Characterization and functional analysis of cis-acting elements of the human farnesyl diphosphate synthetase (FDPS) gene 5′ flanking region

AbstractFarnesyl diphosphate synthetase (FDPS) is a key enzyme in the isoprenoid pathway responsible for cholesterol biosynthesis, post-translational protein modifications and synthesis of steroid hormones, whose expression is regulated by phorbol esters and polyunsaturated fatty acids.Genomic comparison of the 5′ upstream sequence of the FDPS genes identifies conserved binding sites for NF-Y, SP1, SRE3, and YY1 regulatory elements in rat, mouse, dog and chimpanzee. Two additional specific consensus sequences, upstream of the core promoter that had not been analysed previously, are shared only by human and chimpanzee genomes.The work presented here aimed at characterizing these genomic sequence elements in the human FDPS promoter region and their contribution to gene expression. We have characterized functionally the minimal basal promoter of the human FDPS gene by means of deletion mutants and we have identified two cis-acting elements which modulate the FDPS gene expression and are recognized by Pax5 and OCT-1 transcription factors

Wound Repair Capability in EDS Fibroblasts can be Retrieved by Exogenous Type V Collagen

Impaired wound healing is a typical clinical hallmark of Ehlers-Danlos Syndrome (EDS). Mutated fibroblasts from EDS patients, which deposit an abnormal extracellular matrix, showed defective migration resulting in a marked delay in wound repair. The migratory capability remarkably improved in the presence of exogenous type V collagen

Whole-exome sequencing in patients with protein aggregate myopathies reveals causative mutations associated with novel atypical phenotypes

BACKGROUND: Myofibrillar myopathies (MFM) are a subgroup of protein aggregate myopathies (PAM) characterized by a common histological picture of myofibrillar dissolution, Z-disk disintegration, and accumulation of degradation products into inclusions. Mutations in genes encoding components of the Z-disk or Z-disk-associated proteins occur in some patients whereas in most of the cases, the causative gene defect is still unknown. We aimed to search for pathogenic mutations in genes not previously associated with MFM phenotype.METHODS: We performed whole-exome sequencing in four patients from three unrelated families who were diagnosed with PAM without aberrations in causative genes for MFM.RESULTS: In the first patient and her affected daughter, we identified a heterozygous p.(Arg89Cys) missense mutation in LMNA gene which has not been linked with PAM pathology before. In the second patient, a heterozygous p.(Asn4807Phe) mutation in RYR1 not previously described in PAM represents a novel, candidate gene with a possible causative role in the disease. Finally, in the third patient and his symptomatic daughter, we found a previously reported heterozygous p.(Cys30071Arg) mutation in TTN gene that was clinically associated with cardiac involvement.CONCLUSIONS: Our study identifies a new genetic background in PAM pathology and expands the clinical phenotype of known pathogenic mutations

Increased prevalence of unstable HLA-C variants in HIV-1 rapid-progressor patients

HIV-1 infection in the absence of treatment results in progression toward AIDS. Host genetic factors play a role in HIV-1 pathogenesis, but complete knowledge is not yet available. Since less-expressed HLA-C variants are associated with poor HIV-1 control and unstable HLA-C variants are associated with higher HIV-1 infectivity, we investigated whether there was a correlation between the different stages of HIV-1 progression and the presence of specific HLA-C allotypes. HLA-C genotyping was performed using allele-specific PCR by analyzing a treatment-naïve cohort of 96 HIV-1-infected patients from multicentric cohorts in the USA, Canada, and Brazil. HIV-1-positive subjects were classified according to their different disease progression status as progressors (Ps, n = 48), long-term non-progressors (LTNPs, n = 37), and elite controllers (ECs, n = 11). HLA-C variants were classified as stable or unstable according to their binding stability to β2-microglobulin/peptide complex. Our results showed a significant correlation between rapid progression to AIDS and the presence of two or one unstable HLA-C variants (p-value: 0.0078, p-value: 0.0143, respectively). These findings strongly suggest a link between unstable HLA-C variants both at genotype and at allele levels and rapid progression to AIDS. This work provides further insights into the impact of host genetic factors on AIDS progression

