Domenico A. Coviello

Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia.

Three gross rearrangements of the low density lipoprotein receptor (LDL-R) gene were recognized during a survey of 23 unrelated Italian subjects with familial hypercholesterolemia (FH). Restriction endonuclease data were obtained by Southern blotting and hybridization with exon-specific probes. Proband FH-29 is heterozygous for a 4-kb deletion, which eliminates exons 13 and 14. This mutation is similar to that previously reported by other investigators in one Italian homozygous and two British and Canadian heterozygous patients. Proband FH-30 is homozygous for a 5.5-kb insertion caused by a duplication of exons 16 and 17 of the LDL-R gene. LDL-R mRNA isolated from skin fibroblasts of FH-30 was found to be larger than normal mRNA (5.6 versus 5.3 kb), in concordance with the insertion of the 236 nucleotides corresponding to exons 16 and 17. Proband FH-44 was found to have greater than 25-kb deletion, which eliminates the first six exons and the promoter region of the gene. This is the first example of a deletion that eliminates the promoter as well as the ligand-binding domain of the LDL-R gene. In the skin fibroblasts of this patient, the level of LDL-R mRNA was approximately half that found in control fibroblasts. We designate the new mutations found in FH-30 and FH-44 as FHviterbo and FHBologna-1, respectively, after the names of the Italian cities where the two patients were born.

Amplification of c-myc and pvt-1 homologous sequences in acute nonlymphatic leukemia

Leukemic cells with double minute (DM) chromosomes from an ANLL(M1) patient were found to carry 10-15 fold amplified c-myc sequences. The linked pvt-1-like locus was amplified at the same level, suggesting that the c-myc amplicon is at least 300 kb in size.

Phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) up-regulates the expression of estrogen receptors in human THP-1 leukemia cells.

In the present work, we have inspected expression of estrogen receptors (ER) and their regulation by the phorbol diester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in a leukemic cell line, the THP‐1 cells, using multiple experimental approaches. Firstly, ligand binding assay (LBA) revealed that control (unstimulated) THP‐1 cells express type I (high affinity, limited capacity) ER in the nuclear fraction only, whilst treatment of cells with TPA resulted in the appearance of type I ER in the soluble fraction as well, with the 50 ng/ml dose and the 48 h incubation time being the most effective experimental condition. A concomitant increase of type II ER was also seen in both soluble and nuclear cell fractions. Unstimulated THP‐1 cells were found to be ER negative by immunocytochemistry; conversely, cells exposed to 50 ng/ml TPA for 48 h stained positively for ER, with the majority of cells having a strong nuclear staining. Scrutiny of ER mRNA expression using reverse transcriptase‐polymerase chain reaction showed the presence of a wild type ER transcript in both control and TPA‐treated THP‐1 cells, though levels of ER mRNA were found to be comparatively higher in the latter. This combined evidence would imply that the TPA‐induced differentiation of THP‐1 cells is accompanied by the rise of high affinity (type I) ER, suggesting that estrogens may play a role in the regulation of macrophage activity during the inflammatory and/or the immune response. J. Cell. Biochem. 83: 390–400, 2001.

Duplication of exons 13, 14 and 15 of the LDL-receptor gene in a patient with heterozygous familial hypercholesterolemia

SummaryDuring a survey of Italian patients with familial hypercholesterolemia (FH), we identified an FH heterozygous patient with a gross rearrangement of the low density lipoprotein (LDL) receptor gene. Southern blot analysis of the probands DNA digested with restriction enzymes PvuII, BamHI, BglII and XbaI and hybridization with cDNA probes complementary to the 3′ end of the gene revealed the presence of abnormal fragments that were approximately 7 kb larger than their normal counterparts. DNA digestion with other enzymes (EcoRV, NcoI, KpnI and StuI) and hybridization with probes complementary to exons 13–17 generated normal fragments and an abnormal fragment of 6.3–6.8 kb. These results are consistent with the presence of an insertion of approximately 7 kb caused by a duplication of exons 13, 14 and 15. This is a novel mutation that is most probably the result of an unequal crossing-over between repetitive sequences located in intron 12 and intron 15. This novel mutation has been designated FHBologna 2.

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c‐KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c‐KIT+ hAFS‐derived EV (hAFS‐EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS‐EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA‐Cre, SmnF7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS‐EV. In conclusion, this is the first study showing that c‐KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340–1355

Expression and alternative splicing of fibronectin mRNA in human diploid endothelial cells during aging in vitro.

