Ethanol-mediated upregulation of APOA1 gene expression in HepG2 cells is independent of de novo lipid biosynthesis.

BACKGROUND: Moderate alcohol intake in human increases HDL-cholesterol, and has protective effects against cardiovascular disease (CVD). Although de novo lipid synthesis inhibitors are highly effective in lowering total and LDL-cholesterol they have only modest effects on raising HDL-C. A better understanding of the mechanism of ethanol-mediated HDL-C regulation could suggest new therapeutic approaches for CVD. METHODS: Human hepatoblastoma (HepG2) and colorectal epithelial adenocarcinoma (Caco-2) cells were incubated in the presence of varying concentrations of ethanol in the culture medium, with or without addition of de novo lipid synthesis (DNLS) inhibitors (atorvastatin and/or TOFA). ApoA1 protein was measured by Western blot, and RNA of lipid pathway genes APOA1, APOC3, APOA4, APOB100, HMGCR, LDLR, and SREBF2 by quantitative RT-PCR. Lipoproteins (VLDL, LDL, and HDL) and lipids were also monitored. RESULTS: Ethanol stimulated ApoA1 protein (both cytoplasmic and secreted) and APOA1 RNA levels in HepG2 cells in a dose sensitive way, with ~ 50% upregulation at 100 mM ethanol in the medium. The effect was not observed in intestinal-derived Caco-2 cells. DNLS inhibitors did not block the upregulation of ApoA1 RNA by ethanol; TOFA alone produced a modest increase in ApoA1 RNA. Ethanol had no effect on ABCA1 protein levels. Addition of ethanol to the cell medium led to modest increases in de novo synthesis of total cholesterol, cholesteryl esters and triglycerides, and as expected these increases were blocked when the lipid synthesis inhibitors were added. Ethanol stimulated a small increase in HDL and VLDL but not LDL synthesis. Ethanol in the cell medium also induced modest but measurable increases in the RNA of APOC3, APOA4, APOB, LDLR, and HMGCR genes. Unlike APOA1, induction of RNA from APOC3 and APOA4 was also observed in Caco-2 cells as well as HepG2 cells. CONCLUSION: This study has verified the previously reported upregulation of APOA1 by exposure of HepG2, but not Caco-2 cells, to ethanol in the culture medium. It is shown for the first time that the effect is dependent on RNA polymerase II-mediated transcription, but not on de novo biosynthesis of cholesterol or fatty acids, and therefore is not a generalized metabolic response to ethanol exposure. Some other lipid pathway genes are also modulated by ethanol exposure of cells. The results reported here suggest that the proximal signaling molecule leading to increased APOA1 gene expression in response to ethanol exposure may be free acetate or acetyl-CoA. TAKE HOME: Upregulation of ApoA1 gene expression in hepatoma cells in culture, upon exposure to moderate ethanol concentrations in the medium, occurs at the level of RNA and is not dependent on new cholesterol or fatty acid synthesis. The primary signaling molecule may be free acetate or acetyl-CoA. These results are important for understanding the mechanism by which moderate alcohol consumption leads to upregulation of serum HDL-cholesterol in humans, and suggests new approaches to targeting HDL as a risk factor for cardiovascular disease.

Family Based Whole Exome Sequencing Reveals the Multifaceted Role of Notch Signaling in Congenital Heart Disease.

Left-ventricular outflow tract obstructions (LVOTO) encompass a wide spectrum of phenotypically heterogeneous heart malformations which frequently cluster in families. We performed family based whole-exome and targeted re-sequencing on 182 individuals from 51 families with multiple affected members. Central to our approach is the family unit which serves as a reference to identify causal genotype-phenotype correlations. Screening a multitude of 10 overlapping phenotypes revealed disease associated and co-segregating variants in 12 families. These rare or novel protein altering mutations cluster predominantly in genes (NOTCH1, ARHGAP31, MAML1, SMARCA4, JARID2, JAG1) along the Notch signaling cascade. This is in line with a significant enrichment (Wilcoxon, p< 0.05) of variants with a higher pathogenicity in the Notch signaling pathway in patients compared to controls. The significant enrichment of novel protein truncating and missense mutations in NOTCH1 highlights the allelic and phenotypic heterogeneity in our pediatric cohort. We identified novel co-segregating pathogenic mutations in NOTCH1 associated with left and right-sided cardiac malformations in three independent families with a total of 15 affected individuals. In summary, our results suggest that a small but highly pathogenic fraction of family specific mutations along the Notch cascade are a common cause of LVOTO.

