Deep phenotyping of facioscapulohumeral muscular dystrophy type 2 by magnetic resonance imaging.

BACKGROUND AND PURPOSE: The aim was to define the radiological picture of facioscapulohumeral muscular dystrophy 2 (FSHD2) in comparison with FSHD1 and to explore correlations between imaging and clinical/molecular data. METHODS: Upper girdle and/or lower limb muscle magnetic resonance imaging scans of 34 molecularly confirmed FSHD2 patients from nine European neuromuscular centres were analysed. T1-weighted and short-tau inversion recovery (STIR) sequences were used to evaluate the global pattern and to assess the extent of fatty replacement and muscle oedema. RESULTS: The most frequently affected muscles were obliquus and transversus abdominis, semimembranosus, soleus and gluteus minimus in the lower limbs; trapezius, serratus anterior, latissimus dorsi and pectoralis major in the upper girdle. Iliopsoas, popliteus, obturator internus and tibialis posterior in the lower limbs and subscapularis, spinati, sternocleidomastoid and levator scapulae in the upper girdle were the most spared. Asymmetry and STIR hyperintensities were consistent features. The pattern of muscle involvement was similar to that of FSHD1, and the combined involvement of trapezius, abdominal and hamstring muscles, together with complete sparing of iliopsoas and subscapularis, was detected in 91% of patients. Peculiar differences were identified in a rostro-caudal gradient, a predominant involvement of lower limb muscles compared to the upper girdle, and in the higher percentage of STIR hyperintensities in FSHD2. CONCLUSION: This multicentre study defines the pattern of muscle involvement in FSHD2, providing useful information for diagnostics and clinical trial design. Both similarities and differences between FSHD1 and FSHD2 were detected, which is also relevant to better understand the pathogenic mechanisms underlying the FSHD-related disease spectrum.

Follicular occlusion tetrad in a male patient with pachyonychia congenita: clinical and genetic analysis.

The case of a 24-year-old male patient affected by follicular occlusion tetrad (acne conglobata, hidradenitis suppurativa, pilonidal cyst and dissecting cellulitis of the scalp) associated with clinical signs of pachyonychia congenita (PC)-2 (focal palmoplantar keratoderma, plantar pain, onycodystrophy and multiple cysts) is reported. The diagnosis was supported by genetic analysis that showed heterozygous mutation within the exon 1 of KRT17 gene. This case may reflect different expressions of a phenotypic spectrum induced by a common genetic alteration.

Bilateral Retinal Angiomatous Proliferation in a Variant of Retinitis Pigmentosa.

PURPOSE: To describe the first case of bilateral retinal angiomatous proliferation (RAP) in a patient with a variant of retinitis pigmentosa (RP). CASE REPORT: An 85-year-old man with RP presented with visual acuity decrease and metamorphopsia in the left eye (LE). Fundus examination revealed typical signs of RP in both eyes, associated with intraretinal macular hemorrhage in the LE. Multimodal imaging, using Colour fundus Photography, Fluorescein (FA), and Indocyanine Green Angiography (ICGA) as well as Spectral-Domain Optical Coherence Tomography (SD-OCT) and Optical Coherence Tomography Angiography (OCTA), revealed a type 3 neovascular lesion in the involved eye. Genetic testing (NGS analysis) was performed to search for genetic variants correlated with the disease phenotype displayed by the patient. The patient was treated with intravitreal injections of bevacizumab, according to a fixed protocol of bimonthly injections plus a booster dose at second month. After 9 months, he was referred for visual acuity decrease and metamorphopsia in the fellow eye, where SD-OCT/OCTA showed a type 3 neovascular lesion in the right eye (RE). He was scheduled for intravitreal injections of bevacizumab. In both eyes, treatment with intravitreal bevacizumab was successful.

Assessing individual risk for AMD with genetic counseling, family history, and genetic testing.

