Ping-Zi Wen

Genetic loci controlling body fat, lipoprotein metabolism, and insulin levels in a multifactorial mouse model.

We analyzed the inheritance of body fat, leptin levels, plasma lipoprotein levels, insulin levels, and related traits in an intercross between inbred mouse strains CAST/Ei and C57BL/6J. CAST/Ei mice are unusually lean, with only approximately 8% of body weight as fat, whereas C57BL/6J mice have approximately 18% body fat. Quantitative trait locus analysis using > 200 F2 mice revealed highly significant loci (lod scores > 4.3) on chromosomes 2 (three separate loci) and 9 that contribute to mouse fat-pad mass for mice on a high-fat diet. Some loci also influenced plasma lipoprotein levels and insulin levels either on chow or high-fat diets. Two loci for body fat and lipoprotein levels (on central and distal chromosome 2) coincided with a locus having strong effects on hepatic lipase activity, an activity associated with visceral obesity and lipoprotein levels in humans. A locus contributing to plasma leptin levels (lod score 5.3) but not obesity was identified on chromosome 4, near the leptin receptor gene. These data identify candidate regions and candidate genes for studies of human obesity and diabetes, and suggest obesity is highly complex in terms of the number of genetic factors involved. Finally, they support the existence of specific genetic interactions between body fat, insulin metabolism, and lipoprotein metabolism.

High-resolution mapping of gene expression using association in an outbred mouse stock

Quantitative trait locus (QTL) analysis is a powerful tool for mapping genes for complex traits in mice, but its utility is limited by poor resolution. A promising mapping approach is association analysis in outbred stocks or different inbred strains. As a proof of concept for the association approach, we applied whole-genome association analysis to hepatic gene expression traits in an outbred mouse population, the MF1 stock, and replicated expression QTL (eQTL) identified in previous studies of F2 intercross mice. We found that the mapping resolution of these eQTL was significantly greater in the outbred population. Through an example, we also showed how this precise mapping can be used to resolve previously identified loci (in intercross studies), which affect many different transcript levels (known as eQTL “hotspots”), into distinct regions. Our results also highlight the importance of correcting for population structure in whole-genome association studies in the outbred stock.

Dissection of multigenic obesity traits in congenic mouse strains

Previous quantitative trait locus mapping (QTL) identified multigenic obesity (MOB) loci on mouse Chromosome (Chr) 2 that influence the interrelated phenotypes of obesity, insulin resistance, and dyslipidemia. To better localize and characterize the MOB locus, three congenic mouse strains were created. Overlapping genomic intervals from the lean CAST/Ei (CAST) strain were introgressed onto an obesity-susceptible C57BL/6 (BL6) background to create proximal (15 Mb–73 Mb), middle (63 Mb–165 Mb), and distal (83 Mb–182 Mb) congenic strains. The congenic strains showed differences in obesity, insulin, and lipid traits consistent with the original QTL analysis for the locus. Importantly, characterization of the MOB congenics localized the effects of genes that underlie obesity-related traits to an introgressed interval (73–83 Mb) unique to the middle MOB congenic. Conversely, significant differences between the lipid and insulin profiles of the middle and distal MOB congenics implicated the presence of at least two genes that underlie these traits. When fed an atherogenic diet, several traits associated with metabolic syndrome were observed in the distal MOB congenic, while alterations in plasma lipoproteins were observed in the middle MOB congenic strain.

Systems genetics of susceptibility to obesity-induced diabetes in mice

Inbred strains of mice are strikingly different in susceptibility to obesity-driven diabetes. For instance, deficiency in leptin receptor (db/db) leads to hyperphagia and obesity in both C57BL/6 and DBA/2 mice, but only on the DBA/2 background do the mice develop beta-cell loss leading to severe diabetes, while C57BL/6 mice are relatively resistant. To further investigate the genetic factors predisposing to diabetes, we have studied leptin receptor-deficient offspring of an F2 cross between C57BL/6J (db/+) males and DBA/2J females. The results show that the genetics of diabetes susceptibility are enormously complex and a number of quantitative trait loci (QTL) contributing to diabetes-related traits were identified, notably on chromosomes 4, 6, 7, 9, 10, 11, 12, and 19. The Chr. 4 locus is likely due to a disruption of the Zfp69 gene in C57BL/6J mice. To identify candidate genes and to model coexpression networks, we performed global expression array analysis in livers of the F2 mice. Expression QTL (eQTL) were identified and used to prioritize candidate genes at clinical trait QTL. In several cases, clusters of eQTLs colocalized with clinical trait QTLs, suggesting a common genetic basis. We constructed coexpression networks for both 5 and 12 wk old mice and identified several modules significantly associated with clinical traits. One module in 12 wk old mice was associated with several measures of hepatic fat content as well as with other lipid- and diabetes-related traits. These results add to the understanding of the complex genetic interactions contributing to obesity-induced diabetes.

