on chromosome 16. In addition, analysis of the manganese superoxide dismutase locus on chromosome 6 revealed a suggestive linkage result in this sample (P<0.006). Quantitative traits related to FCHL also provided some evidence of linkage to these regions. No evidence of linkage to the lipoprotein lipase gene, the microsomal triglyceride transfer protein gene, or several other genes involved in lipid metabolism was observed. The data suggest that the lecithin:cholesterol acyltransferase and apolipoprotein AI-CIII-AIV loci may act as modifying genes contributing to the expression of FCHL.     

Evidence for a gene influencing fasting LDL cholesterol and triglyceride levels on chromosome 21q

High levels of low-density lipoprotein (LDL) cholesterol, low levels of high-density lipoprotein (HDL) cholesterol, and high levels of triglycerides (TG) are strong predictors of cardiovascular disease risk. Motivated by previous evidence for pleiotropy between cholesterol and TG levels, we conducted bivariate linkage analysis of LDL cholesterol and TG concentration among participants of the Hypertension Genetic Epidemiolgy Network (HyperGEN), one of four networks in the NHLBI sponsored Family Blood Pressure Program Project. All available hypertensive siblings and their first-degree relatives were recruited. Both phenotypes were similarly adjusted for ethnicity, study center, sex, age, age-by-sex interactions, smoking, alcohol consumption, hormone use, diabetes medication use, and waist circumference. Variance component linkage analysis was performed as implemented in SOLAR, using ethnicity-specific marker allele frequencies derived from founders and multipoint IBDs calculated in MERLIN. A maximum genome-wide empirical LOD score of 3.9 was detected on chromosome 21 at 54cM, between markers D21S2055 and D21S1446. This signal overlaps with suggestive and/or significant linkages for total cholesterol, LDL cholesterol, and apolipoprotein B in three other studies and is suggestive of one or more genes on chromosome 21q jointly regulating LDL cholesterol and TG concentration.     

Isolated familial somatotropinomas: establishment of linkage to chromosome 11q13.1-11q13.3 and evidence for a potential second locus at chromosome 2p16-12

The majority of somatotropinomas are sporadic, although a small number occur with a familial aggregation, either as a component of an endocrine neoplasia complex that includes multiple endocrine neoplasia type 1 (MEN-1) and Carney complex (CNC) or as isolated familial somatotropinomas (IFS). IFS is defined as the occurrence of at least two cases of acromegaly or gigantism in a family that does not exhibit MEN-1 or CNC. This rare disease is associated with loss of heterozygosity (LOH) on chromosome 11q13, the locus of the MEN-1 gene, although the MEN-1 sequence and expression appear normal. These data suggest the presence of another tumor suppressor gene located at 11q13 that is important in the control of somatotrope proliferation. To establish linkage of IFS to 11q13 and to define the candidate interval of the IFS gene, we performed haplotype and allelotype analyses on two families with IFS. Collectively, allelic retention in one tumor and a recombinant haplotype in an affected individual mapped the tumor suppressor gene involved in the pathogenesis of IFS to a region of 8.6 cM between polymorphic microsatellite markers D11S1335 and INT-2 located at chromosome 11q13.1-13.3. Maximum two-point LOD scores for five markers within this region were 3.0 or more at theta = 0.0. As somatotropinomas are the predominant pituitary tumor subtype associated with CNC and arise before 30 yr of age, which is strikingly similar to the age at diagnosis for IFS, we explored the possibility that the putative CNC genes might also contribute to the pathogenesis of IFS. Although the genetic defect responsible for the complex is unknown, CNC has been mapped by linkage analysis to chromosomes 2p15-16 and 17q23-24 in different kindreds. Two-point LOD scores less than -2.0 were obtained using marker D17S949 from chromosome 17q23-24, excluding linkage. However, LOD scores of 2.5 were obtained for markers within 2p16-12; therefore, linkage of IFS to chromosome 2p cannot be excluded. This report establishes linkage of the tumor suppressor gene involved in the pathogenesis of IFS to chromosome 11q13.1-13.3 and identifies a potential second locus at chromosome 2p16-12.     

Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon

In a screen for gene copy-number changes in mouse mammary tumors, we identified a tumor with a small 350-kb amplicon from a region that is syntenic to a much larger locus amplified in human cancers at chromosome 11q22. The mouse amplicon contains only one known gene, Yap, encoding the mammalian ortholog of Drosophila Yorkie (Yki), a downstream effector of the Hippo(Hpo)-Salvador(Sav)-Warts(Wts) signaling cascade, recently identified in flies as a critical regulator of cellular proliferation and apoptosis. In nontransformed mammary epithelial cells, overexpression of human YAP induces epithelial-to-mesenchymal transition, suppression of apoptosis, growth factor-independent proliferation, and anchorage-independent growth in soft agar. Together, these observations point to a potential oncogenic role for YAP in 11q22-amplified human cancers, and they suggest that this highly conserved signaling pathway identified in Drosophila regulates both cellular proliferation and apoptosis in mammalian epithelial cells.     

Evidence for a novel rheumatoid arthritis susceptibility locus on chromosome 6p

OBJECTIVE: To investigate whether the large linkage peak on chromosome 6p harbors rheumatoid arthritis (RA) susceptibility loci in addition to the well-characterized HLA-DRB1 gene. METHODS: DNA samples obtained from 377 UK RA affected sibling pair (ASP) families, comprising test (181 ASPs) and replication (196 ASPs) cohorts, were used for linkage analysis. Three hundred eighty-four patients with RA derived from a subset of 192 ASPs were compared with a panel of 288 unrelated healthy controls for association studies. Samples were genotyped for 35 microsatellites and 25 single-nucleotide polymorphisms (SNPs). RESULTS: In the test cohort, the maximum logarithm of odds (LOD) score was obtained over D6S1260 (LOD 7.1). Evidence for linkage to the telomeric portion of the peak was increased in subsets of ASPs in which both individuals had erosive disease or both carried 2 copies of the shared epitope. HLA-A, HLA-DRB1, and 8 additional markers showed evidence of linkage in the presence of association with RA (using the extended transmission disequilibrium test [ETDT]). The positive ETDT result for 2 adjacent markers (D6S1665 and 210901-4) mapping to the telomeric end of the linked region ( approximately 11 Mb from DRB1) was replicated (for D6S1665) in the second cohort of ASPs. Haplotypic overtransmission of pairwise combinations between D6S1665*7 and 210901-4*4 was identified through the TDTPhase program. Multipoint conditional analysis showed this effect to be independent of HLA-DRB1. SNP-based association studies of the region identified a 4-marker haplotype in the DEK gene that was significantly associated with RA (P = 0.009). CONCLUSION: Evidence has been presented for an RA susceptibility locus mapping under the linkage peak on 6p, 11 Mb telomeric of HLA-DRB1. Preliminary association data implicate the gene DEK.     

Genetic linkage and association analysis of COPD-related traits on chromosome 8p

Genome-wide linkage analysis in the Boston Early-Onset Chronic Obstructive Pulmonary Disease (COPD) Study has demonstrated significant evidence of linkage to chromosome 8p for forced expiratory volume in 1 second, an important COPD-related phenotype. In this study, we sought to fine map the linkage peak and to test variants in two candidate genes for association with COPD and related traits. In a variance component linkage analysis on chromosome 8, including seven additional short tandem repeat markers, the logarithm of the odds of linkage score was reduced from 3.30 to 1.80 (at 1 cM). Five single nucleotide polymorphisms (SNPs) in Defensin Beta-1 (DEFB1) were genotyped in the Boston Early-Onset COPD Study families; none was significantly associated. Four SNPs and an insertion-deletion polymorphism in Macrophage Scavenger Receptor-1 (MSR1) were also genotyped in the family-based study. A coding variant (Pro275Ala) was marginally associated with two qualitative airflow obstruction traits (p < or = 0.02). This SNP showed a trend toward association in a case-control study comparing participants in the National Emphysema Treatment Trial to smoker controls (p = 0.07). Despite the reduced support for linkage upon further analysis, it remains possible that chromosome 8p contains a gene that influences COPD susceptibility. There is marginal, though not convincing, evidence for association with MSR1.     

