267 Spanish Exomes Reveal Population-Specific Differences in Disease-Related Genetic Variation.

Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ∼10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms.

An impairment of long distance SOX10 regulatory elements underlies isolated Hirschsprung disease.

A deletion encompassing several SOX10 enhancers was recently identified in a patient presenting with Waardenburg syndrome type 4 (WS4), which is defined as a combination of Hirschsprung disease (HSCR, intestinal aganglionosis) and WS (deafness and pigmentation defects). The expression patterns of some of the known SOX10 enhancers in animal models led to the speculation that endophenotypes of WS4 may be linked to mutations within some of these sequences. The present study investigated deletions and point mutations within four SOX10 enhancers in 144 unexplained isolated HSCR cases. One deletion and two point mutations affecting binding sites for known neural crest transcription factors were identified. In vitro functional analysis revealed that the first point mutation disrupts autoregulation by SOX10, whereas the second affects AP2a and SOX10 synergistic activity. The present findings suggest that the mutations within SOX10 enhancers contribute to isolated HSCR.

Overexpression of DNMT3b target genes during Enteric Nervous System development contribute to the onset of Hirschsprung disease.

Hirschsprung disease (HSCR) is attributed to a failure of neural crest cells (NCCs) to migrate, proliferate, differentiate and/or survive in the bowel wall during embryonic Enteric Nervous System (ENS) development. ENS formation is the result from a specific gene expression pattern regulated by epigenetic events, such DNA methylation by the DNA methyltransferases (DNMTs), among other mechanisms. Specifically, DNMT3b de novo methyltransferase is associated with NCCs development and has been shown to be implicated in ENS formation and in HSCR. Aiming to elucidate the specific mechanism underlying the DNMT3b role in such processes, we have performed a chromatin immunoprecipitation coupled with massively parallel sequencing analysis to identify the DNMT3B target genes in enteric precursor cells (EPCs) from mice. Moreover, the expression patterns of those target genes have been analyzed in human EPCs from HSCR patients in comparison with controls. Additionally, we have carried out a search of rare variants in those genes in a HSCR series. Through this approach we found 9 genes showing a significantly different expression level in both groups. Therefore, those genes may have a role in the proper human ENS formation and a failure in their expression pattern might contribute to this pathology.

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.

BACKGROUND: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. RESULTS: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. CONCLUSIONS: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.

Coenzyme Q is irreplaceable by demethoxy-coenzyme Q in plasma membrane of Caenorhabditis elegans.

A procedure was developed to isolate fractions enriched in plasma membrane from Caenorhabditis elegans. Coenzyme Q9 (Q9) was found in plasma membrane isolated from either wild-type or long-lived qm30 and qm51 clk-1 mutant strains of Caenorhabditis elegans, along with dietary coenzyme Q8 (Q8) and the biosynthetic intermediate demethoxy-Q9 (DMQ9). NADH was able to reduce both Q8 and Q9, but not DMQ9. Our results indicate that DMQ9 cannot achieve the same redox role of Q9 in plasma membrane, suggesting that proportion of all these Q isoforms in plasma membrane must be an important factor in establishing the clk-1 mutant phenotype.

Evaluation of the role of RET polymorphisms/haplotypes as modifier loci for MEN 2, and analysis of the correlation with the type of RET mutation in a series of Spanish patients.

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer syndrome, which is divided into three subtypes: MEN 2A, MEN 2B and familial medullary thyroid cancer (FMTC). Approximately 92% of MEN 2 cases are caused by mutations in exons 10, 11, 13-16 of the RET proto-oncogene. There exists inter- and intra-familial phenotypic variability among the MEN 2 families, even when the disease is caused by the same RET mutation, suggesting a role for genetic modifiers, such as polymorphisms/haplotypes. We have sought to determine the frequency and position of RET germline mutations in a cohort of 114 Spanish probands with any sign of MEN 2, and to search for putative modifier loci. Mutational screening of RET revealed 9 different mutations, present in 26 of the 114 probands (22.8%). In addition, distributions of 8 RET polymorphisms and the haplotypes comprising them, were studied in the context of the families positive for RET mutational screening, in order to evaluate them as genetic modifiers. The relationship between RET mutation type and presence of a polymorphism/haplotype was analyzed. The relationship between the presence of pheochromocytoma (PC) and/or hiperparathyroidism (HPT) in carriers of the same RET mutation, and the genotype for the specific variants was also studied. The results derived from those analyses revealed no associations of any variant/haplotype to a specific mutation or to the clinical presentation. Nevertheless, these observations do not permit us to exclude the possible role of other variants in RET or other related genes, in the final presentation of the disease.