Hyplip2, a New Gene for Combined Hyperlipidemia and Increased Atherosclerosis

Objective—We previously reported the mapping of a quantitative trait locus (QTL) on chromosome 15 contributing to hyperlipidemia in a cross between inbred strains MRL/MpJ (MRL) and BALB/cJ (BALB). Using marker-assisted breeding, we constructed a congenic strain in which chromosome 15 interval from MRL is placed on the genetic background of BALB. The congenic allowed us to confirm the QTL result and to further characterize the properties and location of the underlying gene. Methods and Results—On chow and high-fat (atherogenic) diets, the congenic mice exhibited higher levels of plasma triglycerides and cholesterol than BALB mice. In response to the atherogenic diet, the congenic mice but not BALB mice exhibited a dramatic ≈30-fold increase in atherogenic lesions accompanied by ≈2-fold decrease in high-density lipoprotein cholesterol levels. With respect to atherosclerotic lesions and some lipid parameters, this chromosome 15 gene, designated Hyplip2, exhibited dominant inheritance. Expression array analyses suggested that Hyplip2 may influence inflammatory and bile acid synthesis pathways. Finally, we demonstrated the usefulness of subcongenic strains to narrow the locus (50 Mbp) with the goal of positionally cloning Hyplip2. Conclusions—Our data demonstrate that the Hyplip2 gene significantly contributes to combined hyperlipidemia and increased atherosclerosis in mice.

A locus on the X Chromosome is linked to body length in mice

Linkage between body length (anus to nose (AN) length) and three markers on the mouse X Chromosome was found in an interspecific backcross ((C57BL/6J x Mus spretus) Fl x C57BL/6J), designated BSB. A cross of 409 mice were scored for 148 genetic markers distributed on all chromosomes except the Y Chromosome. Statistical analysis revealed highly significant linkage (LOD score 5.5) between body length and a locus in the middle portion of the X Chromosome, the nearest markers being the microsatellite marker DXMit73 and a farnesyl pyrophosphate locus (Fpsl9) 3.1 cM proximal to DXMit73. The locus explains 10% of the variance in AN length and affects both males and females to about the same extent.

The glutathione peroxidase gene, Gpx1, maps to mouse Chromosome 9

Map position: Prkarla is localized on mouse Chromosome (Chr) 1 1: centromere-Ngfr-5,7(+_l.7) cM-Wnt3-2.1(_+l.O) c M Prkar l a-7.8( + l .9 ) cM-D11Jkn l e-telomere Method of mapping: (AEJ/Gn-a bpH/a bp n x M. spretus)F 1 x AEJ/Gn-a bpn/a bp H interspecific backcross mice [1]. Haplotype analysis is shown in Fig. 1A. Molecular reagents: The following probes were used to detect the specific loci: p5b (Ngfr); pBG14 (Wnt3); p15.4 (D11Jknle); and pRIa (Prkarla) [1,2,3,5]. Allele detection: To detect restriction fragment length polymorphisms (RFLPs) useful for mapping the murine loci under study, genomic DNAs from AEJ/Gn and M. spretus mice were digested with 14 restriction endonucleases, and individual digests were analyzed by Southern blot hybridization with probes for the Prkarla, Wnt3, D11Jknle, and Ngfr loci [1,2,3,5]. RFLPs were detected with the specified enzyme: Prkarla with BglI; Wnt3 with TaqI; Ngfr with PstI; D11Jknle with SacI. The segregation of the M. spretus allele for Prkarla, Wnt3, D11Jknle, and Ngfr was followed in 192 N2 offspring from the interspecific backcross. Linkage of each locus typed in the interspecific backcross was analyzed by calculating the maximum likelihood estimates of linkage parameters as described [4] with the computer program Spretus Madness: Part Deux (developed by Karl Smalley, Jim Averbach, Linda D. Siracusa and Arthur M. Buchberg, Jefferson Cancer Institute, Philadelphia, Pa.). Discussion: cAMP-dependent protein kinase A consists of two isoforms, type I and type II. While the two isoforms share a common catalytic domain, they differ in their cAMP-binding regulatory subunits, termed RI and RII. Prkarla is the gene for the RI subunit of cAMP-dependent protein kinase A. The Prkarla locus was previously known as the Tsel (Tissue-specific extinguisher 1) locus. The PRKAR1A locus maps to human Chr 17 and is responsible for the down-regulation of liver genes in hepatoma x fibro-