PurposeThe goal was to develop a simple model for predicting the individual risk profile for age-related macular degeneration (AMD) on the basis of genetic information, disease family history, and smoking habits.Patients and methodsThe study enrolled 151 AMD patients following specific clinical and environmental inclusion criteria: age >55 years, positive family history for AMD, presence of at least one first-degree relative affected by AMD, and smoking habits. All of the samples were genotyped for rs1061170 (CFH) and rs10490924 (ARMS2) with a TaqMan assay, using a 7500 Fast Real Time PCR device. Statistical analysis was subsequently employed to calculate the real individual risk (OR) based on the genetic data (ORgn), family history (ORf), and smoking habits (ORsm).Results and conclusionThe combination of ORgn, ORf, and ORsm allowed the calculation of the Ort that represented the realistic individual risk for developing AMD. In this report, we present a computational model for the estimation of the individual risk for AMD. Moreover, we show that the average distribution of risk alleles in the general population and the knowledge of parents' genotype can be decisive to assess the real disease risk. In this contest, genetic counseling is crucial to provide the patients with an understanding of their individual risk and the availability for preventive actions.

PCR-based approach for qualitative molecular analysis of six neurotropic pathogens.

In the last few years, polymerase chain reaction analysis is frequently required to improve the detection of pathogen infections in central nervous system as a potential cause of neurological disorders and neuropsychiatric symptoms. The goal of this paper is to set up a fast, cheap and reliable molecular approach for qualitative detection of six neurotropic pathogens. A method based on PCR has been designed and implemented to guarantee the qualitative DNA detection of herpes simplex virus types 1 and 2 (HSVI/II), Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus (RUBV) and Toxoplasma gondii in the cerebrospinal fluid, where otherwise they are barely detectable. Each PCR assay was tested using dilutions of positive controls, which demonstrated a sensitivity allowing to detect up to 102 copies/ml in PCR and 10 copies/ml in real-time PCR for each pathogen. Once been set up, the protocol was applied to evaluate the cerebrospinal fluid from 100 patients with suspected infectious diseases of the central nervous system and 50 patients without any infection. The method allowed to identify 17 positive cerebrospinal fluid with polymerase chain reaction and 22 with real-time PCR (RT-PCR), respectively. Therefore, application of RT PCR allows a fast and sensitive evaluation of neurotropic DNA pathogens in the course of diagnostic routine within neurological units.

Sentinel node mapping with radiotracer alone in vulvar cancer: a five year single-centre experience and literature review.

PURPOSE OF INVESTIGATION: The pathologic status of lymph node represents the most important prognostic factor in vulvar cancer patients, but a complete groin dissection is associated with high post-operative morbidity. Sentinel lymph node (SLN) could be representative of the totality of regional lymph nodes and consequently its biopsy might have a significant impact on clinical management in vulvar cancer patients. MATERIALS AND METHODS: From January 2006 to December 2010 45 patients with vulvar carcinoma are evaluated. Preoperative lymphatic mapping with technetium-99m-labeled nanocolloid was performed in all patients, followed by radioguided intraoperative detection. The detection rate is 100% of patients. All the SLNs were dissected separately for histopathological evaluation and a routine inguinofemoral lymphadenectomy was performed. RESULTS: Nine patients had positive SLNs. In the remaining 36 patients with negative SLNs, one of them showed positive non-SLNs at histological examination. It was the only false negative case in the present series. CONCLUSIONS: Based on literature review, lymphoscintigraphy and sentinel node biopsy under gamma-detecting probe guidance offer a reliable and careful method to identify sentinel node in early vulvar cancer. Taking certain guidelines, SLN biopsy seems to be a safe alternative to inguinofemoral node dissection in order to reduce morbidity of surgical treatment.

Development of an Italian RM Y-STR haplotype database: Results of the 2013 GEFI collaborative exercise.

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural" and "urban" samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes.

Direct PCR: a new pharmacogenetic approach for the inexpensive testing of HLA-B*57:01.

One of the most successful applications of pharmacogenetics research is the genetic screening for HLA-B*57:01, strongly associated with an increased risk to develop hypersensitivity reaction in HIV-positive patients following abacavir administration. Taking into consideration the limits of current genotyping methodologies, we have developed and validated (150 buccal swabs) an inexpensive pharmacogenetic approach for HLA-B*57:01 typing. In our assay DNA extraction and amplification are combined in one single step (direct PCR protocol), which is performed directly on the biological sample without the need of extraction and sequencing passages. The amplicons obtained by direct PCR can be easily separated on the agarose gel under ultraviolet. As per our results, the direct PCR represents a good alternative to the traditional methods of HLA-B*57:01 pharmacogenetic test, especially for those laboratories or countries where currently available approaches are often not available or not affordable. Furthermore it is an innovative approach, promoting a personalized, safer and cost-effective therapy.