Silencing of ubiquinone biosynthesis genes extends life span in Caenorhabditis elegans.

Ubiquinone (coenzyme Q; Q) is a key factor in the mitochondria electron transport chain, but it also functions as an antioxidant and as a cofactor of mitochondrial uncoupling proteins. Furthermore, Q isoforms balance in Caenorhabditis elegans is determined by both dietary intake and endogenous biosynthesis. In the absence of synthesis, withdrawal of dietary Q8 in adulthood extends life span. Thus, Q plays an important role in the aging process and understanding its synthesis acquires a new impetus. We have identified by RNA interference (RNAi) eight genes, including clk-1, involved in ubiquinone biosynthesis in C. elegans feeding animals with dsRNA-containing Escherichia coli HT115 strains. Silenced C. elegans showed lower levels of both endogenous Q9 and Q8 provided by diet, produced less superoxide without a significant modification of mitochondrial electron chain, and extended life span compared with non-interfered animals. E. coli strains harboring dsRNA also interfered with their own Q8 biosynthesis. These findings suggest that more efficient electron transport between a lower amount of Q and electron transport capacity of the mitochondrial complexes leads to less production of reactive oxygen species that contributes to extension of life span in the nematode C. elegans.

Identification of epistatic interactions through genome-wide association studies in sporadic medullary and juvenile papillary thyroid carcinomas.

BACKGROUND: The molecular mechanisms leading to sporadic medullary thyroid carcinoma (sMTC) and juvenile papillary thyroid carcinoma (PTC), two rare tumours of the thyroid gland, remain poorly understood. Genetic studies on thyroid carcinomas have been conducted, although just a few loci have been systematically associated. Given the difficulties to obtain single-loci associations, this work expands its scope to the study of epistatic interactions that could help to understand the genetic architecture of complex diseases and explain new heritable components of genetic risk. METHODS: We carried out the first screening for epistasis by Multifactor-Dimensionality Reduction (MDR) in genome-wide association study (GWAS) on sMTC and juvenile PTC, to identify the potential simultaneous involvement of pairs of variants in the disease. RESULTS: We have identified two significant epistatic gene interactions in sMTC (CHFR-AC016582.2 and C8orf37-RNU1-55P) and three in juvenile PTC (RP11-648k4.2-DIO1, RP11-648k4.2-DMGDH and RP11-648k4.2-LOXL1). Interestingly, each interacting gene pair included a non-coding RNA, providing thus support to the relevance that these elements are increasingly gaining to explain carcinoma development and progression. CONCLUSIONS: Overall, this study contributes to the understanding of the genetic basis of thyroid carcinoma susceptibility in two different case scenarios such as sMTC and juvenile PTC.

Exome sequencing reveals a high genetic heterogeneity on familial Hirschsprung disease.

Hirschsprung disease (HSCR; OMIM 142623) is a developmental disorder characterized by aganglionosis along variable lengths of the distal gastrointestinal tract, which results in intestinal obstruction. Interactions among known HSCR genes and/or unknown disease susceptibility loci lead to variable severity of phenotype. Neither linkage nor genome-wide association studies have efficiently contributed to completely dissect the genetic pathways underlying this complex genetic disorder. We have performed whole exome sequencing of 16 HSCR patients from 8 unrelated families with SOLID platform. Variants shared by affected relatives were validated by Sanger sequencing. We searched for genes recurrently mutated across families. Only variations in the FAT3 gene were significantly enriched in five families. Within-family analysis identified compound heterozygotes for AHNAK and several genes (N = 23) with heterozygous variants that co-segregated with the phenotype. Network and pathway analyses facilitated the discovery of polygenic inheritance involving FAT3, HSCR known genes and their gene partners. Altogether, our approach has facilitated the detection of more than one damaging variant in biologically plausible genes that could jointly contribute to the phenotype. Our data may contribute to the understanding of the complex interactions that occur during enteric nervous system development and the etiopathology of familial HSCR.

Caenorhabditis elegans ubiquinone biosynthesis genes.

