Haploidentical hematopoietic stem cell transplantation in a myelofibrosis patient with primary graft failure

The prognosis of patients affected by myelofibrosis (MF) is usually dismal and allogeneic hematopoietic stem cell transplantation (HSCT) remains the only cure. The number of HSCTs in MF patients has recently increased. However, a major obstacle is still represented by primary graft failure (PGF). Currently there are no definitive guidelines for the treatment of PGF and a second HSCT can be performed only when an allogeneic donor is rapidly available. Herein we report on a MF patient with PGF after an unrelated HSCT, who was rescued by a non-myeloablative, unmanipulated, haploidentical HSCT that resulted in persistent engraftment and bone-marrow fibrosis regression, but not in a long-term disease control. Based on this experience we briefly review the role of different conditioning regimens and hematopoietic stem cell sources in the setting of HSCT for MF patients with PGF. The role of haploidentical donors in MF patients lacking HLAmatched relatives is also discussed

Genetic variation in the Italian population at five tandem repeat loci amplified in vitro: use in paternity testing

A multilocus genotype survey of 190 to 352 chromosomes was performed in Italians. Genomic DNA of five tandem repeat loci was amplified in vitro with the polymerase chain reaction. Variable number of tandem repeat (VNTR) or short tandem repeat loci investigated were: D1S80; AP0B, located in the 3' flanking region of the apolipoprotein B gene; D17S5; F8VWF, located in intron 40 of the von Willebrand factor gene; and D6S89. The repeat motif was from 2 to 70 bp. The polymerase chain reaction product size was from 100 to 1070 bp. Relative allele frequencies exhibited bimodal or complex distributions. The number of alleles detected in the sample varied from 10 for F8VWF to 20 for D1S80. The observed heterozygote frequencies of the loci ranged from 0.75 for D17S5 and F8VWF to 0.83 for D1S80, and were in accord with expected frequencies. No mutations were detected in a total of 566 meioses for the five loci studied. The most frequent genotype for all five loci combined has a frequency of 4.1 x 10(-6). In 90 parental determination cases, D1S80, AP0B, D17S5 and F8VWF gave conclusive information in 39/45 exclusions and in 21/45 attributions

Allele and genotype frequencies of eight DNA polymorphisms in the Italian population

We report the allele and genotype frequencies in Italians of eight unlinked commonly utilized polymorphic loci: HUMTHO1, HUMFES/FPS, HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and GC. Genomic DNA from at least 100 individuals was amplified by PCR and typed after electrophoresis (HUMTHO1, HUMFES/FPS), or reverse dot-blot (HLA-DQA1 and the other five loci). The allelic frequencies determined were compared with available population data. These results are useful for population and individual identification studies. \ua9 1995 Academic Press

New insights into FOXP2 alternative splicing

Study of alternative splicing of FOXP

Haploidentical hematopoietic stem cell transplantation in a myelofibrosis patient with primary graft failure

The prognosis of patients affected by myelofibrosis (MF) is usually dismal and allogeneic hematopoietic stem cell transplantation (HSCT) remains the only cure. The number of HSCTs in MF patients has recently increased. However, a major obstacle is still represented by primary graft failure (PGF). Currently there are no definitive guidelines for the treatment of PGF and a second HSCT can be performed only when an allogeneic donor is rapidly available. Herein we report on a MF patient with PGF after an unrelated HSCT, who was rescued by a non-myeloablative, unmanipulated, haploidentical HSCT that resulted in persistent engraftment and bone-marrow fibrosis regression, but not in a long-term disease control. Based on this experience we briefly review the role of different conditioning regimens and hematopoietic stem cell sources in the setting of HSCT for MF patients with PGF. The role of haploidentical donors in MF patients lacking HLAmatched relatives is also discussed

