MRI and US in the evaluation of fetal anomalies: The need to work together.

PURPOSE: Real-time virtual sonography (RVS) is a new technique that synchronizes real-time ultrasonography (US) and multiplanar reconstructed magnetic resonance imaging (MRI). The purpose of this study was to evaluate the feasibility and ability of RVS to assess the main pathologies in fetuses with suspected US anomalies. METHOD AND MATERIALS: Real-time virtual sonography (Hitachi, HI VISION Ascendus) was offered to 30 patients who had undergone fetal MRI. The acquired MRI image dataset was loaded into the fusion system and displayed together with the real-time US image. The ability of RVS to assess the main anatomical sites and fetal anomalies was evaluated. RESULTS: Real-time virtual sonography was technically possible in all cases. From a total of 30 patients, RVS helped the diagnosis in 10 cases. In 15 cases of encephalic pathology, fusion imaging improved the accuracy of the diagnosis; in the other 5 cases, MRI was superior to US even when using the RVS. CONCLUSION: This is a study on the feasibility and practical use of RVS. Thanks to information from both US and MRI, RVS allowed better identification of the fetal pathologies and improved the performance of the ultrasound examination. In our experience, it was really helpful in pathologies that would benefit from US follow-up.

Array-comparative genomic hybridization analysis in a cohort of 130 children with Autism Spectrum Disorders: A single Center Italian Study.

Introduction: utism spectrum disorder (ASD) is a heterogeneous clinical condition, and its genetic basis is widely confirmed. The chromosomal microarray analysis (CMA) is a first-line diagnostic test that identifies copy number variants (CNVs). Some of these genomic rearrangements are associated with ASD, but the meaning of most of them is still unknown. Materials and methods: We performed a comparative genome hybridization (array-CGH) analysis in 130 children with confirmed ASD. Genetic results were analyzed and compared to clinical phenotype. Results and discussion.: 61/130 children carry CNVs, 44 presenting variants of unknown significance (u-CNVs), and 17 with susceptibility-CNVs (c-CNVs). Clinical evaluation showed no differences in cognitive abilities, language and EEG abnormalities, ASD symptoms among CNVs group and other patients. Finally, we highlight the role of GPHN, IMMP2L and ZMYND11, as ASD susceptibility genes. Conclusions: Our findings underscore the importance of array-CGH in ASD children since new CNVs and emerging genes appear to be associated with different clinical pictures.

Genetic association of HLA-DQB1 and HLA-DRB1 polymorphisms with alopecia areata in the Italian population.

BACKGROUND: Alopecia areata (AA) is a multifactorial disease characterized by hair loss especially from the scalp. As for other autoimmune conditions, the major histocompatibility complex (HLA) region is associated with AA susceptibility. OBJECTIVE: To provide evidence for the association of specific HLA-DQB1 and HLA-DRB1 alleles with AA in an Italian population, using a case-control approach. METHODS: We performed a case-control study to investigate whether HLA-DQB1 and -DRB1 alleles predispose to AA in the Italian population. HLA class II typing was performed in 85 patients with AA and 210 healthy controls from the same ethnic group. RESULTS: An increased frequency of DQB1*03, coding for DQ7 heterodimers, and a decreased rate of the DQB1*06 allele were observed in patients when compared with controls; the greatest and significant difference was in the group of cases with a more severe phenotype [AA>50% patients (more than 50% hair loss) vs. controls, P=4·5×10(-3) , P(c)=0·031, odds ratio (OR) 2·01, 95% confidence interval (CI) 1·22-3·31 and P=2·5×10(-3) , P(c)=0·017, OR 0·22, 95% CI 0·07-0·72, respectively]. DQB1*03, serologically related to DQ8 or coding for DQ9 molecules, was not associated with AA susceptibility. Out of all patients, 65·9% carried DQ7 heterodimers compared with 49·5% of the controls (P=7·3×10(-3) , OR 1·97, 95% CI 1·17-3·32) and DQ7 prevalence rose to 76·3% in patients with AA>50% (P=1·7×10(-3) , OR 3·28, 95% CI 1·48-7·27). No significant difference was found in the distribution of DRB1 variants or phenotypes among cases and controls. CONCLUSION: Our data show a correlation between the HLA-DQB1 locus and the occurrence of AA in Italy supporting DQB1*03(DQ7) as a predisposing allele for the disease and the relevance of the HLA genetic test in the clinical management of AA.

