TFIIH stabilization recovers the DNA repair and transcription dysfunctions in thermo-sensitive trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.

Cockayne syndrome group A and ferrochelatase finely tune ribosomal gene transcription and its response to UV irradiation.

CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens.

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/ß-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.

The polyphenol/saponin-rich Rhus tripartita extract has an apoptotic effect on THP-1 cells through the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: Hyperactivation of mechanistic target of rapamycin (mTOR) signaling pathway is involved in the regulation of cellular growth, proliferation, and more in general, is a common phenomenon in most types of cancers. Thus, natural substances targeting this pathway can be of great therapeutic potential in supporting the treatment of tumor patients. Rhus tripartita (Ucria) Grande is a plant growing in desertic areas which is traditionally used for the treatment of several diseases in Tunisia. In the present work, the biochemical profile of the main compounds present in the plant leaf extract was determined and the anti-leukemic potential of the plant extracts against acute monocytic leukaemia (AML) THP-1 cells was investigated. METHODS: After HPLC identification of some phenolic compounds present in the plant extract and the quantification of saponin content, the cytotoxic effect of Rhus tripartita extracts on THP-1 cell culture was evaluated using the colorimetric MTT assay for cell viability. THP-1 cells were incubated with medium containing the relative IC50 concentrations of total plant extract, saponin extract and some standard compounds (rutin (R); kaempferol (K); mixture of catechin, epicatechin, and epicatechin-gallate (CEEG); ellagic acid (EA). Finally, qRT-PCR and western blotting analysis were used to evaluate the effect of some flavonoids present in a crude extract of polyphenols and the total extract of saponins on cell survival and apoptosis. RESULTS: Analysis of expression level of some gene (PIK3CA, PTEN, AKT1, mTOR, EIF4E, RPS6KB1, and TSC1) involved in the mTOR pathway and the phosphorylation of S6 and AKT proteins allowed to observe that a total Rhus tripartita extract and some of the compounds found in the extract controls THP-1 cell proliferation and apoptosis via regulation of the PI3K-Akt-mTOR signaling pathway. CONCLUSION: Rhus tripartita-induced inhibition of cell cycle and induction of apoptosis may involve the mTOR pathway. Therefore, Rhus tripartita extract may be a useful candidate as a natural anti-cancer drug to support the treatment of AML.

Non-viral gene delivery of the oncotoxic protein NS1 for treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.

Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation.

Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.

Novel biomarkers for primary biliary cholangitis to improve diagnosis and understand underlying regulatory mechanisms.

BACKGROUND AND AIMS: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis. METHODS: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker. RESULTS: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients. CONCLUSIONS: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.

Diagnostic Targeted Sequencing Panel for Hepatocellular Carcinoma Genomic Screening.

Commercially available targeted panels miss genomic regions frequently altered in hepatocellular carcinoma (HCC). We sought to design and benchmark a sequencing assay for genomic screening of HCC. We designed an AmpliSeq custom panel targeting all exons of 33 protein-coding and two long noncoding RNA genes frequently mutated in HCC, TERT promoter, and nine genes with frequent copy number alterations. By using this panel, the profiling of DNA from fresh-frozen (n = 10, 1495×) and/or formalin-fixed, paraffin-embedded (FFPE) tumors with low-input DNA (n = 36, 530×) from 39 HCCs identified at least one somatic mutation in 90% of the cases. Median of 2.5 (range, 0 to 74) and 3 (range, 0 to 76) mutations were identified in fresh-frozen and FFPE tumors, respectively. Benchmarked against the mutations identified from Illumina whole-exome sequencing (WES) of the corresponding fresh-frozen tumors (105×), 98% (61 of 62) and 100% (104 of 104) of the mutations from WES were detected in the 10 fresh-frozen tumors and the 36 FFPE tumors, respectively, using the HCC panel. In addition, 18 and 70 somatic mutations in coding and noncoding genes, respectively, not found by WES were identified by using our HCC panel. Copy number alterations between WES and our HCC panel showed an overall concordance of 86%. In conclusion, we established a cost-effective assay for the detection of genomic alterations in HCC.