Ubiquinone (coenzyme Q, Q) is an essential lipid electron carrier in the mitochondria respiratory chain, and also functions as antioxidant and participates as a cofactor of mitochondrial uncoupling proteins. Caernorhabditis elegans synthesize Q9, but both dietary Q8 intake and endogenous Q9 biosynthesis determine Q balance. Thus, it is of current interest to know the regulatory mechanisms of Q9 biosynthesis in this nematode. Here we review results that leaded to identification of genes involved in Q9 biosynthesis in this nematode using the RNA interference technology. C. elegans coq genes were silenced and depletion of Q content was observed, indicating that the genes related here participate in Q9 biosynthesis. Silenced populations showed an extension of adult life span, probably by the decrease of endogenous oxidative stress produced in mitochondria. We also report the heterologous complementation of C. elegans coq-5 and coq-7 genes in their homologue yeast coq null mutants, leading to restore its ability to growth in non-fermentable sugars. These complemented yeast strains accumulated Q6 but also the intermediate demethoxy-Q6. These findings support the conservative functional homology of these genes.

Hirschsprung's disease as a model of complex genetic etiology.

Hirschsprung disease (HSCR), or aganglionic megacolon, is a developmental disorder characterised by the absence of ganglion cells along variable length of the distal gastrointestinal tract, leading to the most common form of functional intestinal obstruction in neonates and children. Aganglionosis is attributed to a failure of neural crest cells to migrate, proliferate, differentiate or survive during enteric nervous system (ENS) development in the embryonic stage. The incidence of HSCR is estimated at 1/5000 live births and most commonly presents sporadically with reduced penetrance and male predominance, although it can be familial and may be inherited as autosomal dominant or autosomal recessive. In 70% of cases, HSCR occurs as an isolated trait and in the other 30% HSCR is associated with other congenital malformation syndromes. HSCR has a complex genetic etiology with several genes and loci being described as associated with either isolated or syndromic forms. These genes encode for receptors, ligands (especially those participating in the RET and EDNRB signaling transduction pathways), transcriptional factors or other cell elements that are usually involved in the neural crest cell development and migration that give rise to ENS. Nevertheless, the RET proto-oncogene is considered the major disease causing gene in HSCR. A common RET variant within the conserved transcriptional enhancer sequence in intron 1 has been shown to be associated with a great proportion of sporadic cases and could act as a modifier by modulating the penetrance of mutations in other genes and possibly of those mutations in the RET proto-oncogene itself.

A map of human microRNA variation uncovers unexpectedly high levels of variability.

BACKGROUND: MicroRNAs (miRNAs) are key components of the gene regulatory network in many species. During the past few years, these regulatory elements have been shown to be involved in an increasing number and range of diseases. Consequently, the compilation of a comprehensive map of natural variability in a healthy population seems an obvious requirement for future research on miRNA-related pathologies. METHODS: Data on 14 populations from the 1000 Genomes Project were analyzed, along with new data extracted from 60 exomes of healthy individuals from a population from southern Spain, sequenced in the context of the Medical Genome Project, to derive an accurate map of miRNA variability. RESULTS: Despite the common belief that miRNAs are highly conserved elements, analysis of the sequences of the 1,152 individuals indicated that the observed level of variability is double what was expected. A total of 527 variants were found. Among these, 45 variants affected the recognition region of the corresponding miRNA and were found in 43 different miRNAs, 26 of which are known to be involved in 57 diseases. Different parts of the mature structure of the miRNA were affected to different degrees by variants, which suggests the existence of a selective pressure related to the relative functional impact of the change. Moreover, 41 variants showed a significant deviation from the Hardy-Weinberg equilibrium, which supports the existence of a selective process against some alleles. The average number of variants per individual in miRNAs was 28. CONCLUSIONS: Despite an expectation that miRNAs would be highly conserved genomic elements, our study reports a level of variability comparable to that observed for coding genes.

MAX mutations cause hereditary and sporadic pheochromocytoma and paraganglioma.

PURPOSE: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL. DESIGN: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics. RESULTS: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine. CONCLUSIONS: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients.

Novel mutations at RET ligand genes preventing receptor activation are associated to Hirschsprung's disease.

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract. The major susceptibility gene for the disease is the RET proto-oncogene, which encodes a receptor tyrosine kinase activated by the glial cell-derived neurotrophic factor (GDNF) family ligands. We analyzed the coding sequence of GDNF, NTRN, and, for the first time, ARTN and PSPN in HSCR patients and detected several novel variants potentially involved in the pathogenesis of HSCR. In vitro functional analysis revealed that the variant R91C in PSPN would avoid the correct expression and secretion of the mature protein. Moreover, this study also highlighted the role of both this variant and F127L in NRTN in altering RET activation by a significant reduction in phosphorylation. To support the role of PSPN R91C in HSCR phenotype, enteric nervous system (ENS) progenitors were isolated from human postnatal gut tissues and expression of GFRα4, the main co-receptor for PSPN, was demonstrated. This suggests that not only GDNF and NRTN but also PSPN might promote survival of precursor cells during ENS development. In summary, we report for the first time the association of PSPN gene with HSCR and confirm the involvement of NRTN in the disease, with the identification of novel variants in those genes. Our results suggest that the biological consequence of the mutations NTRN F127L and PSPN R91C would be a reduction in the activation of RET-dependent signaling pathways, leading to a defect in the proliferation, migration, and/or differentiation process of neural crest cells within the developing gut and thus to the typical aganglionosis of the HSCR phenotype.

