Variation in complement factor 3 is associated with risk of age-related macular degeneration

The association of variants in complement factors H and B with age-related macular degeneration has led to more intense genetic and functional analysis of the complement pathway. We identify a nonsynonymous coding change in complement factor 3 that is strongly associated with risk of age-related macular degeneration in a large case-control sample.     

Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the most common form of irreversible blindness in developed countries. Variants in the factor H gene (CFH, also known as HF1), which encodes a major inhibitor of the alternative complement pathway, are associated with the risk for developing AMD. Here we test the hypothesis that variation in genes encoding other regulatory proteins of the same pathway is associated with AMD. We screened factor B (BF) and complement component 2 (C2) genes, located in the major histocompatibility complex class III region, for genetic variation in two independent cohorts comprising approximately 900 individuals with AMD and approximately 400 matched controls. Haplotype analyses identify a statistically significant common risk haplotype (H1) and two protective haplotypes. The L9H variant of BF and the E318D variant of C2 (H10), as well as a variant in intron 10 of C2 and the R32Q variant of BF (H7), confer a significantly reduced risk of AMD (odds ratio = 0.45 and 0.36, respectively). Combined analysis of the C2 and BF haplotypes and CFH variants shows that variation in the two loci can predict the clinical outcome in 74% of the affected individuals and 56% of the controls. These data expand and refine our understanding of the genetic risk for AMD.     

Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3'-kinase is essential for male fertility

The c-kit-encoded transmembrane tyrosine kinase receptor for stem cell factor (Kit/SCF-R) is required for normal haematopoiesis, melanogenesis and gametogenesis. However, the roles of individual Kit/SCF-R-induced signalling pathways in the control of developmental processes in the intact animal are completely unknown. To examine the function of SCF-induced phosphatidylinositol (PI) 3'-kinase activation in vivo, we employed the Cre-loxP system to mutate the codon for Tyr719, the PI 3'-kinase binding site in Kit/SCF-R, to Phe in the genome of mice by homologous recombination. Homozygous (Y719F/Y719F) mutant mice are viable. The mutation completely disrupted PI 3'-kinase binding to Kit/SCF-R and reduced SCF-induced PI 3'-kinase-dependent activation of Akt by 90%. The mutation induced a gender- and tissue-specific defect. Although there are no haematopoietic or pigmentation defects in homozygous mutant mice, males are sterile due to a block in spermatogenesis, with initially decreased proliferation and subsequent extensive apoptosis occurring at the spermatogonial stem-cell level. In contrast, female homozygotes are fully fertile. This is the first report so far demonstrating the role of an individual signalling pathway downstream of Kit/SCF-R in the intact animal. It provides the first in vivo model for male sterility caused by a discrete signalling pathway defect affecting early germ cells.     

Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.     

Abnormal TNF activity in Timp3-/- mice leads to chronic hepatic inflammation and failure of liver regeneration

Tumor-necrosis factor (TNF), a pleiotropic cytokine, triggers physiological and pathological responses in several organs. Here we show that deletion of the mouse gene Timp3 resulted in an increase in TNF-alpha converting enzyme activity, constitutive release of TNF and activation of TNF signaling in the liver. The increase in TNF in Timp3(-/-) mice culminated in hepatic lymphocyte infiltration and necrosis, features that are also seen in chronic active hepatitis in humans. This pathology was prevented when deletion of Timp3 was combined with Tnfrsf1a deficiency. In a liver regeneration model that requires TNF signaling, Timp3(-/-) mice succumbed to liver failure. Hepatocytes from Timp3(-/-) mice completed the cell cycle but then underwent cell death owing to sustained activation of TNF. This hepatocyte cell death was completely rescued by a neutralizing antibody to TNF. Dysregulation of TNF occurred specifically in Timp3(-/-), and not Timp1(-/-) mice. These data indicate that TIMP3 is a crucial innate negative regulator of TNF in both tissue homeostasis and tissue response to injury.     

A mutation in stratifin is responsible for the repeated epilation (Er) phenotype in mice

Stratifin (Sfn, also called 14-3-3sigma) is highly expressed in differentiating epidermis and mediates cell cycle arrest. Sfn is repressed in cancer, but its function during development is uncharacterized. We identified an insertion mutation in the gene Sfn in repeated epilation (Er) mutant mice by positional cloning. Er/+ mice expressed a truncated Sfn protein, which probably contributes to the defects in Er/Er and Er/+ epidermis and to cancer development in Er/+ mice.     

Mice mutant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis

Atrioventricular and semilunar valve abnormalities are common birth defects, but how cardiac valvulogenesis is directed remains largely unknown. During studies of genetic interaction between Egfr, encoding the epidermal growth factor receptor, and Ptpn11, encoding the protein-tyrosine-phosphatase Shp2, we discovered that Egfr is required for semilunar, but not atrioventricular, valve development. Although unnoticed in earlier studies, mice homozygous for the hypomorphic Egfr allele waved-2 (Egfrwa2/wa2) exhibit semilunar valve enlargement resulting from over-abundant mesenchymal cells. Egfr-/- mice (CD1 background) have similar defects. The penetrance and severity of the defects in Egfrwa2/wa2 mice are enhanced by heterozygosity for a targeted mutation of exon 2 of Ptpn11 (ref. 3). Compound (Egfrwa2/wa2:Ptpn11+/-) mutant mice also show premature lethality. Electrocardiography, echocardiography and haemodynamic analyses showed that affected mice develop aortic stenosis and regurgitation. Our results identify the Egfr and Shp2 as components of a growth-factor signalling pathway required specifically for semilunar valvulogenesis, support the hypothesis that Shp2 is required for Egfr signalling in vivo, and provide an animal model for aortic valve disease.     

Mutations in PCSK9 cause autosomal dominant hypercholesterolemia

Autosomal dominant hypercholesterolemia (ADH; OMIM144400), a risk factor for coronary heart disease, is characterized by an increase in low-density lipoprotein cholesterol levels that is associated with mutations in the genes LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B). We mapped a third locus associated with ADH, HCHOLA3 at 1p32, and now report two mutations in the gene PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) that cause ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase), a newly identified human subtilase that is highly expressed in the liver and contributes to cholesterol homeostasis.     

Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis

The polo-like kinase Plk4 (also called Sak) is required for late mitotic progression, cell survival and postgastrulation embryonic development. Here we identified a phenotype resulting from Plk4 haploinsufficiency in Plk4 heterozygous cells and mice. Plk4+/- embryonic fibroblasts had increased centrosomal amplification, multipolar spindle formation and aneuploidy compared with wild-type cells. The incidence of spontaneous liver and lung cancers was approximately 15 times high in elderly Plk4+/- mice than in Plk4+/+ littermates. Using the in vivo model of partial hepatectomy to induce synchronous cell cycle entry, we determined that the precise regulation of cyclins D1, E and B1 and of Cdk1 was impaired in Plk4+/- regenerating liver, and p53 activation and p21 and BubR1 expression were suppressed. These defects were associated with progressive cell cycle delays, increased spindle irregularities and accelerated hepatocellular carcinogenesis in Plk4+/- mice. Loss of heterozygosity occurs frequently (approximately 60%) at polymorphic markers adjacent to the PLK4 locus in human hepatoma. Reduced Plk4 gene dosage increases the probability of mitotic errors and cancer development.     

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.