Ulerythema ophryogenes, a rare and often misdiagnosed syndrome: analysis of an idiopathic case.

Keratosis pilaris (KP) is a follicular hyperkeratosis disorder which is frequently detected in the adult population (44%), mostly in female adolescents (80%). It is a genetic autodominant dermatosis with variable penetrance, but no specific gene association has been determined, even though association to the presence of chromosome 18p deletion has been reported in some cases. We report the case of a 51-year-old Caucasian woman affected by keratosis pilaris gradually progressing with age and with a story of multiple abortions. Standard karyotype and CGH array analyses did not reveal any genetic abnormality. Virological analyses detected the presence of HPV 36 DNA inside the dorsum biopsy, leading to hypothesize its involvement in the evolution of the lesion. Clinical history and patient examination led the diagnosis of an idiopathic case of Ulerythema ophryogenes. The analysis of more cases could be useful to verify the involvement of cutaneous HPV in the progression of the clinical manifestation of the KP variants.

Single-nucleotide polymorphisms in 3'-untranslated region inducible costimulator gene and the important roles of miRNA in alopecia areata.

Background: Alopecia areata (AA) spares the stem cell compartment and attacks only the base of the hair follicle, which is surrounded by infiltrating lymphocytes. AA is associated with polymorphisms in immune-related genes and with decreased function of CD4+CD25+ T regulatory (Treg) cells. Treg function is modulated by the costimulatory molecules, like inducible costimulator (ICOS) that are crucial in orienting T cell differentiation and function so that they strongly impact on the immunologic decision between tolerance or autoimmunity development. Objective: The aim of our study was to investigate the possible association of AA with single-nucleotide polymorphisms (SNP) present in the ICOS 3'-untranslated region (3'UTR) region and to elucidate how SNPs modulate ICOS gene expression by affecting miRNA binding sites. Methods: This is a case-control study performed in 184 patients with AA and 200 controls. ICOS gene and miRNA expression were analyzed by real-time polymerase chain reaction. Results: The genotype carrying the rs4404254(C) [p = 0.012, OR (95% CI): 0.5 (0.3-0.8)] and rs4675379(C) [p = 0.015, OR (95% CI): 0.3 (0.1-0.8)] 3' UTR alleles was more frequently observed in AA patients than in controls and correlated with a reduced ICOS expression. miR-1276 significantly suppressed ICOS expression by binding to the 3'UTR of ICOS mRNA. Also, we observed that, miR-101 and miR-27b are upregulated, while miR-103 and miR-2355-3p are downregulated in peripheral blood mononuclear cells of AA patients compared to controls. Conclusion: Our data show that rs4404254 and rs4675379 SNPs of ICOS gene are associated with AA and also reveal that the presence of rs4404254 polymorphism correlates with ICOS post-transcriptional repression by microRNA binding.

Giant scrotal elephantiasis: an idiopathic case.

Scrotal elephantiasis is very rare disease in industrialized countries, where it is mainly due to surgery, irradiation or malignancies. It can be defined as idiopathic only when the possible congenital, infectious and compressive causes are excluded. We report a case of massive scrotal lymphoedema in an adult Caucasian patient, in Italy. He presented an extremely voluminous scrotal mass measuring 50 x 47 x 13 cm (weight 18 kg), which extended below his knees, invalidating all his daily activities. The patient was hospitalized in order to undergo to surgical treatment. Although genetic causes were searched and the possible role of infectious agents and compressive factors was evaluated, no etiology was ascertained. Histopathologic examination showed non-specific chronic inflammation, confirming the diagnosis of idiopathic elephantiasis. One year after surgical treatment, the patient is healthy without recurrence signs.

Triplet repeat mutations in human disease.