Functional and clinical relevance of novel mutations in a large cohort of patients with Cockayne syndrome.

BACKGROUND: Cockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8/CSA or ERCC6/CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS. METHODS AND RESULTS: We assigned 39 patients to the ERCC8/CSA and 85 to the ERCC6/CSB genes. Most of the genetic variants were truncations. The missense variants were distributed non-randomly with concentrations in relatively short regions of the respective proteins. Our analyses revealed several hotspots and founder mutations in ERCC6/CSB. Although no unequivocal genotype-phenotype relationships could be made, patients were more likely to have severe clinical features if the mutation was downstream of the PiggyBac insertion in intron 5 of ERCC6/CSB than if it was upstream. Also a higher proportion of severely affected patients was found with mutations in ERCC6/CSB than in ERCC8/CSA. CONCLUSION: By identifying >70 novel homozygous or compound heterozygous genetic variants in 124 patients with CS with different disease severity and ethnic backgrounds, we considerably broaden the CSA and CSB mutation spectrum responsible for CS. Besides providing information relevant for diagnosis of and genetic counselling for this devastating disorder, this study improves the definition of the puzzling genotype-phenotype relationships in patients with CS.

The Role of Long Non-Coding RNAs in Hepatocarcinogenesis.

Whole-transcriptome analyses have revealed that a large proportion of the human genome is transcribed in non-protein-coding transcripts, designated as long non-coding RNAs (lncRNAs). Rather than being "transcriptional noise", increasing evidence indicates that lncRNAs are key players in the regulation of many biological processes, including transcription, post-translational modification and inhibition and chromatin remodeling. Indeed, lncRNAs are widely dysregulated in human cancers, including hepatocellular carcinoma (HCC). Functional studies are beginning to provide insights into the role of oncogenic and tumor suppressive lncRNAs in the regulation of cell proliferation and motility, as well as oncogenic and metastatic potential in HCC. A better understanding of the molecular mechanisms and the complex network of interactions in which lncRNAs are involved could reveal novel diagnostic and prognostic biomarkers. Crucially, it may provide novel therapeutic opportunities to add to the currently limited number of therapeutic options for HCC patients. In this review, we summarize the current status of the field, with a focus on the best characterized dysregulated lncRNAs in HCC.

Genomic Analysis Revealed New Oncogenic Signatures in TP53-Mutant Hepatocellular Carcinoma.

The TP53 gene is the most commonly mutated gene in human cancers and mutations in TP53 have been shown to have either gain-of-function or loss-of-function effects. Using the data generated by The Cancer Genome Atlas, we sought to define the spectrum of TP53 mutations in hepatocellular carcinomas (HCCs) and their association with clinicopathologic features, and to determine the oncogenic and mutational signatures in TP53-mutant HCCs. Compared to other cancer types, HCCs harbored distinctive mutation hotspots at V157 and R249, whereas common mutation hotspots in other cancer types, R175 and R273, were extremely rare in HCCs. In terms of clinicopathologic features, in addition to the associations with chronic viral infection and high Edmondson grade, we found that TP53 somatic mutations were less frequent in HCCs with cholestasis or tumor infiltrating lymphocytes, but were more frequent in HCCs displaying necrotic areas. An analysis of the oncogenic signatures based on the genetic alterations found in genes recurrently altered in HCCs identified four distinct TP53-mutant subsets, three of which were defined by CTNNB1 mutations, 1q amplifications or 8q24 amplifications, respectively, that co-occurred with TP53 mutations. We also found that mutational signature 12, a liver cancer-specific signature characterized by T>C substitutions, was prevalent in HCCs with wild-type TP53 or with missense TP53 mutations, but not in HCCs with deleterious TP53 mutations. Finally, whereas patients with HCCs harboring deleterious TP53 mutations had worse overall and disease-free survival than patients with TP53-wild-type HCCs, patients with HCCs harboring missense TP53 mutations did not have worse prognosis. In conclusion, our results highlight the importance to consider the genetic heterogeneity among TP53-mutant HCCs in studies of biomarkers and molecular characterization of HCCs.

High expression of HOXA13 correlates with poorly differentiated hepatocellular carcinomas and modulates sorafenib response in in vitro models.

