Detection of mosaicism for segmental and whole chromosome imbalances by targeted sequencing.

Mosaic segmental and whole chromosome copy number alterations are postzygotic variations known to be associated with several disorders. We have previously presented an efficient targeted sequencing approach to simultaneously detect point mutations and copy number variations (CNVs). In this study, we evaluated the efficiency of this approach to detect mosaic CNVs, using seven postnatal and 19 tumor samples, previously characterized by chromosomal microarray analyses (CMA). These samples harbored a total of 28 genomic imbalances ranging in size from 0.68 to 171 Mb, and present in 10-80% of the cells. All CNV regions covered by the platform were correctly identified in postnatal samples, and only seven out of 19 CNVs from tumor samples were not identified either because of a lack of target probes in the affected genomic regions or an absence of minimum reads for an alteration call. These results demonstrate that, in a research setting, this is a robust approach for detecting mosaicism in cases of segmental and whole chromosome alterations. Although the current sequencing platform presented a resolution similar to genomic microarrays, it is still necessary to further validate this approach in a clinical setting in order to replace CMA and sequencing analyses by a single test.

Congenital chromoanagenesis in the routine postnatal chromosomal microarray analyses.

Chromosomal microarray analyses (CMA) have greatly increased both the yield and diagnostic accuracy of postnatal analysis; it has been used as a first-tier cytogenetic test in patients with intellectual disability, autism spectrum disorder, and multiple congenital abnormalities. During the last 15 years, we performed CMA in approximately 8,000 patients with neurodevelopmental and/or congenital disorders, of which 13 (0.16%) genetically catastrophic complex chromosomal rearrangements were identified. These ultrarare rearrangements showed clustering of breakpoints, characteristic of chromoanagenesis events. Al1 13 complex events display underlying formation mechanisms, originating either by a synchronization of the shattering of clustered chromosome regions in which regional asynchrony of DNA replication may be one of the main causes of disruption. We provide an overview of the copy number profiling in these patients. Although several previous studies have suggested that chromoanagenesis is often a genetic disease source in postnatal diagnostic screening, due to either the challenge of clinical interpretation of these complex rearrangements or the limitation of microarray resolution relative to the small size and complexity of chromogenic induced chromosome abnormalities, bringing further attention and to study its occurrence in the clinical setting is extremely important.

Insight into the mechanisms and consequences of recurrent telomere capture associated with a sub-telomeric deletion.

A complex mosaicism of the short arm of chromosome 1 detected by SNP microarray analysis is described in a patient presenting a 4-Mb 1p36 terminal deletion and associated phenotypic features. The array pattern of chromosome 1p displayed an intriguing increase in divergence of the SNP heterozygote frequency from the expected 50% from the centromere towards the 1p36 breakpoint. This suggests that various overlapping segments of UPD were derived by somatic recombination between the 1p homologues. The most likely explanation was the occurrence of a series of events initiated in either a gamete or an early embryonic cell division involving a 1pter deletion rapidly followed by multiple telomere captures, resulting in additive, stepped increases in frequency of homozygosity towards the telomere. The largest segment involved the entire 1p, and at least four other capture events were observed, indicating that at least five independent telomere captures occurred in separate cell lineages. The determination of breakpoint position by detection of abrupt changes in B-allele frequency using a moving window analysis demonstrated that they were identical in blood and saliva, the tissues available for analysis. We developed a model to explain the interaction of parameters determining the mosaic clones and concluded that, while number, size, and position of telomere captures were important initiating determinants, variation in individual clone frequencies was the main contributor to mosaic differences between tissues. All previous reports of telomere capture have been restricted to single events. Other cases involving multiple telomere capture probably exist but require investigation by SNP microarrays for their detection.

Chromosomal microarray analyses from 5778 patients with neurodevelopmental disorders and congenital anomalies in Brazil.

Chromosomal microarray analysis (CMA) has been recommended and practiced routinely since 2010 both in the USA and Europe as the first-tier cytogenetic test for patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. However, in Brazil, the use of CMA is still limited, due to its high cost and complexity in integrating the results from both the private and public health systems. Although Brazil has one of the world's largest single-payer public healthcare systems, nearly all patients referred for CMA come from the private sector, resulting in only a small number of CMA studies in Brazilian cohorts. To date, this study is by far the largest Brazilian cohort (n = 5788) studied by CMA and is derived from a joint collaboration formed by the University of São Paulo and three private genetic diagnostic centers to investigate the genetic bases of neurodevelopmental disorders and congenital abnormalities. We identified 2,279 clinically relevant CNVs in 1886 patients, not including the 26 cases of UPD found. Among detected CNVs, the corresponding frequency of each category was 55.6% Pathogenic, 4.4% Likely Pathogenic and 40% VUS. The diagnostic yield, by taking into account Pathogenic, Likely Pathogenic and UPDs, was 19.7%. Since the rational for the classification is mostly based on Mendelian or highly penetrant variants, it was not surprising that a second event was detected in 26% of those cases of predisposition syndromes. Although it is common practice to investigate the inheritance of VUS in most laboratories around the world to determine the inheritance of the variant, our results indicate an extremely low cost-benefit of this approach, and strongly suggest that in cases of a limited budget, investigation of the parents of VUS carriers using CMA should not be prioritized.