Different mRNAs for fibronectin arise from the variable processing of a single primary transcript. We used ribonuclease protection assay to investigate the changes occurring in fibronectin expression and the alternative splicing of mRNA precursor during aging in vitro of human diploid endothelial cells. Senescent endothelial cells release more protein and contain 4-5-fold more fibronectin mRNA than young cells. The pattern of alternative splicing of fibronectin mRNA, with the EDA and the CS1 segments largely included (35% and 77%, respectively) and the EDB segment undetectable, correlates well with previous studies at the protein level both in vitro and in vivo. No changes in the splicing pattern of fibronectin mRNA precursor were detected during endothelial cellular senescence. The increased expression of fibronectin in senescent cells may be a result of the activity of interleukin-1 alpha, which is overexpressed in senescent endothelial cells. It could be also important in vivo during aging and in atherosclerotic lesions.

Use of three dna polymorphisms of the ldl receptor gene in the diagnosis of familial hypercholesterolemia

SummaryFamilial hypercholesterolemia (FH) is an autosomal dominant metabolic disorder caused by several different mutations in the low density lipoprotein (LDL) receptor gene. This large number of different mutations, often undetectable in Southern blotting, makes it impossible directly to diagnose the disease. However, restriction fragment length polymorphisms (RFLPs) can be used to follow the inheritance of the defective gene in FH families. In the present study, we report the use of three RFLPs, detected by PvuII, ApaLI and AvaII restriction enzymes, to determine the haplotypes of normal and defective LDL receptor genes in 61 families with FH and in 128 normal individuals. Two of the nine haplotypes were significantly more often associated with the affected genes, whereas one was significantly less frequent. Although none of the associations was strong enough to allow diagnosis in individuals, it was possible, using the three RFLPs, to identify the haplotype of the affected gene in 57 families and to carry out unequivocal diagnosis in 67% of the cases. In four families, PvuII and AvaII detected an abnormal fragment co-segregating with the disease, thus increasing the percentage of diagnosis to 73.4% of the cases.

The human amniotic fluid stem cell secretome effectively counteracts doxorubicin-induced cardiotoxicity.

The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity.

Next-generation sequencing and its initial applications for molecular diagnosis of systemic auto-inflammatory diseases

Objectives Systemic auto-inflammatory disorders (SAIDs) are a heterogeneous group of monogenic diseases sharing a primary dysfunction of the innate immune system. More than 50% of patients with SAID does not show any mutation at gene(s) tested because of lack of precise clinical classification criteria and/or incomplete gene screening. To improve the molecular diagnosis and genotype interpretation of SAIDs, we undertook the development of a next-generation sequencing (NGS)-based protocol designed to simultaneous screening of 10 genes. Methods Fifty patients with SAID, already genotyped for the respective causative gene(s), were massively sequenced for the coding portions of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12, NOD2, PSTPIP1, IL1RN, LPIN2 and PSMB8. Three different bioinformatic pipelines (Ion Reporter, CLC Bio Genomics Workbench, GATK-based in-house workflow) were compared. Results Once resulting variants were compared with the expected mutation list, no workflow turned out to be able to detect all the 79 variants known in the 50 DNAs. Additional variants were also detected, validated by Sanger sequencing and compared to assess true and false positive detection rates of the three workflows. Finally, the overall clinical picture of 34 patients was re-evaluated in the light of the new mutations found. Conclusions The present gene panel has resulted suitable for molecular diagnosis of SAIDs. Moreover, genotype–phenotype correlation has confirmed that the interpretation of NGS data in patients with an undefined inflammatory phenotype is remarkably difficult, thus supporting the need of evidence-based and validated clinical criteria to be used concurrently with the genetic analysis for the final diagnosis and classification of patients with SAIDs.

The Genoa experience of prenatal diagnosis in NF1

Type 1 neurofibromatosis (NF1) is an autosomal dominant disorder with an incidence of about 1 in 3500 live births. Symptoms are highly variable from a few cafè‐au‐lait spots and axillary freckling to plexiform neurofibromas, optic gliomas, pseudarthrosis, and malignancy. Since disease causing mutations are dispersed throughout the gene, prenatal diagnosis is usually performed in familial cases by linkage analysis and rarely by direct characterization of the mutation. We have characterized 48 families and have performed four prenatal diagnoses. In three cases, the linkage analysis was carried out using informative markers. A direct approach using the protein truncation test (PTT) and sequencing was performed in one case in which a R1947X mutation was identified. The extreme variability of the phenotypic expression of the NF1 gene makes reproductive decisions in NF1 families very difficult, as molecular diagnosis cannot predict clinical expression of the disease. The psychological management of the couple is therefore difficult. In two of the three examined families the reproductive choices were not influenced by the specific manifestations of the disease in that family. Copyright