Maria Margollicci

Spermatogenesis in a man with complete deletion of USP9Y.

Deletions in the azoospermia factor region AZFa on the human Y chromosome and, more specifically, in the region that encompasses the ubiquitin-specific peptidase 9, Y-linked gene USP9Y have been implicated in infertility associated with oligospermia and azoospermia. We have characterized in detail a deletion in AZFa that results in an absence of USP9Y in a normospermic man and his brother and father. The association of this large deletion with normal fertility shows that USP9Y, hitherto considered a candidate gene for infertility and azoospermia, does not have a key role in male reproduction. These results suggest that it may not be necessary to consider USP9Y when screening the Y chromosome of infertile or subfertile men for microdeletions.

Chitotriosidase and soluble IL‐2 receptor: Comparison of two markers of sarcoidosis severity

Background. Sarcoidosis is a multisystemic granulomatous disease with an unpredictable clinical course characterized by accumulation of activated proliferating T lymphocytes and mononuclear phagocytes in affected organs. Aims and methods. The aims of this study were to describe the clinical, radiological and immunological features of a population of sarcoidosis patients followed at the Sarcoidosis Regional Centre in Siena and to analyse chitotriosidase and sIL‐2R concentrations in serum of these patients in order to understand their potential as disease markers. Results. Chitotriosidase and sIL‐2R concentrations in serum of sarcoidosis patients were found to be significantly higher than in healthy controls (p<0.01) and a positive correlation between the two markers was documented for the first time. Moreover, chitotriosidase and sIL‐2R were expressed differently in different radiographic stages of the disease. Conclusion. Chitotriosidase and sIL‐2R are two markers of sarcoidosis of different origin, the values of which show a correlation in these patients; they are easily detectable in serum and could be useful clinical markers of progression.

Chitotriosidase Activity in the Serum of Patients with Sarcoidosis and Pulmonary Tuberculosis

Background: Human chitotriosidase is a chitinase selectively expressed by activated macrophages. An increase in chitotriosidase activity was previously described by us in the serum and bronchoalveolar lavage of sarcoidosis patients. Objective: The aim of the present study was to analyze serum chitotriosidase activity in a larger number of sarcoidosis patients to verify the reported increase with respect to controls and to compare serum chitotriosidase levels in patients with sarcoidosis and tuberculosis, two granulomatous disorders of different etiology. Methods: Chitotriosidase activity was measured in the serum of 96 sarcoidosis patients, 15 pulmonary tuberculosis patients and 30 healthy controls. Results: We found significantly higher serum chitotriosidase activity in sarcoidosis patients than controls (p < 0.01) and in sarcoidosis patients than tuberculosis patients (p < 0.01), confirming a striking elevation of chitotriosidase activity (>10 times greater than normal) in pulmonary sarcoidosis patients. This is the first time that chitotriosidase activity has been analyzed in the serum of patients with pulmonary tuberculosis; it was found to be significantly lower than in sarcoidosis patients and not significantly greater than in controls. Conclusion: Although the mechanisms leading to the increase in chitotriosidase activity in sarcoidosis are still unknown, this enzyme may be specifically involved in the pathogenesis of the disease. Further studies with a greater number of patients are needed to confirm these results and to determine whether chitotriosidase could be a marker with diagnostic or prognostic value in sarcoidosis.

VARS2 and TARS2 Mutations in Patients with Mitochondrial Encephalomyopathies

By way of whole‐exome sequencing, we identified a homozygous missense mutation in VARS2 in one subject with microcephaly and epilepsy associated with isolated deficiency of the mitochondrial respiratory chain (MRC) complex I and compound heterozygous mutations in TARS2 in two siblings presenting with axial hypotonia and severe psychomotor delay associated with multiple MRC defects. The nucleotide variants segregated within the families, were absent in Single Nucleotide Polymorphism (SNP) databases and are predicted to be deleterious. The amount of VARS2 and TARS2 proteins and valyl‐tRNA and threonyl‐tRNA levels were decreased in samples of afflicted patients according to the genetic defect. Expression of the corresponding wild‐type transcripts in immortalized mutant fibroblasts rescued the biochemical impairment of mitochondrial respiration and yeast modeling of the VARS2 mutation confirmed its pathogenic role. Taken together, these data demonstrate the role of the identified mutations for these mitochondriopathies. Our study reports the first mutations in the VARS2 and TARS2 genes, which encode two mitochondrial aminoacyl‐tRNA synthetases, as causes of clinically distinct, early‐onset mitochondrial encephalopathies.