FORGE Canada Consortium: outcomes of a 2-year national rare-disease gene-discovery project.

Inherited monogenic disease has an enormous impact on the well-being of children and their families. Over half of the children living with one of these conditions are without a molecular diagnosis because of the rarity of the disease, the marked clinical heterogeneity, and the reality that there are thousands of rare diseases for which causative mutations have yet to be identified. It is in this context that in 2010 a Canadian consortium was formed to rapidly identify mutations causing a wide spectrum of pediatric-onset rare diseases by using whole-exome sequencing. The FORGE (Finding of Rare Disease Genes) Canada Consortium brought together clinicians and scientists from 21 genetics centers and three science and technology innovation centers from across Canada. From nation-wide requests for proposals, 264 disorders were selected for study from the 371 submitted; disease-causing variants (including in 67 genes not previously associated with human disease; 41 of these have been genetically or functionally validated, and 26 are currently under study) were identified for 146 disorders over a 2-year period. Here, we present our experience with four strategies employed for gene discovery and discuss FORGE's impact in a number of realms, from clinical diagnostics to the broadening of the phenotypic spectrum of many diseases to the biological insight gained into both disease states and normal human development. Lastly, on the basis of this experience, we discuss the way forward for rare-disease genetic discovery both in Canada and internationally.

The case for locus-specific databases.

Locus-specific databases are the most useful repositories of the sequence information underlying medical genetic conditions and, for this reason, they need our continued support.

Germline mutations in MAP3K6 are associated with familial gastric cancer.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Recessive TTN truncating mutations define novel forms of core myopathy with heart disease.

Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

Disruption of CLPB is associated with congenital microcephaly, severe encephalopathy and 3-methylglutaconic aciduria.

BACKGROUND: The heterogeneous group of 3-methylglutaconic aciduria disorders includes several inborn errors of metabolism that affect mitochondrial function through poorly understood mechanisms. We describe four newborn siblings, from a consanguineous family, who showed microcephaly, small birth weight, severe encephalopathy and 3-methylglutaconic aciduria. Their neurological examination was characterised by severe hypertonia and the induction of prolonged clonic movements of the four limbs upon minimal tactile stimulation. METHODS AND RESULTS: Using homozygosity mapping and exome sequencing, we identified a homozygous truncating mutation (p.I562Tfs*23) in CLPB segregating with the disease in this family. CLPB codes for a member of the family of ATPases associated with various cellular activities (AAA(+) proteins) whose function remains unknown. We found that CLPB expression is abolished in fibroblasts from the patients. To investigate the function of this gene, we interfered with the translation of the zebrafish clpb orthologue using an antisense morpholino. The clpb morphants showed an abnormal touch-evoked response with increased swim velocity and tail beat frequency. This motor phenotype is reminiscent of that observed in the patients and is suggestive of increased excitability in neuronal circuits. Interestingly, knocking down clpb reduced the number of inhibitory glycinergic interneurons and increased a population of excitatory glutamatergic neurons in the spinal cord. CONCLUSIONS: Altogether, our study suggests that disruption of CLPB causes a novel form of neonatal encephalopathy associated with 3-methylglutaconic aciduria.

Mutations in C5ORF42 cause Joubert syndrome in the French Canadian population.

