A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms.

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model.

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration. The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies.

Genetics of cardiomyopathies: novel perspectives with next generation sequencing.

Cardiomyopathies are a heterogeneous group of primary diseases of the myocardium usually of genetic origin and with familial presentation. The identification of multiple genetic causes for these diseases has opened a new window for early diagnosis, understanding of their natural history and improvement in risk stratification and management. However, in the past years, the clinical application of genetics has been limited by the prohibiting cost and restricted yield of the available genotyping technologies. The emergence of Next Generation Sequencing (NGS) has completely changed this scenario. This group of sequencing technologies allow the evaluation of hundreds or even thousands of genes in parallel at an affordable cost. Now the challenge is not genotyping per se but the interpretation of the complex results that NGS generates. In this paper we review the main aspects related to the application and impact of Next Generation Sequencing in the study of cardiomyopathies: technology, analysis procedures, bioinformatics, clinical validation and interpretation of results.

Phenotype and prognostic correlations of the converter region mutations affecting the ß myosin heavy chain.

OBJECTIVES: The prognostic value of genetic studies in cardiomyopathies is still controversial. Our objective was to evaluate the outcome of patients with cardiomyopathy with mutations in the converter domain of ß myosin heavy chain (MYH7). METHODS: Clinical characteristics and survival of 117 affected members with mutations in the converter domain of MYH7 were compared with 409 patients described in the literature with mutations in the same region. RESULTS: Twenty-five mutations were evaluated (9 in our families including 3 novel (Ile730Asn, Asp717Gly and Arg719Pro)). Clinical diagnoses were hypertrophic (n=407), dilated (n=15), non-compaction (n=4) and restrictive (n=5) cardiomyopathies, unspecified cardiomyopathy (n=11), sudden death (n=50) and 35 healthy carriers. One hundred eighty-four had events (cardiovascular death or transplant). Median event-free survival was 50±2 years in our patients and 53±3 years in the literature (p=0.27). There were significant differences in the outcome between mutation: Ile736Thr had fewer events than other mutations in the region (p=0.01), while Arg719Gln (p<0.01) had reduced event-free survival. CONCLUSIONS: Mutations in the converter region are generally associated with adverse prognosis although there are differences between mutations. The identification of a mutation in this particular region provides important prognostic information that should be considered in the clinical management of affected patients.