Quantitative lymphatic vessel trait analysis suggests Vcam1 as candidate modifier gene of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic debilitating disease resulting from a complex interaction of multiple genetic factors with the environment. To identify modifier genes of IBD, we used an F2 intercross of IBD-resistant C57BL/6J-Il10(-/-) mice and IBD-susceptible C3H/HeJBir-Il10(-/-) (C3Bir-Il10(-/-)) mice. We found a prominent involvement of lymphatic vessels in IBD and applied a scoring system to quantify lymphatic vascular changes. Quantitative trait locus (QTL) analyses revealed a large-effect QTL on chromosome 3, mapping to an interval of 43.6 Mbp. This candidate interval was narrowed by fine mapping to 22 Mbp, and candidate genes were analyzed by a systems genetics approach that included quantitative gene expression profiling, search for functional polymorphisms, and haplotype block analysis. We identified vascular adhesion molecule 1 (Vcam1) as a candidate modifier gene in the interleukin 10-deficient mouse model of IBD. Importantly, VCAM1 protein levels were increased in susceptible C3H/HeJ mice, compared with C57BL/6J mice; systemic blockade of VCAM1 in C3Bir-Il10(-/-) mice reduced their inflammatory lymphatic vessel changes. These results indicate that genetically determined expression differences of VCAM1 are associated with susceptibility to colon inflammation, which is accompanied by extensive lymphatic vessel changes. VCAM1 is, therefore, a promising therapeutic target for IBD.

An initial linkage map of the West Nile Virus vector Culex tarsalis.

We have constructed the first genetic linkage map for the North American arboviral vector Culex tarsalis. 120 F(2) offspring from a cross between two colonies were genotyped using 25 microsatellites and six inter-simple sequence repeat (ISSR) markers. We resolved four linkage groups which likely correspond to two full-length chromosomes and two arms of the final chromosome. The longest linkage group contains the sex locus and corresponds to chromosome 3. Recombination rates around the sex locus were dramatically higher in females compared to males. The majority of microsatellite loci share sequence identity with regions of the Culex quinquefasciatus genome, whose assembly should aid in anchoring linkage groups to physical chromosomes. This map will aid in identification of loci involved with variable phenotypes in C. tarsalis including WNV susceptibility.

An extension of the regression of offspring on mid-parent to test for association and estimate locus-specific heritability: the revised ROMP method.

The Regression of Offspring on Mid-Parent (ROMP) method is a test of association between a quantitative trait and a candidate locus. ROMP estimates the trait heritability and the heritability attributable to a locus and requires genotyping the offspring only. In this study, the theory underlying ROMP was revised (ROMP(rev)) and extended. Computer simulations were used to determine the type I error and power of the test of association, and the accuracy of the locus-specific heritability estimate. The ROMP(rev) test had good power at the 5% significance level with properly controlled type I error. Locus-specific heritability estimates were, on average, close to simulated values. For non-zero locus-specific heritability, the proposed standard error was downwardly biased, yielding reduced coverage of 95% confidence intervals. A bootstrap approach with proper coverage is suggested as a second step for loci of interest. ROMP(rev) was applied to a study of cardiovascular-related traits to illustrate its use. An association between polymorphisms within the fibrinogen gene cluster and plasma fibrinogen was detected (p < 0.005) that accounted for 29% of the estimated fibrinogen heritability. The ROMP(rev) method provides a computationally fast and simple way of testing for association and obtaining accurate estimates of locus-specific heritability while minimizing the genotyping required.

Genetic regulation of plasma von Willebrand factor levels: quantitative trait loci analysis in a mouse model.

