The ‘Fondazione Smith Kline’ Italy, with its “Autism Research Area”, subsequently spun out as a separate Foundation (“ITAN - Italian Autism Network”), has sponsored a multi-centric project to recruit and assess autism individuals and their first-degree relatives to create a repository of genomic DNA, plasma, peripheral RNA and lymphoblastoid cell lines (LCLs). Extensive clinical data and medical history of autistic subjects and their first-degree relatives have also been stored in the database. The goal of the project is to build a biorepository that interacts with a database of extensive clinical information collected electronically at each recruiting sites, enabling genetic, genomic and proteomic research on ASD. Although the target research areas are multidisciplinary (from genetic and biomarker research, to epidemiological studies), the design of the project was driven mainly by its genetic goals. The project aims at collecting small families formed by a proband (ASD child), a sibling when available, and their parents. The collection of families would allow performing family-based genetic association studies as well as the screening of genetic variants in cases and affected and unaffected first-degree relatives.
Recruiting sites and ethical issues
The biorepository is hosted by the Department of Neurosciences, Biomedicine and Movement Sciences at the University of Verona, Italy. Recruitment has begun in 2008 and has been conducted thus far at thirteen different Italian sites (Verona, Acireale (Catania), Bari, Bologna, Brescia, Cagliari, Lecco, Napoli, Padova, Pisa, Rimini, Roma, and Troina (Enna).
Approximately half (48%) of the patients recruited were referred by the primary care physician, 31% by a tertiary care center, and the rest by schools, or outpatient clinics.
A study protocol for the clinical assessment and sample management was written and agreed by the main centers and founding members of the network. Candidate recruiting sites were selected among clinical centers with a demonstrated ability to assess autism families as described in the project protocol. The study and its protocol were approved by the Azienda Ospedaliera Universitaria Integrata of Verona Ethic Review Board. Furthermore, each recruiting site obtained independent approval to conduct the project by their respective local ethical review committees. Eighteen sites joined the Italian Autism Network, with thirteen actively recruiting. All adult subjects participating in this project gave their written consent (or the consent for their chidren) to donate biological samples and clinical and demographic information to participate in this study; assent to participate to this study from the children was obtained whenever possible.
Cases, parents and, whenever available, sibling and/or non-affected individuals identified as controls, were recruited. Based on the selection criteria and the family composition, different type of families can be summarized through the pedigree analysis. We used the R package kinship2 with the pedigree shrinking function to gain a metric of pedigree structure in terms of bitSize (a measure is defined as 2 * # NonFounders - # Founders, see ).
Inclusion and exclusion criteria
A proband child or affected sibling was eligible for recruitment when all the following inclusion criteria apply: 1) at least 4 years of age at the time of the interview, 2) having at least one parent or legal guardian giving voluntary written consent for their children to take part to the research project, 3) meeting DSM-IV criteria for Autistic Disorder or Asperger’s Disorder, or Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), 4) reaching score cut-off in ADI-R. Siblings of the proband were recruited when available, and defined as unaffected, if they met the following criteria: 1) at least 4 years of age at the time of recruitment, 2) devoid of any comorbid psychiatric illness as determined by: i) the Child Behavioural Checklist (CBCL) or by the Kiddie Schedule for Affective Disorders and Schizophrenia for children (Kiddie-SADS), ii) absence of social skills impairment as determined by the Social Responsiveness Scale (SRS), iii) the Broader Phenotype Autism Symptom Scale (BPASS) (administered to evaluate the presence of autism spectrum symptoms). Individuals were not eligible for recruitment if any of the following criteria apply: 1) history of serious head injury, encephalitis or tumors, 2) age above 18; 3) Diagnoses of Childhood Disintegrative Disorder or Rett Syndrome, diagnosis of known ASD-related genetic syndromes or presence of severe mental retardation (IQ < 20) that would compromise the validity of the diagnosis of autism.
