Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Quantitative trait loci modifying cardiac atrial septal morphology and risk of patent foramen ovale in the mouse.

Atrial septal defect (ASD) is a common congenital heart disease (CHD) occurring in 5 to 7 per 10,000 live births. Mutations in 5 human genes (NKX2.5, TBX5, GATA4, MYHC, ACTC) are known to cause dominant ASD, but these account for a minority of cases. Human and mouse data suggest that ASD exists in an anatomical continuum with milder septal variants patent foramen ovale (PFO) and atrial septal aneurysm, strongly associated with ischemic stroke and migraine. We have previously shown in inbred mice that the incidence of PFO strongly correlates with length of the interatrial septum primum, defining a quantitative trait underlying PFO risk. To better understand genetic causation of atrial septal abnormalities, we mapped quantitative trait loci (QTL) influencing septal morphology using mouse strains (QSi5 and 129T2/SvEms) maximally informative for PFO incidence and 3 quantitative septal anatomical traits including septum primum length. [QSi5x129T2/SvEms]F2 intercross animals (n=1437) were phenotyped and a whole genome scan performed at an average 17-cM interval. Statistical methodology scoring PFO as a binary phenotype was developed as a confirmatory mapping technique. We mapped 7 significant and 6 suggestive QTL modifying quantitative phenotypes, with 4 supported by binary analysis. Quantitative traits, although strongly associated with PFO (P<0.001), correlated poorly with each other and in all but 1 case QTL for different traits were nonoverlapping. Thus, multiple anatomical processes under separate genetic control contribute to risk of PFO. Our findings demonstrate the feasibility of modeling the genetic basis of common CHD using animal genetic and genomic technologies.

Development of a highly fecund inbred strain of mice.

A highly fecund inbred mouse line has been established from the Quackenbush Swiss (QS) outbred strain by full-sib inbreeding combined with selection for high number of pups born alive (NBA) and low interlitter interval (ILI). After more than 50 generations of inbreeding and selection, this line, named QSi5, has an NBA of 13.4 and an ILI of 29 days, averaged over the first four parities, and a total productivity of 50.7 NBA. With its exceptional reproductive performance, this line will be very useful in the creation of resources (including advanced intercross lines) for analysis of quantitative trait loci for a wide range of traits, and for the cost-effective creation of congenic lines.

Quantitative trait loci for steady-state platelet count in mice.

Platelet count in humans is a strongly genetically regulated trait, with approximately 85% of the interindividual variance in platelet numbers attributable to genetic factors. Inbred mouse strains also have strain-specific platelet count ranges. As part of a project to identify novel factors that regulate platelet count, we identified two inbred mouse strains, CBA/CaH and QSi5, with substantial differences in platelet count (mean values of 581 vs. 1062 x 10(9)/L). An F(2) intercross resource of 1126 animals was bred from these two parental strains for a genomewide scan for quantitative trait loci (QTL) for platelet count. QTL were identified on MMU1 (LOD 6.8, p < 0.0005) and MMU11 (LOD 11.2, p < 0.0005) by selectively genotyping animals from the extremes of the F(2) platelet count distribution. Three other QTL of suggestive statistical significance were also detected on MMU7, 13, and 17. It is noteworthy that no QTL were detected in the vicinity of the genes encoding thrombopoietin ( Thpo), and its receptor ( c-Mpl), both known to influence platelet production. Comparison of gene expression levels between the parental mouse strains by microarrays also showed little difference in the mRNA levels of these known candidate genes. These results represent the first published use of a genetic linkage-based approach in a mouse model toward the identification of genetic factors that regulate platelet count.