Triplet repeats are the sites of mutation in three human heritable disorders, spinal and bulbar muscular atrophy (SBMA), fragile X syndrome, and myotonic dystrophy (DM). These repeats are GC-rich and highly polymorphic in the normal population. Fragile X syndrome and DM are examples of diseases in which premutation alleles cause little or no disease in the individual, but give rise to significantly amplified repeats in affected progeny. This newly identified mechanism of mutation has, so far, been identified in two of the most common heritable disorders, fragile X syndrome and DM, and one rare disease, SBMA.

Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.

The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with the adult form of myotonic dystrophy.

An unstable triplet repeat in a gene related to myotonic muscular dystrophy.

Synthetic oligonucleotides containing GC-rich triplet sequences were used in a scanning strategy to identify unstable genetic sequences at the myotonic dystrophy (DM) locus. A highly polymorphic GCT repeat was identified and found to be unstable, with an increased number of repeats occurring in DM patients. In the case of severe congenital DM, the paternal triplet allele was inherited unaltered while the maternal, DM-associated allele was unstable. These studies suggest that the mutational mechanism leading to DM is triplet amplification, similar to that occurring in the fragile X syndrome. The triplet repeat sequence is within a gene (to be referred to as myotonin-protein kinase), which has a sequence similar to protein kinases.

Androgen insensitivity syndrome.

OBJECTIVE: We provide a review of the literature about the Androgen Insensitivity Syndrome (AIS), its onset and associated developmental anomalies and the genetic alterations causing it. MATERIALS AND METHODS: We searched PubMed with a larger emphasis on the physiology, genetics and current management of AIS. RESULTS: AIS is an X-linked recessive Disorder of Sex Development (DSD). It is caused by mutations of the Androgen Receptor, and their large amount and heterogeneity (missense and nonsense mutations, splicing variants, deletions, and insertions) are responsible for the wide spectrum of possible phenotypes of patients, divided into Partial AIS (PAIS) and Complete AIS (CAIS). Once the clinical and laboratory investigations have laid the foundation for a diagnostic hypothesis, it is important to identify the actual karyotype of the individual and search for the mutation in the Androgen Receptor to diagnose with certainty the syndrome. Alternatively, in the absence of such evidence, the diagnosis should more properly be an AIS-like condition, which we describe as well in our report. CONCLUSIONS: The management of this DSD is based on pharmacotherapies, surgery and psychological support: all of them must be directed to facilitate the patient's life, considering his/her sexual identity.

Structure and expression of the human ubiquitin fusion-degradation gene (UFD1L).

We report the genomic organization, RNA and protein expression patterns of the gene encoding for the human homolog of the yeast ubiquitin fusion-degradation protein-1 (UFD1L). This enzyme is involved in a ubiquitin-dependent proteolytic pathway (UFD), firstly described in yeast. The human UFD1L gene is organized into 12 exons ranging in size from 33 to 161 bp. Sequence analysis of the 5'-flanking region of the gene revealed a high GC content, multiple CCAAT-binding motifs, CREB, CFT, and AP-2 sites. RNA transcripts were detected in all tissues and cell lines examined, including thymus, thymocytes, T- and B-cells, fibroblasts, chorionic villi, and amniocytes. In Western blot, the UFD1L antibody demonstrated the presence of multiple protein isoforms in all the tested tissues. Expression profile and promoter characteristics suggest UFD1L is a housekeeping gene with implications in the pathogenesis of DiGeorge/velo-cardio-facial syndrome, due to 22q11.2 deletions.

Association between the human glycoprotein PC-1 gene and elevated glucose and insulin levels in a paired-sibling analysis.