Joubert syndrome (JBTS) is an autosomal-recessive disorder characterized by a distinctive mid-hindbrain malformation, developmental delay with hypotonia, ocular-motor apraxia, and breathing abnormalities. Although JBTS was first described more than 40 years ago in French Canadian siblings, the causal mutations have not yet been identified in this family nor in most French Canadian individuals subsequently described. We ascertained a cluster of 16 JBTS-affected individuals from 11 families living in the Lower St. Lawrence region. SNP genotyping excluded the presence of a common homozygous mutation that would explain the clustering of these individuals. Exome sequencing performed on 15 subjects showed that nine affected individuals from seven families (including the original JBTS family) carried rare compound-heterozygous mutations in C5ORF42. Two missense variants (c.4006C>T [p.Arg1336Trp] and c.4690G>A [p.Ala1564Thr]) and a splicing mutation (c.7400+1G>A), which causes exon skipping, were found in multiple subjects that were not known to be related, whereas three other truncating mutations (c.6407del [p.Pro2136Hisfs*31], c.4804C>T [p.Arg1602*], and c.7477C>T [p.Arg2493*]) were identified in single individuals. None of the unaffected first-degree relatives were compound heterozygous for these mutations. Moreover, none of the six putative mutations were detected among 477 French Canadian controls. Our data suggest that mutations in C5ORF42 explain a large portion of French Canadian individuals with JBTS.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.

Mutations in NFKB2 and potential genetic heterogeneity in patients with DAVID syndrome, having variable endocrine and immune deficiencies.

BACKGROUND: DAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated. METHODS: We ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies. RESULTS: Mutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC). CONCLUSIONS: We confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function.

Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia.

BACKGROUND: Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. METHODS: We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. RESULTS: Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. CONCLUSIONS: Based on our genetic results, TTC7A is the likely causal gene for MIA.

Disruption of TBC1D7, a subunit of the TSC1-TSC2 protein complex, in intellectual disability and megalencephaly.

BACKGROUND: Mutations in TSC1 or TSC2 cause the tuberous sclerosis complex (TSC), a disorder characterised by the development of hamartomas or benign tumours in various organs as well as the variable presence of epilepsy, intellectual disability (ID) and autism. TSC1, TSC2 and the recently described protein TBC1D7 form a complex that inhibits mTORC1 signalling and limits cell growth. Although it has been proposed that mutations in TBC1D7 might also cause TSC, loss of its function has not yet been documented in humans. METHODS AND RESULTS: We used homozygosity mapping and exome sequencing to study a consanguineous family with ID and megalencephaly but without any specific features of TSC. We identified only one rare coding variant, c.538delT:p.Y180fsX1 in TBC1D7, in the regions of homozygosity shared by the affected siblings. We show that this mutation abolishes TBC1D7 expression and is associated with increased mTORC1 signalling in cells of the affected individuals. CONCLUSIONS: Our study suggests that disruption of TBC1D7 causes ID but without the other typical features found in TSC. Although megalencephaly is not commonly observed in TSC, it has been associated with mTORC1 activation. Our observation thus reinforces the relationship between this pathway and the development of megalencephaly.

Identification and biochemical characterization of a novel mutation in DDX11 causing Warsaw breakage syndrome.

Mutations in the gene encoding the iron-sulfur-containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C-induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA-dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity.

A novel rearrangement of occludin causes brain calcification and renal dysfunction.

Pediatric intracranial calcification may be caused by inherited or acquired factors. We describe the identification of a novel rearrangement in which a downstream pseudogene translocates into exon 9 of OCLN, resulting in band-like brain calcification and advanced chronic kidney disease in early childhood. SNP genotyping and read-depth variation from whole exome sequencing initially pointed to a mutation in the OCLN gene. The high degree of identity between OCLN and two pseudogenes required a combination of multiplex ligation-dependent probe amplification, PCR, and Sanger sequencing to identify the genomic rearrangement that was the underlying genetic cause of the disease. Mutations in exon 3, or at the 5-6 intron splice site, of OCLN have been reported to cause brain calcification and polymicrogyria with no evidence of extra-cranial phenotypes. Of the OCLN splice variants described, all make use of exon 9, while OCLN variants that use exons 3, 5, and 6 are tissue specific. The genetic rearrangement we identified in exon 9 provides a plausible explanation for the expanded clinical phenotype observed in our individuals. Furthermore, the lack of polymicrogyria associated with the rearrangement of OCLN in our patients extends the range of cranial defects that can be observed due to OCLN mutations.

Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4.

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.

Mutation in the gene encoding ubiquitin ligase LRSAM1 in patients with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.

De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia.

Schizophrenia likely results from poorly understood genetic and environmental factors. We studied the gene encoding the synaptic protein SHANK3 in 285 controls and 185 schizophrenia patients with unaffected parents. Two de novo mutations (R1117X and R536W) were identified in two families, one being found in three affected brothers, suggesting germline mosaicism. Zebrafish and rat hippocampal neuron assays revealed behavior and differentiation defects resulting from the R1117X mutant. As mutations in SHANK3 were previously reported in autism, the occurrence of SHANK3 mutations in subjects with a schizophrenia phenotype suggests a molecular genetic link between these two neurodevelopmental disorders.

Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.

Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.

Juvenile hemochromatosis is an early-onset autosomal recessive disorder of iron overload resulting in cardiomyopathy, diabetes and hypogonadism that presents in the teens and early 20s (refs. 1,2). Juvenile hemochromatosis has previously been linked to the centromeric region of chromosome 1q (refs. 3-6), a region that is incomplete in the human genome assembly. Here we report the positional cloning of the locus associated with juvenile hemochromatosis and the identification of a new gene crucial to iron metabolism. We finely mapped the recombinant interval in families of Greek descent and identified multiple deleterious mutations in a transcription unit of previously unknown function (LOC148738), now called HFE2, whose protein product we call hemojuvelin. Analysis of Greek, Canadian and French families indicated that one mutation, the amino acid substitution G320V, was observed in all three populations and accounted for two-thirds of the mutations found. HFE2 transcript expression was restricted to liver, heart and skeletal muscle, similar to that of hepcidin, a key protein implicated in iron metabolism. Urinary hepcidin levels were depressed in individuals with juvenile hemochromatosis, suggesting that hemojuvelin is probably not the hepcidin receptor. Rather, HFE2 seems to modulate hepcidin expression.

Genomic Sequence of Canadian Chenopodium berlandieri: A North American Wild Relative of Quinoa.

Chenopodium berlandieri (pitseed goosefoot) is a widespread native North American plant, which was cultivated and consumed by indigenous peoples prior to the arrival of European colonists. Chenopodium berlandieri is closely related to, and freely hybridizes with the domesticated South American food crop C. quinoa. As such it is a potential source of wild germplasm for breeding with C. quinoa, for improved quinoa production in North America. The C. berlandieri genome sequence could also be a useful source of information for improving quinoa adaptation. To this end, we first optimized barcode markers in two chloroplast genes, rbcL and matK. Together these markers can distinguish C. berlandieri from the morphologically similar Eurasian invasive C. album (lamb's quarters). Second, we performed whole genome sequencing and preliminary assembly of a C. berlandieri accession collected in Manitoba, Canada. Our assembly, while fragmented, is consistent with the expected allotetraploid structure containing diploid Chenopodium sub-genomes A and B. The genome of our accession is highly homozygous, with only one variant site per 3-4000 bases in non-repetitive sequences. This is consistent with predominant self-fertilization. As previously reported for the genome of a partly domesticated Mexican accession of C. berlandieri, our genome assembly is similar to that of C. quinoa. Somewhat unexpectedly, the genome of our accession had almost as many variant sites when compared to the Mexican C. berlandieri, as compared to C. quinoa. Despite the overall similarity of our genome sequence to that of C. quinoa, there are differences in genes known to be involved in the domestication or genetics of other food crops. In one example, our genome assembly appears to lack one functional copy of the SOS1 (salt overly sensitive 1) gene. SOS1 is involved in soil salinity tolerance, and by extension may be relevant to the adaptation of C. berlandieri to the wet climate of the Canadian region where it was collected. Our genome assembly will be a useful tool for the improved cultivation of quinoa in North America.