Expression of PROKR1 and PROKR2 in human enteric neural precursor cells and identification of sequence variants suggest a role in HSCR.

BACKGROUND: The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. PRINCIPAL FINDINGS: In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. CONCLUSIONS: Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.

Polymorphisms in the genes encoding the 4 RET ligands, GDNF, NTN, ARTN, PSPN, and susceptibility to Hirschsprung disease.

PURPOSE: Hirschsprung disease (HSCR) is a developmental disorder caused by a failure of neural crest cells to migrate, proliferate, and/or differentiate during the enteric nervous system development. It presents a multifactorial, nonmendelian pattern of inheritance, with several genes playing some role in its pathogenesis. Its major susceptibility gene is the RET protooncogene, which encodes a receptor tyrosine kinase activating several key signaling pathways in the enteric nervous system development. Given the pivotal role of RET in HSCR, the genes encoding their ligands (GDNF, NRTN, ARTN, and PSPN) are also good candidates for the disease. METHODS: We have performed a case-control study using Taqman technology to evaluate 10 polymorphisms within these genes, as well as haplotypes comprising them, as susceptibility factors for HSCR. RESULTS: No differences were found in the allelic frequencies of the variants or in the haplotype distribution between patients and controls. In addition, no particular association was detected of the variants/haplotypes to any demographic/clinical parameters within the group of patients. CONCLUSION: These data would be consistent with the lack of association between these polymorphisms and HSCR, although they do not permit to completely discard a possible role of other variants within these genes in the disease. Moreover, because the gene-by-gene approach does not take into account the polygenic nature of HSCR disease, it would be interesting to investigate sets of variants in many other different susceptibility loci described for HSCR, which may permit to consider possible interactions among susceptibility genes.

NTF-3, a gene involved in the enteric nervous system development, as a candidate gene for Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital disorder caused by a failure of neural crest cells to migrate, proliferate, and/or differentiate during the enteric nervous system (ENS) development. The requirement of the NTF-3/TrkC signaling for the proper development of the ENS, together with the evidences presented by animal models, led us to investigate the involvement of NTF-3 gene in HSCR. We performed both a mutational screening of NTF-3 and a complete evaluation of 3 polymorphisms as genetic susceptibility factors for HSCR. We identified a novel sequence variant, G76R, present in 2 different patients and absent in controls. We postulate that this variation could generate a lack of mature functional NTF-3 proteins in neural crest cell precursors; thus, altering the NTF-3/TrkC signaling pathway and influencing in the adequate ENS development. Although these results do not provide complete assurance of the involvement of this gene in HSCR, given the polygenic nature of the disease and its etiology, investigation of the genes encoding protein members of the signaling pathways governing the ENS development could provide new key findings in the elucidation of this complex disease.

A complex additive model of inheritance for Hirschsprung disease is supported by both RET mutations and predisposing RET haplotypes.

PURPOSE: The RET proto-oncogene is considered to be the major susceptibility gene involved in Hirschsprung disease. Traditional RET germline mutations account for a small subset of Hirschsprung disease patients, but several studies have shown that there is a specific haplotype of RET associated with the sporadic forms of Hirschsprung disease. We have investigated for RET germline mutations and analyzed the RET haplotypic distribution in carriers versus noncarriers of RET germline mutations. METHODS: We have screened the coding region of RET in 106 Spanish Hirschsprung disease patients using dHPLC technology. Statistical comparisons of the distribution of RET haplotypes between sporadic patients with and without a RET germline mutation were performed. RESULTS: Nine novel germline mutations and one previously described were identified. A significant over-transmission of the "Hirschsprung disease haplotype" was detected when comparing transmitted versus nontransmitted alleles in the group of Hirschsprung disease triads without mutation. However, no distortion of the transmission of alleles was found in the group of mutated families. CONCLUSIONS: These results would be concordant with a complex additive model of inheritance. The whole findings seem to suggest that low-penetrance mutations would be necessary but not sufficient and the additional presence of the "Hirschsprung disease haplotype" could contribute to the manifestation of the disease.