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1.  Putting Pleiotropy and Selection into Context Defines a New Paradigm for Interpreting Genetic Data 
Background
Natural selection shapes many human genes, including some related to complex diseases. Understanding how selection affects genes, especially pleiotropic ones, may be important in evaluating disease associations and the role played by environmental variation. This may be of particular interest for genes with antagonistic roles that cause divergent patterns of selection. The lectin like low-density lipoprotein 1 receptor (LOX-1), encoded by OLR1, is exemplary. It has antagonistic functions in the cardiovascular and immune systems as the same protein domain binds oxidized LDL and bacterial cell wall proteins - the former contributing to atherosclerosis, the latter presumably protecting from infection. We studied patterns of selection in this gene, in humans and non-human primates, to determine whether variable selection can lead to conflicting results in CVD association studies.
Methods and Results
We analyzed sequences from 11 non-human primate species as well as SNP and sequence data from multiple human populations. Results indicate that the derived allele is favored across primate lineages (probably due to recent positive selection). However, both the derived and ancestral alleles were maintained in human populations, especially European ones (possibly due to balancing selection derived from LOX-1's dual roles). Balancing selection likely reflects response to diverse environmental pressures among humans.
Conclusions
These data indicate that differential selection patterns, within and between species, in OLR1 render association studies difficult to replicate even if the gene is etiologically connected to CVD. Selection analyses can identify genes exhibiting gene-environment interactions critical for unraveling disease association.
doi:10.1161/CIRCGENETICS.113.000126
PMCID: PMC3889706  PMID: 23616601
lipoproteins; immune system; genetics; LOX-1 receptor; evolution
2.  Human placenta-derived neurospheres are susceptible to transformation after extensive in vitro expansion 
Introduction
The cancer stem cell model links neoplastic cells with normal stem cell biology, but little is known on how normal stem cells are transformed into cancer stem cells.
Methods
To investigate the processes underlying the transformation of normal stem cells we developed in vitro a cancer stem cell model from human amniotic and chorionic placenta membranes. In this model we studied the expression of specific stem cell molecules by flow cytometry, and genes, by real time RT-PCR. Microscopy immunfluorescence was employed to investigate the proliferative and differentiation patterns. Fluorescence microscopy and FACS were employed to investigate the proliferative and differentiation patterns. To evaluate the tumorigenic potential of our model we injected the cells into NOD.CB17-Prkdcscid/NCrHsd mice.
Results
Normal human stem cells from amniotic and chorionic placenta membranes were converted into neural cell lineages, under specific conditions, to form secondary neurospheres with a capacity for self-renewal. After extensive in vitro culture, these cells underwent spontaneous transformations and acquired a neuroblastoma (NB)-like phenotype with an elevated proliferative potential that is comparable to established neuroblastoma cell lines. The ability of these cells to transform their phenotype was evidenced by increased clonogenic ability in vitro; by augmented expression level of certain proliferation- and transformation-related genes (e.g., CCNA2, MYCN, ENPP2, GRIA3, and KIT); by the presence of multinucleated and hyperdiploid cells. We further demonstrated that the transformed phenotype is an NB by measuring the expression of NB-specific markers, disialoganglioside GD2 and N-Myc proteins.
Conclusions
We have developed a cancer stem cell model starting from normal human stem cells derived from amniotic and chorionic placenta membranes. These cells are able to differentiate into neural cell lineages and to undergo spontaneous transformations and acquire an NB-like phenotype.
doi:10.1186/scrt444
PMCID: PMC4055136  PMID: 24754904
3.  Common sequence variants in the LOXL1 gene in pigment dispersion syndrome and pigmentary glaucoma 
BMC Ophthalmology  2014;14:52.
Background
Single nucleotide polymorphisms (SNPs) within the LOXL1 gene are associated with pseudoesfoliation syndrome and pseudoesfoliation glaucoma. The aim of our study is to investigate a potential involvement of LOXL1 gene in the pathogenesis of pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG).