Hepatocellular carcinoma (HCC) represents the fifth and ninth cause of mortality among male and female cancer patients, respectively and typically arises on a background of a cirrhotic liver. HCC develops in a multi-step process, often encompassing chronic liver injury, steatosis and cirrhosis eventually leading to the malignant transformation of hepatocytes. Aberrant expression of the class I homeobox gene family (HOX), a group of genes crucial in embryogenesis, has been reported in a variety of malignancies including solid tumors. Among HOX genes, HOXA13 is most overexpressed in HCC and is known to be directly regulated by the long non-coding RNA HOTTIP. In this study, taking advantage of a tissue microarray containing 305 tissue specimens, we found that HOXA13 protein expression increased monotonically from normal liver to cirrhotic liver to HCC and that HOXA13-positive HCCs were preferentially poorly differentiated and had fewer E-cadherin-positive cells. In two independent cohorts, patients with HOXA13-positive HCC had worse overall survival than those with HOXA13-negative HCC. Using HOXA13 immunohistochemistry and HOTTIP RNA in situ hybridization on consecutive sections of 16 resected HCCs, we demonstrated that HOXA13 and HOTTIP were expressed in the same neoplastic hepatocyte populations. Stable overexpression of HOXA13 in liver cancer cell lines resulted in increased colony formation on soft agar and migration potential as well as reduced sensitivity to sorafenib in vitro. Our results provide compelling evidence of a role for HOXA13 in HCC development and highlight for the first time its ability to modulate response to sorafenib.

Overexpression of parkin rescues the defective mitochondrial phenotype and the increased apoptosis of Cockayne Syndrome A cells.

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEß). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEß) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

TFIIH-dependent MMP-1 overexpression in trichothiodystrophy leads to extracellular matrix alterations in patient skin.

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP.

From laboratory tests to functional characterisation of Cockayne syndrome.

The significant progress made over the last few years on the pathogenesis of Cockayne syndrome (CS) greatly improved our knowledge on several aspects crucial for development and ageing, demonstrating that this disorder, even if rare, represents a valuable tool to clarify key aspects of human health. Primary cells from patients have been instrumental to elucidate the multiple roles of CS proteins and to approach the dissection of the complex interplay between repair and transcription that is central to the CS clinical phenotype. Here we discuss the results of the cellular assays applied for confirmation of the clinical diagnosis as well as the results of genetic and molecular studies in DNA repair defective patients. Furthermore, we provide a general overview of recent in vivo and in vitro studies indicating that both CSA and CSB proteins are involved in distinct aspects of the cellular responses to UV and oxidative stress, transcription and regulation of gene expression, chromatin remodelling, redox balance and cellular bioenergetics. In light of the literature data, we will finally discuss how inactivation of specific functional roles of CS proteins may differentially affect the phenotype, thus explaining the wide range in type and severity of symptoms reported in CS patients.

SPANX-B and SPANX-C (Xq27 region) gene dosage analysis in Sicilian patients with melanoma.

The incidence of melanoma has dramatically increased in many countries (it is 4.5 cases every 100 000 inhabitants in Sicily) and Xq27 region contains genes important in cancer like the SPANX (sperm protein associated with the nucleus in the X chromosome) gene family. These genes, made up of two exons separated by an intron of about 650 base pair, are expressed in sperm cells and in many tumours, including melanoma. These observations suggested that SPANX genes, or some of them, may be involved in melanoma development. The aim of this study was to investigate the genetic variability of SPANX-B and SPANX-C in a sample of Sicilian male population including patients with melanoma of the skin and controls. A total of 99 patients were enrolled in this study. They included: 17 male patients with cutaneous melanoma and 82 normal males. Semiquantitative fluorescent multiplex PCR dosage analysis was carried out to identify the variety of classes of SPANX-B and SPANX-C genes. Sixteen and 13 genetic classes were detected for SPANX-B and SPANX-C genes, respectively. A statistical significant difference for a particular class of SPANX-C gene was found comparing patients with melanoma and controls (P=0.011). Further investigations should be conducted to confirm these observations and to evaluate the possible implication of other genes of the region Xq27-28 in melanoma.