Serine/arginine-rich splicing factors (SRSF) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins on mitochondria activity has already been shown for SRSF6 but SRSF4 altered expression has never been reported as disease causing. An 8-year-old patient admitted to the Hematology Unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance on the SRSF4 gene (p.R235W) found by Whole Genome Sequencing analysis and inherited by the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression compared to HD and altered mitochondrial function and energetic metabolism, which appeared associated with low mTOR phosphorylation and an imbalance in proteins regulating biogenesis (i.e., CLUH) and mitochondrial dynamics (i.e., DRP1, OPA1). Transfection with the wt-SRSF4 gene restored the mitochondrial function. In conclusion, this study shows that the de-scribed variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to cells’ mitochondrial function impairment. Since mitochondrial function is crucial for hematopoietic stem cells maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, SRSF4 mutation could have substantially contribute to the clinical phenotype of our patient.
The TLX2 (HOX11L1, Ncx, Enx) and PHOX2B genes encode transcription factors crucial in the development of neural-crest-derived cells, leading to ANS (autonomic nervous system) specific neuronal lineages. Moreover, they share a similar expression pattern and are both involved in downstream steps of BMP (bone morphogenetic protein) signalling. In an attempt to reconstruct the gene network sustaining the correct development of the ANS, we have undertaken an in vitro experimental strategy to identify direct upstream regulators of the TLX2 gene. After characterizing a sequence displaying enhancer property in its 5' flanking region, we confirmed the functional link between the human PHOX2B and TLX2 genes. Transient transfections and electrophoretic-mobility-shift assays suggested that PHOX2B is able to bind the cell-specific element in the 5' regulatory region of the TLX2 gene, determining its transactivation in neuroblastoma cells. Such interaction was also confirmed in vivo by means of chromatin immunoprecipitation assay and, in addition, up-regulation of endogenous TLX2 mRNA level was demonstrated following PHOX2B over-expression, by quantitative real-time PCR. Finally, PHOX2B proteins carrying mutations responsible for CCHS (congenital central hypoventilation syndrome) development showed a severe impairment in activating TLX2 expression, both in vitro and in vivo. Taken together, these results support the PHOX2B-TLX2 promoter interaction, suggesting a physiological role in the transcription-factor cascade underlying the differentiation of neuronal lineages of the ANS during human embryogenesis.
Alexander disease (ALXDRD) is a rare neurodegenerative disorder of astrocytes resulting from pathogenic variants in the GFAP gene. The genotype-phenotype correlation remains elusive due to the variable expressivity of clinical manifestations. In an attempt to clarify the effects of GFAP variants in ALXDRD, numerous studies were collected and analyzed. In particular, we systematically searched for GFAP variants associated with ALXDRD and collected information on the location within the gene and protein, prediction of deleteriousness/pathogenicity, occurrence, sex and country of origin of patients, DNA source, genetic testing, and clinical signs. To identify possible associations, statistical analyses and meta-analyses were applied, thus revealing a higher than expected percentage of adult patients with ALXDRD. Furthermore, substitution of Arginine, the most frequently altered residue among the 550 predominantly missense causative GFAP variants collected, were mostly de novo and more prevalent in early-onset forms of ALXDRD. The effect of defective splicing in modifying the impact of GFAP variants on the age of onset of ALXDRD was also postulated after evaluating the distribution of the corresponding deleterious predictive values. In conclusion, not only previously unrecognized genotype-phenotype correlations were revealed in ALXDRD, but also subtle mechanisms could explain the variable manifestations of the ALXDRD clinical phenotype.
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