We studied whether there is an association between the single nucleotide polymorphism c.533A>C (K121Q) in the glycoprotein PC-1 gene and features of the metabolic syndrome in case-control and intrafamily association studies in 922 subjects from Finland and Sweden. No difference was observed in the Q allele frequency between control subjects and type 2 diabetic subjects (12.9 vs. 15.1%). The QK genotype was associated with higher fasting plasma glucose (FPG) concentrations than the KK genotype in type 2 diabetic patients (P <0.001) and their relatives (P <0.05). A permutation test of siblings discordant for the QK and KK genotypes also showed that the nondiabetic siblings with the QK genotype had higher FPG (6.1 +/- 2.0 vs. 5.4 +/- 0.6 mmo/l, P <0.001) and fasting insulin (7.0 +/- 3.6 vs. 4.8 +/- 2.6 mU/l, P <0.05) concentrations than the carriers of the KK genotype. In addition, diabetic siblings with the QK genotype had higher systolic blood pressure (147.0 +/- 18.0 vs. 140.0 +/- 18.7 mmHg, P <0.05) and higher fasting (9.9 +/- 3.0 vs. 8.8 +/- 2.8 mmol/l, P <0.05) and 2-h plasma glucose (17.3 +/- 8.5 vs. 12.9 +/- 4.2 mmol/l, P < 0.05) concentrations than the diabetic carriers of the KK genotype. The present study shows that, although the Q allele of the human glycoprotein PC-1 gene is associated with surrogate measures of insulin resistance, it may not be enough to increase the susceptibility to type 2 diabetes.

The Q allele variant (GLN121) of membrane glycoprotein PC-1 interacts with the insulin receptor and inhibits insulin signaling more effectively than the common K allele variant (LYS121).

When overexpressed, the membrane glycoprotein PC-1 may play a role in human insulin resistance through the inhibition of insulin receptor (IR) autophosphorylation. A PC-1 variant (K121Q, with lysine 121 replaced by glutamine) is also associated with whole-body insulin resistance when not overexpressed. To better understand the effects of the Q allele on IR function and downstream signaling, we transfected cultured cells with cDNAs for either the Q or the K alleles. In human MCF-7 cells, the Q allele was severalfold more effective (P < 0.05-0.01) than the K allele in reducing insulin stimulation of IR autophosphorylation, insulin receptor substrate-1 phosphorylation, phosphatidylinositol 3-kinase activity, glycogen synthesis, and cell proliferation. Similar data on IR autophosphorylation inhibition were also obtained in mouse R-/hIR and human HEK 293 cell lines. In transfected MCF-7 cells, 125I-labeled insulin binding and IR content were unchanged, and PC-1 overexpression did not influence IGF-1 stimulation of IGF-1 receptor autophosphorylation. Both the Q and K alleles directly interacted with the IR, as documented by coimmunoprecipitation assays. This interaction was greater for the Q allele than for the K allele (P < 0.01), suggesting that direct PC-1-IR interactions are important for the PC-1 inhibitory effect on insulin signaling. In conclusion, the Q allele has stronger inhibitory activity on IR function and insulin action than the more common K allele, and this is likely a consequence of the intrinsic characteristics of the molecule, which more strongly interacts with the IR.

The K121Q variant of the human PC-1 gene is not associated with insulin resistance or type 2 diabetes among Danish Caucasians.

The human plasma-cell membrane differentiation antigen-1 (PC-1) has been shown to inhibit insulin receptor tyrosine kinase activity. Recently, a K121Q polymorphism in the human PC-1 gene was found in a Sicilian population and was shown to be strongly associated with insulin resistance. The objectives of the present investigation were to examine in the Danish Caucasian population whether the K121Q variant was associated with type 2 diabetes or, in glucose-tolerant subjects, with impaired whole-body insulin sensitivity. We genotyped 404 Danish type 2 diabetic patients and found that the allele frequency of the variant was 0.14 (95% CI 0.12-0.16), whereas the allele frequency was 0.16 (95% CI 0.13-0.19) among 237 matched glucose-tolerant control subjects (P = 0.6). In the control subjects, there were no significant differences among wild-type, heterozygous, or homozygous subjects in regard to 1) serum insulin and plasma glucose levels at fasting, 60 min, or 120 min during an oral glucose tolerance test (OGTT) or 2) the estimates of insulin resistance obtained from the homeostasis model assessment (HOMA). Furthermore, we investigated the impact of the variant in 2 other Danish population samples that comprised 356 young healthy subjects and 226 glucose-tolerant offspring of type 2 diabetic probands, respectively. In all of the study populations, the polymorphism was not associated with an altered insulin sensitivity index as estimated from an intravenous glucose tolerance test in combination with an intravenous injection of tolbutamide. In addition, among the 226 offspring, the variations in serum insulin and serum C-peptide responses measured during an OGTT were not related to the PC-1 genotype. In conclusion, the K121Q polymorphism of the human PC-1 gene is not associated with type 2 diabetes or insulin resistance among Danish Caucasians.