Alternative splicing regulation of human FOXP2 speech gene

FOXP2 gene encodes a forkhead transcription factor linked to speech and language disorders (SPCH1). Three major FOXP2 spliced variants were previously described: a full-length (FOXP2-FL), a transcript starting at exon 4 (FOXP2-III) and an isoform truncated at exon 10 (FOXP2-10+) lacking the DNA binding domain. This study aims to investigate the functional role of FOXP2 variants in cellular models and characterize the RNA-binding proteins involved in transcript maturation. Of the three transcripts, only FOXP2-FL and FOXP2-III were translated into peptides in Hek293 cells. In silico analysis revealed clusters of putative binding sites for Polypyrimidine-Tract Binding protein 1 (PTBP1) in intron sequences flanking exons 3b and 11 of FOXP2. PTBP1 is a hnRNP (heterogeneous nuclear ribonucleoprotein) that mainly regulates exon-skipping. We overexpressed PTBP1 in Hek293 cells and looked at FOXP2 transcripts modulation by RT-PCR and qPCR. No significant change was observed in ratios between FOXP2 variants, but we detected a novel transcript excluding exon 11 (FOXP2-\u39411), which lacks the first of the two nuclear localization signal sequences (NLS1) of FOXP2-FL. Based on this finding we checked for endogenous FOXP2-\u39411 transcript in a panel human cell lines and we found it was expressed in H4 glioblastoma cells. We are in process to verify the translation of FOXP2-\u39411 into peptide: the lack of NLS1 may determine conformation changes influencing FOXP2 dimerization and/or DNA binding and therefore its function in a tissue-specific manner. For further investigations on FOXP2 splicing mediated by PTBP1, we depleted endogenous PTBP1 in Hek293 cells using small interfering RNA (siRNAs). By western blot analysis, we found a significant upregulation of FOXP2-FL, thus supporting the hypothesis that PTBP1 might control FOXP2 expression

"The parental origin of hydatidiform moles and blighted ova: molecular probing with hypervariable DNA polymorphisms"

10nonenoneE.TRABETTI; R.GALAVOTTI; L.ZANINI; E.ZARDINI; N.ZATTI; F.BERNARDI; A.NOTARANGELO; A.CROCE; PF.PIGNATTI; GASPARINI P.E., Trabetti; R., Galavotti; L., Zanini; E., Zardini; N., Zatti; F., Bernardi; A., Notarangelo; A., Croce; Pignatti, P. F.; Gasparini, Paol

Functional characterization of alternatively spliced variants of human FOXP2 speech gene.

FOXP2 gene encodes a forkhead transcription factor recently linked to autism spectrum disorders (ASDs). Three major FOXP2 spliced variants have been described: a full-length (FOXP2-FL), a shorter transcript starting at exon 4 (FOXP2-III) and an isoform truncated at exon 10 (FOXP2-10+). FOXP2-10+ mRNA has a short 3'-UTR and encodes a protein lacking the DNA binding domain, whose transient expression was detected during mouse brain development. This study aims to investigate the functional role of FOXP2 variants in cellular models and characterize the RNA-binding proteins involved in transcript maturation. In Hek293 cells, of the three co-expressed transcripts, only FOXP2-FL and FOXP2-III were translated into peptides, questioning the functional role of FOXP2-10+ transcript. In silico analysis of FOXP2 gene revealed clusters of putative binding sites for Polypyrimidine-Tract Binding protein 1 (PTBP1), a regulator of exon-skipping events in tissue differentiation, including neuronal development. PTBP1 consensus sites were located in intron sequences flanking two exons of FOXP2 and, interestingly, within the 3'-UTR of FOXP2-FL. Based on these findings, we overexpressed PTBP1 in Hek293 cells and analyzed FOXP2 transcript profile. By qPCR, no significant change was observed in ratios between FOXP2 variants, but we detected a novel transcript excluding exon 11 (FOXP2-\u39411), lacking a nuclear localization signal sequence. Of note, intron sequences flanking exon 11 contain putative PTBP1 binding sites. Currently, we are investigating FOXP2 transcript expression upon silencing of endogenous PTBP1 in Hek293 cells and during mouse brain development. We propose PTBP1 as a regulator of FOXP2 transcripts maturation or stability