Alazami syndrome: the first case of papillary thyroid carcinoma.

Alazami syndrome (MIM#615071) is a rare developmental disorder caused by biallelic variants in the LARP7 gene. Hallmark features include short stature, global developmental delay, and distinctive facial features. To date, 23 patients from 11 families have been reported in the literature. Here we describe a 19-year-old man who, in association with the typical features of Alazami syndrome, was diagnosed at the age of 14 years with papillary thyroid carcinoma, harboring the somatic BRAF V600E mutation. Whole exome sequencing revealed two novel LARP7 variants in compound heterozygosity, whereas only common variants were detected in genes associated with familial nonmedullary thyroid cancer (MIM#188550). LARP7 acts as a tumor suppressor in breast and gastric cancer, and possibly, according to recent studies, in thyroid tumors. Since thyroid cancer is rare among children and adolescents, we hypothesize that the LARP7 variants identified in our patient are responsible for both Alazami syndrome and tumor susceptibility. We also provide an overview of the clinical findings in all Alazami syndrome patients reported to date and discuss the possible pathogenetic mechanism that may underlie this condition, including the role of LARP7 in tumor susceptibility.

The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

Uncoupling of satellite DNA and centromeric function in the genus Equus.

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

Sexual Dysfunction: A Neglected and Overlooked Issue in Adult GH Deficiency: The Management of AGHD Study.

CONTEXT: Although sexuality influences well-being and quality of life (QoL), studies on sexual dysfunction (SD) in adult growth hormone deficiency (AGHD) patients are lacking. OBJECTIVE: To investigate the prevalence of SD in AGHD patients grouped according to recombinant human growth hormone (r-hGH) therapy. DESIGN: Prospective, cross-over, 24 months, monocentric study. SETTING: Real-life clinical setting in a tertiary, endocrinological center. PATIENTS: 83 AGHD patients (31 women, 52 men, mean age 56.3 ± 14.7 years) were enrolled according to stringent criteria. INTERVENTIONS: Patients already on long-term r-hGH therapy (Group 1, n = 32) vs untreated (Group 2, n = 51). MAIN OUTCOME MEASURES: Serum hormones, QoL Satisfaction in Hypopituitarism (QLS-H) and QoL Assessment of GHD in Adults (QoL-AGHDA) questionnaires for QoL, Index for Erectile Function-15 (IIEF-15) in men, and Female Sexual Function Index (FSFI) in women for SD. RESULTS: The overall prevalence of SD was 71.2% (60% men, 89% women). All IIEF-15 scores were lower (P = 0.001) and erectile dysfunction was more prevalent in Group 2 (75%) than Group 1 (35%). IGF-1 was correlated to scores of all IIEF-15 domains, particularly with that of erectile function (EF) (R2=0.123, P = 0.019). EF domain score correlated with QLS-H (P < 0.005) and QoL-AGHDA (P = 0.001). Despite the high prevalence of female SD also in untreated AGHD women, FSFI scores did not correlate with IGF-1 levels and QoL scores. CONCLUSIONS: SD is highly prevalent in AGHD patients, especially in those untreated. SD represents an overlooked and neglected issue in AGHD, regardless the contribution of sexual life on QoL. The evaluation of sexual function should be integrated in the global assessment of AGHD patients.

Expression and clinicopathological role of miR146a in thyroid follicular carcinoma.

PURPOSE: Dysregulation of microRNA expression has been involved in the development and progression of follicular thyroid carcinoma (FTC). The aim of this work was to study the expression of miRNA146a in FTC and the association with clinicopathological features of the disease. METHODS: Thirty-eight patients affected by FTC were included in the study. Twenty patients carrying follicular thyroid adenoma (FA) were also enroled as the benign counterpart of FTC. Total RNA including miRNA146a was extracted from formalin-fixed paraffin-embedded (FFPE) pairs of affected/unaffected tissue and its expression was assessed by real-time PCR. Two selected target genes, TRAF6 (tumour necrosis factor receptor-associated factor 6) and IRAK1 (Il-1 receptor-associated kinase 1/2), were also analysed. RESULTS: miR146a expression in FTC tissue was overall not downregulated in malignant versus unaffected tissue, but its expression was inversely correlated with clinicopathological features of FTCs at diagnosis. A decreased expression of miR146a became apparent in FTC thyroid tissue of widely compared to minimally invasive tumours. However, miR146a expression differences between contralateral unaffected tissue (extra-FTC) and FTC were not observed regardless of clinicopathological features. IRAK1, a known target for miR146a, was upregulated in FTC and the increase was mainly appreciable in Hurtle FTC variant. Unexpectedly, miR146a did not correlate with TRAF6 showing an inverse trend compared to IRAK1 although both genes regulate the activity of nuclear factor- kB (NF-kB). CONCLUSION: The results of this study indicate that downregulation of miR146a, inversely correlated with clinicopathological features of FTCs at diagnosis and suggest a possible involvement of miR146a in FTC development. IRAK1 over-expression in FTC may be related to tumour development/progression. In vitro experiments are needed to support this hypothesis.