GM2 gangliosidosis variant B1 neuroradiological findings.

Abstract. Variant B1 is a rare type of GM2 gangliosidosis. Clinically, it shows a wide spectrum of forms ranging from infantile to juvenile. We report the first magnetic resonance imaging (MRI) findings from three patients affected by GM2 gangliosidosis variant B1, two presenting with the infantile form and one with the juvenile form. The MRI appearances of the two patients with the infantile form disease are congruent with those reported for the early-onset type of both Tay-Sachs and Sandhoff diseases, and are characterized by early involvement of the basal ganglia and thalamus with cortical atrophy appearing later. In contrast, the patient with the juvenile form of variant B1 showed progressive cortical and white-matter atrophy of the supratentorial structures and, to a lesser extent, the infratentorial structures. No basal ganglia or thalamic anomalies were observed. Because in the adult forms of both Tay-Sachs and Sandhoff diseases a progressive cerebellar atrophy represents the only abnormality detectable, it appears that an MRI pattern peculiar to GM2 gangliosidosis can be defined. This pattern ranges from the basal ganglia injury associated with the early and severe demyelination process noted in the infantile form of the disease, to cerebellar atrophy with no supratentorial anomalies in the adult form. An “intermediate” MRI picture, with cortical atrophy and mild cerebellar atrophy, but without basal ganglia impairment, can be observed in the juvenile form. In addition, our investigations suggest that MRI abnormalities in GM2 gangliosidosis correlate with the clinical form of the disease rather than with the biochemical variant of the enzymatic defect.

Late-onset MNGIE without peripheral neuropathy due to incomplete loss of thymidine phosphorylase activity

Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmoplegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1 gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramitochondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neuropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, including a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase activity may induce a late-onset and incomplete MNGIE phenotype.

HLA-DQ typing in the diagnostic algorithm of celiac disease

OBJECTIVE celiac disease (CD) is an immune-mediated chronic inflammatory disease associated with HLA-DQ2 and DQ8 molecules. We evaluated the role of HLA in the CD diagnostic algorithm in order to contribute to the development of practical indications for the use of HLA typing. MATERIAL AND METHODS we selected 317 subjects typed for DR-DQ genes. CD was present in 123 patients, and 89 were included in the study; a control sample of 70 healthy individuals was recruited. RESULTS 64% of patients with CD carried DQ2 heterodimer (α5β2), 13.5% carried DQ8 heterodimer without DQ2, 21.4% only showed β2 chain and 1.1% were positive for DQ2 α5 chain. The only presence of α5 chain did not predispose to CD, while DQB1*02 allele resulted more frequent than in other reports, pointing out the intrinsic correlation between β2 chain and CD. In the case-control study we observed a progression of increased risk, ranging from 1:7 for HLA-DQ2 homozygous to 1:85 for DQ8 heterozygous subjects. Overall, 8,6% of first degree family members were affected, exclusively in presence of HLA-DQ2, -DQ8 or DQB1*02, and CD was significantly more frequent among siblings than parents. Finally, considering the different patterns of clinical presentation among the HLA-DQ risk classes identified we found no relationship between CD clinical presentation and HLA-DQ risk categories. CONCLUSIONS our results strengthen the evidence that HLA-DQ status strongly influences the development of CD and demonstrate that knowledge of a patients HLA-DQ genotype allows to establish clinically relevant genetic risk profiles.

Postictal serum nucleotidases activities in patients with epilepsy

Adenosine, a potent anticonvulsant, can be produced in the body by the hydrolysis of adenine nucleotides through the action of ecto- or soluble nucleotidases. Changes in nucleotide hydrolysis occur after pentylenetetrazol-induced epileptic events. We evaluated serum ATP, ADP and AMP hydrolysis rates and soluble nucleotide phosphodiesterase (PDEase) activity at 5, 10, 15, 30 and 60 min, and 12h following an epileptic event. Fifteen patients (seven female, eight male; mean age 15.5 years) were included in the study. The type of seizure was generalized in four patients and was localization related in the remaining 11. There were no differences in adenine nucleotide hydrolysis rates between patients and healthy subjects in the interictal stage. In comparison with controls, ATP, ADP and AMP hydrolysis rates were significantly increased at 5 min (53+/-1.4%, 79.2+/-2.8% and 37.0+/-2.6%, respectively) and up to 30 min following the epileptic event. In contrast to ADP and AMP, ATP hydrolysis remained significantly increased at 60 min (71.4+/-1.6%), returning to the basal level after 12h. Serum PDEase activity was also significantly higher in the patients than in healthy subjects, peaking at 15 min (61+/-2.9%) and remaining significantly increased up to 60 min (4.6+/-1.2%) following the epileptic episode. Globally, the variations in the postictal serum ADP hydrolysis rate almost overlapped those of AMP hydrolysis, whereas changes in the ATP hydrolysis rate overlapped those of PDEase activity. The clinical significance of this elevation in postictal soluble serum nucleotidase activity remains to be clarified. However, it is possible to hypothesize that the higher nucleotidase activity might play a role in the modulation of epileptic events.