Microsatellite association mapping of a primary osteoarthritis susceptibility locus on chromosome 6p12.3–q13

Objective

To test a high density of microsatellite markers from within a primary osteoarthritis (OA) locus on chromosome 6 for association with OA as a means of narrowing and focusing our search for the susceptibility gene.

Methods

One hundred forty‐six families, each with 2 or more women concordant for primary OA (ascertained by total hip replacement), were genotyped for 36 microsatellite markers from within a narrow interval at 6p12.3–q13 which we had previously shown to be linked to OA. Each marker was tested for linkage and for association, the latter by means of the transmission disequilibrium test and by a case–control analysis.

Results

The highest 2‐point logarithm of odds (LOD) score was 4.8, with 11 markers having LOD scores ≥2.0. Several markers demonstrated evidence of association, in particular, a cluster of markers positioned within or near the functional candidate gene BMP5.

Conclusion

Our linkage data reinforce the evidence of a major susceptibility locus on chromosome 6. We had previously failed to detect an association with BMP5 using gene‐based single‐nucleotide polymorphisms. The association data reported here prompt us to speculate that the chromosome 6 susceptibility may be coded for by cis‐acting polymorphism in the regulatory elements of this gene, rather than by variation in its protein coding sequence.

     

Lack of support for the presence of an osteoarthritis susceptibility locus on chromosome 6p

OBJECTIVE: To replicate, in a Northern Irish population, the previously reported association between a locus on chromosome 6 and hip osteoarthritis (OA). METHODS: Patients with hip OA were identified from a registry of patients who had undergone total hip replacement surgery over an 8-year period at a single large orthopedic unit in Northern Ireland. Patients identified as index cases were contacted by mail and asked to reply only if another family member also had undergone total hip replacement surgery. Using this approach, we identified 288 sibling pairs concordant for primary hip OA. DNA was extracted from peripheral blood, and microsatellite markers were amplified by polymerase chain reaction and subsequently genotyped. RESULTS: No evidence of linkage to this region was demonstrated by either 2-point analysis or multipoint analysis of 17 microsatellites. CONCLUSION: The reported association between a locus on chromosome 6 and hip OA could not be confirmed in this population. Different methods of ascertainment and phenotyping of OA may contribute to the current inability to replicate genetic associations for hip OA.     

Deletions of chromosome 5q13.3 and 17p loci cooperate in myeloid neoplasms

Nonrandom interstitial deletions and monosomy of chromosomes 5, 7, and 17 in refractory myelodysplasia (MDS) and acute myelogenous leukemia (AML) suggest a multistep pathway that culminates in aggressive clinical course. Because cytogenetic studies frequently identify chromosome 5 and 17 deletions within a single clone, we searched for allele loss for 5q loci and TP53 gene mutations in the same leukemic samples. Cosegregating deletions of chromosomes 5 and 17 were found to specifically include the 5q13.3 interval between the loci D5S672 and D5S620/D5S626, a locus hypothesized to harbor a tumor suppressor gene(1) and the TP53 gene on 17p. A rare patient with secondary refractory MDS and an unbalanced translocation [der(5;17)], which resulted in deletions of the 5q13.3-qter and 17p loci, provided clues on the sequence of genetic alterations. Serial molecular analysis of this patient revealed a dysplastic clone with der(5;17), which gave rise to a leukemic clone on acquiring an inactivating mutation of TP53. Our findings are consistent with functional cooperation between a putative tumor suppressor gene at 5q13.3 that contributes toward the progression of early stages of MDS, and the TP53 gene when mutated, causes transformation to AML. (Blood. 2000;95:2138-2143)     

Rhabdomyosarcoma-associated locus and MYOD1 are syntenic but separate loci on the short arm of human chromosome 11.

The MYOD1 locus is preferentially expressed in skeletal muscle and at higher levels in its related neoplasm, rhabdomyosarcoma. We have combined physical mapping of the human locus with meiotic and physical mapping in the mouse, together with synteny homologies between the two species, to compare the physical relationship between MYOD1 and the genetically ascertained human rhabdomyosarcoma-associated locus. We have determined that the myogenic differentiation gene is tightly linked to the structural gene for the M (muscle) subunit of lactate dehydrogenase in band p15.4 on human chromosome 11 and close to the p and Ldh-1 loci in the homologous region of mouse chromosome 7. Because the rhabdomyosarcoma locus maps to 11p15.5, MYOD1 is very unlikely to be the primary site of alteration in these tumors. Further, these analyses identify two syntenic clusters of muscle-associated genes on the short arm of human chromosome 11, one in the region of rhabdomyosarcoma locus that includes IGF2 and TH and the second the tightly linked MYOD1 and LDHA loci, which have been evolutionarily conserved in homologous regions of both the mouse and the rat genomes.     

