Identification of putative genetic modifying factors that influence the development of Papillon-Lefévre or Haim-Munk syndrome phenotypes.

BACKGROUND: Papillon-Lefévre syndrome (PLS; OMIM 245000) and Haim-Munk syndrome (HMS; OMIM 245010), which are both characterized by palmoplantar hyperkeratosis and periodontitis, are phenotypic variants of the same disease caused by mutations of the cathepsin C (CTSC) gene. AIM: To identify putative genetic modifying factors responsible for the differential development of the PLS or HMS phenotypes, we investigated two Hungarian patients with different phenotypic variants (PLS and HMS) but carrying the same homozygous nonsense CTSC mutation (c.748C/T; p.Arg250X). METHODS: To gain insights into phenotype-modifying associations, whole exome sequencing (WES) was performed for both patients, and the results were compared to identify potentially relevant genetic modifying factors. RESULTS: WES revealed two putative phenotype-modifying variants: (i) a missense mutation (rs34608771) of the SH2 domain containing 4A (SH2D4A) gene encoding an adaptor protein involved in intracellular signalling of cystatin F, a known inhibitor of the cathepsin protein, and (ii) a missense variant (rs55695858) of the odorant binding protein 2A (OBP2A) gene, influencing the function of the cathepsin protein through the glycosyltransferase 6 domain containing 1 (GLT6D1) protein. CONCLUSION: Our study contributes to the accumulating evidence supporting the clinical importance of phenotype-modifying genetic factors, which have high potential to aid the elucidation of genotype-phenotype correlations and disease prognosis.

Atypical neurofibromatosis type 1 with unilateral limb hypertrophy mimicking overgrowth syndrome.

Neurofibromatosis type 1 (NF1; OMIM 162200), a dominantly inherited multitumor syndrome, results from mutations in the Neurofibromin 1 (NF1) gene. We present the case of a Hungarian woman with the clinical phenotype of NF1 over her whole body and the clinical features of unilateral overgrowth involving her entire left leg. This unusual phenotype suggested either the atypical form of NF1 or the coexistence of NF1 and overgrowth syndrome. Direct sequencing of the genomic DNA isolated from peripheral blood revealed a novel frameshift mutation (c.5727insT, p.V1909fsX1912) in the NF1 gene. Next-generation sequencing of 50 oncogenes and tumour suppressor genes, performed on the genomic DNAs isolated from tissue samples and peripheral blood, detected only wild-type sequences. Based on these results, we concluded that the patient is affected by an unusual phenotype of NF1, and that the observed unilateral overgrowth of the left leg might be a rare consequence of the identified c.5727insT mutation.

HuCOP1 contributes to the regulation of DNA repair in keratinocytes.

We have previously demonstrated that the E3 ligase Human Constitutive Photomorphogenic Protein (huCOP1) is expressed in human keratinocytes and negatively regulates p53. The MutS homolog 2 (MSH2) protein plays a central role in DNA MMR mechanism and is implicated in the cellular response to anticancer agents, such as cisplatin. Our aim was to clarify whether huCOP1 plays a role in DNA MMR by affecting MSH2 protein level in human keratinocytes. To define the role of huCOP1 in DNA mismatch repair, we determined whether huCOP1 affects MSH2 abundance. MSH2 protein level was detected by immunocytochemical staining using a keratinocyte cell line in which the expression level of huCOP1 was stably decreased (siCOP1). To investigate whether huCOP1 silencing influences cisplatin-induced cell death, control and siCOP1 keratinocyte cells were treated with increasing concentrations of cisplatin and cell viability was recorded after 48 and 96 h. Stable silencing of huCOP1 in human keratinocytes resulted in a reduced level of MSH2 protein. huCOP1 silencing also sensitized keratinocytes to the interstrand crosslinking inducer cisplatin. Our results indicate that decreased huCOP1 correlates with lower MSH2 levels. These protein level changes lead to increased sensitivity toward cisplatin treatment, implicating that huCOP1 plays a positive role in maintaining genome integrity in human keratinocytes.

One mutation, two phenotypes: a single nonsense mutation of the CTSC gene causes two clinically distinct phenotypes.