The sacral chordoma margin

[Objective]: Aim of the manuscript is to discuss how to improve margins in sacral chordoma. [Background]: Chordoma is a rare neoplasm, arising in half cases from the sacrum, with reported local failure in >50% after surgery. [Methods]: A multidisciplinary meeting of the “Chordoma Global Consensus Group” was held in Milan in 2017, focusing on challenges in defining and achieving optimal margins in chordoma with respect to surgery, definitive particle radiation therapy (RT) and medical therapies. This review aims to report on the outcome of the consensus meeting and to provide a summary of the most recent evidence in this field. Possible new ways forward, including on-going international clinical studies, are discussed. [Results]: En-bloc tumor-sacrum resection is the cornerstone of treatment of primary sacral chordoma, aiming to achieve negative microscopic margins. Radical definitive particle therapy seems to offer a similar outcome compared to surgery, although confirmation in comparative trials is lacking; besides there is still a certain degree of technical variability across institutions, corresponding to different fields of treatment and different tumor coverage. To address some of these questions, a prospective, randomized international study comparing surgery versus definitive high-dose RT is ongoing. Available data do not support the routine use of any medical therapy as (neo)adjuvant/cytoreductive treatment. [Conclusion]: Given the significant influence of margins status on local control in patients with primary localized sacral chordoma, the clear definition of adequate margins and a standard local approach across institutions for both surgery and particle RT is vital for improving the management of these patients

Identificazione ed analisi della regione 5' del gene umano per l'enzima farnesil difosfato sintetasi (FDPS).

Il gene FDPS (OMIM #134629; 1q22) codifica per Farnesil Difosfato Sintetasi, enzima del pathway colesterogenico cellulare, che catalizza la formazione di geranil- e farnesil-pirofosfato. L\u2019interesse per la regolazione della sua espressione rientra nell\u2019ambito dei nostri studi sulla variabilit\ue0 del carattere densit\ue0 minerale ossea, poich\ue8 esso \ue8, negli osteoclasti, il principale target molecolare degli aminobifosfonati (N-BPs), inibitori del riassorbimento osseo (Kavanagh et al., 2006). Ceppi mutanti di Dyctiostelium discoideum che sovraesprimono FDPS sono resistenti all\u2019azione di N-BPs (Grove et al., 2000). Obiettivi del nostro studio sono: 1) la caratterizzazione della regione promotrice dell\u2019espressione del gene umano; 2) l\u2019identificazione di trascritti alternativi e la loro distribuzione tissutale; 3) la ricerca di varianti polimorfiche nelle regioni regolatrici che influenzino il livello di espressione genica e quindi potenzialmente anche la risposta individuale ai bifosfonati, largamente impiegati nel trattamento dell\u2019osteoporosi. 1) Abbiamo isolato dal genoma umano una regione di 718 pb, comprensive del putativo sito di inizio della trascrizione (TSS) fino al 5\u2019 del primo esone del gene FDPS. L\u2019analisi in silico della regione (contenuto in GC del 55%) rivela la presenza di una TATA box e una CAAT box e siti putativi per fattori di trascrizione quali Sp1, Oct-1, NF-Y, YY1. L\u2019analisi funzionale della regione, eseguita mediante saggi di attivit\ue0 luciferasica in cellule HeLa trasfettate, identifica una attivit\ue0 basale di espressione nella regione prossimale -382/ +118 relativi al sito TSS. E\u2019 in corso la caratterizzazione mediante mutagenesi sito specifica delle sequenze regolative in cis, identificate in silico nel promotore del gene. 2) L\u2019analisi mediante RT-PCR con primer specifici per sequenze esoniche del gene FDPS, eseguita su RNA provenienti da tessuti umani, ha identificato almeno due varianti relative al primo esone, espresse diversamente nei tessuti adulti di fegato, rene, pancreas, milza, timo, prostata, testicolo e ovaia. 3) La ricerca dei polimorfismi nella regione in 5\u2019 al gene FDPS \ue8 stata effettuata tramite sequenziamento della regione fino a 718 bp a monte del sito putativo di inizio della trascrizione in un campione di 28 soggetti. Nessuna variante polimorfica \ue8 stata evidenziata da questa analisi. Al momento stiamo vagliando il possibile ruolo funzionale del polimorfismo intragenico rs2297480 (introne 1). Bibliografia Grove JE, Brown RJ, Watts DJ (2000). J Bone Min Res 15: 971-81. Kavanagh KL, Guo K, Dunford et al., (2006) Proc. Natl. Acad. Sci USA 103: 7829-34

A novel mutation in ABCD1 unveils different clinical phenotypes in a family with adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. The disease is the consequence of mutations in the ABCD1 gene that encodes the peroxisomal membrane protein ALDP which is involved in the transmembrane transport of very long-chain fatty acids. We describe a family with six members carrying a novel heterozygous mutation IVS4+2T>A (c.1393+2T>A) of the ABCD1 gene, highlighting the wide range of phenotypic manifestations of ALD and the importance of genetic screening before any pregnancy in asymptomatic women whose carrier status is unknown