RESUMO

Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct imprinted disorders characterized by genetic abnormalities at 15q11-q13. Early diagnosis of both syndromes provides improved treatment and accurate genetic counseling. Whole blood (WB) is the most common DNA source of many methodologies to detect PWS and AS, however, the need of WB makes a massive screening difficult in newborns due to economic and technical limitations. The aim of this study was to adapt a Methylation-sensitive High-Resolution Melting (MS-HRM) approach from dried blood spot (DBS) samples, assessing the different DNA isolation techniques and diagnostic performance. Over a 1-year period, we collected 125 DBS cards, of which 45 had already been diagnosed by MS-HRM (20 PWS, 1 AS, and 24 healthy individuals). We tested three different DBS-DNA extraction techniques assessing the DNA concentration and quality, followed by MS-HRM and statistical comparison. Each DBS-DNA extraction method was capable of accuracy in detecting all PWS and AS individuals. However, the efficiency to detect healthy individuals varied according to methodology. In our experience, DNA extracted from DBS analyzed by the MS-HRM methodology provides an accurate approach for genetic screening of imprinting related disorders in newborns, offering several benefits compared to traditional whole blood methods.

Assuntos

RESUMO

A síndrome de Prader-Willi (SPW) é associada a distúrbios neurológicos, comportamentais e diversas deficiências hormonais, incluindo o hormônio do crescimento (GH). Embora o tratamento de reposição do GH melhore a composição corporal, o crescimento e o quadro clínico geral, esta não é uma cura e suas bases clínicas ainda são desconhecidas na síndrome. A SPW ocorre por três mecanismos moleculares: deleção paterna da região 15q11-q13; dissomia uniparental materna 15; ou defeitos de imprinting. A linhagem celular GH3 de pituitária de rato foi utilizada por ser um modelo bem estudado de células secretoras de GH e prolactina. Utilizando o sistema CRISPR-Cas9, realizamos a deleção completa da região ortóloga da SPW de 3,2 Mb na linhagem GH3, produzindo o genótipo da síndrome in vitro, para investigar quais segmentos gênicos da SPW estão envolvidos na regulação do GH. A investigação de off-targets por sequenciamento Sanger revelou desde reparos sem alteração nucleotídica até grandes rearranjos nos flancos dos sítios-alvo dos gRNAs, inclusive com inserções não previstas de DNA exógeno. Nossos dados ressaltam a necessidade de projetar cuidadosamente as condições experimentais e caracterizar minuciosamente os materiais genéticos obtidos pelo sistema CRISPR-Cas9. A análise do DNA no flanco proximal da região da SPW em ratos demonstrou sequências que podem representar o marco inicial do silenciamento gênico por imprinting na região, assim como ocorre próximo a UBE3A em humanos na porção distal da região ortóloga. Os quatro modelos de sublinhagens SPW-Knockout gerados neste trabalho com deleções de 3,2 Mb envolvendo toda a região da SPW permitirão compreender melhor a relação entre os genes da síndrome e as vias moleculares envolvidas na regulação do GH, além da sua utilização como modelos em novos estudos sobre a SPW. O gene SNORD107 surgiu como o principal candidato a regular o GH dentro da região da SPW, podendo formar um complexo ribonucleoprotéico que exerceria função regulatória pós-transcricional na cadeia de produção do GH.

Prader-Willi syndrome (PWS) is associated with neurodevelopmental and behavioral abnormalities and numerous hormonal deficiencies, including growth hormone (GH). Although GH replacement treatment improves body composition, growth, and the overall clinical presentation, it is not a cure and its clinical basis is still unknown in the syndrome. PWS occurs by three molecular mechanisms: paternal deletion of the 15q11-q13 region; maternal uniparental disomy 15; or imprinting defects. The rat pituitary GH3 cell line is a well-studied model of GH and prolactin-secreting cells. Using the CRISPR-Cas9 system, we performed the complete deletion of the 3.2 Mb PWS orthologous region in GH3 cells, generating the syndrome genotype in vitro to investigate which PWS genes are involved in GH regulation. Off-target analysis by Sanger sequencing revealed perfect breakpoint repairs, but also large rearrangements on the flanking sites of the gRNA targets, including unexpected insertions of exogenous DNA. These data highlight the need to carefully design the experimental conditions and fully characterize the genetic materials obtained by CRISPR-Cas9. DNA analysis on the proximal flank of the PWS region in rats has shown sequences that can mark the initial borders of genomic imprinting, as it occurs close to UBE3A in humans in the distal portion of the orthologous region. The four PWS-Knockout models generated in this work with 3.2 Mb deletions involving the entire PWS region will allow a better understanding of the relationship between PWS genes and the molecular pathways involved in GH regulation and can be used as models in new PWS studies. The SNORD107 gene has emerged as the main candidate to regulate GH within the PWS region and may form a ribonucleoprotein complex with a post-transcriptional regulatory function in the GH production pathway.

