Geographic distribution and regional origin of 272 cystic fibrosis mutations in European populations. The Biomed CF Mutation Analysis Consortium

The geographic distribution of 272 cystic fibrosis (CF) mutations has been studied by assessing the origin of 27,177 CF chromosomes from 29 European countries and three countries from the North of Africa. The most common mutations are delta F308 (66.8%), G542X (2.6%), N1303K (1.6%), G551D (1.5%) and W1282X (1.0%). The delta F508 mutation has the highest frequency in Denmark (87.2%) and the lowest in Algeria (26.3%). Mutation G542X is common in the Mediterranean countries, with a mean frequency of 6.1%. N1303K is found in most of the western and Mediterranean countries and has the highest frequency in Tunisia (17.2%). The wide distribution of these mutations suggests an ancient origin. G551D is common in north-west and central Europe, but is uncommon in other parts of Europe. W1282X has the highest frequency in Israel (36.2%), being also common in most Mediterranean countries and north Africa. Seventeen mutation have frequencies between 0.1 and 0.9%, 1717-1G-->A (0.83%), R553X (0.75%), R1162X (0.51%), 621 + 1G-->T (0.54%) and 2183AA-->G (0.36%), being the most common ones. Some mutations reach relatively high frequencies in some extended geographic regions, such as mutation 394delTT in northern Europe (1.1-28.8%), R117H in northwestern Europe (1.3-3.0%), R553X in central Europe (1.1-24.4%), 1717-1G-->A in Belgium and France (1.1-5.3%), and 2183AA-->G in Italy and Greece (3.2%). Other mutations are only common in small regions: T338I (Sardinia), 711 + 1G-->T (Tunisia), R1162X (Algeria and north of Italy), 1609delCA (east of Spain), 1811 + 1.6kbA-->G (southeastern Spain), R1066C (Portugal), S549R (Algeria), R334W (Crete), 621 + 1G-->T (Central Greece), 3849 + 10kbC-->T (Israel), 2789 + 5G-->A (south of Greece), 451 + 1G--A (Israel), R347P (south of Bulgaria), 1677delTA (south of Bulgaria and Turkey), G85E (south of Greece), R347H (Turkey), 3905insT (Switzerland), 1078delT (Brittany), 1898 + 1G-->A (Wales), A455E (The Netherlands), delta I507 (Brittany), 3659delC (Sweden) and R560T (northern Ireland). Most of these mutations must have an origin and diffusion in the specific European population subgroup. Overall 55 mutations are common in one or several countries or regions of Europe and 217 mutations are rare with relative frequencies of lower than 1% in any of these regions and countries. This information might facilitate mutation analysis of CF in the different regions of Europe

SMA human iPSC-derived motor neurons show perturbed differentiation and reduced miR-335-5P expression

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells

Analysis of intracellular distribution and apoptosis involvement of the Ufd1l gene product by over-expression studies

UFD1L is the human homologue of the yeast ubiquitin fusion degradation 1 (Ufd1) gene and maps on chromosome 22q11.2 in the typically deleted region (TDR) for DiGeorge/velocardiofacial syndromes (DGS/VCFS). In yeast, Ufd1 protein is involved in a degradation pathway for ubiquitin fused products (UFD pathway). Several studies have demonstrated that Ufd1 is a component of the Cdc48-Ufd1-Npl4 multiprotein complex which is active in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for protein degradation or even more specific processing. The multiprotein complex Cdc48-Ufd-Npl4 is also active in mammalian cells. The biochemical role of UFD1L protein in human cells is unknown, even though the interaction between UFD1L and NPL4 proteins has been maintained. In order to clarify this issue, we examined the intracellular distribution of the protein in different mammalian cells and studied its involvement in the Fas and ceramide factors-mediated apoptotic pathways. We established that in mammalian cells, Ufd1l is localized around the nucleus and that it does not interfere with Fas-and ceramide-mediated apoptosis

Small fragment homologous replacement: evaluation of factors influencing modification efficiency in an eukaryotic assay system

Homologous Replacement is used to modify specific gene sequences of chromosomal DNA in a process referred to as "Small Fragment Homologous Replacement", where DNA fragments replace genomic target resulting in specific sequence changes. To optimize the efficiency of this process, we developed a reporter based assay system where the replacement frequency is quantified by cytofluorimetric analysis following restoration of a stably integrated mutated eGFP gene in the genome of SV-40 immortalized mouse embryonic fibroblasts (MEF-SV-40). To obtain the highest correction frequency with this system, several parameters were considered: fragment synthesis and concentration, cell cycle phase and methylation status of both fragment and recipient genome. In addition, different drugs were employed to test their ability to improve technique efficiency. SFHR-mediated genomic modification resulted to be stably transmitted for several cell generations and confirmed at transcript and genomic levels. Modification efficiency was estimated in a range of 0.01-0.5%, further increasing when PARP-1 repair pathway was inhibited. In this study, for the first time SFHR efficiency issue was systematically approached and in part addressed, therefore opening new potential therapeutic ex-vivo applications

Targeted Next Generation Sequencing in patients with Myotonia Congenita

INTRODUCTION: Myotonia Congenita (MC) is a nondystrophic skeletal muscle disease characterized by muscle stiffness, weakness, delayed skeletal relaxation and hypertrophic muscle. The disease can be inherited as dominant or recessive. More than 130 mutations in CLCN1 gene have been identified. MATERIALS AND METHODS: We analyzed the entire coding region and exon-intron boundaries of the CLCN1 gene in 40 MC patients. Samples already Sanger-sequenced were successively evaluated by Next Generation Sequencing (NGS), on Ion Torrent PGM. Moreover, additional 15 patients were sequenced directly by NGS. RESULTS: NGS allowed us to identify all CLCN1 mutations except those located within exon 3, demonstrating a 96% of sensitivity. Due to primer design, one SNP (exactly rs7794560) also failed to be detected. Our results enlarge the spectrum of CLCN1 mutations and showed a novel approach for molecular analysis of MC

MicroRNA genetic variations: association with type 2 diabetes.