BACKGROUND: The genetic factors responsible for the wide variation in plasma von Willebrand factor (VWF) levels observed among individuals are largely unknown, although these genes are also likely to contribute to variability in the severity of von Willebrand disease (VWD) and other bleeding and thrombotic disorders. We have previously mapped two genes contributing to the regulation of plasma VWF levels in mice (Mvwf1 on chromosome 11 and Mvwf2 on chromosome 6). OBJECTIVE: To identify additional quantitative trait loci (QTL) contributing to the genetic regulation of murine plasma VWF levels. METHODS: To map genetic loci contributing to the > 7-fold difference in plasma VWF levels between two mouse strains (A/J and CASA/RkJ), high-density individual genotyping and R/qtl analyses were applied to a previously generated set of approximately 200 F2 mice obtained from an intercross of these two inbred lines. RESULTS: Genomic loci for two additional candidate VWF modifier genes were identified: Mvwf3 on chromosome 4 and Mvwf4 on chromosome 13. These loci demonstrate primarily epistatic effects when co-inherited with two CASA/RkJ Vwf alleles, although Mvwf4 may also exert a small, independent, additive effect. CONCLUSIONS: Mvwf3 and Mvwf4, combined with the effect of Mvwf2, explain approximately 45% of the genetic variation in plasma VWF level among the A/J and CASA/RkJ strains. Mvwf3 and Mvwf4 exhibit homology of synteny to three human chromosomal segments (on chromosomes 1, 5 and 6) previously reported by the Genetic Analysis of Idiopathic Thrombophilia (GAIT) study, suggesting that orthologs of Mvwf3 and Mvwf4 may also encode important VWF modifier genes in humans.

Predisposition to efficient mammary tumor metastatic progression is linked to the breast cancer metastasis suppressor gene Brms1.

Tumor metastasis is one of the most important clinical aspects of neoplastic disease because patient mortality is frequently attributable to disseminated rather than primary tumors. However, it still is not possible to definitively distinguish those individuals at high risk for disseminated disease, who would benefit from aggressive adjuvant therapy, from the low-risk patients who might be spared the side effects of additional anticancer therapy. To identify factors that predispose toward metastatic disease, we have used a genetic approach. Using a highly metastatic model of mammary cancer, we identified previously inbred mouse strains (DBA/2J, NZB/B1NJ, and I/LnJ) that harbor genetic factors that significantly suppress metastatic efficiency. In this study, we report the results of four experiments to localize the genetic map locations of the metastasis efficiency modifier genes. One statistically significant locus was identified on proximal Chr 19 designated Mtes1. Secondary candidate intervals were detected on Chrs 6, 9, 13, and 17. Interestingly, Mtes1 colocalizes with the murine orthologue of the human breast cancer metastasis suppressor gene Brms1, suggesting that allelic variants of Brms1 might be responsible for the metastasis suppression observed.

Loss of imprinting of insulin-like growth factor-II (IGF2) gene in distinguishing specific biologic subtypes of Wilms tumor.

BACKGROUND: Loss of imprinting (LOI) of the insulin-like growth factor-II (IGF2) gene, an epigenetic alteration associated with expression of the normally silent maternal allele, was observed first in Wilms tumor. Although LOI has subsequently been detected in most adult tumors, the biologic role of LOI in cancer remains obscure. We analyzed the imprinting status of Wilms tumors with respect to pathologic subtype, stage, and patient's age at diagnosis and examined the expression of genes potentially affected by LOI. METHODS: Of 60 Wilms tumors examined, 25 were informative for an ApaI polymorphism in the IGF2 gene, allowing analysis of allele-specific gene expression, and could be classified by pathologic subtype. Gene expression was measured quantitatively by real-time polymerase chain reaction, and pathologic analysis was blinded for genetic status. All statistical tests were two-sided. RESULTS: We observed LOI of IGF2 in nine (90%) of 10 Wilms tumors classified as having a pathologic subtype associated with a later stage of renal development and in only one (6.7%) of 15 Wilms tumors with a pathologic subtype associated with an earlier stage of renal development (P< .001). LOI was associated with a 2.2-fold increase (95% confidence interval [CI] = 1.6-fold to 3.1-fold) in IGF2 expression (P< .001). Children whose Wilms tumors displayed LOI of IGF2 were statistically significantly older at diagnosis (median = 65 months; interquartile range [IQR] = 47-83 months) than children whose tumors displayed normal imprinting (median = 24 months; IQR = 13-35 months; P< .001). CONCLUSIONS: These data demonstrate a clear relationship between LOI and altered expression of IGF2 in Wilms tumors and provide a molecular basis for understanding the divergent pathogenesis of this cancer. Analysis of LOI could provide a valuable molecular tool for the classification of Wilms tumor.

Review of statistical methods for QTL mapping in experimental crosses.

Identification of quantitative trait loci (QTLs) in experimental animals is critical for understanding the biochemical bases of complex traits, and thus for the identification of drug targets. The author reviews the basic statistical methods for mapping QTLs in experimental crosses and comments on a number of the statistical issues to consider in the application of these methods.

