Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.     

Pyruvate kinase deficiency in mice protects against malaria

The global health impact of malaria is enormous, with an estimated 300-500 million clinical cases and 1 million annual deaths. In humans, initial susceptibility to infection with Plasmodium species, disease severity and ultimate outcome of malaria (self-healing or lethal) are under complex genetic control. Alleles associated with sickle cell anemia, beta-thalassemia and deficiency in glucose-6-phosphate dehydrogenase have a protective effect against malaria and may have been retained by positive selection in areas of endemic malaria. Likewise, genetic variations in erythrocyte antigens and levels of host cytokines affect type and severity of disease. A mouse model of infection with Plasmodium chabaudi was used to study the genetic component of malaria susceptibility. Segregation analyses in informative F2 crosses derived from resistant C57BL/6J and susceptible A/J, C3H and SJL strains using extent of blood stage replication of the parasite and survival as traits mapped three P. chabaudi resistance (Char) loci on chromosomes 9 (Char1), 8 (Char2) and 17 (Char3, MHC-linked). Recombinant congenic strains AcB55 and AcB61 are unusually resistant to malaria despite carrying susceptibility alleles at Char1 and Char2. Malaria resistance in AcB55 and AcB61 is associated with splenomegaly and constitutive reticulocytosis, is inherited in an autosomal recessive fashion and is controlled by a locus on chromosome 3 (Char4). Sequencing of candidate genes from the Char4 region identified a loss-of-function mutation (269T-->A, resulting in the amino acid substitution I90N) in the pyruvate kinase gene (Pklr) that underlies the malaria resistance in AcB55 and AcB61. These results suggest that pyruvate kinase deficiency may similarly protect humans against malaria.     

Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity

Mutations in SPINK5, encoding the serine protease inhibitor LEKTI, cause Netherton syndrome, a severe autosomal recessive genodermatosis. Spink5(-/-) mice faithfully replicate key features of Netherton syndrome, including altered desquamation, impaired keratinization, hair malformation and a skin barrier defect. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1 due to stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme-like hyperactivity. This leads to defective stratum corneum adhesion and resultant loss of skin barrier function. Profilaggrin processing is increased and implicates LEKTI in the cornification process. This work identifies LEKTI as a key regulator of epidermal protease activity and degradation of desmoglein 1 as the primary pathogenic event in Netherton syndrome.     

A homozygous stop codon in the lysyl hydroxylase gene in two siblings with Ehlers-Danlos syndrome type VI.

Ehlers-Danlos syndrome (EDS) is characterized by joint hypermobility, alterations in the skin and additional signs of connective tissue involvement. EDS type VI was the first connective tissue disorder for which a specific defect in collagen metabolism was identified, namely a deficiency of lysyl hydroxylase activity. We now report a homozygous single basepair substitution converting the CGA codon (Arg319) to a TGA termination codon in two siblings with EDS type VI. The healthy parents, who are first cousins, and two of the three healthy siblings of the patients are heterozygous. The mutation leads to an almost complete absence of lysyl hydroxylase activity in extracts derived from fibroblasts of the patients.     

Fras1 deficiency results in cryptophthalmos,renal agenesis and blebbed phenotype in mice

Loss of tight association between epidermis and dermis underlies several blistering disorders and is frequently caused by impaired function of extracellular matrix (ECM) proteins. Here we describe a new protein in mouse, Fras1, that is specifically detected in a linear fashion underlying the epidermis and the basal surface of other epithelia in embryos. Loss of Fras1 function results in the formation of subepidermal hemorrhagic blisters as well as unilateral or bilateral renal agenesis during mouse embryogenesis. Postnatally, homozygous Fras1 mutants have fusion of the eyelids and digits and unilateral renal agenesis or dysplasia. The defects observed in Fras1-/- mice phenocopy those of the existing bl (blebbed) mouse mutants, which have been considered a model for the human genetic disorder Fraser syndrome. We show that bl/bl homozygous embryos are devoid of Fras1 protein, consistent with the finding that Fras1 is mutated in these mice. In sum, our data suggest that perturbations in the composition of the extracellular space underlying epithelia could account for the onset of the blebbed phenotype in mouse and Fraser syndrome manifestation in human.     

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.     

Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1

Genomic imprinting is an epigenetic modification that results in expression from only one of the two parental copies of a gene. Differences in methylation between the two parental chromosomes are often observed at or near imprinted genes. Beckwith-Wiedemann syndrome (BWS), which predisposes to cancer and excessive growth, results from a disruption of imprinted gene expression in chromosome band 11p15.5. One third of individuals with BWS lose maternal-specific methylation at KvDMR1, a putative imprinting control region within intron 10 of the KCNQ1 gene, and it has been proposed that this epimutation results in aberrant imprinting and, consequently, BWS1, 2. Here we show that paternal inheritance of a deletion of KvDMR1 results in the de-repression in cis of six genes, including Cdkn1c, which encodes cyclin-dependent kinase inhibitor 1C. Furthermore, fetuses and adult mice that inherited the deletion from their fathers were 20-25% smaller than their wildtype littermates. By contrast, maternal inheritance of this deletion had no effect on imprinted gene expression or growth. Thus, the unmethylated paternal KvDMR1 allele regulates imprinted expression by silencing genes on the paternal chromosome. These findings support the hypothesis that loss of methylation in BWS patients activates the repressive function of KvDMR1 on the maternal chromosome, resulting in abnormal silencing of CDKN1C and the development of BWS.     

Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice

Huntington disease (HD), an autosomal dominant, progressive neurodegenerative disorder, is caused by an expanded CAG repeat sequence leading to an increase in the number of glutamine residues in the encoded protein. The normal CAG repeat range is 5-36, whereas 38 or more repeats are found in the diseased state; the severity of disease is roughly proportional to the number of CAG repeats. HD shows anticipation, in which subsequent generations display earlier disease onsets due to intergenerational repeat expansion. For longer repeat lengths, somatic instability of the repeat size has been observed both in human cases at autopsy and in transgenic mouse models containing either a genomic fragment of human HD exon 1 (ref. 9) or an expanded repeat inserted into the endogenous mouse gene Hdh (ref. 10). With increasing repeat number, the protein changes conformation and becomes increasingly prone to aggregation, suggesting important functional correlations between repeat length and pathology. Because dinucleotide repeat instability is known to increase when the mismatch repair enzyme MSH2 is missing, we examined instability of the HD CAG repeat by crossing transgenic mice carrying exon 1 of human HD (ref. 16) with Msh2-/- mice. Our results show that Msh2 is required for somatic instability of the CAG repeat.     

Candidate lung tumor susceptibility genes identified through whole-genome association analyses in inbred mice

We performed a whole-genome association analysis of lung tumor susceptibility using dense SNP maps ( approximately 1 SNP per 20 kb) in inbred mice. We reproduced the pulmonary adenoma susceptibility 1 (Pas1) locus identified in previous linkage studies and further narrowed this quantitative trait locus (QTL) to a region of less than 0.5 Mb in which at least two genes, Kras2 (Kirsten rat sarcoma oncogene 2) and Casc1 (cancer susceptibility candidate 1; also known as Las1), are strong candidates. Casc1 knockout mouse tumor bioassays showed that Casc1-deficient mice were susceptible to chemical induction of lung tumors. We also found three more genetic loci for lung adenoma development. Analysis of one of these candidate loci identified a previously uncharacterized gene Lasc1, bearing a nonsynonymous substitution (D102E). We found that the Lasc1 Glu102 allele preferentially promotes lung tumor cell growth. Our findings demonstrate the prospects for using dense SNP maps in laboratory mice to refine previous QTL regions and identify genetic determinants of complex traits.     

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.     

Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency to neurodevelopmental abnormalities in mice

Williams syndrome is a neurodevelopmental disorder caused by the hemizygous deletion of 1.6 Mb on human chromosome 7q11.23. This region comprises the gene CYLN2, encoding CLIP-115, a microtubule-binding protein of 115 kD. Using a gene-targeting approach, we provide evidence that mice with haploinsufficiency for Cyln2 have features reminiscent of Williams syndrome, including mild growth deficiency, brain abnormalities, hippocampal dysfunction and particular deficits in motor coordination. Absence of CLIP-115 also leads to increased levels of CLIP-170 (a closely related cytoplasmic linker protein) and dynactin at the tips of growing microtubules. This protein redistribution may affect dynein motor regulation and, together with the loss of CLIP-115-specific functions, underlie neurological alterations in Williams syndrome.     

Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused by mutations in the human type VII collagen gene (COL7A1). Individuals with DEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for DEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. In this study, we delivered and expressed full-length type VII collagen using a self-inactivating minimal lentivirus-based vector. Transduction of lentiviral vectors containing the COL7A1 transgene into recessive DEB (RDEB) keratinocytes and fibroblasts (in which type VII collagen was absent) resulted in persistent synthesis and secretion of type VII collagen. Unlike RDEB parent cells, the gene-corrected cells had normal morphology, proliferative potential, matrix attachment and motility. We used these gene-corrected cells to regenerate human skin on immune-deficient mice. Human skin regenerated by gene-corrected RDEB cells had restored expression of type VII collagen and formation of anchoring fibrils at the dermal-epidermal junction in vivo. These studies demonstrate that it is possible to restore type VII collagen gene expression in RDEB skin in vivo.