BACKGROUND: Papillon-Lefévre syndrome (PLS; OMIM 245000) and Haim-Munk syndromes (HMS; OMIM 245010) are phenotypic variants of the same rare disease caused by mutations of the cathepsin C (CTSC) gene, and they exhibit autosomal recessive inheritance. AIMS: To identify diseases caused by mutations of the CTSC gene in two Hungarian patients and to perform haplotype analysis to elucidate any familial relationship between them. METHODS: Mutation screening and polymorphism analysis were performed by direct sequencing of the CTSC gene. RESULTS: Mutation screening of the CTSC gene from the two patients revealed the presence of the same homozygous nonsense mutation (c.748C/T; p.Arg250X). However, one patient exhibited the PLS phenotype and the other the HMS phenotype. Although these patients were not aware that they were related, haplotype analysis, especially the genotypes of the rs217116 and the rs217115 polymorphisms, clearly indicated that the patients carry the same haplotype, whereas the unrelated healthy controls carried several different haplotypes. CONCLUSIONS: Our results demonstrate that PLS and HMS are phenotypic variants of the same disease and, additionally, exclude the presence of a putative genetic modifier factor within the CTSC gene that is responsible for the development of the two phenotypes. We suggest that this putative genetic modifier factor is located outside the CTSC gene, or alternatively, that the development of the different phenotypes is the consequence of different environmental or lifestyle factors.

BRAFV600E mutation in cutaneous lesions of patients with adult Langerhans cell histiocytosis.

BACKGROUND: Langerhans cell histiocytosis (LCH) is characterized by the proliferation of pathologic Langerhans cells. The disease can develop in any age and can affect almost any organ. Cutaneous involvement is frequent in LCH. The recent demonstration of the activating, oncogenic BRAFV600E gene mutation in LCH samples strongly supports the neoplastic origin of the disease. OBJECTIVES: Our aim was to analyse the clinical data of the patients and whether BRAFV600E mutation is present in skin lesions of patients with adult onset LCH, and to investigate whether the BRAFV600E mutation status has any effect on the clinical presentation and the outcome of the disease. METHODS: We diagnosed and treated 15 adult LCH patients in the period of 1987-2012 and collected their clinical data. Three of our patients suffered from skin involvement and 12 patients had multiorgan disease (five patients out of the multisystem group died). Eleven formalin-fixed paraffin-embedded skin samples from 10 patients were available for BRAFV600E mutation analysis. RESULTS: Among the 11 examined samples, 6 contained the BRAFV600E mutation (54.5%). Our results indicate that in the adult group of LCH patients the presence of BRAFV600E mutation is similar to what was previously suggested in case of the childhood forms, at least as far as skin lesions are concerned. The BRAF mutation status of our patients does not seem to correlate with the extent and/or the outcome of the disease. CONCLUSION: Our results support the neoplastic origin of LCH and suggest that skin lesions of LCH are sufficient for the diagnosis of the disease and for assessing its BRAF status. In addition, analysis of BRAF status of patients with LCH can lead to the administration of new targeted therapies which may provide better disease control and prognosis.

UVB-dependent changes in the expression of fast-responding early genes is modulated by huCOP1 in keratinocytes.

Ultraviolet (UV) B is the most prominent physical carcinogen in the environment leading to the development of various skin cancers. We have previously demonstrated that the human ortholog of the Arabidopsis thaliana constitutive photomorphogenesis 1 (COP1) protein, huCOP1, is expressed in keratinocytes in a UVB-regulated manner and is a negative regulator of p53 as a posttranslational modifier. However, it was not known whether huCOP1 plays a role in mediating the UVB-induced early transcriptional responses of human keratinocytes. In this study, we report that stable siRNA-mediated silencing of huCOP1 affects the UVB response of several genes within 2 h of irradiation, indicating that altered huCOP1 expression sensitizes the cells toward UVB. Pathway analysis identified a molecular network in which 13 of the 30 examined UVB-regulated genes were organized around three central proteins. Since the expression of the investigated genes was upregulated by UVB in the siCOP1 cell line, we hypothesize that huCOP1 is a repressor of the identified pathway. Several members of the network have been implicated previously in the pathogenesis of non-melanoma skin cancers; therefore, clarifying the role of huCOP1 in these skin diseases may have clinical relevance in the future.

Thrombosis and risk factors in female patients with a rare acquired thrombophilia: chronic myeloproliferative disorder - polycythaemia vera and essential thrombocythaemia.

