Analysis of the genetic basis of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome.

PFAPA syndrome is the most common autoinflammatory syndrome in children from Western countries. In spite of its strong familial clustering, its genetic basis and inheritance pattern are still unknown. We performed a comprehensive genetic study on 68 individuals from 14 families. Linkage analysis suggested a susceptibility locus on chromosome 8, but direct molecular sequencing did not support this initial statistical finding. Exome sequencing revealed the absence of any gene that was mutated in all patients. Exhaustive screening of genes involved in other autoinflammatory syndromes or encoding components of the human inflammasome showed no DNA variants that could be linked to PFAPA molecular pathology. Among these, the previously-reported missense mutation V198M in the NLRP3 gene was clearly shown not to co-segregate with PFAPA. Our results on this relatively large cohort indicate that PFAPA syndrome is unlikely to be a monogenic condition. Moreover, none of the several genes known to be involved in inflammation or in autoinflammatory disorders seem to be relevant, alone, to its etiology, suggesting that PFAPA results from oligogenic or complex inheritance of variants in multiple disease genes and/or non-genetic factors

Desmocollin 1 and desmoglein 1 expression in human epidermis and keratinizing oral mucosa: a comparative immunohistochemical and molecular study

Epidermis and keratinizing oral mucosa (KOM) are effective barriers against a wide spectrum of insults. The optimal form of protection provided by each epithelium is determined also by the molecular composition of desmosomes. Up to now, the expression of the "skin type" desmosomal cadherins, i.e. desmocollin 1 (Dsc1) and desmoglein 1 (Dsg1), was correlated with the morphological features of keratinocyte terminal differentiation in epidermis, but not in KOM. The aim of the present study was to investigate Dsc1 and Dsg1 expression in adult human KOM compared to epidermis. Biopsies of epidermis and KOM were obtained from young healthy adults (n=6) and simultaneously processed for immunofluorescence analysis, post-embedding immunogold electron microscopy (immunogold EM), and RT-PCR analysis. For molecular biology analysis, as a negative control, we considered human fibroblasts. By immunofluorescence and immunogold EM, Dsc1 labeling was not detected in any suprabasal layer of KOM, but it was present in the upper spinous/granular layers of epidermis. Immunofluorescence and transmission electron microscopy analysis showed that (i) Dsg1 expression was evident in the spinous, granular, and horny layer of the oral epithelium and (ii) Dsg1 immunoreactivity was always lower in desmosomes between oral keratinocytes than in all epidermal junctions. RT-PCR analysis confirmed that in KOM Dsc1 gene expression was undetectable. On the whole, these observations suggest a weakened adhesion in KOM, allowing oral keratinocytes to undergo a faster transition throughout the living layers of the epithelium. The intrinsic and specific regulation of the molecular composition of desmosomes can contribute in defining a specific keratinocyte phenotype in KOM and in epidermis

AutoMap is a high performance homozygosity mapping tool using next-generation sequencing data.

Homozygosity mapping is a powerful method for identifying mutations in patients with recessive conditions, especially in consanguineous families or isolated populations. Historically, it has been used in conjunction with genotypes from highly polymorphic markers, such as DNA microsatellites or common SNPs. Traditional software performs rather poorly with data from Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS), which are now extensively used in medical genetics. We develop AutoMap, a tool that is both web-based or downloadable, to allow performing homozygosity mapping directly on VCF (Variant Call Format) calls from WES or WGS projects. Following a training step on WES data from 26 consanguineous families and a validation procedure on a matched cohort, our method shows higher overall performances when compared with eight existing tools. Most importantly, when tested on real cases with negative molecular diagnosis from an internal set, AutoMap detects three gene-disease and multiple variant-disease associations that were previously unrecognized, projecting clear benefits for both molecular diagnosis and research activities in medical genetics

Conjunctival Melanoma Targeted Therapy: MAPK and PI3K/mTOR Pathways Inhibition.

To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells. The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining. A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested. The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant

Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia Defects.

Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa

Early epidermal response after a single dose of gamma-rays in organotypic culture of human breast skin

BACKGROUND: Skin reaction is the most common side-effect of radiation therapy. Radiation-induced dermal fibrosis has been characterized histologically, but little is known about the epidermis overlying fibronecrotic lesions. OBJECTIVES: To characterize the epidermal response 24 h after a single clinically relevant dose of gamma-rays in cultured human breast skin. METHODS: Biopsies obtained from cosmetic surgery (n = 7) were placed epidermis upwards in a Transwell system, and were exposed to a single dose of gamma-irradiation (2 Gy). A parallel set of nonirradiated skin fragments was incubated under the same conditions. Both irradiated and nonirradiated fragments were harvested 24 h after irradiation and processed for light microscopy and molecular biology analysis. A quantitative analysis of cell proliferation was performed after 5-bromo-2'-deoxyuridine incorporation. Cytokeratin 10 (CK10) and desmocollin 1 (Dsc1) expression was evaluated by immunofluorescence. Dsc1 and transforming growth factor (TGF)-beta1 gene expression was measured by reverse transcriptase-polymerase chain reaction analysis. RESULTS: The mean percentage inhibition of epidermal proliferation in irradiated samples was 53.7% (P < 0.01, paired Student's t-test). The inhibition of cell proliferation was significant in five of seven samples (P < 0.05, unpaired Student's t-test). Normal cell architecture was found in irradiated samples. Throughout the epithelial compartment, the distribution patterns of CK10 and Dsc1 were comparable in nonirradiated and irradiated fragments. Condensation of CK10 filaments suggested a cytoskeletal rearrangement in irradiated samples. Dsc1 and TGF-beta1 mRNA levels were, respectively, reduced and unmodified 24 h after irradiation. CONCLUSIONS: A perturbation of epidermal homeostasis occurs as early as 24 h after a single dose of gamma-rays. Our immunofluorescence observations indicate that keratinocyte terminal differentiation is not yet affected at the protein level 24 h after exposure to gamma-rays. The lack of an inverse relationship between TGF-beta1 gene expression and epidermal proliferation, together with decreased Dsc1 gene expression, may represent the early molecular basis for the development of the late effects of radiotherapy observed many months/years after radiotherapy. Our findings set the stage for further investigation of the best time to begin topical treatment at the start of radiation therapy