Association of KIF3A, but not OVOL1 and ACTL9, with atopic eczema in Italian patients.

BACKGROUND: Atopic eczema (AE) (OMIM %603165) is the most common chronic inflammatory skin disease characterized by xerosis, pruritus, and erythematous lesions with increased transepidermal water loss. It's a complex disease due to the interaction between environmental and genetics factors. To date, different loci have been related to the disease. OBJECTIVES: To verify the association, between AE and rs479844, rs2164983, and rs2897442, target for OVOLI (11q13), ACTL9 (19p13.2), and in KIF3A (5q31) genes in the Italian population. Recently, these SNPs have been validated as associated to the disease. METHODS: A case-control study testing a cohort of 359 AE cases and 778 controls. RESULTS: We confirmed the association between rs2897442 in KIF3A gene and the disease at both allele and genotype level (P-value: 4.8 × 10(-4) and P-value: 6.3 × 10(-4), respectively). The C allele of the SNP showed an Odds Ratio (OR) of 1.46 (95% CI 1.18-1.82), moreover the CC genotype achieved an OR of 2.77 (95% CI 1.66-4.61). We failed to reveal association between AE and the other two SNPs tested. CONCLUSIONS: Our study indicated KIF3A as a novel gene implicated in the development of AE in the Italian population.

Population data for 17 Y-chromosome STRs in a sample from Apulia (Southern Italy).

The 17 Y-STR loci included in the AmpFLSTR Yfiler PCR Amplification Kit were analyzed in 98 unrelated healthy males from Apulia (Southern Italy). A total of 97 different haplotypes were identified, of which 96 haplotypes were unique and 1 occurred twice. Allele frequencies for each Y-STR locus in pooled sample and estimated value of gene diversity (GD) were evaluated. The lowest value of GD was observed for DYS392 (0.126) and the highest one (0.936) for DYS385. The HD (haplotype diversity) for the studied Y-STR set showed a value of 0.9994, with an HMP (haplotype match probability) value of 0.0006, while the overall DC was 98.98%. Microvariant alleles were found for the DYS458 and DYS385 markers and sequenced. Furthermore, Φ(st)-based genetic distance computation and pair-wise analysis of molecular variance (AMOVA) test were carried out. When comparing our population with the Apulia sample previously investigated, the AMOVA analysis detected no evidence for significant differentiation. The comparison with all Italian populations submitted to the YHRD website showed no relevant differences with all Southern Italian populations (San Giorgio La Molara, Belvedere, Trapani and Catania) and significant genetic deviation with all Northern Italian populations (Udine, Biella, La Spezia, Modena, Ravenna, Marche and North Sardinia). Moreover, the other populations and meta-populations belonging to the whole Mediterranean area (Croatia, Macedonia, Albania, Greece, Turkey, Israel, Libya, Tunisia, Algeria, Morocco and Spain) were different from our Apulia sample. The data were submitted to YHRD.

Allele frequencies of the new European Standard Set (ESS) loci in a population of Apulia (Southern Italy).

Allele frequencies of five miniSTRs loci (D1S1656, D2S441, D12S391, D10S1248 and D22S1045) included in the new European Standard Set (ESS) were calculated from a sample of 150 unrelated individuals from Apulia, a Region of Southern Italy. Two different PCR Amplification Kits were used, in order to evaluate the concordance of the genotypes. The results obtained with the two kits showed no differences in all genotype profiles. No deviation from Hardy-Weinberg expectations was detected at either locus. Moreover genetic analysis using Fst estimation showed no evidence for differentiation at the five new loci between Apulia and Italian populations. The high levels of polymorphisms of the analyzed markers in the Apulian population allow to confirm that these markers are useful tools in paternity and forensic analysis from degraded DNA samples.

The 2011 GeFI collaborative exercise. Concordance study, proficiency testing and Italian population data on the new ENFSI/EDNAP loci D1S1656, D2S441, D10S1248, D12S391, D22S1045.