OBJECTIVE: In polycythaemia vera (PV) and essential thrombocythaemia (ET), the life expectancy of the patients is greatly affected by thrombotic events. An investigation was performed of the potential association of PV/ET, and thrombotic complications with cardiovascular (CV) risk factors, a leukocyte count at the haematological diagnosis > 11.1 G/L, and the JAK2V617F mutation. PATIENTS AND METHODS: In the period 1998-2011, 128 women with a median age of 62 years were enrolled. RESULTS: The risk of thrombotic events before the diagnosis was 32.8% (42/128), while in the follow-up period it was 10.2% (13/128). The difference in the probability of thrombosis-free survival between those with at most one CV risk factor and those with two or more CV risk factors was significant (p = 0.005). The presence of two or more CV risk factors (univariate: p = 0.011; multivariate: relative risk: 4.728, 95% CI 1.312-17.040; p = 0.018) significantly increased the risk of thrombosis. Univariate analyses revealed that high blood pressure (p = 0.001), hyperlipidaemia (p = 0.005) and cigarette smoking (p = 0.051) were associated with a significantly higher risk of thrombosis. Analyses of the influence of the leukocyte count (univariate: p = 0.424; multivariate: relative risk: 1.407, 95% CI 0.359-5.507; p = 0.624) and the JAK2V617F mutation (univariate: p = 0.367; multivariate: relative risk: 1.428, 95% CI 0.316-6.460; p = 0.643) on subsequent thrombotic complications resulted in a non-signicant tendency. CONCLUSIONS: Female patients who display CV risk factors (high blood pressure, hyperlipidaemia and/or cigarette smoking) and PV or ET may well be at a higher risk of thrombotic events and require special consideration as concerns as the prevention and management of thrombotic events.

Role of the TNFA -308G > A polymorphism in the genetic susceptibility to acne vulgaris in a Sicilian population.

BACKGROUND: Acne vulgaris is a common and clinically well-characterized skin disease that affects a great proportion of the general population and thus, is a major public health problem. The aim of the present study was to investigate whether TNFA -308 G > A polymorphism might be involved in the pathogenesis of acne in a population from Sicily. METHODS: A total of 74 patients with acne and of 88 healthy control subjects from Catania, Italy were examined in the present study. TNFA -308 G > A polymorphisms using the PCR-RFLP method were determined in DNA extracted from buccal swabs. RESULTS: When controls were compared to acne patients, their genotype distributions, respectively G/G: 64.3%, G/A: 35.7% and G/G: 74.0%, G/A: 26.0%, were shown to be different, although not statistically significant (p = 0.191). A significant protective association between the TNFA -308 GA genotype and acne in males (p = 0.027; OR95% CI: 0.288; 0.094-0.889) was shown. CONCLUSIONS: The present results suggest that TNFA -308 polymorphism may contribute to acne susceptibility, as suggested by the protective effect of the G/A phenotype in the males of the Sicilian cohort. Further studies in larger groups, investigating the TNFA -308G/A or other polymorphisms of this gene in acne patients may be helpful to clarify the pathogenesis of the disease.

Detection of a rare CDKN2A intronic mutation in a Hungarian melanoma-prone family and its role in splicing regulation.

BACKGROUND: The major locus for melanoma predisposition is the cell cycle regulatory CDKN2A gene on chromosome 9p21. However, the frequency of germline coding mutations of the CDKN2A gene is lower than expected in melanoma-prone families linked to chromosome 9p21. OBJECTIVES: To investigate whether the rare IVS1+37 G/C intronic mutation of the CDKN2A gene, recently identified in a Hungarian melanoma-prone family, influences mRNA splicing regulation. METHODS: CDKN2A minigenes containing the wild-type and the mutant intronic sequence were created and transfected into HeLa cells with the aim of studying the mRNA transcripts. RESULTS: The results revealed the emergence of a differential splicing pattern from the wild-type and the mutant minigene, suggesting that this mutation may alter the splicing of CDKN2A primary mRNA and therefore might have a pathogenetic role in familial melanoma. CONCLUSIONS: We believe that these results confirm the importance of the identification and characterization of CDKN2A intronic mutations with a view to improving our understanding of the pathogenesis, and explain why the frequency of germline coding mutations of the CDKN2A gene is lower than expected in melanoma-prone families linked to chromosome 9p21.

Simultaneous adeno- and squamous cell carcinoma with different phenotypic profiles in a rat model of chronic gastroesophageal reflux.