Methods
A cohort of Caucasian origin of 84 unrelated and clinically well-characterised patients with PDS/PG and 200 control subjects were included in the study. Genomic DNA from whole blood was extracted and the coding and regulatory regions of LOXL1 gene were risequenced in both patients and controls to identify unknown sequence variations. Genotype and haplotype analysis were performed with UNPHASED software. The expression levels of LOXL1 were determined on c-DNA from peripheral blood lymphocytes by quantitative real-time RT-PCR.
Results
A significant allele association was detected for SNP rs2304722 within the fifth intron of LOXL1 (Odds ratio (OR = 2.43, p-value = 3,05e-2). Haplotype analysis revealed the existence of risk and protective haplotypes associated with PG-PDS (OR = 3.35; p-value = 1.00e-5 and OR = 3.35; p-value = 1.00e-4, respectively). Expression analysis suggests that associated haplotypes can regulate the expression level LOXL1.
Conclusions
Haplotypes of LOXL1 are associated with PG-PDS independently from rs1048661, leading to a differential expression of the transcript.
doi:10.1186/1471-2415-14-52
PMCID: PMC4028282  PMID: 24739284
Pigment dispersion syndrome; Pigmentary glaucoma; Genetics; LOXL1
4.  An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy 
Nature genetics  2013;45(8):947-950.
DNA polymerase delta, whose catalytic subunit is encoded by POLD1, is responsible for lagging strand DNA synthesis during DNA replication1. It achieves this with high fidelity due to its intrinsic 3′ to 5′ exonuclease activity, which confers proofreading ability. Missense mutations in the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancer2. Here we report a recurring heterozygous single amino acid deletion at the polymerase active site of POLD1 that abolishes DNA polymerase activity but only mildly impairs 3′ to 5′ exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This suggests that perturbation of function of the ubiquitously expressed POLD1 polymerase has surprisingly tissue-specific effects in man, and argues for an important role for POLD1 function in adipose tissue homeostasis.
doi:10.1038/ng.2670
PMCID: PMC3785143  PMID: 23770608
5.  Overexpression of CUGBP1 in Skeletal Muscle from Adult Classic Myotonic Dystrophy Type 1 but Not from Myotonic Dystrophy Type 2 
PLoS ONE  2013;8(12):e83777.
Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested.
doi:10.1371/journal.pone.0083777
PMCID: PMC3869793  PMID: 24376746
6.  Genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1 
Strange, Amy | Capon, Francesca | Spencer, Chris CA | Knight, Jo | Weale, Michael E | Allen, Michael H | Barton, Anne | Band, Gavin | Bellenguez, Céline | Bergboer, Judith GM | Blackwell, Jenefer M | Bramon, Elvira | Bumpstead, Suzannah J | Casas, Juan P | Cork, Michael J | Corvin, Aiden | Deloukas, Panos | Dilthey, Alexander | Duncanson, Audrey | Edkins, Sarah | Estivill, Xavier | Fitzgerald, Oliver | Freeman, Colin | Giardina, Emiliano | Gray, Emma | Hofer, Angelika | Hüffmeier, Ulrike | Hunt, Sarah E | Irvine, Alan D | Jankowski, Janusz | Kirby, Brian | Langford, Cordelia | Lascorz, Jesús | Leman, Joyce | Leslie, Stephen | Mallbris, Lotus | Markus, Hugh S | Mathew, Christopher G | McLean, WH Irwin | McManus, Ross | Mössner, Rotraut | Moutsianas, Loukas | Naluai, Åsa T | Nestle, Frank O | Novelli, Giuseppe | Onoufriadis, Alexandros | Palmer, Colin NA | Perricone, Carlo | Pirinen, Matti | Plomin, Robert | Potter, Simon C | Pujol, Ramon M | Rautanen, Anna | Riveira-Munoz, Eva | Ryan, Anthony W | Salmhofer, Wolfgang | Samuelsson, Lena | Sawcer, Stephen J | Schalkwijk, Joost | Smith, Catherine H | Ståhle, Mona | Su, Zhan | Tazi-Ahnini, Rachid | Traupe, Heiko | Viswanathan, Ananth C | Warren, Richard B | Weger, Wolfgang | Wolk, Katarina | Wood, Nicholas | Worthington, Jane | Young, Helen S | Zeeuwen, Patrick LJM | Hayday, Adrian | Burden, A David | Griffiths, Christopher EM | Kere, Juha | Reis, André | McVean, Gilean | Evans, David M | Brown, Matthew A | Barker, Jonathan N | Peltonen, Leena | Donnelly, Peter | Trembath, Richard C
Nature genetics  2010;42(11):985-990.
