Genetica molecolare dell'autismo: studi di associazione ed analisi di geni candidati

Autism is a neurodevelpmental disorder characterized by impaired verbal communication, limited reciprocal social interaction, restricted interests and repetitive behaviours. Twin and family studies indicate a large genetic contribution to ASDs (Autism Spectrum Disorders). During my Ph.D. I have been involved in several projects in which I used different genetic approaches in order to identify susceptibility genes in autism on chromosomes 2, 7 and X: 1)High-density SNP association and CNV analysis of two Autism Susceptibility Loci. The International Molecular Genetic Study of Autism Consortium (IMGSAC) previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we evaluated the patterns of linkage disequilibrium (LD) and the distribution of haplotype blocks, utilising data from the HapMap project, across the two strongest peaks of linkage on chromosome 2 and 7. More than 3000 SNPs have been selected in each locus in all known genes, as well as SNPs in non-genic highly conserved sequences. All markers have been genotyped to perform a high-density association analysis and to explore copy number variation within these regions. The study sample consisted of 127 and 126 multiplex families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Association and CNV analysis implicated several new genes, including IMMP2L and DOCK4 on chromosome 7 and ZNF533 and NOSTRIN on the chromosome 2. Particularly, my contribution to this project focused on the characterization of the best candidate gene in each locus: On the AUTS5 locus I carried out a transcript study of ZNF533 in different human tissues to verify which isoforms and start exons were expressed. High transcript variability and a new exon, never described before, has been identified in this analysis. Furthermore, I selected 31 probands for the risk haplotype and performed a mutation screen of all known exons in order to identify novel coding variants associated to autism. On the AUTS1 locus a duplication was detected in one multiplex family that was transmitted from father to an affected son. This duplication interrupts two genes: IMMP2L and DOCK4 and warranted further analysis. Thus, I performed a screening of the cohort of IMGSAC collection (285 multiplex families), using a QMPSF assay (Quantitative Multiplex PCR of Short fluorescent Fragments) to analyse if CNVs in this genic region segregate with autism phenotype and compare their frequency with a sample of 475 UK controls. Evidence for a role of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family. 2)Analysis of X chromosome inactivation. Skewed X chromosome inactivation (XCI) is observed in females carrying gene mutations involved in several X-linked syndromes. We aimed to estimate the role of X-linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 164 affected girls. The study sample included families from different european consortia. I analysed the XCI inactivation pattern in a sample of italian mothers from singletons families with ASD and also a control groups (144 adult females and 40 young females). We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X-linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X-linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27-Xq28 region, with a peak Z score of 1.75 close to rs719489. In this region FMR1 and MECP2 have been associated in some cases with austim and therefore represent candidates for the disorder. I performed a mutation screen of MECP2 in 33 unrelated probands from IMGSAC and italian families, showing XCI skewness. Recently, Xq28 duplications including MECP2, have been identified in families with MR, with asymptomatic carrier females showing extreme (>85%) skewing of XCI. For these reason I used the sample of probands from X-skewed families to perform CNV analysis by Real-time quantitative PCR. No duplications have been found in our sample. I have also confirmed all data using as alternative method the MLPA assay (Multiplex Ligation dependent Probe Amplification). 3)ASMT as functional candidate gene for autism. Recently, a possible involvement of the acetylserotonin O-methyltransferase (ASMT) gene in susceptibility to ASDs has been reported: mutation screening of the ASMT gene in 250 individuals from the PARIS collection revealed several rare variants with a likely functional role; Moreover, significant association was reported for two SNPs (rs4446909 and rs5989681) located in one of the two alternative promoters of the gene. To further investigate these findings, I carried out a replication study using a sample of 263 affected individuals from the IMGSAC collection and 390 control individuals. Several rare mutations were identified, including the splice site mutation IVS5+2T>C and the L326F substitution previously reported by Melke et al (2007), but the same rare variants have been found also in control individuals in our study. Interestingly, a new R319X stop mutation was found in a single autism proband of Italian origin and is absent from the entire control sample. Furthermore, no replication has been found in our case-control study typing the SNPs on the ASMT promoter B.