A polymorphism (K121Q) of the human glycoprotein PC-1 gene coding region is strongly associated with insulin resistance.

The genes responsible for insulin resistance are poorly defined. Plasma cell differentiation antigen (PC-1) glycoprotein inhibits insulin receptor signaling and is associated with insulin resistance. We describe here a novel polymorphism in exon 4 of the PC-1 gene (K121Q) and demonstrate that it is strongly associated with insulin resistance in 121 healthy nonobese (BMI <30 kg/m2) nondiabetic (by oral glucose tolerance test [OGTT]) Caucasians from Sicily. Compared with 80 KK subjects, Q allele carriers (n = 41, 39 KQ and 2 QQ) showed higher glucose and insulin levels during OGTT (P < 0.001 by two-way analysis of variance) and insulin resistance by euglycemic clamp (M value = 5.25 +/- 1.38 [n = 24] vs. 6.30 +/- 1.39 mg x kg(-1) x min(-1) [n = 49], P = 0.005). Q carriers had higher risk of being hyperinsulinemic and insulin resistant (odds ratio [CI]: 2.99 [1.28-7.0], P < 0.001). Insulin receptor autophosphorylation was reduced (P < 0.01) in cultured skin fibroblasts from KQ versus KK subjects. Skeletal muscle PC-1 content was not different in 11 KQ versus 32 KK subjects (33 +/- 16.1 vs. 17.5 +/- 15 ng/mg protein, P = 0.3). These results suggest a cause-effect relationship between the Q carrying genotype and the insulin resistance phenotype, and raise the possibility that PC-1 genotyping could identify individuals who are at risk of developing insulin resistance, a condition that predisposes to type 2 diabetes and coronary artery disease.

A PC-1 amino acid variant (K121Q) is associated with faster progression of renal disease in patients with type 1 diabetes and albuminuria.

Insulin resistance characterizes type 1 diabetes in patients with albuminuria. A PC-1 glycoprotein amino acid variant, K121Q, is associated with insulin resistance. We examined the impact of the PC-1 K121Q variant on the rate of decline of the glomerular filtration rate (GFR) by creatinine clearance derived from the Cockroft-Gault formula in 77 type 1 diabetic patients with albuminuria who were followed for an average of 6.5 years (range 2.5-15). Patients carrying the Q allele (n = 22; 20 with KQ and 2 with QQ genotypes) had a faster GFR decline than those patients with the KK genotype (n = 55) (median 7.2 vs. 3.7 ml x min(-1) x year(-1); range 0.16 to 16.6 vs. -3.8 to 16.0 ml x min(-1) x year(-1); P < 0.001). Significantly more patients carrying the Q allele belonged to the highest tertile of GFR decline (odds ratio = 5.7, 95% CI 4.1-7.2, P = 0.02). Levels of blood pressure, HbA1c, and albuminuria were comparable in the two genotype groups. Albuminuria (P = 0.001), mean blood pressure (P = 0.046), and PC-1 genotype (P = 0.036) independently correlated with GFR decline. Because all patients were receiving antihypertensive treatment, the faster GFR decline in the patients carrying the Q allele could be the result of reduced sensitivity to the renoprotective effect of antihypertensive therapy. PC-1 genotyping identifies type 1 diabetic patients with a faster progression of diabetic nephropathy.

A cluster of three single nucleotide polymorphisms in the 3'-untranslated region of human glycoprotein PC-1 gene stabilizes PC-1 mRNA and is associated with increased PC-1 protein content and insulin resistance-related abnormalities.