Serum Chitotriosidase Levels in Patients with Allergic and Non-Allergic Asthma

Since the data did not have a normal distribution (Shapiro-Wilk’s test), statistical differences were detected by the Kruskal-Wallis test (p !0.05. ) Serum concentrations of chitotriosidase were 48.72 8 31 nmol/h/ml in allergic asthmatics, 51.5 8 29 nmol/h/ml in non-allergic asmathics and 21.42 8 11 nmol/h/ml in controls ( fig. 1 ). Significant differences were found between the concentrations of chitotriosidase in asthmatics and controls (p ! 0.05 ). Concentra-tions of chitotriosidase greater than normal values were observed in 60% of allergic and 63% of non-allergic asthmatics. No signifi-cant differences were found between chitotriosidase concentra-tions in allergic and non-allergic patients, although the highest concentrations of chitotriosidase were found in 4 patients with documented allergy to dust and moulds. . a l tj e l ec Tre [8] s tudied serum chitotriosidase activity in vari-ous lung diseases, finding concentrations of chitotriosidase in a small population of asthmatics similar to ours. Unfortunately this study did not divide patients according to their history of allergy or specify whether any groups of patients had higher chitotriosi-dase concentrations. Asthma is a complex inflammatory disorder of the airways characterized by bronchial hyper-reactivity to different stimuli, and its pathogenesis is not always completely clear. Chitinases and chitinase-like proteins have recently been proposed as molecules involved in the pathogenesis of asthma with a possible role as prognostic biomarkers [1–6] . Higher serum concentrations of YKL-40 (chitinase-3-like-1), a ‘mammalian chitinase-like protein’, have been reported [2, 4, 5] in asthma patients and hyper-responsive subjects than in con-trols. The YKL-40 gene has been associated with genetic suscep-tibility to the disease. Acid mammalian chitinase (AMCase) has been studied in animal models of airway inflammation [3] and AMCase gene expression has been investigated in detail in BAL fluid macrophages and epithelial brushing from asthma patients [1, 5] . Another member of the chitinase 1 family not yet studied in detail in asthma is chitotriosidase, an enzyme produced by acti-vated macrophages, structurally similar to YKL-40 but having chitinolytic activity [7, 8] . In the last 5 years our research group has investigated the role of chitotriosidase in the pathogenesis of different inflammatory lung diseases, such as sarcoidosis [9] . Here we analyzed serum concentrations of chitotriosidase in al-lergic and non-allergic asthma patients and in a group of controls to evaluate its potential involvement in the different pathways of inflammation occurring in this disease.Ch itotriosidase concentrations were measured in serum sam-ples from 23 patients with allergic asthma, 19 non-allergic asth-matics and 35 healthy controls by a fluorimetric method (Sigma, St. Louis, Mo., USA). Selected patients with PC20 methacholine less than 8 mg/ml were followed at our centre for almost 5 years. They were divided into allergic and non-allergic groups according to medical history, prick test and RAST test results. All patients performed lung function tests, chest X-ray and total IgE analysis at the time of serum sampling. Statistical analysis was performed using Windows software (2005) Graph Pad Prism version 4.0.

A second MNGIE patient without typical mitochondrial skeletal muscle involvement

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by mutations in the gene encoding thymidine phosphorylase (TYMP). Clinically, MNGIE is characterized by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. Most MNGIE patients have signs of mitochondrial dysfunction in skeletal muscle at morphological and enzyme level, as well as mitochondrial DNA depletion, multiple deletions and point mutations. A case without mitochondrial skeletal muscle involvement and with a TYMP splice-acceptor site mutation (c. 215–1 G>C) has been reported. Here, we describe an Italian patient with the same mutation and without mitochondrial skeletal muscle involvement, suggesting a possible genotype–phenotype correlation.