Diagnosis was established by experienced child psychiatrists after completing full neurological, psychiatric and physical examination, and after reviewing and completing the Autism Diagnostic Interview – Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) that were administered by trained and certified clinicians. The cognitive level of children was assessed whenever possible through either one of the following scales: Wechsler Intelligence Scale for Children, Wechsler Preschool and Primary Scale of Intelligence, Leiter International Performance Scale or Griffiths Mental Developmental Scale. Additional structured evaluation included the Children’s Global Assessment Scale (CGAS) and, for subjects with suspect of Asperger’s Disorder, the Krug Asperger’s Disorder Index (KADI). The KADI is an individually administered, norm-referenced screening instrument that provides useful information for determining the diagnosis of Asperger’s Disorder. It is standardized for use with individuals 6 through 21 years of age. Instrumental and laboratory examination of the fragile X (FRAX) expansions and major cytogenetic abnormalities were conducted for all the autism individuals and their siblings. MRI scan and awake and nap EEG were evaluated in all patients, if they were not available from previous assessments conducted at a close time.
Parents of the probands and siblings of age 18 and above were assessed as follows: i) Cognitive level: four items of the WAIS (arithmetic, vocabulary, block design, picture arrangement); ii) Language: Token test; iii) Autism spectrum symptoms: BPASS Broader Phenotype Autism Symptom Scale (BPASS); iv) Psychopathology: SCID screening interview.
Non-affected siblings of less and 18 years of age recruited were assessed with medical, psychiatric and neurologic examination. For each non-affected sibling, cognitive levels are established via the four items of the Wechsler Intelligence Scale for Children (WISC) that has shown to produce a reliable measure of IQ . The four items include the arithmetic, vocabulary, block design, and picture arrangement. Language competence was assessed with the Peabody Picture Vocabulary test (PPVT-R), for ages between 4 and 12, and the Token Test for age between 12 and 18. Social Responsiveness Scale (SRS) was also administered to evaluate socialization. Psychopathology in non-affected siblings was assessed via the Child Behavioural Checklist (CBCL). The following definitions were used to define the presence of psychopathology: T-score: normal < 65; at risk: 66–74; pathological: > 75 (2.5 SD from normal average) if scores less above 75 (presence of psychopathology) the Kiddie-SADS interview is administered to diagnose the psychiatric interview.
Sample collection, handling and storage
The Italian Autism Network collected DNA, plasma, RNA and EBV transformed lymphocytes in the biorepository from each of the individuals assessed. From all individuals eligible to participate in this project (as either cases or unaffected relatives) for whom a signed informed consent was obtained, the following peripheral blood samples were taken: 1) 2 × 6-mL in ACD blood tubes for DNA extraction; 2) 1 × 6 mL ACD tube for extracting lymphocytes to be transformed with Epstein Barr virus (EBV) in LCLs; 3) 1 × 6-mL in EDTA tube to obtain plasma aliquots for proteomic analysis, and 4) 2 × 2.5-mL in PAX gene tubes for obtaining RNA. Plasma aliquots (0.2 mL) were prepared by centrifugation of EDTA tubes and stored at -80 °C. Blood-derived samples for RNA extraction were prepared by gently mixing PAX gene tubes, storing them for 2 h at room temperature, cooling them at –20o and later at -80 °C to be ready for shipment. Samples for DNA extraction and cell lines transformation were shipped at room temperature to the biorepository on the same day of collection. Frozen samples (plasma, blood samples for RNA) were shipped via express courier in dry ice to the biorepository.
Upon delivery at the biorepository, Plasma aliquots and PAX gene tubes were stored at -80 °C. DNA extraction was performed using the Puregene Blood Kit (Gentra Systems, Minneapolis, MN, US), a modified salting-out precipitation method, following the manufacturer’s instructions. Each DNA sample was then quality controlled and quantified using NanoDrop ND 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, US). RNA samples from lymphocytes were isolated with QIAcube system, quantified with NanoDrop and analyzed for quality control by use of the RNA 6000 NanoLabChip (Agilent Technologies, Santa Clara, CA, US). The biorepository also transformed the lymphocytes with EBV into lines that were and then stored in Liquid Nitrogen. All relevant data regarding the samples are entered into a computerized system that allows matching the information of the samples with the clinical data stored in the centralized clinical database.
Data security procedure for the clinical database
All blood, plasma, cell lines, DNA and RNA samples are coded and stored under secure conditions with restricted access according to the Italian privacy policies required for keeping secure sensitive data. The clinical information collected at each site is entered into an encrypted system and stored in a database. Codes corresponding to the research subjects are known only by the clinician responsible for the research project, who obtained the informed consent from the parents of affected subjects and any sibling at each recruiting site (Site Principal Investigator), and by the clinical personnel directly involved in the assessment of the research subject, as required by standard clinical practice.