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.

Identification of a chromosome 8p locus for early-onset coronary heart disease in a French Canadian population.

Susceptibility to coronary heart disease (CHD) has long been known to exhibit familial aggregation, with heritability estimated to be greater than 50%. The French Canadian population of the Saguenay-Lac Saint-Jean region of Quebec, Canada is descended from a founder population that settled this region 300-400 years ago and this may provide increased power to detect genes contributing to complex traits such as CHD. Probands with early-onset CHD, defined by angiographically determined coronary stenosis, and their relatives were recruited from this population (average sibship size of 6.4). Linkage analysis was performed following a genome-wide microsatellite marker scan on 42 families with 284 individuals. Nonparametric linkage (NPL) analysis provided suggestive evidence for a CHD susceptibility locus on chromosome 8 with an NPL score of 3.14 (P=0.001) at D8S1106. Linkage to this locus was verified by fine mapping in an enlarged sample of 50 families with 320 individuals. This analysis provided evidence of linkage at D8S552 (NPL score=3.53, P=0.0003), a marker that maps to the same location as D8S1106. Candidate genes in this region, including macrophage scavenger receptor 1, farnesyl-diphosphate farnesyltransferase 1, fibrinogen-like 1, and GATA-binding protein 4, were resequenced in all coding exons in both affected and unaffected individuals. Association studies with variants in these and five other genes did not identify a disease-associated mutation. In conclusion, a genome-wide scan and additional fine mapping provide evidence for a locus on chromosome 8 that contributes to CHD in a French Canadian population.

Meckel syndrome in the Hutterite population is actually a Joubert-related cerebello-oculo-renal syndrome.

Meckel syndrome (MKS) is a rare lethal autosomal recessive disorder characterized by the presence of occipital encephalocele, cystic kidneys, fibrotic changes of the liver and polydactyly. Joubert syndrome (JS)-related disorders (JSRDs) or cerebello-oculo-renal syndromes (CORS) are a group of recessively inherited conditions characterized by a molar tooth sign (MTS) on cranial MRI, a set of core clinical features (developmental delay/mental retardation, hypotonia, ataxia, episodic breathing abnormalities, abnormal eye movements) and variable involvement of other systems including renal, ocular, central nervous system, craniofacial, hepatic, and skeletal. A significant clinical overlap between MKS and JSRD/CORS has been recognized in the literature. We describe a group of 10 Hutterite patients, of which 7 had been previously diagnosed with MKS, with a JSRD. Clinical features include variable early mortality, cognitive handicap, a characteristic dysmorphic facial appearance, hypotonia, ataxia, abnormal breathing pattern, nystagmus, and MTS on MRI. Additional features include occipital encephalocele, posterior fossa fluid collections resembling Dandy-Walker malformation, hydrocephalus, coloboma, and renal disease. This JSRD is a recognizable dysmorphic syndrome characterized by hypertelorism, deep-set eyes, down-slanting palpebral fissures, ptosis, arched eyebrows with medial sparseness, square nasal tip, short philtrum with tented upper lip, open mouth with down-turned corners, and posteriorly rotated low-set ears. Renal disease is present in 70% of patients and is characterized by cystic kidneys, abnormalities in renal function and hypertension. Homozygous deletions of NPHP1 and the known loci for JS/JSRD and MKS were excluded by identity-by-descent mapping studies suggesting that this condition in the Hutterites represents yet another locus for a JSRD.

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Transmission ratio distortion in the myotonic dystrophy locus in human preimplantation embryos.

One form of myotonic dystrophy, dystrophia myotonica 1 (DM1), is caused by the expansion of a (CTG)(n) repeat within the dystrophia myotonica-protein kinase (DMPK) gene located in chromosome region 19q13.3. Unaffected individuals carry alleles with repeat size (CTG)(5-37), premutation carriers (CTG)(38-49) and DM1 affected individuals (CTG)(50-6,000). Preferential transmission both of expanded repeats from DM1-affected parents and larger DMPK alleles in the normal-size range have been reported in live-born offspring. To determine the moment in development when transmission ratio distortion (TRD) for larger normal-size DMPK alleles is generated, the transmission from heterozygous parents with one repeat within the (CTG)(5-18) range (Group I repeat) and the other within the (CTG)(19-37) range (Group II repeat) to human preimplantation embryos was analysed. A statistically significant TRD of 59% (95% confidence interval of 54-64) in favour of Group II repeats from both mothers and fathers was observed in preimplantation embryos, which remained significant when female embryos were considered separately. In contrast, no significant TRD was detected for repeats from informative Group I/Group I parents. Our analysis showed that Group II repeats specifically were preferentially transmitted in human preimplantation embryos. We suggest that TRD, in Group II repeats at the DMPK locus, is likely to result from events occurring at or around the time of fertilisation.

SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPi-IIc in maintaining phosphate homeostasis.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness, and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. We performed a genomewide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH. The disease mapped to a 1.6-Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaP(i)-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaP(i)-IIc protein in the first membrane-spanning domain and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound heterozygous missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. Individuals of the investigated kindreds who were heterozygous for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. We conclude that NaP(i)-IIc has a key role in the regulation of phosphate homeostasis.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Autosomal recessive cerebellar hypoplasia in the Hutterite population.

Cerebellar hypoplasia is a rare malformation caused by a variety of etiologies. It usually manifests clinically as non-progressive cerebellar ataxia with or without mental retardation. We further characterize a syndrome of autosomal recessive cerebellar hypoplasia in the Hutterite population, referred to as dysequilibrium syndrome (DES). We reviewed 12 patients (eight females, four males; age range 4 to 33 y) with this syndrome. Patients were examined and underwent a standard set of investigations to characterize better the clinical features, natural history, and neuroimaging of this syndrome. DES is an autosomal recessive disorder with distinct clinical features including global developmental delay, late ambulation (after age 6 y), truncal ataxia, and a static clinical course. Neuroimaging is characterized by hypoplasia of the inferior portion of the cerebellar hemispheres and vermis, and mild simplification of cortical gyri.

Molecular biology of hereditary diabetes insipidus.

The identification, characterization, and mutational analysis of three different genes-the arginine vasopressin gene (AVP), the arginine vasopressin receptor 2 gene (AVPR2), and the vasopressin-sensitive water channel gene (aquaporin 2 [AQP2])-provide the basis for understanding of three different hereditary forms of "pure" diabetes insipidus: Neurohypophyseal diabetes insipidus, X-linked nephrogenic diabetes insipidus (NDI), and non-X-linked NDI, respectively. It is clinically useful to distinguish two types of hereditary NDI: A "pure" type characterized by loss of water only and a complex type characterized by loss of water and ions. Patients who have congenital NDI and bear mutations in the AVPR2 or AQP2 genes have a "pure" NDI phenotype with loss of water but normal conservation of sodium, potassium, chloride, and calcium. Patients who bear inactivating mutations in genes (SLC12A1, KCNJ1, CLCNKB, CLCNKA and CLCNKB in combination, or BSND) that encode the membrane proteins of the thick ascending limb of the loop of Henle have a complex polyuro-polydipsic syndrome with loss of water, sodium, chloride, calcium, magnesium, and potassium. These advances provide diagnostic and clinical tools for physicians who care for these patients.

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.

Segregation of urine calcium excretion in families ascertained for nephrolithiasis: evidence for a major gene.

BACKGROUND: The quantitative genetics of urine calcium excretion has not been established. It is a trait of interest because hypercalciuria is commonly found in subjects with nephrolithiasis. The aim of this study was to model the segregation of this trait in a sample of French-Canadian families ascertained through a stone former. METHODS: Major gene, polygenic, and mixed models were fit to 24-hour urine calcium excretion from 567 individuals in 221 nuclear families, while simultaneously taking into account gender, age at examination, body mass index (BMI), and the use of thiazide drugs. The nuclear families were extracted from 154 pedigrees, some of which were four generations, with at least two siblings with a history of calcium stones. RESULTS: All the proposed genetic models fit the data significantly better than the null model. The most parsimonious model was the mixed codominant/polygenic model but it was statistically indistinguishable from the single-gene codominant model. In both of these models the heritability attributable to the major gene was estimated to be 0.58. CONCLUSION: Our results suggest that a major gene with a relatively large effect on variation in urine calcium excretion is segregating in French-Canadian families with stone formers. This implies that the power of quantitative trait segregation analysis of urine calcium excretion may be increased in these families, and results indicate that it should be feasible to genetically map the quantitative trait locus.

A locus for Bowen-Conradi syndrome maps to chromosome region 12p13.3.