Assuntos

RESUMO

Prader-Willi syndrome (PWS) is a complex imprinting disorder related to genomic errors that inactivate paternally-inherited genes on chromosome 15q11-q13 with severe implications on endocrine, cognitive and neurologic systems, metabolism, and behavior. The absence of expression of one or more genes at the PWS critical region contributes to different phenotypes. There are three molecular mechanisms of occurrence: paternal deletion of the 15q11-q13 region; maternal uniparental disomy 15; or imprinting defects. Although there is a clinical diagnostic consensus criteria, DNA methylation status must be confirmed through genetic testing. The endocrine system can be the most affected in PWS, and growth hormone replacement therapy provides improvement in growth, body composition, and behavioral and physical attributes. A key feature of the syndrome is the hypothalamic dysfunction that may be the basis of several endocrine symptoms. Clinical and molecular complexity in PWS enhances the importance of genetic diagnosis in therapeutic definition and genetic counseling. So far, no single gene mutation has been described to contribute to this genetic disorder or related to any exclusive symptoms. Here we proposed to review individually disrupted genes within the PWS critical region and their reported clinical phenotypes related to the syndrome. While genes such as MKRN3, MAGEL2, NDN, or SNORD115 do not address the full spectrum of PWS symptoms and are less likely to have causal implications in PWS major clinical signs, SNORD116 has emerged as a critical, and possibly, a determinant candidate in PWS, in the recent years. Besides that, the understanding of the biology of the PWS SNORD genes is fairly low at the present. These non-coding RNAs exhibit all the hallmarks of RNA methylation guides and can be incorporated into ribonucleoprotein complexes with possible hypothalamic and endocrine functions. Also, DNA conservation between SNORD sequences across placental mammals strongly suggests that they have a functional role as RNA entities on an evolutionary basis. The broad clinical spectrum observed in PWS and the absence of a clear genotype-phenotype specific correlation imply that the numerous genes involved in the syndrome have an additive deleterious effect on different phenotypes when deficiently expressed.

RESUMO

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. This malignancy shows a wide spectrum of clinical outcome and its prognosis is conditioned by manifold biological and genetic factors. We investigated the tumor genetic profile and clinical data of 29 patients with NB by multiplex ligation-dependent probe amplification (MLPA) to assess therapeutic risk. In 18 of these tumors, MYCN status was assessed by fluorescence in situ hybridization (FISH). Copy number variation was also determined for confirming MLPA findings in two 6p loci. We found 2p, 7q and 17q gains, and 1p and 11q losses as the most frequent chromosome alterations in this cohort. FISH confirmed all cases of MYCN amplification detected by MLPA. In view of unexpected 6p imbalance, copy number variation of two 6p loci was assessed for validating MLPA findings. Based on clinical data and genetic profiles, patients were stratified in pretreatment risk groups according to international consensus. MLPA proved to be effective for detecting multiple genetic alterations in all chromosome regions as requested by the International Neuroblastoma Risk Group (INRG) for therapeutic stratification. Moreover, this technique proved to be cost effective, reliable, only requiring standard PCR equipment, and attractive for routine analysis. However, the observed 6p imbalances made PKHD1 and DCDC2 inadequate for control loci. This must be considered when designing commercial MLPA kits for NB. Finally, four patients showed a normal MLPA profile, suggesting that NB might have a more complex genetic pattern than the one assessed by presently available MLPA kits.

Assuntos