doi:10.1038/ng.694
PMCID: PMC3749730  PMID: 20953190
7.  PTX3 genetic variation and dizygotic twinning in The Gambia: could pleiotropy with innate immunity explain common dizygotic twinning in Africa? 
Annals of human genetics  2012;76(6):454-463.
Dizygotic (DZ) twinning has a genetic component and is common among sub-Saharan Africans; in The Gambia its frequency is up to 3% of live births. Variation in Pentraxin 3 (PTX3), a soluble pattern recognition receptor that plays an important role both in humoral innate immunity and in female fertility, has been associated with resistance to M. tuberculosis infection and to P. aeruginosa infection in cystic fibrosis patients. We tested whether PTX3 variants in Gambian women associate with DZ twinning, by genotyping five PTX3 single nucleotide polymorphisms (SNPs) in 130 sister pairs (96 full sibs and 34 half sibs) who had DZ twins. We found that two, three and five SNP haplotypes differed in frequency between twinning mothers and those without a history of twinning (from p = 0.006 to 3.03e-06 for two SNP and three SNP haplotypes, respectively). Twinning mothers and West African tuberculosis-controls from a previous study shared several frequent haplotypes. Most importantly, our data are consistent with the previously reported association of PTX3 and female fertility in a West African sample from Ghana. Taken together, these results indicate that selective pressure on PTX3 variants that affect the innate immune response to infectious agents, could also produce the observed high incidence of DZ twinning in Gambians.
doi:10.1111/j.1469-1809.2012.00723.x
PMCID: PMC3731069  PMID: 22834944
dizygotic twinning; fertility; innate immunity; Pentraxin 3; The Gambia; Africa
8.  CFTR gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR) 
Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, “Small Fragment Homologous Replacement (SFHR)”, has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely last any measurable CFTR-dependent chloride efflux The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.
PMCID: PMC3725395  PMID: 17981772
Homologous Replacement; Real-Time PCR; SFHR; Embryonic Stem Cells; CFTR
10.  Haplotypes in IL-8 Gene Are Associated to Age-Related Macular Degeneration: A Case-Control Study 
PLoS ONE  2013;8(6):e66978.
Background
Age-related macular degeneration (AMD) is the main cause of blindness in the developed world. The etiology of AMD is multifactorial due to the interaction between genetic and environmental factors. IL-8 has a role in inflammation and angiogenesis; we report the genetic characterization of IL-8 allele architecture and evaluate the role of SNPs or haplotypes in the susceptibility to wet AMD, case-control study.
Methods
Case-control study including 721 AMD patients and 660 controls becoming from Italian population. Genotyping was carried out by Real Time-PCR. Differences in the frequencies were estimated by the chi-square test. Direct sequencing was carried out by capillary electrophoresis trough ABI3130xl.
Results
rs2227306 showed a p–value of 4.15*10−5 and an Odds Ratio (OR) for T allele of 1.39 [1.19–1.62]. After these positive results, we sequenced the entire IL-8 regulatory and coding regions of 60 patients and 30 controls stratified for their genotype at rs2227306. We defined two different haplotypes involving rs4073 (A/T), rs2227306 (C/T), rs2227346 (C/T) and rs1126647 (A/T): A-T-T-T (p-value: 2.08*10−9; OR: 1.68 [1.43–1.97]) and T-C-C-A (p-value: 7.07*10−11; OR: 0.60 [0.51–0.70]). To further investigate a potential functional role of associated haplotypes, we performed an expression study on RNA extracted from whole blood of 75 donors to verify a possible direct correlation between haplotype and gene expression, failing to reveal significant differences.