Glycoprotein PC-1 inhibits insulin signaling and, when overexpressed, plays a role in human insulin resistance. Mechanisms of PC-1 overexpression are unknown. We have identified a haplotype in the 3'-untranslated region of the PC-1 gene that may modulate PC-1 expression and confer an increased risk for insulin resistance. Individuals from Sicily, Italy, carrying the "P" haplotype (i.e., a cluster of three single nucleotide polymorphisms: G2897A, G2906C, and C2948T) were at higher risk (P < 0.01) for insulin resistance and had higher (P < 0.05) levels of plasma glucose and insulin during an oral glucose tolerance test and higher levels of cholesterol, HDL cholesterol, and systolic blood pressure. They also had higher (P < 0.05-0.01) PC-1 protein content in both skeletal muscle and cultured skin fibroblasts. In CHO cells transfected with either P or wild-type cDNA, specific PC-1 mRNA half-life was increased for those transfected with P (t/2 = 3.73 +/- 1.0 vs. 1.57 +/- 0.2 h; P < 0.01). In a population of different ethnicity (Gargano, East Coast Italy), patients with type 2 diabetes (the most likely clinical outcome of insulin resistance) had a higher P haplotype frequency than healthy control subjects (7.8 vs. 1.5%, P < 0.01), thus replicating the association between the P allele and the insulin resistance-related abnormalities observed among Sicilians. In conclusion, we have identified a possible molecular mechanism for PC-1 overexpression that confers an increased risk for insulin resistance-related abnormalities.

LGI1 gene mutation screening in sporadic partial epilepsy with auditory features.

Partial epilepsy with auditory features occasionally segregates in families as an autosomal dominant trait. In some families mutations in the leucine-rich glioma inactivated (LGI1) gene have been identified. Sporadic cases might harbour either denovo or low-penetrant LGI1 mutations, which will substantially alter the family risk for epilepsy. We selected sixteen sporadic patients with cryptogenic temporal lobe epilepsy and partial seizures with auditory features. We compared clinical features of these patients with those of published autosomal dominant family cases. We screened these patients for LGI1 mutations. Comparing the sporadic patients with the published familial cases no difference in either the primary auditory features or in the other associated epileptic manifestations was identified. Sequence analysis of the whole LGI1 gene coding regions in sporadic patients did not reveal changes in the LGI1 gene. The genetic analysis demonstrates that LGI1 is not a major gene for sporadic cases of partial epilepsy with auditory features at least in the Italian population. Screening of sporadic patients for LGI1 mutations appears not useful in genetic counselling of these patients.

Mapping of a new autosomal dominant non-syndromic hearing loss locus (DFNA43) to chromosome 2p12.

Hearing impairment (HI) is the most frequent sensory defect with wide genetic heterogeneity. Approximately 80% of genetic hearing loss is non-syndromic and 15-25% of exhibit autosomal dominant inheritance. We analysed an Italian three generation family in which non-syndromic hearing impairment is transmitted as an autosomal dominant trait. Onset of HI in all affected subjects occurred in the second decade of life, with subsequent gradual progression from moderate to profound loss. HI was bilateral and symmetrical, involving all frequencies. After exclusion of the known DFNA loci with markers from the Hereditary Hearing Loss Homepage (URL: http://dnalab-www.uia.ac.be/dnalab/hhh), a genome wide scan was carried out using 358 highly informative microsatellite markers. Significant linkage (Zmax=4.21, theta=0) was obtained with chromosome 2p12 markers. The results were confirmed by multipoint analysis (Zmax=4.51), using the location score method. Haplotype analysis defined a 9.6 cM disease gene interval on chromosome 2 without overlap with the other identified loci. Fine mapping and identification of candidate genes are in progress.

A novel PTPN11 mutation in LEOPARD syndrome.

PTPN11 gene mutations are common to both patients with Noonan (NS) and LEOPARD syndrome (LS). So far only two recurrent mutations have been identified in LS patients by different research groups, i.e., Tyr279Cys and Thr468Met. In this work we describe the third PTPN11 mutation that has been found in a single LS patient. The mutation (c.1517A>C) substitutes a proline for a glutamine at amino acid 506 (Gln506Pro) in the phosphatase domain (PTP) of the PTPN11 peptide SHP2. This region is a mutation hotspot. Changes at amino acids 501 to 504 cause NS. Gln506Pro is predicted, by modeling analysis, to seriously disrupt the normal contacts between the regulating N-SH2 and the active PTP domains, leading to hyperactivity of the phosphatase. This report demonstrates that rarer mutations other than Tyr279Cys and Thr468Met can be found in LS patients and the need of screening the whole gene in those negative for the commonest mutations.