Mastocitosi cutanee: il mastocitoma in età pediatrica

Background: Mastocytosis is a rare disease involving mast cells (MC) and their CD34+ progenitors. According to the WHO consensus classification, cutaneous mastocytosis (CM) is considered a benign disease confined to the skin, preferentially seen in young children with a marked tendency to regress spontaneously. Aim of our study was the long-term assessment of the outcome of solitary (SM) and multiple (MM) mastocytomas in a pediatric population. Materials and methods: From January 1996 to December 2010, 241 pediatric patients with a diagnosis of CM were followed-up at the outpatient division of pediatric dermatology of the University of Bologna. We focused our retrospective evaluation on patients affected by SM or MM. We collected, through the analysis of medical records and with a telephone questionnaire for patients and their families, information on clinical aspects of the disease evolution and on the efficacy of topical steroid therapy. Results: Over the 241 considered patients we recorded: SM or MM in 176 (73%) pts., urticaria pigmentosa in 53 (22%) pts., telangiectasia macularis eruptiva perstans in 9 (4%) pts., diffuse CM in 2 (0,9%) pts. and polymorph CM in 1 (0,4%) pt. On 176 children affected by SM or MM (97 M vs. 79 F), 130 (74%) patients were followed-up with a mean of 56,3 (r. 4-142) months. A satisfactory outcome was recorded in 99 (76%) cases of whom 52 (53%) treated with topic steroids. Mean time to complete regression was 16.4 m. on treated patients vs. 34.7 m. on non treated patients (p=0,001). Conclusions: From our study emerged that resolution of the disease is independent from therapy, but the time to regression and to complete recovery of the coetaneous lesions is faster and favored by the application of topic steroid with an improvement of the quality of life for children and their families.

Altered cardiovascular rhythmicity in children with white coat and ambulatory hypertension

Adults with ambulatory hypertension or white coat hypertension (WCH) display abnormal cardiovascular rhythms. We studied cardiovascular rhythms by Fourier analysis of 24-h ambulatory blood pressure (BP) measurement profiles in 129 hypertensive children, 54 children with WCH, and 146 age-, height-, and gender-matched healthy subjects. The day/night mean arterial pressure ratio was lower in hypertensive and patients with WCH compared with controls (1.13 versus 1.16 versus 1.21, respectively; p < 0.0001). Eighty-five percent of controls were dippers compared with 74% of WCH (n.s.) and 64% of patients with ambulatory hypertension (p < 0.0001). The prevalence of 24-h rhythms was similar among the groups, but prevalence of 12-h BP rhythms was increased in hypertensive (67%) and WCH (72%) compared with controls (51%, p < 0.0001). The amplitudes of the 24-, 8-, and 6-h BP rhythms were reduced in hypertensive and WCH compared with controls (p < 0.05). Hypertensive and patients with WCH displayed delayed 24-, 12-, 8-, 6-h acrophases in comparison with controls (p < 0.05). In conclusion, hypertensive children exhibit abnormal cardiovascular rhythmicity compared with controls, especially a higher prevalence of nondipping compared with normotensive children. Abnormalities in patients with WCH are intermediate between healthy children and patients with ambulatory hypertension.

Conjunctival Proliferation After a Mild Pepper Spray Injury in a Young Child

PURPOSE To report a case of conjunctival proliferation in a 2.5-year-old boy after initial evidence of a mild chemical injury after ocular exposure to pepper spray (oleoresin capsicum). METHODS Case report with ophthalmologic and histologic findings. RESULTS A child presented with mild conjunctival injection and chemosis without any corneal erosion after direct exposure to pepper spray. Three weeks later, a significant conjunctival proliferation was found at the limbus, which was refractory to treatment with topical corticosteroids. Finally, proliferative tissue was surgically excised without clinical recurrence during 2 months of follow-up. CONCLUSIONS We hypothesize that the young age of the patient may have been an important factor for the severe conjunctival proliferation in comparison to a mainly uncomplicated course of pepper spray injuries in most adults. We recommend the use of topical antiinflammatory treatment even in apparently mild pepper spray injuries, especially in young children.

Exhaled nitric oxide in symptomatic children at preschool age predicts later asthma

Prediction of asthma in young children with respiratory symptoms is hampered by the lack of objective measures applicable in clinical routine. In this prospective study in a preschool children cohort, we assessed whether the fraction of exhaled nitric oxide (FeNO), a biomarker of airway inflammation, is associated with asthma at school age.