Conclusions
These results suggest a possible secondary role of IL-8 gene in the development of the disease. This paper outlines the importance of association between inflammation and AMD. Moreover IL-8 is a new susceptibility genomic biomarker of AMD.
doi:10.1371/journal.pone.0066978
PMCID: PMC3686762  PMID: 23840568
11.  Identification of fifteen new psoriasis susceptibility loci highlights the role of innate immunity 
Nature genetics  2012;44(12):1341-1348.
Summary
To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of three genome-wide association studies (GWAS) and two independent datasets genotyped on the Immunochip, involving 10,588 cases and 22,806 controls in total. We identified 15 new disease susceptibility regions, increasing the number of psoriasis-associated loci to 36 for Caucasians. Conditional analyses identified five independent signals within previously known loci. The newly identified shared disease regions encompassed a number of genes whose products regulate T-cell function (e.g. RUNX3, TAGAP and STAT3). The new psoriasis-specific regions were notable for candidate genes whose products are involved in innate host defense, encoding proteins with roles in interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C), and NF-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.
doi:10.1038/ng.2467
PMCID: PMC3510312  PMID: 23143594
12.  The Pharmacogenomic HLA Biomarker Associated to Adverse Abacavir Reactions: Comparative Analysis of Different Genotyping Methods 
Current Genomics  2012;13(4):314-320.
Many pharmacogenomic biomarkers (PGBM) were identified and translated into clinical practice, affecting the usage of drugs via label updates. In this context, abacavir is one of the most brilliant examples of pharmacogenetic studies translated into clinical practice. Pharmacogenetic studies have revealed that abacavir HSRs are highly associated with the major histocompatibility complex class I. Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results the abacavir label has been modified: the European Medicines Agency (EMA) and the FDA recommend/suggested that the administration of abacavir must be preceded by a specific genotyping test. The HLA locus is extremely polymorphic, exhibiting many closely related alleles, making it difficult to discriminate HLA-B*57:01 from other related alleles, and a number of different molecular techniques have been developed recently to detect the presence of HLA-B*57:01. In this review, we provide a summary of the available techniques used by laboratories to genotype HLA-B*57:01, outlining the scientific and pharmacoeconomics pros and cons.
doi:10.2174/138920212800793311
PMCID: PMC3394119  PMID: 23204921
HLA-B*57:01; abacavir; hypersensitivity reaction (HSR); pharmacogenomics.
14.  Familial partial lipodystrophy, mandibuloacral dysplasia and restrictive dermopathy feature barrier-to-autointegration factor (BAF) nuclear redistribution 
Cell Cycle  2012;11(19):3568-3577.
Prelamin A processing impairment is a common feature of a restricted group of rare genetic alterations/disorders associated with a wide range of clinical phenotypes. Changes in histone posttranslational modifications, alterations in non-histone chromatin proteins and chromatin disorganization have been specifically linked to impairment of specific, distinct prelamin A processing steps, but the molecular mechanism involved in these processes is not yet understood . In this study, we show that the accumulation of wild-type prelamin A detected in restrictive dermopathy (RD), as well as the accumulation of mutated forms of prelamin A identified in familial partial lipodystrophy (FPLD) and mandibuloacral dysplasia (MADA), affect the nuclear localization of barrier-to-autointegration factor (BAF), a protein able to link lamin A precursor to chromatin remodeling functions. Our findings, in accordance with previously described results, support the hypothesis of a prelamin A involvement in BAF nuclear recruitment and suggest BAF-prelamin A complex as a protein platform usually activated in prelamin A-accumulating diseases. Finally, we demonstrate the involvement of the inner nuclear membrane protein emerin in the proper localization of BAF-prelamin A complex.
doi:10.4161/cc.21869
PMCID: PMC3478308  PMID: 22935701
BAF; BANF1; prelamin A; lamin A/C; laminopathies; emerin; EDMD1
15.  p53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells 
PLoS ONE  2012;7(9):e45129.
Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.
doi:10.1371/journal.pone.0045129
PMCID: PMC3446967  PMID: 23028800
16.  Meta-analysis confirms the LCE3C_LCE3B deletion as a risk factor for psoriasis in several ethnic groups and finds interaction with HLA-Cw6 
A multicenter meta-analysis including data from 9389 psoriasis patients and 9477 control subjects was performed to investigate the contribution of the deletion of genes LCE3C and LCE3B, involved in skin barrier defense, to psoriasis susceptibility in different populations. The study confirms that the deletion of LCE3C and LCE3B is a common genetic factor for susceptibility to psoriasis in European populations [OROverall = 1.21 (1.15–1.27)], and for the first time directly demonstrated the deletion's association with psoriasis in [Chinese OR = 1.27 (1.16–1.34); Mongolian OR = 2.08 (1.44–2.99)] populations. The analysis of the HLA-Cw6 locus showed significant differences in the epistatic interaction with the LCE3C and LCE3B deletion in at least some European populations, indicating epistatic effects between these two major genetic contributors to psoriasis. The study highlights the value of examining genetic risk factors in multiple populations to identify genetic interactions, and indicates the need of further studies to understand the interaction of the skin barrier and the immune system in susceptibility to psoriasis.
doi:10.1038/jid.2010.350
PMCID: PMC3386316  PMID: 21107349
17.  Altered chromatin organization and SUN2 localization in mandibuloacral dysplasia are rescued by drug treatment 
Histochemistry and Cell Biology  2012;138(4):643-651.
Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane–centrosome interplay and nuclear movement may be affected in MADA fibroblasts.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-012-0977-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-012-0977-5
PMCID: PMC3432780  PMID: 22706480
Mandibuloacral dysplasia type A (MADA); Prelamin A forms; SUN2; Heterochromatin defects; Statins; Trichostatin A
18.  The empowerment of translational research: lessons from laminopathies 
The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.
doi:10.1186/1750-1172-7-37
PMCID: PMC3458975  PMID: 22691392
Laminopathies; Emery-Dreifuss Muscular Dystrophy; Dilated Cardiomyopathy with Conduction Defects; Mandibuloacral Dysplasia; Familial Partial Lipodystrophy Type 2; Hutchinson-Gilford Progeria Syndrome; Rare Diseases; Networking activity; interdisciplinary approach to diseases
19.  IPLEX Administration Improves Motor Neuron Survival and Ameliorates Motor Functions in a Severe Mouse Model of Spinal Muscular Atrophy 
Molecular Medicine  2012;18(1):1076-1085.
Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder and the first genetic cause of death in childhood. SMA is caused by low levels of survival motor neuron (SMN) protein that induce selective loss of α-motor neurons (MNs) in the spinal cord, resulting in progressive muscle atrophy and consequent respiratory failure. To date, no effective treatment is available to counteract the course of the disease. Among the different therapeutic strategies with potential clinical applications, the evaluation of trophic and/or protective agents able to antagonize MNs degeneration represents an attractive opportunity to develop valid therapies. Here we investigated the effects of IPLEX (recombinant human insulinlike growth factor 1 [rhIGF-1] complexed with recombinant human IGF-1 binding protein 3 [rhIGFBP-3]) on a severe mouse model of SMA. Interestingly, molecular and biochemical analyses of IGF-1 carried out in SMA mice before drug administration revealed marked reductions of IGF-1 circulating levels and hepatic mRNA expression. In this study, we found that perinatal administration of IPLEX, even if does not influence survival and body weight of mice, results in reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in SMA mice. Additionally, we show that phenotypic changes observed are not SMN-dependent, since no significant SMN modification was addressed in treated mice. Collectively, our data indicate IPLEX as a good therapeutic candidate to hinder the progression of the neurodegenerative process in SMA.
doi:10.2119/molmed.2012.00056
PMCID: PMC3474434  PMID: 22669476
20.  MCP1 SNPs and Pulmonary Tuberculosis in Cohorts from West Africa, the USA and Argentina: Lack of Association or Epistasis with IL12B Polymorphisms 
PLoS ONE  2012;7(2):e32275.