Estimation of allele frequencies with data on sibships.

Allele frequencies are generally estimated with data on a set of unrelated individuals. In genetic studies of late-onset diseases, the founding individuals in pedigrees are often not available, and so one is confronted with the problem of estimating allele frequencies with data on related individuals. We focus on sibpairs and sibships, and compare the efficiency of four methods for estimating allele frequencies in this situation: (1) use the data for one individual from each sibship; (2) use the data for all individuals, ignoring their relationships; (3) use the data for all individuals, taking proper account of their relationships, considering a single marker at a time; and (4) use the data for all individuals, taking proper account of their relationships, considering a set of linked markers simultaneously. We derived the variance of estimator 2, and showed that the estimator is unbiased and provides substantial improvement over method 1. We used computer simulation to study the performance of methods 3 and 4, and showed that method 3 provides some improvement over method 2, while method 4 improves little on method 3.

Olfactory receptor-gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements.

The olfactory receptor (OR)-gene superfamily is the largest in the mammalian genome. Several of the human OR genes appear in clusters with > or = 10 members located on almost all human chromosomes, and some chromosomes contain more than one cluster. We demonstrate, by experimental and in silico data, that unequal crossovers between two OR gene clusters in 8p are responsible for the formation of three recurrent chromosome macrorearrangements and a submicroscopic inversion polymorphism. The first two macrorearrangements are the inverted duplication of 8p, inv dup(8p), which is associated with a distinct phenotype, and a supernumerary marker chromosome, +der(8)(8p23.1pter), which is also a recurrent rearrangement and is associated with minor anomalies. We demonstrate that it is the reciprocal of the inv dup(8p). The third macrorearrangment is a recurrent 8p23 interstitial deletion associated with heart defect. Since inv dup(8p)s originate consistently in maternal meiosis, we investigated the maternal chromosomes 8 in eight mothers of subjects with inv dup(8p) and in the mother of one subject with +der(8), by means of probes included between the two 8p-OR gene clusters. All the mothers were heterozygous for an 8p submicroscopic inversion that was delimited by the 8p-OR gene clusters and was present, in heterozygous state, in 26% of a population of European descent. Thus, inversion heterozygosity may cause susceptibility to unequal recombination, leading to the formation of the inv dup(8p) or to its reciprocal product, the +der(8p). After the Yp inversion polymorphism, which is the preferential background for the PRKX/PRKY translocation in XX males and XY females, the OR-8p inversion is the second genomic polymorphism that confers susceptibility to the formation of common chromosome rearrangements. Accordingly, it may be possible to develop a profile of the individual risk of having progeny with chromosome rearrangements.

Comparison of human genetic and sequence-based physical maps.

Recombination is the exchange of information between two homologous chromosomes during meiosis. The rate of recombination per nucleotide, which profoundly affects the evolution of chromosomal segments, is calculated by comparing genetic and physical maps. Human physical maps have been constructed using cytogenetics, overlapping DNA clones and radiation hybrids; but the ultimate and by far the most accurate physical map is the actual nucleotide sequence. The completion of the draft human genomic sequence provides us with the best opportunity yet to compare the genetic and physical maps. Here we describe our estimates of female, male and sex-average recombination rates for about 60% of the genome. Recombination rates varied greatly along each chromosome, from 0 to at least 9 centiMorgans per megabase (cM Mb(-1)). Among several sequence and marker parameters tested, only relative marker position along the metacentric chromosomes in males correlated strongly with recombination rate. We identified several chromosomal regions up to 6 Mb in length with particularly low (deserts) or high (jungles) recombination rates. Linkage disequilibrium was much more common and extended for greater distances in the deserts than in the jungles.

Multigenic control of Listeria monocytogenes susceptibility in mice.

We have used a novel quantitative trait locus model to study the genetics of survival of F2 progeny of susceptible BALB/cByJ and resistant C57BL/6ByJ mice that have been infected with Listeria monocytogenes. This allowed us to map modifiers of L. monocytogenes susceptibility to chromosomes 5 and 13.

Genotyping for human whole-genome scans: past, present, and future.