The aim of our study was to investigate the incidence of duodeno-gastroesophageal reflux-induced malignant transformation in a series of duodeno-esophageal anastomosis operations in rats. This surgical method provides a model for reflux-induced esophageal pathologies, without carcinogen administration. The study design included the follow-up of 31 cases. Thirty weeks of duodeno-gastroesophageal reflux disease significantly increased the risk of the development of Barrett's esophagus, and reflux-induced esophageal adenocarcinoma formation was evident in four animals. In one of these particular cases, a superficial squamous cell cancer was noted in close vicinity to the adenocarcinoma formation. For further analysis, a detailed immunohistochemical staining protocol was used. The immunophenotypes revealed cyclin D1 expression (nuclear positivity in 35% of all the squamous cells), p53 protein accumulation (50% nuclear positivity), with a low expression of cox-2, and negative c-erbB2 staining in the squamous carcinoma cells. The specialized intestinal metaplasia and mucinous adenocarcinoma cells exhibited exclusively diffuse cox-2 positivity (90% of all glandular cells) and weak focal c-erbB2 (5%) staining, without cyclin D1 expression or p53 protein accumulation. Real-time polymerase chain reaction was applied to quantify the abundance of p53, cyclin D1 and cox-2 mRNAs in this biopsy. The most dramatic changes were observed in the level of expression of cyclin D1 (a 9.08-fold expression as compared with the non-treated esophagus samples), while the p53 and cox-2 gene expressions were increased by 1.61 and 2.45-fold, respectively, relative to the non-treated samples. The results afford evidence of the simultaneous activation of more than one possible carcinogenetic pathway in experimental gastroesophageal reflux disease. Synchronous neoplasm formation with different growth pattern characteristics is a rarity in humans, and this phenomenon suggests that the presented model is a suitable means of mimicking the whole spectrum of human gastroesophageal reflux disease pathology.

Moxifloxacin versus standard therapy in patients with pneumonia hospitalized after failure of preclinical anti-infective treatment.

The failure rate of primary empirical anti-infective treatment of community-acquired pneumonia is reported to range between 2 and 7%. These patients are subject to a greater risk of intensive medical treatment and a higher mortality rate than patients who respond to primary treatment. We investigated 63 patients in a "real life scenario" who were admitted to the hospital after failure of primary outpatient therapy for community-acquired pneumonia. Thirty-three patients received intravenous standard therapy (betalactam 14, macrolide 3, levofloxacin 6, doxycycline 1, combinations 9 patients) while 30 patients were treated with intravenous moxifloxacin. The oral antibiotic pretreatment that failed most frequently was clarithromycin (n = 25), followed by amoxicillin/clavulanic acid (n = 16), cefixime (n = 10), cefuroxime/axetil (n = 5), doxycycline (3), cefpodoxime, and ciprofloxacin (2 each). There were no differences between the two groups in respect of age, gender, numbers of patients in nursing homes, numbers of patients with different underlying diseases (chronic bronchitis, coronary heart disease, diabetes mellitus, smoking, etc.), severity of pneumonia at the time of admission, numbers of patients requiring intensive care, and lethality. The group that underwent standard therapy experienced failure of the empirical intra-hospital antibiotic therapy more often during therapy [10 (30%) patients vs 2 (6%) in the moxifloxacin group, p = 0.009] and clinical failure of treatment on day 28 after initiation of therapy [7 (21%) patients vs 2 (6%) in the moxifloxacin group, p = 0.003]. In cases of failure of empirical preclinical antibiotic treatment for community-acquired pneumonia, subsequent intrahospital treatment with moxifloxacin is more successful than standard therapy in our study reflecting a "real life scenario".

Characterization of tomato PHYB1 and identification of molecular defects in four mutant alleles.

The structure of the gene encoding the apoprotein of phytochrome B (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5' RACE at -116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225.

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.

A 268 bp upstream sequence mediates the circadian clock-regulated transcription of the wheat Cab-1 gene in transgenic plants.

We previously reported that the expression of the wheat Cab-1 gene is regulated by an endogenous circadian rhythm and by the photoreceptor phytochrome both in wheat and in transgenic tobacco plants. To define regulatory elements necessary for the circadian rhythm-regulated Cab-1 gene expression, we now analysed the fluctuation of steady-state mRNA levels in a series of 5' deletion mutants in transgenic tobacco plants. We found that the expression of a deletion mutant containing 211 bp upstream sequence still exhibited circadian rhythm. Furthermore we show that an enhancer-like sequence of the Cab-1 promoter (from -357 to -90) can endow a chimaeric gene consisting of a truncated 35S promoter (from -90 to +8) and the bacterial beta-glucuronidase (GUS) gene with circadian clock-regulated gene expression. Finally we demonstrate by nuclear run-off experiments that the transcription rates of the Cab genes in wheat oscillate in a rhythmic manner, with a periodicity of approximately 24 hours. Consistent with our previous findings these results (i) indicate that the expression of the wheat Cab-1 gene is regulated mainly at the transcription level and (ii) identify a short promoter region between -211 and -90 that is responsible for the circadian clock-regulated gene expression.