The 2011 collaborative exercise of the ISFG Italian Working Group GeFI was aimed at validating the five ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. The protocol required to type at least 50 multilocus profiles from locally resident individuals and two blind bloodstains in duplicate (i.e., using at least two different commercial kits), and to send the electropherograms to the Organizing Committee. Nineteen laboratories distributed across Italy participated, collecting a total of 960 samples. Full concordance was found for the five new miniSTRs as observed from the comparison of 13,150 alleles. The inspection of the electropherograms allowed the identification of a very limited number of mistypings in the miniSTR genotypes thus contributing to the establishment of an high quality Italian database of frequencies.

Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells.

Alveolar type II pneumocytes (ATII cells) are considered putative alveolar stem cells. Since no treatment is available to repair damaged epithelium and prevent lung fibrosis, novel approaches to induce regeneration of injured alveolar epithelium are desired. The objective of this study was to assess both the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATII cells and the ability of committed HUES-3 cells (HUES-3-ATII cells) to recover in vivo a pulmonary fibrosis model obtained by silica-induced damage. In vitro differentiated HUES-3-ATII cells displayed an alveolar phenotype characterised by multi-lamellar body and tight junction formation, by the expression of specific markers such as surfactant protein (SP)-B, SP-C and zonula occludens (ZO)-1 and the activity of cystic fibrosis transmembrane conductance regulator-mediated chloride ion transport. After transplantation of HUES-3-ATII cells into silica-damaged mice, histological and biomolecular analyses revealed a significant reduction of inflammation and fibrosis markers along with lung function improvement, weight recovery and increased survival. The persistence of human SP-C, human nuclear antigen and human DNA in the engrafted lungs indicates that differentiated cells remained engrafted up to 10 weeks. In conclusion, cell therapy using HUES-3 cells may be considered a promising approach to lung injury repair.

Effects of paraquat and capsaicin on the expression of genes related to inflammatory, immune responses and cell death in immortalized human HaCat keratinocytes.

The present experiments were aimed to characterize in immortalized human HaCat keratinocytes the gene expression induced by paraquat and capsaicin, two agents known to induce cell death or to affect inflammatory and pain pathways, respectively. In particular, the following set of genes were analysed by qRealtime PCR: CXCL10,CXCL11, IL-10 (inflammatory and immune responses), TP73, BCL2, (apoptotic and anti-apoptotic genes), MMP9 (proteolysis), SOD-1, BAK-1 and CAT (peroxysomal and microsomal oxidation pathways). In this way, we were able to differentiate the two toxins since they had a different profile of gene expression. In fact, paraquata was found to activate set of genes involved in inflammatory (CXL10,CXL11 and IL-10), and cell death (BCL2, BAK-1, MMP9) pathways. Another specific site of action of paraquat was represented by an activation of the gene involved in SOD-1 transcription. On the contrary, capsaicin was found to produce only an up-regulation of BCL2, an anti-apoptotic gene and MMP9, whereas no significant changes were reported in genes involved in inflammatory and immune responses. Finally, in comparison to previous experiments carried out with TNF-alpha and IL-1beta, we have shown that paraquat produced a similar pattern of activation of set of genes involved both in inflammation and apoptosis.

Effects of TNF-α and IL-1 ß on the activation of genes related to inflammatory, immune responses and cell death in immortalized human HaCat keratinocytes.

The present experiments were designed to characterize by microarray analysis the transcriptional responses of human keratinocytes (HaCat) to TNF-α and IL-1 ß, given alone or in combination, in order to better understand the mechanisms underlying inflammatory, immune responses and cell death in which both cytokines play a pathophysiological role. Significant differences in the percentage and quality of genes dysregulated by TNF-α and IL-1 ß were shown. Both cytokines activated a series of genes involved in inflammatory, immune response as well as in cell death. In our experimental conditions, TNF-α, in contrast to IL-1 ß, did not induce a significant level of apoptosis in keratinocytes. However, given together both cytokines produced a significant decrease in apoptotic cells and synergistic transcriptional response which was due to the activation of several specific genes occurring after application of each cytokine. TNF-α and IL-1 ß evoked apoptotic effect and transcriptional responses were linked to the stimulation of their specific receptors since a pre-treatment with monoclonal antibodies vs TNF-α and/or IL-1 ß receptors was able to significantly reduce them.