Abstract MicroRNAs are small single-stranded molecules that have emerged as important genomic regulators in different pathways. Different studies have shown that they are implicated in the metabolism and glucose homeostasis, and therefore, they could also be involved in the pathogenesis of metabolic disorders such as type 2 diabetes (T2DM). The aim of this study was to verify whether genetic variations in candidate microRNA (miRNA or miR) genes could contribute to T2DM susceptibility. We have selected 13 miRNAs as candidate loci according to literature data and to a computational analysis. MicroRNA genes were analyzed by direct sequencing in a cohort of 163 Italian T2DM patients and 185 healthy controls. We identified 6 novel variants never described before and 9 SNPs already described in databases. Five newly identified variants were found only in the cases group. We performed a case/control association study to test the associations of particular alleles/genotypes of identified SNPs with the disease. Two polymorphisms were associated with T2DM susceptibility: in particular, the G allele of rs895819 in hsa-mir-27a has shown a significantly protective effect (OR = 0.58 and P = 0.008), while the G allele of rs531564 in hsa-mir-124a appears to be a risk allele (OR = 2.15, P = 0.008). This is the first report indicating that genetic polymorphisms in miRNA regions could contribute to T2DM susceptibility

Variants in MHY7 Gene Cause Arrhythmogenic Cardiomyopathy

Background: Arrhythmogenic Cardiomyopathy (ACM) is a disease of the cardiac muscle, characterized by frequent ventricular arrhythmias and functional/ structural abnormalities, mainly of the right ventricle. To date, 20 different genes have been associated with ACM and the majority of them encode for desmosomal proteins. In this study, we describe the characterization of two novel variants in MHY7 gene, segregating in two ACM families. MYH7 encodes for myosin heavy chain β (MHC-β) isoform, involved in cardiac muscle contractility. Method and Results: In family A, the autopsy revealed ACM with biventricular involvement in both the proband and his father. In family B, the proband had been diagnosed as affected by ACM and implanted with implantable cardioverter defibrillator (ICD), due to ECG evidence of monomorphic ventricular tachycardia after syncope. After clinical evaluation, a molecular diagnosis was performed using a NGS custom panel. The two novel variants identified predicted damaging, located in a highly conserved domain: c. 2630T>C is not described while c.2609G>A has a frequency of 0.00000398. In silico analyses evaluated the docking characteristics between proteins using the Haddock2.2 webserver. Conclusions: Our results reveal two variants in sarcomeric genes to be the molecular cause of ACM, further increasing the genetic heterogeneity of the disease; in fact, sarcomeric variants are usually associated with HCM phenotype. Studies on the role of sarcomere genes in the pathogenesis of ACM are surely recommended in those ACM patients negative for desmosomal mutation screening

Common polymorphisms in MIR146a, MIR128a and MIR27a genes contribute to neuropathy susceptibility in type 2 diabetes.

Abstract Diabetic polyneuropathy (DPN) and cardiovascular autonomic neuropathy (CAN) are common type 2 diabetes complications with a large inter-individual variability in terms of clinical manifestations and severity. Our aim was to evaluate a possible involvement of genetic polymorphisms in miRNA regions in the susceptibility to DPN and CAN. Nine polymorphisms in miRNA genes were studied in a sample of 132 type 2 diabetes patients (T2D) analysed for DPN and 128 T2D patients analysed for CAN. A genotype–phenotype correlation analysis was performed. The T allele of rs11888095 single nucleotide polymorphism (SNP) in MIR128a was significantly associated with a higher risk (ORadj = 4.89, Padj = 0.02), whereas the C allele of rs2910164 SNP in MIR146a was associated with a lower risk to develop DPN (ORadj = 0.49, Padj = 0.09), respectively. A multivariate logistic regression analysis confirmed that both SNPs contribute to DPN (p\0.001 and p = 0.01 for MIR128a and MIR146a, respectively). MIR128a SNP significantly contributed also to DPN score (p = 0.026). Rs895819 SNP in MIR27a was significantly associated with a higher risk to develop early CAN (Padj = 0.023 and ORadj = 3.43). The rs2910164 SNP in MIR146a showed a protective effect respect to early CAN (Padj = 0.052, ORadj = 0.32) and to confirmed CAN (Padj = 0.041, ORadj = 0.13). The same SNP resulted significantly associated with a lower CAN score and a higher E/I (p = 0.002 and p = 0.003, respectively). In conclusion, we described associations of MIR128a and MIR146a SNPs with DPN susceptibility and of MIR146a and MIR27a SNPs with CAN susceptibility. This is the first study showing that genetic variability in miRNA genes could be involved in diabetic neuropathies susceptibility