Mapping a novel locus for familial atrial fibrillation on chromosome 10p11-q21.

BACKGROUND: Atrial Fibrillation (AF), the most common cardiac arrhythmia, is a significant public health problem in the United States, affecting approximately 2.2 million Americans. Recently, several chromosomal loci and genes have been found to be associated with familial AF. However, in most other AF cases, the genetic basis is still poorly understood. OBJECTIVE: The purpose of this study was to investigate the molecular basis of familial AF in a Dutch kindred group. METHODS: We analyzed a four-generation Dutch family in which AF segregated as an autosomal dominant trait. After the exclusion of linkage to 10q22-24, 6q14-16, 5p13, KCNQ1, KCNE2, KCNJ2 and some ion-channel-associated candidate genes, a genome-wide linkage scan using 398 microsatellite markers was performed. RESULTS: Two-point logarithms of odds (LOD) scores >1 at recombination fraction [theta] = 0.00 and a haplotype segregating with the disorder were demonstrated only across regions of chromosome 10. Subsequent fine mapping gave a maximum two-point LOD score of 4.1982 at D10S568 at [theta] = 0.00. Distinct recombination in several individuals narrowed the shared region among all affected individuals to 16.4 cM on the Genethon map (flanking markers: D10S578 and D10S1652), which corresponds to chromosome 10p11-q21. Thirteen candidate genes residing in this region, which could be associated with AF, were screened. No mutation has been found in their coding regions including the intron splice regions. CONCLUSION: We identify a novel locus for AF on chromosome 10p11-q21, which provides further evidence of genetic heterogeneity in this arrhythmia.     

Locus for elevated apolipoprotein B levels on chromosome 1p31 in families with familial combined hyperlipidemia

Familial combined hyperlipidemia (FCH), a common cause of premature coronary artery disease, is genetically complex and poorly understood. Recently, a major locus on chromosome 1q21-23 exhibiting highly significant linkage was identified in Finnish FCH families by use of a parametric analysis. We now report highly significant evidence of linkage (maximum LOD score 3.8, recombination fraction 0) of an important FCH phenotype, elevated apolipoprotein B (apoB) levels, to a distinctly separate locus on chromosome 1p31 in Dutch pedigrees. ApoB is the major protein on very low density and low density lipoproteins, and elevated apoB levels have been used as a surrogate trait for FCH. Additional microsatellite markers in the 1p31 region were genotyped, and evidence of linkage improved (maximum LOD score 4.7) in a multipoint analysis of two markers in the peak region. The leptin receptor gene resides within this locus and is involved in obesity and insulin/glucose homeostasis. However, there was no evidence of an association between leptin receptor and apoB levels, raising the possibility that another gene on this chromosomal region contributes to elevated apoB levels in this Dutch population. This is one of the first loci identified for apoB levels in humans and is the second major locus implicated in the genetic etiology of FCH.     

Evidence for linkage of familial Diamond-Blackfan anemia to chromosome 8p23.3-p22 and for non-19q non-8p disease

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia that usually presents early in infancy and is inherited in 10% to 20% of cases. Linkage analysis has shown that DBA in many of both dominant and recessive DBA families mapped to chromosome 19q13.2 leading to the cloning of a gene on chromosome 19q13.2 that encodes a ribosomal protein, RPS19. However, subsequently, mutations of the RPS19 gene have only been identified in 25% of all patients with DBA. This study analyzed 14 multiplex DBA families, 9 of which had 19q13.2 haplotypes inconsistent with 19q linkage. A genome-wide search for linked loci suggested the presence of a second DBA locus in a 26.4-centimorgan (cM) interval on human chromosome 8p. Subsequently, 24 additional DBA families were ascertained and all 38 families were analyzed with additional polymorphic markers on chromosome 8p. In total, 18 of 38 families were consistent with linkage to chromosome 8p with a maximal LOD score with heterogeneity of 3.55 at D8S277 assuming 90% penetrance. The results indicate the existence of a second DBA gene in the 26.4-cM telomeric region of human chromosome 8p23.3-p22, most likely within an 8.1-cM interval flanked by D8S518 and D8S1825. Seven families were inconsistent with linkage to 8p or 19q and did not reveal mutations in the RPS19 gene, suggesting further genetic heterogeneity. (Blood. 2001;97:2145-2150)     

