Estudio genético de dos fenotipos óseos: osteocondromatosis múltiple y alta masa ósea

[spa]Mi trabajo de tesis está compuesto por dos estudios diferenciados. Por un lado, el análisis molecular de la Osteocondromatosis Múltiple (MO) en pacientes españoles y latinoamericanos. El uso combinado de dos métodos complementarios en la búsqueda de mutaciones (detección de la dosis génica y análisis de la secuencia de ADN) ha permitido descubrir la causa de MO en el 95% de los pacientes españoles y en el 83% de los pacientes latinoamericanos analizados. Se han genotipado los exones y las regiones intrónicas flanqueantes de los genes causantes de la MO, EXT1 y EXT2, en 39 pacientes españoles y 27 latinoamericanos. Se ha identificado la mutación causal en 37 de los pacientes españoles, 29 en EXT1 y 8 en EXT2, 18 de las cuales no habían sido descritas previamente. Tras el análisis mutacional en los pacientes latinoamericanos se ha identificado la mutación causal en 18 de ellos. El análisis mediante MLPA ha permitido descubrir mutaciones de tipo mosaico y se ha confirmado que este tipo de mutaciones también pueden dar lugar a MO. También se ha realizado un estudio de la correlación genotipo-fenotipo en estos pacientes. Este estudio ha permitido determinar que los pacientes españoles con mutaciones missense tienen menor número de osteocondromas que los pacientes con otro tipo de mutaciones. En los pacientes latinoamericanos no se ha podido establecer ninguna correlación entre el grado de severidad de la MO y el gen mutado. Por otra parte, mi tesis aborda el estudio genético molecular del fenotipo alta masa ósea (HBM) en probandos españolas. Se encontraron 13 casos con este fenotipo en los que se analizaron los exones relevantes del gen LRP5, descrito como causante de la HBM y de su inhibidor DKK1. No se encontraron mutaciones en las regiones analizadas del gen LRP5. En una probando con HBM se ha identificado un cambio missense en DKK1 (p.Y74F), no descrito previamente, que podría ser la causa del fenotipo. Se realizó un estudio de 55 loci autosómicos asociados con la densidad mineral ósea (DMO) para comprobar si las probandos tienen un mayor número de alelos protectores frente a la pérdida de masa ósea. En la mayoría de los casos de HBM estudiados, los niveles de DMO se distribuyen inversamente al número de alelos de riesgo de osteoporosis. El único caso en el que esto no se cumple es el que presenta el mayor valor de Z-score y su alta masa ósea podría explicarse por una variante genética rara y penetrante. Por otro lado, se pudo disponer de osteoblastos primarios de dos probandos con HBM y de 5 muestras control y se realizó un análisis transcriptómico de estas células para analizar la expresión diferencial de genes relevantes en el metabolismo óseo en este tipo celular. En el estudio de expresión en osteoblastos primarios se ha observado una correlación negativa entre el valor de Z-score y la expresión de IL6R, DLX3, TWIST1 y PPARG. Este estudio transcriptómico apunta a que tanto un aumento en los niveles de RUNX2 como una disminución en la cantidad de SOX6 podrían tener un papel en algunos casos de HBM. Teniendo en cuenta los resultados obtenidos, se propone un modelo de heterogeneidad genética para la HBM, en la que hay casos debidos a efectos pequeños y aditivos de diversos genes y otros causados principalmente por mutaciones en un único gen.[eng]There are two different studies that compose my thesis. The first one is a molecular analysis of multiple osteochondromas (MO) in Spanish and Latin American patients. MO is a genetically heterogeneous disease caused by mutations in two genes: EXT1 and EXT2. On sequencing all exons and flanking regions of those two genes in the samples of 39 unrelated patients, 37 pathogenic mutations were identified. Twenty-nine different mutations were found in the EXT1 gene, while 8 were found in EXT2. Eighteen out of the 37 mutations were novel. After the mutational analysis in Latin American patients the causative mutation in 18 of them has been identified. MLPA analysis has revealed mosaic mutations and it has been confirmed that such mutations can also lead to MO. Also a study of genotype-phenotype correlation in these patients was made. On the second one, my thesis addresses the molecular genetic study of high bone mass phenotype (HBM). Thirteen cases were found with this phenotype and the relevant exons of LRP5, described as HBM causative gene, and DKK1, that is LRP5 inhibitor, were sequenced. No mutations in the relevant exons of LRP5 were found. A rare missense change in DKK1 was found in one woman (p.Y74F). Fifty-five BMD SNPs were genotyped in the HBM cases to obtain risk scores for each individual. Z-scores were negatively correlated with these risk scores, with a single exception, which may be explained by a rare penetrant genetic variant. An expression analysis in primary osteoblasts from two HBM cases and five controls was carried out. It showed that IL6R, DLX3, TWIST1 and PPARG were negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of heterogeneity and the additive effects of several genes for the HBM phenotype

A broad spectrum of genomic changes in Latinamerican patients with EXT1/EXT2-CDG

Multiple osteochondromatosis (MO), or EXT1/EXT2-CDG, is an autosomal dominant O-linked glycosylation disorder characterized by the formation of multiple cartilage-capped tumors (osteochondromas). In contrast, solitary osteochondroma (SO) is a non-hereditary condition. EXT1 and EXT2, are tumor suppressor genes that encode glycosyltransferases involved in heparan sulfate elongation. We present the clinical and molecular analysis of 33 unrelated Latin American patients (27 MO and 6 SO). Sixty-three percent of all MO cases presented severe phenotype and two malignant transformations to chondrosarcoma (7%). We found the mutant allele in 78% of MO patients. Ten mutations were novel. The disease-causing mutations remained unknown in 22% of the MO patients and in all SO patients. No second mutational hit was detected in the DNA of the secondary chondrosarcoma from a patient who carried a nonsense EXT1 mutation. Neither EXT1 nor EXT2 protein could be detected in this sample. This is the first Latin American research program on EXT1/EXT2-CDG

Genetic analysis of high bone mass cases from the BARCOS cohort of spanish postmenopausal women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas

Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel

Genetic analysis of high bone mass cases from the BARCOS cohort of spanish postmenopausal women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas

Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel

Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas

Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel

Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas

Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel

Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas

Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel