RESUMEN
Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.
Asunto(s)
Epistasis Genética/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética , Enfermedad de Hirschsprung/genética , Proteínas Proto-Oncogénicas c-ret/genética , Semaforinas/genética , Animales , Secuencia de Bases , Estudio de Asociación del Genoma Completo , Ratones , Datos de Secuencia Molecular , Semaforinas/deficiencia , Semaforinas/metabolismo , Análisis de Secuencia de ADNRESUMEN
Despite it has been reported that several loci are involved in Hirschsprung's disease, the molecular basis of the disease remains yet essentially unknown. The study of collective properties of modules of functionally-related genes provides an efficient and sensitive statistical framework that can overcome sample size limitations in the study of rare diseases. Here, we present the extension of a previous study of a Spanish series of HSCR trios to an international cohort of 162 HSCR trios to validate the generality of the underlying functional basis of the Hirschsprung's disease mechanisms previously found. The Pathway-Based Analysis (PBA) confirms a strong association of gene ontology (GO) modules related to signal transduction and its regulation, enteric nervous system (ENS) formation and other processes related to the disease. In addition, network analysis recovers sub-networks significantly associated to the disease, which contain genes related to the same functionalities, thus providing an independent validation of these findings. The functional profiles of association obtained for patients populations from different countries were compared to each other. While gene associations were different at each series, the main functional associations were identical in all the five populations. These observations would also explain the reported low reproducibility of associations of individual disease genes across populations.
Asunto(s)
Enfermedad de Hirschsprung/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Enfermedad de Hirschsprung/genética , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Hirschsprung disease (HSCR) genetics is a paradigm for the study and understanding of multigenic disorders. Association between Down syndrome and HSCR suggests that genetic factors that predispose to HSCR map to chromosome 21. To identify these additional factors, we performed a dose-dependent association study on chromosome 21 in Down syndrome patients with HSCR. Assessing 10,895 SNPs in 26 Caucasian cases and their parents led to identify two associated SNPs (rs2837770 and rs8134673) at chromosome-wide level. Those SNPs, which were located in intron 3 of the DSCAM gene within a 19 kb-linkage disequilibrium block region were in complete association and are consistent with DSCAM expression during enteric nervous system development. We replicated the association of HSCR with this region in an independent sample of 220 non-syndromic HSCR Caucasian patients and their parents. At last, we provide the functional rationale to the involvement of DSCAM by network analysis and assessment of SOX10 regulation. Our results reveal the involvement of DSCAM as a HSCR susceptibility locus, both in Down syndrome and HSCR isolated cases. This study further ascertains the chromosome-scan dose-dependent methodology used herein as a mean to map the genetic bases of other sub-phenotypes both in Down syndrome and other aneuploidies.
Asunto(s)
Moléculas de Adhesión Celular/genética , Cromosomas Humanos Par 21 , Síndrome de Down/genética , Enfermedad de Hirschsprung/genética , Sitios de Unión , Conexinas/genética , Redes Reguladoras de Genes , Estudios de Asociación Genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Humanos , Polimorfismo de Nucleótido Simple , Unión Proteica , Proteína beta1 de Unión ComunicanteRESUMEN
Hirschsprung disease (HSCR, aganglionic megacolon) is a complex and heterogeneous disease with an incidence of 1 in 5000 live births. Despite the multifactorial determination of HSCR in the vast majority of cases, there is a monogenic subgroup for which private rare RET coding sequence mutations with high penetrance are found (45% of HSCR familial cases). An asymmetrical parental origin is observed for RET coding sequence mutations with a higher maternal inheritance. A parent-of-origin effect is usually assumed. Here we show that a differential reproductive rate for males and females also leads to an asymmetrical parental origin, which was never considered as a possible explanation till now. In the case of HSCR, we show a positive association between penetrance of the mutation and parental transmission asymmetry: no parental transmission asymmetry is observed in sporadic RET CDS mutation carrier cases for which penetrance of the mutation is low, whereas a parental transmission asymmetry is observed in affected sib-pairs for which penetrance of the mutation is higher. This allows us to conclude that the explanation for this parental asymmetry is that more severe mutations have resulted in a differential reproductive rate between male and female carriers.
Asunto(s)
Enfermedad de Hirschsprung/genética , Patrón de Herencia , Proteínas Proto-Oncogénicas c-ret/genética , Reproducción/genética , Alelos , Femenino , Frecuencia de los Genes , Humanos , Masculino , Tasa de Mutación , Sistemas de Lectura Abierta , Linaje , Penetrancia , Factores Sexuales , HermanosRESUMEN
Next-generation sequencing (NGS) technologies can be a boon to human mutation detection given their high throughput: consequently, many genes and samples may be simultaneously studied with high coverage for accurate detection of heterozygotes. In circumstances requiring the intensive study of a few genes, particularly in clinical applications, a rapid turn around is another desirable goal. To this end, we assessed the performance of the bench-top 454 GS Junior platform as an optimized solution for mutation detection by amplicon sequencing of three type 3 semaphorin genes SEMA3A, SEMA3C, and SEMA3D implicated in Hirschsprung disease (HSCR). We performed mutation detection on 39 PCR amplicons totaling 14,014 bp in 47 samples studied in pools of 12 samples. Each 10-hr run was able to generate â¼75,000 reads and â¼28 million high-quality bases at an average read length of 371 bp. The overall sequencing error was 0.26 changes per kb at a coverage depth of ≥20 reads. Altogether, 37 sequence variants were found in this study of which 10 were unique to HSCR patients. We identified five missense mutations in these three genes that may potentially be involved in the pathogenesis of HSCR and need to be studied in larger patient samples.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedad de Hirschsprung/genética , Mutación Missense , Semaforinas/genética , Alelos , Secuencia de Bases , Estudios de Casos y Controles , Cromosomas Humanos Par 7/genética , ADN , Exones , Femenino , Heterocigoto , Enfermedad de Hirschsprung/diagnóstico , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Semaforinas/químicaRESUMEN
Individuals with Down syndrome (DS) display a 40-fold greater risk of Hirschsprung disease (HSCR) than the general population of newborns implicating chromosome 21 in HSCR etiology. Here we demonstrate that the RET enhancer polymorphism RET+9.7 (rs2435357:C>T) at chromosome 10q11.2 is associated with HSCR in DS individuals both by transmission disequilibrium (P=0.0015) and case-control (P=0.0115) analysis of matched cases. Interestingly, the RET+9.7 T allele frequency is significantly different between individuals with DS alone (0.26+/-0.04), HSCR alone (0.61+/-0.04), and those with HSCR and DS (0.41+/-0.04), demonstrating an association and interaction between RET and chromosome 21 gene dosage. This is the first report of a genetic interaction between a common functional variant (rs2435357) and a not infrequent copy number error (chromosome 21 dosage) in two human developmental disorders.
Asunto(s)
Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 21/genética , Síndrome de Down/complicaciones , Elementos de Facilitación Genéticos , Enfermedad de Hirschsprung/complicaciones , Proteínas Proto-Oncogénicas c-ret/genética , Estudios de Casos y Controles , Síndrome de Down/genética , Enfermedad de Hirschsprung/genética , Humanos , Repeticiones de Microsatélite/genética , No Disyunción Genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome.
Asunto(s)
Adaptación Biológica/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Retículo Endoplásmico/fisiología , Estrés Fisiológico/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/fisiología , Ratones , Modelos Biológicos , Modelos Teóricos , Fenotipo , Desnaturalización Proteica/fisiología , Pliegue de Proteína , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
The UDP-glucose:glycoprotein glucosyltransferase (UGT) is a central player of glycoprotein quality control in the endoplasmic reticulum (ER). UGT reglucosylation of nonnative glycopolypeptides prevents their release from the calnexin cycle and secretion. Here, we compared the fate of a glycoprotein with a reversible, temperature-dependent folding defect in cells with and without UGT1. Upon persistent misfolding, tsO45 G was slowly released from calnexin and entered a second level of retention-based ER quality control by forming BiP/GRP78-associated disulfide-bonded aggregates. This correlated with loss in the ability to correct misfolding. Deletion of UGT1 did not affect the stringency of ER quality control. Rather, it accelerated release from calnexin and transfer to the second ER quality control level, but it did so after an unexpectedly long lag, showing that cycling in the calnexin chaperone system is not frenetic, as claimed by existing models, and is fully activated only upon persistent glycoprotein misfolding.
Asunto(s)
Calnexina/metabolismo , Glucosiltransferasas/fisiología , Glicoproteínas/química , Glicoproteínas/metabolismo , Pliegue de Proteína , alfa-Glucosidasas/fisiología , Animales , Línea Celular , Cistina/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Eliminación de Gen , Glucosiltransferasas/genética , Glicoproteínas/fisiología , Glicosilación , Calor , Ratones , Desnaturalización Proteica , Células Madre/enzimología , Células Madre/metabolismo , alfa-Glucosidasas/genéticaRESUMEN
The eukaryotic cell monitors the fidelity of protein folding in the endoplasmic reticulum and only permits properly folded and/or assembled proteins to transit to the Golgi compartment in a process termed "quality control." An endoplasmic reticulum (ER) lumenal sensor for quality control is the UDP-glucose:glycoprotein glucosyltransferase that targets unfolded glycoproteins for transient, calcium-dependent glucosylation. This modification mediates glycoprotein interaction with the folding machinery comprised of calnexin or calreticulin in conjunction with ERp57. Two human UGT homologues, HUGT1 and HUGT2, exist that share 55% identity. The highest degree of identity resides in the COOH-terminal 20% of these proteins, the putative catalytic domain of HUGT1. However, only HUGT1 displays the expected functional activity. The contribution of the NH2-terminal remainder of HUGT1 to glucosyltransferase function is presently unknown. In this report we demonstrate that HUGT2 is localized to the ER in a manner that overlaps the distribution of HUGT1. Analysis of a series of HUGT1 and HUGT2 chimeric proteins demonstrated that the carboxyl-terminal region of HUGT2 contains a catalytic domain that is functional in place of the analogous portion of HUGT1. Whereas neither catalytic domain displayed detectable activity when expressed alone, co-expression of either catalytic domain with the noncatalytic amino-terminal portion of HUGT1 conferred UDP-Glc binding and transfer of glucose that was specific for unfolded glycoprotein substrates. The results indicate that the amino-terminal 80% of HUGT1 is required for activation of the catalytic domain, whereas the homologous portion of HUGT2 cannot provide this function.
Asunto(s)
Proteínas de Transporte de Monosacáridos/química , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Sitios de Unión , Western Blotting , Células COS , Dominio Catalítico , Reactivos de Enlaces Cruzados/farmacología , Cisteína/química , Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Glicosilación , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Eukaryotic cells coordinate protein-folding reactions in the endoplasmic reticulum with gene expression in the nucleus and messenger RNA translation in the cytoplasm. As the rate of protein synthesis increases, protein folding can be compromised, so cells have evolved signal-transduction pathways that control transcription and translation -- the 'unfolded protein response'. Recent studies indicate that these pathways also coordinate rates of protein synthesis with nutrient and energy stores, and regulate cell differentiation to survive nutrient-limiting conditions or to produce large amounts of secreted products such as hormones, antibodies or growth factors.