A Gene for Autosomal Recessive Limb-Girdle Muscular Dystrophy in Manitoba Hutterites Maps to Chromosome Region 9q31-q33: Evidence for Another Limb-Girdle Muscular Dystrophy Locus

SummaryCharacterized by proximal muscle weakness and wasting, limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of clinical disorders. Previous reports have documented either autosomal dominant or autosomal recessive modes of inheritance, with genetic linkage studies providing evidence for the existence of at least 12 distinct loci. Gene products have been identified for five genes responsible for autosomal recessive forms of the disorder. We performed a genome scan using pooled DNA from a large Hutterite kindred in which the affected members display a mild form of autosomal recessive LGMD. A total of 200 markers were used to screen pools of DNA from patients and their siblings. Linkage between the LGMD locus and D9S302 (maximum LOD score 5.99 at recombination fraction .03) was established. Since this marker resides within the chromosomal region known to harbor the gene causing Fukuyama congenital muscular dystrophy (FCMD), we expanded our investigations, to include additional markers in chromosome region 9q31-q34.1. Haplotype analysis revealed five recombinations that place the LGMD locus distal to the FCMD locus. The LGMD locus maps close to D9S934 (maximum multipoint LOD score 7.61) in a region that is estimated to be ∼4.4 Mb (Genetic Location Database composite map). On the basis of an inferred ancestral recombination, the gene may lie in a 300-kb region between D9S302 and D9S934. Our results provide compelling evidence that yet another gene is involved in LGMD; we suggest that it be named “LGMD2H.

Bmp and Nodal Independently Regulate lefty1 Expression to Maintain Unilateral Nodal Activity during Left-Right Axis Specification in Zebrafish

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning

Exacerbated leishmaniasis caused by a viral endosymbiont can be prevented by immunization with Its viral capsid

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities

Limb-Girdle Muscular Dystrophy Type 2H Associated with Mutation in TRIM32, a Putative E3-Ubiquitin–Ligase Gene

Limb-girdle muscular dystrophy type 2H (LGMD2H) is a mild autosomal recessive myopathy that was first described in the Manitoba Hutterite population. Previous studies in our laboratory mapped the causative gene for this disease to a 6.5-Mb region in chromosomal region 9q31-33, flanked by D9S302 and D9S1850. We have now used additional families and a panel of 26 microsatellite markers to construct haplotypes. Twelve recombination events that reduced the size of the candidate region to 560 kb were identified or inferred. This region is flanked by D9S1126 and D9S737 and contains at least four genes. Exons of these genes were sequenced in one affected individual, and four sequence variations were identified. The families included in our study and 100 control individuals were tested for these variations. On the basis of our results, the mutation in the tripartite-motif–containing gene (TRIM32) that replaces aspartate with asparagine at position 487 appears to be the causative mutation of LGMD2H. All affected individuals were found to be homozygous for D487N, and this mutation was not found in any of the controls. This mutation occurs in an NHL (named after the proteins NCL1, HT2A, and LIN-41) domain at a position that is highly conserved. NHL domains are known to be involved in protein-protein interactions. Although the function of TRIM32 is unknown, current knowledge of the domain structure of this protein suggests that it may be an E3-ubiquitin ligase. If proven, this represents a new pathogenic mechanism leading to muscular dystrophy

Linkage of Tuberculosis to Chromosome 2q35 Loci, Including NRAMP1, in a Large Aboriginal Canadian Family

An epidemic of tuberculosis occurred in a community of Aboriginal Canadians during the period 1987–89. Genetic and epidemiologic data were collected on an extended family from this community, and the evidence for linkage to NRAMP1, a candidate gene for susceptibility to mycobacterial diseases, was assessed. Individuals were grouped into risk (liability) classes based on vaccination, age, previous disease, and tuberculin skin-test results. Under the assumption of a dominant mode of inheritance and a relative risk of 10, which is associated with the high-risk genotypes, a maximum LOD score of 3.81 was observed for linkage between a tuberculosis-susceptibility locus and D2S424, which is located just distal to NRAMP1, in chromosome region 2q35. Significant linkage was also observed between a tuberculosis-susceptibility locus and a haplotype of 10 NRAMP1 intragenic variants. No linkage to the major histocompatibility-complex region on chromosome 6p was observed, despite distortion of transmission from one member of the oldest couple to their affected offspring. The ability to assign individuals to risk classes was crucial to the success of this study

Susceptibility to progressive Cryptococcus neoformans pulmonary infection is regulated by loci on mouse chromosomes 1 and 9.

Genetic factors that regulate the pathogenesis of pneumonia caused by the fungus Cryptococcus neoformans are poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection with C. neoformans ATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice following C. neoformans pulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10, and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance allele at Cnes4 and a susceptibility allele at Cnes5. These studies reveal additional genetic complexity of the host response to C. neoformans that is associated with divergent patterns of pulmonary inflammation

Cystic fibrosis mutations in North American populations of French ancestry: Analysis of Quebec French-Canadian and Louisiana Acadian families

A 3-bp deletion (ΔF508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. ΔF508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of ΔF508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with ΔF508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without ΔF508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-Lac St. Jean population was 1/895 live-born infants

Homozygous Deletion of the Very Low Density Lipoprotein Receptor Gene Causes Autosomal Recessive Cerebellar Hypoplasia with Cerebral Gyral Simplification

An autosomal recessive syndrome of nonprogressive cerebellar ataxia and mental retardation is associated with inferior cerebellar hypoplasia and mild cerebral gyral simplification in the Hutterite population. An identity-by-descent mapping approach using eight patients from three interrelated Hutterite families localized the gene for this syndrome to chromosome region 9p24. Haplotype analysis identified familial and ancestral recombination events and refined the minimal region to a 2-Mb interval between markers D9S129 and D9S1871. A 199-kb homozygous deletion encompassing the entire very low density lipoprotein receptor (VLDLR) gene was present in all affected individuals. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. To our knowledge, this syndrome represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect

Three Families with Autosomal Dominant Nephrogenic Diabetes Insipidus Caused by Aquaporin-2 Mutations in the C-Terminus

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763–772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812–818del). The wild-type AQP2 is predicted to be a 271–amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330–333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%–17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2