C syndrome - what do we know and what could the future hold?

In 1969 Opitz et al. described two siblings with a new syndrome, which they called ‘C syndrome of multiple congenital abnormalities’ and was presented as a ‘probably private syndrome’. After this first description, new cases appeared with highly similar phenotypes and a new syndrome, known as C Syndrome, Opitz C Syndrome or Opitz Trigonocephaly Syndrome (OCS; MIM # 211,750) was firmly established

The ASXL1 mutation p.Gly646Trpfs*12 found in a Turkish boy with Bohring-Opitz syndrome

Bohring‐Opitz syndrome (BOS, MIM #605039) is a rare and severe disease characterized mainly by intrauterine growth retardation, feeding difficulties, severe to profound developmental delay, nonspecific brain abnormalities, microcephaly, flexion at the elbows with ulnar deviation and flexion of the wrists and metacarpophalangeal joints (known as BOS posture) and distinctive facial features.1 Heterozygous ASXL1 truncating mutations have been identified as the main cause of BOS.1, 2 A recent publication 3 called the attention to the fact that mutations associated with BOS are also present in the ExAC (Exome Aggregation Consortium) database.4 As ASXL1 is one of the genes most commonly mutated during hematopoietic clonal expansion of cells, the authors hypothesized that the presence of this mutation in public databases could be due to somatic mosaicism, and they could confirm the hypothesis by manual examination of the ExAC WES reads

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty-two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794-bp deletion (c.532-37-736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions. © 2011 Wiley-Liss, Inc.Fil: Cozar, Mónica. Universidad de Barcelona; EspañaFil: Urreizti, Roser. Universidad de Barcelona; EspañaFil: Vilarinho, Laura. Instituto de Genética Médica Jacinto Magalhaes; PortugalFil: Grosso, Carola. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Estudios de las Metabolopatías Congénitas; ArgentinaFil: Dodelson de Kremer, Raquel. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Estudios de las Metabolopatías Congénitas; ArgentinaFil: Asteggiano, Carla Gabriela. Gobierno de la Provincia de Córdoba. Ministerio de Salud. Hospital de Niños de la Santísima Trinidad; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dalmau, Jaime. Hospital Infantil La Fe; EspañaFil: García, Ana María. Hospital Infantil La Fe; EspañaFil: Vilaseca, María Antonia. Hospital Sant Joan de De ́u; EspañaFil: Grinberg Vaisman, Daniel Raúl. Universidad de Barcelona; EspañaFil: Balcells, Susana. Universidad de Barcelona; Españ

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty‐two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794‐bp deletion (c.532−37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8‐bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions

MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones

Background MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. Methods Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). Results Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. Conclusions We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy

De Novo PORCN and ZIC2 mutations in a highly consanguineous family

We present a Turkish family with two cousins (OC15 and OC15b) affected with syndromic developmental delay, microcephaly, and trigonocephaly but with some phenotypic traits distinct between them. OC15 showed asymmetrical skeletal defects and syndactyly, while OC15b presented with a more severe microcephaly and semilobal holoprosencephaly. All four progenitors were related and OC15 parents were consanguineous. Whole Exome Sequencing (WES) analysis was performed on patient OC15 as a singleton and on the OC15b trio. Selected variants were validated by Sanger sequencing. We did not identify any shared variant that could be associated with the disease. Instead, each patient presented a de novo heterozygous variant in a different gene. OC15 carried a nonsense mutation (p.Arg95*) in PORCN, which is a gene responsible for Goltz-Gorlin syndrome, while OC15b carried an indel mutation in ZIC2 leading to the substitution of three residues by a proline (p.His404_Ser406delinsPro). Autosomal dominant mutations in ZIC2 have been associated with holoprosencephaly 5. Both variants are absent in the general population and are predicted to be pathogenic. These two de novo heterozygous variants identified in the two patients seem to explain the major phenotypic alterations of each particular case, instead of a homozygous variant that would be expected by the underlying consanguinity

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