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- Validation of Rare Structural Variants in Portuguese Azoospermic PatientsPublication . Seabra, Catarina; Carvalho, Filipa; Gonçalves, João; Matthiesen, Rune; Fernandes, Susana; Ana, Neto; de Sousa, Mário; Barros, Alberto; Amorim, António; Donald, Conrad; Lopes, AlexandraAzoospermia affects approximately 15% of infertile males. Despite considerable research efforts in the last decades, in the majority of cases the cause remains unidentified. Chromosomal abnormalities and Yq microdeletions have been thoroughly studied, yet only account for 17% of azoospermic men. In fact, little is known about the contribution of the hemizygous X-linked and autosomal genes to male infertility. This study focuses on the validation of rare deletions encompassing candidate genes on the X chromosome and on the autosomes, previously identified by Affymetrix 6.0 SNP Array, in a cohort of 166 Portuguese individuals with severe spermatogenic impairment (non-obstructive azoospermia and severe oligozoospermia). As expected, the protein-coding genes CXORF48, and MAGEA8, as well as a miRNA (hsa-mir-4330) could not be amplified by PCR from the single X chromosome of the patients suspected of carrying deletions. These rearrangements will be further validated by aCGH (array Comparative Genomic Hybridization). Additionally, by MLPA analysis on 11p13 we confirmed a large deletion (~1Mb) spanning the WT1 gene - a conserved transcription factor known to play a crucial role in gonadal differentiation. A retrospective clinical evaluation of this patient revealed partial gonadal dysgenesis, consistent with a causal role for the newly discovered deletion. These results reveal new candidate genes for a role in spermatogenic pathways and suggest that haploinsufficiency of proteins important for the development of the male reproductive system can lead to spermatogenic dysfunction.
- Analysis of the divergences in the nonsense-mediated mRNA decay mechanism among two globin transcripts highly conservedPublication . Pereira, Francisco JC; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs, or nonsense codons), which has been highly conserved during evolution. Knowing that human a- and b-globin genes descend from a common ancestral, the aim of this work was to understand if both encoded mRNAs carrying a nonsense mutation, share parallel NMD profiles. We have previously shown that human b-globin mRNAs carrying a PTC located in close proximity to the translation initiation AUG codon escape NMD. This was called the “AUG-proximity effect”. The present work illustrates that the extension of the AUG-proximity effect, i.e. to what position in the open reading frame (ORF) an AUG-proximal PTC does not trigger NMD, is different between human a- and b-globin mRNAs. Remarkably, our data also demonstrate that, contrary to what occurs in the b-globin transcripts, a-globin mRNAs carrying an AUG-proximal PTC allow for efficient translation re-initiation, although it only partially explains their NMD resistance. In addition, our results reveal that in the a- and b-globin transcripts, the extension of the AUG-proximity effect is determined by the ORF sequence. Furthermore, we show how the mRNA secondary structure, which is affected by the ORF sequence, determines the AUG-proximity effect extension. Our data point out that the time taken to translate the short ORF, affected by its sequence and stability, besides being involved in modulating translation re-initiation, also plays an important role in establishing the extension of the AUG-proximity effect. Thus, although both mRNAs are evolutionarily related they show different features in their NMD response.