A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

Ex vivo fluorescence confocal microscopy: the first application for real-time pathological examination of prostatic tissue.

OBJECTIVE: To report the first application of ex vivo fluorescence confocal microscopy (FCM) - a novel optical technology that is capable of providing fast microscopic imaging of unfixed tissue specimens- in the urological field assessing its diagnostic accuracy for non neoplastic and cancerous prostate tissue (prostatic adenocarcinoma) compared to the 'gold standard' histopathological diagnoses. PATIENTS AND METHODS: In all, 89 specimens from 13 patients with clinically localised prostate cancer were enrolled into the study. All patients underwent robot-assisted laparoscopic radical prostatectomy with fresh prostatic tissue biopsies taken at the end of each intervention using an 18-G biopsy punch. Specimens were randomly assigned to the three collaborating pathologists for evaluation. Intra- and inter-observer agreement was tested by the means of Cohen's κ. The diagnostic performance was evaluated on receiver operating characteristic curve analysis. RESULTS: The overall diagnostic agreement between FCM and histopathological diagnoses was substantial with a 91% correct diagnosis (κ = 0.75) and an area under the curve of 0.884 (95% confidence interval 0.840-0.920), 83.33% sensitivity, and 93.53% specificity. CONCLUSION: FCM seems to be a promising tool for enhanced specimens' reporting performance, given its simple application and very rapid microscopic image generation (<5 min/specimen). This technique may potentially be used for intraoperative pathological specimens' analysis.

Targeted Next Generation Sequencing in patients with Myotonia Congenita.

INTRODUCTION: Myotonia Congenita (MC) is a nondystrophic skeletal muscle disease characterized by muscle stiffness, weakness, delayed skeletal relaxation and hypertrophic muscle. The disease can be inherited as dominant or recessive. More than 130 mutations in CLCN1 gene have been identified. MATERIALS AND METHODS: We analyzed the entire coding region and exon-intron boundaries of the CLCN1 gene in 40 MC patients. Samples already Sanger-sequenced were successively evaluated by Next Generation Sequencing (NGS), on Ion Torrent PGM. Moreover, additional 15 patients were sequenced directly by NGS. RESULTS: NGS allowed us to identify all CLCN1 mutations except those located within exon 3, demonstrating a 96% of sensitivity. Due to primer design, one SNP (exactly rs7794560) also failed to be detected. Our results enlarge the spectrum of CLCN1 mutations and showed a novel approach for molecular analysis of MC.

Influence of lifestyle habits, nutritional status and insulin resistance in NAFLD.

Non alcoholic fatty liver disease (NAFLD) is associated with obesity, diabetes and insulin resistance (IR). The aim of our study was to assess the relationship between IR, anthropometry, lifestyle habits, resting energy expenditure (REE) and degree of fatty liver at ultrasound in 48 overweight patients with NAFLD as compared to 24 controls without fatty liver, matched for age. Nutritional status, alcohol intake and physical activity were assessed by skinfold thickness measurements, a 7-day diary, and SenseWear armband (SWA). REE was assessed by both SWA (REE-SWA) and a Vmax metabolic cart (REE-Vmax). Fatty liver was measured by US and the Doppler Power Index was calculated. IR was assessed using the HOMA index. There was significant correlation between waist circumference, HOMA, Doppler power index and fatty liver grade at US. Multivariate analysis showed that alteration of waist circumference, Doppler power index, and HOMA were the major significant predictors of fatty liver. Our data demonstrated a significant association between NAFLD and central adiposity and IR.

Effect of intense military training on body composition.

Individuals in a structural physical training program can show beneficial changes in body composition, such as body fat reduction and muscle mass increase. This study measured body composition changes by using 3 different techniques-skinfold thickness (SF) measurements, air displacement plethysmography (BOD-POD), and dual-energy x-ray absorptiometry (DXA)-during 9 months of intense training in healthy young men engaged in military training. Twenty-seven young men were recruited from a special faction of the Italian Navy. The program previewed three phases: ground combat, sea combat, and amphibious combat. Body composition was estimated at the beginning, in the middle, and at the end of the training. After the subjects performed the ground combat phase, body composition variables significantly decreased: body weight (P < 0.05), fat-free mass (FFM) (P < 0.001), and fat mass (FM) (P < 0.03). During the amphibious combat phase, body weight increased significantly (P < 0.01), mainly because of an increase in FFM (P < 0.001) and a smaller mean decrease in FM. There was a significant difference (P < 0.05) in circumferences and SF at various sites after starting the training course. Bland-Altman analysis did not show any systematic difference between FM and FFM measured with the 3 different techniques on any occasion. On any visit, FFM and FM correlation measured by BOD-POD (P = 0.90) and DXA was significantly greater than measured by SF. A significant difference was found in body mass index (BMI) measured during the study. BOD-POD and SF, compared with DXA, provide valid and reliable measurement of changes in body composition in healthy young men engaged in military training. In conclusion, the findings suggest that for young men of normal weight, changes in body weight alone and in BMI are not a good measure to assess the effectiveness of intense physical training programs, because lean mass gain can masquerade fat weight loss.