Long-term anti-tumour necrosis factor therapy reverses the progression of carotid intima-media thickness in female patients with active rheumatoid arthritis.

The objective of the study is to evaluate the effect of TNF inhibition on carotid thickness over a 2-year period. 144 women with RA diagnosed according to ACR criteria, without clinical evidence of cardiac and/or vascular disease were enrolled and compared with 78 matched controls. All patients received methotrexate (15­20 mg weekly) for 3 months. Responders (n = 79) continued to be treated with methotrexate, non-responders (n = 40) moved to methotrexate plus a TNF alpha antagonist. Echosonographic studies of carotids were obtained before and after 2-year follow-up. A significant decrease of ca-IMT was observed in anti-TNF-treated patients (P < 0.001); on the other hand, no significant variation of ca-IMT was observed after 2 years in MTX-treated patients. Our study indicates that anti-TNF blocking agents, but not methotrexate, are capable of reducing IMT of carotid arteries in female RA patients in a 2-year follow-up.

"The Linosa Study": epidemiological and heritability data of the metabolic syndrome in a Caucasian genetic isolate.

BACKGROUND AND AIMS: Growing evidence suggests that the metabolic syndrome (MetS) has both a genetic and environmental basis. To evaluate the possibility of a further genetic analysis, we estimated prevalence rates and heritabilities for the MetS and its individual traits in the adult population of Linosa, a small and isolated Italian Island in the southern-central part of the Mediterranean Sea. METHODS AND RESULTS: The Linosa Study (LiS) group consisted of 293 Caucasian native subjects from 51 families (123 parents; 170 offsprings). The MetS was defined according to NCEP/ATP III criteria and the following prevalence rates were calculated: hyperglycaemia 20.3%; central obesity 34.9%; hypertension 43.4%; hypertriglyceridaemia 29.9%; "low HDL" 56.6%; MetS 29.9%. Waist circumference was significantly related to all the quantitative parameters included in the NCEP/ATP III MetS definition. The MetS showed a heritability of 27% (p=0.0012) and among its individual components, treated as continuous and discrete traits, heritability ranged from 10% for blood glucose to 54% for HDL-cholesterol. Among MetS subtypes, the clustering of central obesity, hypertriglyceridaemia and "Iow HDL" had the highest heritability (31%; p<0.001). CONCLUSION: These data showed high prevalence rates for the MetS and its related traits in an isolated and small Caucasian population. The appreciable heritability estimates for the MetS and some of its components/clusters in the LiS population might support the observation of genetic factors underlying the pathogenesis of the MetS and encourage further analysis to identify new susceptibility genes.

Divergent phenotype of two siblings human leukocyte antigen identical, affected by nonclassical and classical congenital adrenal hyperplasia caused by 21-hydroxylase deficiency.

CONTEXT: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders most often caused by enzyme 21-hydroxylase deficiency. Most mutations causing enzymatic deficiency are generated by recombinations between the active gene CYP21 and the pseudogene CYP21P. Only 1-2% of affected alleles result from spontaneous mutations. The phenotype of CAH varies greatly, usually classified as classical or nonclassical, depending on variable degree in 21-hydroxylase activity. Here we report a divergent phenotype of two human leukocyte antigen identical siblings, affected by nonclassical and classical CAH caused by 21-hydroxylase deficiency due to different genotype. PATIENTS AND METHODS: Using direct sequencing method and Southern blot, we studied two children (one male and one female), affected, respectively, by nonclassical and classical CAH and their parents. RESULTS: The mother was heterozygous for the Q318X mutation, and the father was heterozygous for the V281L mutation. The brother was a compound heterozygote for the mutations V281L and Q318X, whereas the proband was compound heterozygote for the Q318X mutation and a large conversion. The two children are human leukocyte antigen identical (A*02;B*14;DRB1*01/A*33;B*14;DRB1*03). CONCLUSIONS: Different phenotype of the proband is the result of compound heterozygosity for the maternal mutation Q318X and a de novo large conversion.