Bowen-Conradi syndrome (BCS) is a lethal autosomal recessive disorder with an estimated incidence of 1 in 355 live births in the Hutterite population. A few cases have been reported in other populations. Here, we report the results of a genome-wide scan and fine mapping of the BCS locus in Hutterite families. By linkage and haplotype analysis the BCS locus was mapped to a 3.5 cM segment (1.9 Mbp) in chromosome region 12p13.3 bounded by F8VWF and D12S397. When genealogical relationships among the families were taken into account in the linkage analysis, the evidence for linkage was stronger and the number of potentially linked regions was reduced to one. Under the assumption that all the Hutterite patients were identical by descent for a disease-causing mutation, haplotype analysis was used to infer likely historical recombinants and thereby narrow the candidate region to a chromosomal segment shared in common by all the affected children. This study also demonstrates that BCS and cerebro-oculo-facial-skeletal syndrome (COFS) are genetically distinct.

The most common mutation in FKRP causing limb girdle muscular dystrophy type 2I (LGMD2I) may have occurred only once and is present in Hutterites and other populations.

Limb girdle muscular dystrophy (LGMD) is common in the Hutterite population of North America. We previously identified a mutation in the TRIM32 gene in chromosome region 9q32, causing LGMD2H in approximately two-thirds of the 60 Hutterite LGMD patients studied to date. A genomewide scan was undertaken in five families who did not show linkage to the LGMD2H locus on chromosome 9. A second LGMD locus, LGMD2I, was identified in chromosome region 19q13.3, and the causative mutation was identified as c.826C>A (L276I), a missense mutation in the FKRP gene. A comparison of the clinical characteristics of the two LGMD patient groups in this population reveals some differences. LGMD2I patients generally have an earlier age at diagnosis, a more severe course, and higher serum creatine kinase (CK) levels. In addition, some of these patients show calf hypertrophy, cardiac symptoms, and severe reactions to general anesthesia. None of these features are present among LGMD2H patients. A single common haplotype surrounding the FKRP gene was identified in the Hutterite LGMD2I patients. An identical core haplotype was also identified in 19 other non-Hutterite LGMD2I patients from Europe, Canada, and Brazil. The occurrence of this mutation on a common core haplotype suggests that L276I is a founder mutation that is dispersed among populations of European origin.

Cell-biologic and functional analyses of five new Aquaporin-2 missense mutations that cause recessive nephrogenic diabetes insipidus.

Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a G>A nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein. The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.

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.

Hereditary hypophosphatemic rickets with hypercalciuria is not caused by mutations in the Na/Pi cotransporter NPT2 gene.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a renal phosphate (Pi) wasting disease first described in an extended Bedouin kindred, is characterized by hypophosphatemia, elevated serum 1,25-dihydroxyvitamin D levels, hypercalciuria, rickets, and osteomalacia. Correction of all abnormalities, except for renal Pi wasting, can be achieved by oral Pi supplementation. These findings and the demonstration that mice that are homozygous for the disrupted Na/Pi cotransporter gene Npt2 exhibit many of the biochemical features of HHRH suggested that mutations in the human orthologue NPT2 might be responsible for HHRH. The NPT2 gene in affected individuals from the Bedouin kindred and four small families was screened for mutations to test this hypothesis. No putative disease-causing mutation was found. Two single nucleotide polymorphisms (SNP), a silent substitution in exon 7 and a nucleotide substitution in intron 4, were identified, and neither consistently segregated with HHRH in the Bedouin kindred. Linkage analysis indicated that the two NPT2 intragenic SNP as well as five microsatellite markers in the NPT2 gene region were not linked to HHRH in the Bedouin kindred. Therefore, this is evidence to exclude NPT2 as a candidate gene for HHRH in the families that were studied.

Report of 33 novel AVPR2 mutations and analysis of 117 families with X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease caused by mutations in the arginine vasopressin receptor 2 gene (AVPR2). Thirty-three novel AVPR2 mutations were identified in 62 families that were not included in our previous studies. This study describes the diversity of mutations observed in a total of 117 families, the number of affected people at the time of diagnosis, skewed X chromosome inactivation in severely affected females, the inferred parental origin of de novo mutations, and it provides estimates of incidence. Among 117 families, there were 82 different putative disease-causing mutations. Based on haplotype analysis, it can be inferred that when the same AVPR2 mutation is identified in different families that were not known to be related, the mutations most likely arose independently. More than half of the families had only one affected male; two families presented with a severely affected female and no family history of NDI. A de novo mutation arose during oogenesis in the mother in 20% of isolated cases. The estimate of about 8.8 per million male live births of the incidence of X-linked NDI in the province of Quebec, Canada may be representative of the general population except in Nova Scotia and New Brunswick, where the incidence is more than six times higher. Documentation of the diversity of mutations will assist in revealing the full spectrum of clinical variation. Discussion of genetic and population genetic aspects of X-linked NDI may contribute to early diagnosis and treatment.