Efficient and effective whole-genome 10-cM short tandem repeat polymorphism (STRP) scans are now available. Doubling or tripling STRP density to an average spacing of 3-5 cM is readily achievable. However, if typing costs for diallelic polymorphisms can be brought close to, or preferably less than, one-third those of STRPs, then diallelics may gradually supplement or supplant STRPs in whole-genome scans. The power of higher density genome scans for gene map ping by association and for many other research and clinical applications is great. It would be wise to continue investing heavily for many years in genotyping technology.

Genetic and physical mapping of the locus for autosomal dominant renal Fanconi syndrome, on chromosome 15q15.3.

Autosomal dominant renal Fanconi syndrome is a genetic model for the study of proximal renal tubular transport pathology. We were able to map the locus for this disease to human chromosome 15q15.3 by genotyping a central Wisconsin pedigree with 10 affected individuals. After a whole-genome scan with highly polymorphic simple sequence repeat markers, a maximum LOD score of 3.01 was calculated for marker D15S659 on chromosome 15q15.3. Linkage and haplotype analysis for an additional 24 markers flanking D15S659 narrowed the interval to approximately 3 cM, with the two highest single-point LOD scores observed being 4.44 and 4.68 (for D15S182 and D15S537, respectively). Subsequently, a complete bacterial artificial chromosome contig was constructed, from the High Throughput Genomic Sequence Database, for the region bounded by D15S182 and D15S143. The identification of the gene and gene product altered in autosomal dominant renal Fanconi syndrome will allow the study of the physiology of proximal renal tubular transport.

Genomewide scan for prostate cancer-aggressiveness loci.

The aggressiveness of prostate cancer (PCa) varies widely: some tumors progress to invasive, potentially life-threatening disease, whereas others stay latent for the remainder of an individual's lifetime. The mechanisms resulting in this variability are not yet understood, but they are likely to involve both genetic and environmental influences. To investigate genetic factors, we conducted a genomewide linkage analysis of 513 brothers with PCa, using the Gleason score, which reflects tumor histology, as a quantitative measure of PCa aggressiveness. To our knowledge, this is the first time that a measure of PCa aggressiveness has been directly investigated as a quantitative trait in a genomewide scan. We employed a generalized multipoint Haseman-Elston linkage-analysis approach that regresses the mean-corrected cross product between the brothers' Gleason scores on the estimated proportion of alleles shared by brothers identical by descent at each marker location. Our results suggest that candidate regions on chromosomes 5q, 7q, and 19q give evidence for linkage to PCa-aggressiveness genes. In particular, the strongest signals detected in these regions were at the following markers (with corresponding P values): for chromosome 5q31-33, between markers D5S1480 and D5S820 (P=.0002); for chromosome 7q32, between markers D7S3061 and D7S1804 (P=.0007); and, for chromosome 19q12, at D19S433 (P=.0004). This indicates that one or more of these candidate regions may contain genes that influence the progression of PCa from latent to invasive disease. Identification of such genes would be extremely valuable for elucidation of the mechanism underlying PCa progression and for determination of treatment in men in whom this disease has been diagnosed.

Characterization of human crossover interference.

We present an analysis of crossover interference over the entire human genome, on the basis of genotype data from more than 8,000 polymorphisms in eight CEPH families. Overwhelming evidence was found for strong positive crossover interference, with average strength lying between the levels of interference implied by the Kosambi and Carter-Falconer map functions. Five mathematical models of interference were evaluated: the gamma model and four versions of the count-location model. The gamma model fit the data far better than did any of the other four models. Analysis of intercrossover distances was greatly superior to the analysis of crossover counts, in both demonstrating interference and distinguishing between the five models. In contrast to earlier suggestions, interference was found to continue uninterrupted across the centromeres. No convincing differences in the levels of interference were found between the sexes or among chromosomes; however, we did detect possible individual variation in interference among the eight mothers. Finally, we present an equation that provides the probability of the occurrence of a double crossover between two nonrecombinant, informative polymorphisms.

Genome-wide variation in recombination in female meiosis: a risk factor for non-disjunction of chromosome 21.