Seasonal variation of semen parameters correlates with environmental temperature and air pollution: A big data analysis over 6 years.

BACKGROUND: Male fertility is progressively declining in many developed countries, but the relationship between male infertility and environmental factors is still unclear. OBJECTIVES: To assess the influence of environmental temperature and air pollution on semen parameters, using a big-data approach. METHODS: A big data analysis of parameters related to 5131 men, living in a province of Northern Italy and undergoing semen analyses between January 2010 and March 2016 was performed. Ambient temperature was recorded on the day of analysis and the 90 days prior to the analysis and the average value of particulate matter (PM) and NO2 in the year of the test. All data were acquired by geocoding patients residential address. A data warehouse containing 990,904,591 data was generated and analysed by multiple regressions. RESULTS: 5573 semen analyses were collected. Both maximum and minimum temperatures registered on the day of collection were inversely related to total sperm number (p < .001), non-progressive motility (NPrM) (p < .005) and normal forms (p < .001). Results were confirmed considering temperature in the 30 and 60 days before collection, but not in the 90 days before collection. Total sperm number was lower in summer/autumn (p < .001) and was inversely related with daylight duration (p < .001). PM10 and PM2.5 were inversely related to PrM (p < .001 and p < .005) and abnormal forms (p < .001). CONCLUSIONS: This is the first evaluation of the relationship between male fertility-related parameters and environment using a big-data approach. A seasonal change in semen parameters was found, with a fluctuation related to both temperature and daylight duration. A negative correlation between air pollution and semen quality is suggested. Such seasonal and environmental associations should be considered when assessing changes of male fertility-related parameters over time.

New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining.

The non-homologous end-joining (NHEJ) pathway is a mechanism to repair DNA double strand breaks, which can introduce mutations at repair sites. We constructed new cellular systems to specifically analyze sequence modifications occurring at the repair site. In particular, we looked for the presence of telomeric repeats at the repair junctions, since our previous work indicated that telomeric sequences could be inserted at break sites in germ-line cells during primate evolution. To induce specific DNA breaks, we used the I-SceI system of Saccharomyces cerevisiae or digestion with restriction enzymes. We isolated human and hamster cell lines containing the I-SceI target site integrated in a single chromosomal locus and we exposed the cells to a continuous expression of the I-SceI endonuclease gene. Additionally, we isolated human cell lines that expressed constitutively the I-SceI endonuclease and we introduced the target site on an episomal plasmid stably transfected into the cells. These strategies allowed us to recover repair junctions in which the I-SceI target site was modified at high frequency (100% in hamster cells and about 70% in human cells). Finally, we analyzed junctions produced on an episomal plasmid linearized by restriction enzymes. In all the systems studied, sequence analysis of individual repair junctions showed that deletions were the most frequent modifications, being present in more than 80% of the junctions. On the episomal plasmids, the average deletion length was greater than at intrachromosomal sites. Insertions of nucleotides or deletions associated with insertions were rare events. Junction organization suggested different mechanisms of formation. To check for the insertion of telomeric sequences, we screened plasmid libraries representing about 3.5 x 10(5) junctions with a telomeric repeat probe. No positive clones were detected, suggesting that the addition of telomeric sequences during double strand break repair in somatic cells in culture is either a very rare event or does not occur at all.

Telomerase in differentiated thyroid cancer: promoter mutations, expression and localization.

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm.

Evolutionary movement of centromeres in horse, donkey, and zebra.

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years). These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.