The monocyte chemotactic protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of monocytes to M. tuberculosis infection sites, and previous studies have reported that genetic variants in MCP1 are associated with differential susceptibility to pulmonary tuberculosis (PTB). We examined eight MCP1 single nucleotide polymorphisms (SNPs) in a multi-ethnic, case-control design that included: 321 cases and 346 controls from Guinea-Bissau, 258 cases and 271 controls from The Gambia, 295 cases and 179 controls from the U.S. (African-Americans), and an additional set of 237 cases and 144 controls of European ancestry from the U.S. and Argentina. Two locus interactions were also examined for polymorphisms in MCP1 and interleukin 12B (IL12B), another gene implicated in PTB risk. Examination of previously associated MCP1 SNPs rs1024611 (−2581A/G), rs2857656 (−362G/C) and rs4586 (+900C/T) did not show evidence for association. One interaction between rs2857656 and IL12B SNP rs2288831 was observed among Africans but the effect was in the opposite direction in Guineans (OR = 1.90, p = 0.001) and Gambians (OR = 0.64, p = 0.024). Our data indicate that the effect of genetic variation within MCP1 is not clear cut and additional studies will be needed to elucidate its role in TB susceptibility.
doi:10.1371/journal.pone.0032275
PMCID: PMC3288089  PMID: 22384203
21.  Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System 
PLoS ONE  2012;7(2):e30851.
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.
doi:10.1371/journal.pone.0030851
PMCID: PMC3281040  PMID: 22359552
22.  Association between OLR1 K167N SNP and Intima Media Thickness of the Common Carotid Artery in the General Population 
PLoS ONE  2012;7(2):e31086.
Background and Purpose
The lectin-like oxidised LDL receptor-1 (OLR1) gene encodes a scavenger receptor implicated in the pathogenesis of atherosclerosis. Although functional roles have been suggested for two variants, epidemiological studies on OLR1 have been inconsistent.
Methods - We tested the association between the non-synonymous substitution K167N (rs11053646) and intima media thickness of the common carotid artery (CCA-IMT) in 2,141 samples from the Progression of Lesions in the Intima of the Carotid (PLIC) study (a prospective population-based study).
Results
Significantly increased IMT was observed in male carriers of the minor C (N) allele compared to GC and GG (KN and KK) genotype. Functional analysis on macrophages suggested a decreased association to Ox-LDL in NN carriers compared to KN and KK carriers which is also associated with a reduced OLR1 mRNA expression. Macrophages from NN carriers present also a specific inflammatory gene expression pattern compared to cells from KN and KK carriers.
Conclusions
These data suggest that the 167N variant of LOX-1 receptor affects the atherogenic process in the carotid artery prior to evidence of disease through an inflammatory process.
doi:10.1371/journal.pone.0031086
PMCID: PMC3276570  PMID: 22347434
23.  Novel mutations of TCOF1 gene in European patients with treacher Collins syndrome 
BMC Medical Genetics  2011;12:125.
Background
Treacher Collins syndrome (TCS) is one of the most severe autosomal dominant congenital disorders of craniofacial development and shows variable phenotypic expression. TCS is extremely rare, occurring with an incidence of 1 in 50.000 live births. The TCS distinguishing characteristics are represented by down slanting palpebral fissures, coloboma of the eyelid, micrognathia, microtia and other deformity of the ears, hypoplastic zygomatic arches, and macrostomia. Conductive hearing loss and cleft palate are often present. TCS results from mutations in the TCOF1 gene located on chromosome 5, which encodes a serine/alanine-rich nucleolar phospho-protein called Treacle. However, alterations in the TCOF1 gene have been implicated in only 81-93% of TCS cases.
Methods
In this study, the entire coding regions of the TCOF1 gene, including newly described exons 6A and 16A, were sequenced in 46 unrelated subjects suspected of TCS clinical indication.
Results
Fifteen mutations were reported, including twelve novel and three already described in 14 sporadic patients and in 3 familial cases. Moreover, seven novel polymorphisms were also described. Most of the mutations characterised were microdeletions spanning one or more nucleotides, in addition to an insertion of one nucleotide in exon 18 and a stop mutation. The deletions and the insertion described cause a premature termination of translation, resulting in a truncated protein.