Social Policy Facing the Problems of Youth Employment

Rumiana Stoilova (Bulgaria). Social Policy Facing the Problems of Youth Employment. Ms. Stoilova is a researcher in the Institute of Sociology in Sofia and worked on this project from October 1996 to September 1998. This project involved collecting both statistical and empirical data on the state of youth employment in Bulgaria, which was then compared with similar data from other European countries. One significant aspect was the parallel investigation of employment and unemployment, which took as a premise the continuity of professional experience where unemployment is just a temporary condition caused by external and internal factors. These need to be studied and changed on a systematic basis so as to create a more favourable market situation and to improve individuals' resources for improving their market opportunities. A second important aspect of the project was an analysis of the various entities active on the labour market, including government and private institutions, associations of unemployed persons, of employers or of trade unions, all with their specific legal powers and interests, and of the problems in communication between these. The major trends in youth unemployment during the period studied include a high proportion of the registered unemployed who are not eligible for social assistance, a lengthening of the average period of unemployment, an increase in the percentage of people who are unemployed for the first time and an increasing percentage of these who are not eligible for assistance, particularly among newly registered young people. At the same time the percentage of those for who work has been found is rising and during the last three years an increasing number of the unemployed have started some independent economic activity. Regional differences are also considerable and in the case of the Haskovo region represent a danger of losing the youngest generation, with resulting negative demographic effects. One major weakness of the existing institutional structure is the large scale of the black labour market, with clear negative implications for the young people drawn into it. The role of non-governmental organisations in providing support and information for the unemployed is growing and the government has recently introduced special preferences for organisations offering jobs to unemployed persons. Social policy in the labour market has however been largely restricted to passive measures, mostly because of the risk that poverty poses to people continuously excluded from the labour market. Among the active measures taken, well over half are concerned with providing jobs for the unemployed and there are very limited programmes for providing or improving qualifications. The nature of youth employment in Bulgaria can be seen in the influence of sustained structures (generation) and institutions (family and school). Ms. Stoilova studied the situation of the modern generation through a series of profiles, mostly those of continuously unemployed and self-employed persons, but also distinguishing between students and the unemployed, and between high school and university students. The different categories of young people were studied in separate mini-studies and the survey was carried out in five town in order to gather objective and subjective information on the state of the labour market in the different regions. She conducted interviews with several hundred young people covering questions of family background, career plans, attitudes to the labour situation and government measures to deal with it, and such questions as independence, mobility, attitude to work, etc. The interviews with young people unemployed for a long period of time show the risk involved in starting work and its link with dynamics of economic development. Their approval of structural reforms, of the financial restrictions connected with the introduction of a currency board and the inevitability of unemployment was largely declarative. The findings indicate that the continuously unemployed need practical knowledge and skills to "translate" the macroeconomic realities in concrete alternatives of individual work and initiative. The unemployed experience their exclusion from the labour market not only as a professional problem but also as an existential threat, of poverty, forced mobility and dependence on their parents' generation. The exclusion from the market of goods and services means more than just exercising restraint in their consumption, as it places restrictions on their personal development. Ms. Stoilova suggests that more efficient ways of providing financial aid and mobilisation are needed to counteract the social disintegration and marginalisation of the continuously unemployed. In measuring the speed of reform, university students took both employment opportunities and the implementation of the meritocratic principle in employment into account. When offered a hypothetical choice between a well-paid job and work in one's own profession, 62% would prefer opt for the well-paid job and for working for a company that offered career opportunities rather than employment in a family or own company. While most see the information gained during their studies as useful and interesting, relatively few see their education as competitive on a wider level and many were pessimistic about employment opportunities based on their qualifications. Very similar attitudes were found among high school students, with differences being due rather to family and personal situations. The unemployed, on the other hand, placed greater emphasis on possibilities of gaining or improving qualifications on a job and for the opportunities it would offer for personal contacts. High school students tend to attribute more significance to opportunities for personal accomplishment. A significant difference that five times fewer high school students were willing to work for state-owned companies, and many fewer expected to find permanent employment or to find a job in the area where they lived, Within the family situation, actual support for children seems to be higher than the feelings of confidence expressed in interviews. The attitudes of the families towards past experience seems to be linked with their ability to cope with the difficulties of the present, with those families which show an optimistic and active attitude towards the future having a greater respect for parents experience and tolerance in communication between parents and children.