Altered recombination patterns along non-disjoined chromosomes is the first molecular correlate identified for non-disjunction in humans. To understand better the factors related to this correlate, we have asked to what extent is recombination altered in an egg with a disomic chromosome: are patterns limited to the non-disjoined chromosome or do they extend to the entire cell? More specifically, we asked whether there is reduced recombination in the total genome of an egg with a non-disjoined chromosome 21 and no detectable recombination. We chose this subclass of non-disjoined chromosomes to enrich potentially for extremes in recombination. We found a statistically significant cell-wide reduction in the mean recombination rate in these eggs with non-disjoined chromosomes 21; no specific chromosomes were driving this effect. Most importantly, we found that this reduction was consistent with normal variation in recombination observed among eggs. Thus, given that recombination is a multifactorial trait, these data suggest that when the number of genome-wide recombination events is less than some threshold, specific chromosomes may be at an increased risk for non-disjunction. Further studies are required to confirm these results, to determine the importance of genetic and environmental factors that regulate recombination and to determine their impact on non-disjunction.

A genome screen of multiplex sibships with prostate cancer.

Analysis of a genome screen of 504 brothers with prostate cancer (CaP) who were from 230 multiplex sibships identified five regions with nominally positive linkage signals, on chromosomes 2q, 12p, 15q, 16p, and 16q. The strongest signal in these data is found on chromosome 16q, between markers D16S515 and D16S3040, a region suspected to contain a tumor-suppressor gene. On the basis of findings from previous genome screens of families with CaP, three preplanned subanalyses were carried out, in the hope of increasing the subgroup homogeneity. Subgroups were formed by dividing the sibships into a group with a positive family history (FH+) that met criteria for "hereditary" CaP (n=111) versus those which did not meet the criteria (n=119) and by dividing the families into those with a mean onset age below the median (n=115) versus those with a mean onset age above the median (n=115). A separate subanalysis was carried out for families with a history of breast cancer (CaB+ [n=53]). Analyses of these subgroups revealed a number of potentially important differences in regions that were nonsignificant when all the families were analyzed together. In particular, the subgroup without a positive family history (FH-) had a signal in a region that is proximal to the putative site of the HPC1 locus on chromosome 1, whereas the late-age-at-onset group had a signal on 4q. The CaB+ subgroup revealed a strong linkage signal at 1p35.1.

Cleaning genotype data.

The identification of genes contributing to variation in complex phenotypes requires genetic data of high fidelity. Thus, the identification of pedigree and genotyping errors is a crucial prerequisite to the analysis of data from a genome scan for disease genes. The problem has been given little attention in most gene hunting papers; the focus has often been on eliminating mendelian inconsistencies in order that the analysis may proceed, rather than on achieving the best possible data. Though a number of computer programs are available to assist in the identification of genotyping and pedigree errors, the process is still not completely automated. While the Collaborative Study on the Genetics of Alcoholism (COGA) data set for GAW11 is completely compatible with Mendel's rules, there are still some errors present. We inspected the COGA data for the presence of additional errors, and identified five possible pedigree errors.

Long homozygous chromosomal segments in reference families from the centre d'Etude du polymorphisme humain.

Using genotypes from nearly 8,000 short tandem-repeat polymorphisms typed in eight of the reference families from the Centre d'Etude du Polymorphisme Humain (CEPH), we identified numerous long chromosomal segments of marker homozygosity in many CEPH individuals. These segments are likely to represent autozygosity, the result of the mating of related individuals. Confidence that the complete segment is homozygous is gained only with markers of high density. The longest segment in the eight families spanned 77 cM and included 118 homozygous markers. All individuals in family 884 showed at least one segment of homozygosity: the father and mother were homozygous in 8 and 10 segments with an average length of 13 and 16 cM, respectively, and covering a total of 105 and 160 cM, respectively. The progeny in family 884 were homozygous over 5-16 segments with average length 11 cM. The progeny in family 102 were homozygous over 4-12 segments with average length 19 cM. Of the 100 individuals in the other six families, 1 had especially long homozygous segments, and 19 had short but significant homozygous segments. Our results indicate that long homozygous segments are common in humans and that these segments could have a substantial impact on gene mapping and health.

Method for constructing confidently ordered linkage maps.

We describe a method for identifying, from a comprehensive genetic map, the most dense framework of confidently ordered markers. The approach uses the number of observed recombination events between each pair of markers, and finds the largest subset of markers for which adjacent loci are separated by at least one recombination. We illustrate the approach using a short region of chromosome 7p.