Conclusion
This study confirms that almost all the TCOF1 pathogenic mutations fall in the coding region and lead to an aberrant protein.
doi:10.1186/1471-2350-12-125
PMCID: PMC3199234  PMID: 21951868
Treacher Collins syndrome; TCOF1 mutations; microdeletions; microinsertions
24.  Deletion of the late cornified envelope (LCE) 3B and 3C genes as a susceptibility factor for psoriasis 
Nature genetics  2009;41(2):211-215.
Psoriasis is a common inflammatory skin disease with a prevalence of 2% to 3% in Caucasians1. In a genome-wide search for copy number variants (CNV) using a sample pooling approach we have identified a deletion comprising LCE3B and LCE3C, members of the late cornified envelope (LCE) gene cluster2. The absence of LCE3B and LCE3C (LCE3C-LCE3B-del) is significantly associated (p=1.38E-08) with risk of psoriasis in 2,831 samples from Spain, The Netherlands, Italy and the USA, and in a family-based study (p=5.4E-04). LCE3C-LCE3B-del is tagged by rs4112788 (r2=0.93), which is also strongly associated with psoriasis (p<6.6E-09). LCE3C-LCE3B-del shows epistatic effects with the HLA-Cw6 allele on the development of psoriasis in Dutch samples, and multiplicative effects in the other samples. LCE expression can be induced in normal epidermis by skin barrier disruption and is strongly expressed in psoriatic lesions, suggesting that compromised skin barrier function plays a role in psoriasis susceptibility.
doi:10.1038/ng.313
PMCID: PMC3128734  PMID: 19169253
25.  Oxidized LDL Receptor 1 (OLR1) as a Possible Link between Obesity, Dyslipidemia and Cancer 
PLoS ONE  2011;6(5):e20277.
Recent studies have linked expression of lectin-like ox-LDL receptor 1 (OLR1) to tumorigenesis. We analyzed microarray data from Olr1 knockout (KO) and wild type (WT) mice for genes involved in cellular transformation and evaluated effects of OLR1 over-expression in normal mammary epithelial cells (MCF10A) and breast cancer cells (HCC1143) in terms of gene expression, migration, adhesion and transendothelial migration. Twenty-six out of 238 genes were inhibited in tissues of OLR1 KO mice; the vast majority of OLR1 sensitive genes contained NF-κB binding sites in their promoters. Further studies revealed broad inhibition of NF-kB target genes outside of the transformation-associated gene pool, with enrichment themes of defense response, immune response, apoptosis, proliferation, and wound healing. Transcriptome of Olr1 KO mice also revealed inhibition of de novo lipogenesis, rate-limiting enzymes fatty acid synthase (Fasn), stearoyl-CoA desaturase (Scd1) and ELOVL family member 6 (Elovl6), as well as lipolytic phospholipase A2 group IVB (Pla2g4b). In studies comparing MCF10A and HCC1143, the latter displayed 60% higher OLR1 expression. Forced over-expression of OLR1 resulted in upregulation of NF-κB (p65) and its target pro-oncogenes involved in inhibition of apoptosis (BCL2, BCL2A1, TNFAIP3) and regulation of cell cycle (CCND2) in both cell lines. Basal expression of FASN, SCD1 and PLA2G4B, as well as lipogenesis transcription factors PPARA, SREBF2 and CREM, was higher in HCC1143 cells. Over-expression of OLR1 in HCC1143 cells also enhanced cell migration, without affecting their adherence to TNFα-activated endothelium or transendothelial migration. On the other hand, OLR1 neutralizing antibody inhibited both adhesion and transmigration of untreated HCC1143 cells. We conclude that OLR1 may act as an oncogene by activation of NF-kB target genes responsible for proliferation, migration and inhibition of apoptosis and de novo lipogenesis genes.
doi:10.1371/journal.pone.0020277
PMCID: PMC3102697  PMID: 21637860

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