Loss of heterozygosity of chromosome 8p and 11p in the dysplastic nodule and hepatocellular carcinoma

BACKGROUND AND AIM: In hepatocarcinogenesis, both de novo and multistep pathways have been suggested, and in the latter a dysplastic nodule is the proposed precancerous lesion. But genetic changes involved in the dysplastic nodule are not well understood. In this study, we tried to determine whether allelic loss of the chromosome 8p and/or 11p could be involved in the development of the dysplastic nodule and/or hepatocellular carcinoma. Platelet-derived growth factor-receptor beta-like tumor suppressor gene (PRLTS) and deletion in liver cancer-1 tumor suppressor gene are located at 8p21.3-p22. The hepatitis B virus integration site and WT1 tumor suppressor gene are located at 11p13. METHODS: We therefore studied loss of heterozygosity (LOH) of chromosome 8p21.3-p22 and 11p13 in 22 dysplastic nodules and 21 hepatocellular carcinomas. The samples, microdissected from paraffin-embedded tissues, were examined using a polymerase chain reaction-based LOH assay using microsatellite markers. RESULTS: Loss of heterozygosity was detected for chromosome 8p21.3-p22 in nine (40.9%) of 22 dysplastic nodules and in eight (42.1%) of 19 hepatocellular carcinomas. D8S261, located adjacent to PRLTS, showed most frequent LOH: 28.6% in dysplastic nodule and 40.0% in hepatocellular carcinoma. Loss of heterozygosity on chromosome 11p13 was found in three (15.8%) of 19 dysplastic nodules and in six (31.6%) of 19 hepatocellular carcinomas. Loss of heterozygosity of D11S995 and D11S907 was found in 33.3% and 7.1% of dysplastic nodules, and 8.3% and 44.4% of hepatocellular carcinomas, respectively. CONCLUSION: These results suggest that at least one putative tumor suppressor gene involved in the development and progression of hepatocellular carcinoma may be located on 8p21.3-p22 and 11p13. Particularly, PRLTS might be related to an early genetic event of hepatocarcinogenesis.     

Identification of a prostate cancer susceptibility locus on chromosome 7q11-21 in Jewish families

Results from over a dozen prostate cancer susceptibility genome-wide scans, encompassing some 1,500 hereditary prostate cancer families, indicate that prostate cancer is an extremely heterogeneous disease with multiple loci contributing to overall susceptibility. In an attempt to reduce locus heterogeneity, we performed a genomewide linkage scan for prostate cancer susceptibility genes with 36 Jewish families, which represent a stratification of hereditary prostate cancer families with potentially increased locus homogeneity. The 36 Jewish families represent a combined dataset of 17 Jewish families from the Fred Hutchinson Cancer Research Center-based Prostate Cancer Genetic Research Study dataset and 19 Ashkenazi Jewish families collected at Johns Hopkins University. All available family members, including 94 affected men, were genotyped at markers distributed across the genome with an average interval of <10 centimorgans. Nonparametric multipoint linkage analyses were the primary approach, although parametric analyses were performed as well. Our strongest signal was a significant linkage peak at 7q11-21, with a nonparametric linkage (NPL) score of 3.01 (P = 0.0013). Simulations indicated that this corresponds to a genomewide empirical P = 0.006. All other regions had NPL P values >/=0.02. After genotyping additional markers within the 7q11-21 peak, the NPL score increased to 3.35 (P = 0.0004) at D7S634 with an allele-sharing logarithm of odds of 3.12 (P = 0.00007). These studies highlight the utility of analyzing defined sets of families with a common origin for reducing locus heterogeneity problems associated with studying complex traits.