Single Nucleotide Polymorphisms in Cellular Drug Transporters Are Associated with Intolerance to Antiretroviral Therapy in Brazilian HIV-1 Positive Individuals.

Adverse reactions are the main cause of treatment discontinuation among HIV+ individuals. Genes related to drug absorption, distribution, metabolism and excretion (ADME) influence drug bioavailability and treatment response. We have investigated the association between single nucleotide polymorphisms (SNPs) in 29 ADME genes and intolerance to therapy in a case-control study including 764 individuals. Results showed that 15 SNPs were associated with intolerance to nucleoside and 11 to non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), and 8 to protease inhibitors (PIs) containing regimens under alpha = 0.05. After Bonferroni adjustment, two associations remained statistically significant. SNP rs2712816, at SLCO2B1 was associated to intolerance to NRTIs (ORGA/AA = 2.37; p = 0.0001), while rs4148396, at ABCC2, conferred risk of intolerance to PIs containing regimens (ORCT/TT = 2.64; p = 0.00009). Accordingly, haplotypes carrying rs2712816A and rs4148396T alleles were also associated to risk of intolerance to NRTIs and PIs, respectively. Our data reinforce the role of drug transporters in response to HIV therapy and may contribute to a future development of personalized therapies.

Genetic variability of Herpailurus yagouaroundi, Puma concolor and Panthera onca (Mammalia, Felidae) studied using Felis catus microsatellites

We used four microsatellite loci (Fca08, Fca45, Fca77 and Fca96) from the domestic cat, Felis catus, to investigate genetic variability in specimens of Herpailurus yagouaroundi (jaguarundi, otter cat, eyra), Puma concolor (cougar, mountain lion, puma) and Panthera onca (jaguar) held in various Brazilian zoos. Samples of DNA from the cats were PCR amplified and then sequenced before being analyzed using the CERVUS program. Our results show a mean polymorphic information content (PIC) of 0.83 for H. yagouaroundi, 0.66 for P. concolor and 0.69 for P. onca and a mean of 10.3 alleles for the Fca08 locus, 5.3 for Fca 45, 9 for Fca 77 and 14 for Fca 96. These results indicate a relatively high level of genetic diversity for the specimens studied.

Expressäo relativa de fator semelhante a insulina (IGFI) e receptor do homômonio folículo estimulante (FSHR) em folículos e tecido ovariano de Bos primigenius (Nelore) / Relative expression of insulin like growth factor I (IGFI) and follicle stimulating hormone receptor (FSHR) in follicles and ovarian tissue from Bos primigenius indicus (Nelore)

O aperfeiçoamento das técnicas que objetivam a exploraçäo do potencial reprodutivo das fêmeas requer a compreensäo mais ampla dos mecanismos de controle de desenvolvimento folicular. Uma alternativa de estudo nesta esfera, é a quantificaçäo da expressäo relativa de genes envolvidos nos processos de recrutamento, seleçäo e desenvolvimento folicular, pelo emprego da técnica de transcriçäo - reversa associado a reaçäo em cadeia pela polimerase (RT - PCR). O presente trabalho objetivou quantificar a expressäo relativa dos genes insulin-like growth factor I (IGF-I) e do receptor do hormônio folículo estimulante (FSHR), tendo como controle interno o gene da gliceraldeído 3-fosfato desidrogenase (GAPDH). Foram utilizados ovários bovinos de animais de matadouro em diferentes fases do ciclo estral. O RNA total dos folículos e tecido ovarianos foi purificado por TRIZOL. As reaçöes de RT-PCR foram realizadas com o "kit" SuperScriptTM First-Strand. Os produtos de PCR foram analisados em gel de agarose e as bandas submetidas à análise densitométrica. Todos os genes foram amplificados observando-se a curva exponencial de amplificaçäo, a validaçäo do método foi realizada através de análise de regressäo, sendo estabelecido o coeficiente de amplificaçäo (E). A expressäo relativa de mRNA para cada gene de interesse foi calculada pela fórmula estabelecida por Prelle et al.12. Em todos os tecidos analisados, todos os genes foram expressos, sobressaltando-se diferenças nos diferentes ciclos estudados. Com relaçäo os dados referentes ao coeficiente de amplificaçäo (E), observou-se tanto para gene controle (GAPDH), como para o gene IGF-I concordância nos valores encontrados para as diferentes classes analisadas. Quanto ao gene IGF-I, a interpretaçäo dos achados para a expressäo relativa de mRNA pode está relacionada ao caráter constitutivo dessa proteína ou devido os transcritos näo serem dependentes dos níveis de FSH. Observou-se diferenças na expressäo relativa de mRNA de FSHR entre as classes de tecidos analisados, o que pode ser explicado pela variaçäo do número de receptores nas células da granulosa nas diferentes fases do ciclo estral. Pode-se perceber a partir desse estudo que a técnica de RT-PCR semi quantitativo é de grande importância biotecnológica, possibilitando auxílio na compreensäo da dinâmica folicular. Entretanto esses estudos devem ser ampliados com outros genes para melhor compreensäo das etapas fisiológicas envolvidas na foliculogênese