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CORRELATIONS BETWEEN THE GENETICAL AND CLINICAL IMPLICATIONS IN AUTISM: A PRELIMINARY STUDY

Autor: Laura Nussbaum Luminiţa Ageu Axinia Corcheş Bianca Micu Șerbu Simona Dumitriu Nicoleta Andreescu Maria Puiu
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Autism spectrum disorders (ASD) are a constellation of neurodevelopmental disorders characterized by the deficits in social reciprocity and language/communication ability, the presence of restricted interests and repetitive behaviors.
Since almost two thirds of the genes in the genome are involved in brain development by participating in different ways and mechanisms, it becomes obvious why neurodevelopmental processes that lead to multifactorial disorders are extremely complex; therefore the candidate genes for autism search revealed the most interesting results.
Patients with a clinical diagnosis of ASD, made by board-certificated child psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders-V (DSM-V), will be recruited. Clinical diagnosis will be confirmed by the use of standardized diagnostic tools, considered the “gold standard” – SCQ, ADI, ADOS for clinical diagnosis.
The genetic study of autism spectrum disorders will include genetic markers and determine CNV 15q region by qPCR. The project will generate social and economic effects, deliverables that will result in the assessment and monitoring protocols TSA, adding a touch of classical control techniques and enhancing the quality of treatment, the evident benefit of patients.

Background

Autism spectrum disorders (ASD) are a constellation of neurodevelopmental disorders characterized by the deficits in social reciprocity and language/communication ability and the presence of restricted interests and repetitive behaviors. The prevalence of ASD was estimated approximately as 1 per 110 children, with a male-to-female ratio of approximately 4:1, without race and geographical variations. Recent genetic studies have begun to identify genes likely to be involved in the etiology of these disorders complex [2].
Since almost two thirds of the genes in the genome are involved in brain development by participating in different ways and mechanisms, it becomes obvious why neurodevelopmental processes that lead to multifactorial disorders are extremely complex; therefore the candidate genes for autism search revealed the most interesting results [13]. Because of the multifactorial etiology, the study requires an interdisciplinary, integrative approach.
We begin from the fact that the recent observations have shown that the expression of genes that shape neuronal synaptic connectivity between different regions of the brain, polymorphisms in these genes and their combinations are currently investigated in neurodevelopmental disorders and pervasive symptoms [9]. The article analyzes the GABRB3 gene polymorphisms which encodes a subunit of the GABAA receptor B3 in the pediatric population with ASD, Prader Willi Syndrome (PWS) and Angelman syndrome (AS) and a randomized control group. Genotypes identified will be assessed against established standardized diagnostic tools scores – ADI-R, ADOS, SCQ.
This line of research has not yet been developed and so far there is no study in our population. Identifying correlations between SNPs, GABRB3 expression and phenotype of patients, can bring valuable data about the clinical course and response to antiepileptic therapy group associating epilepsy.
Highlighting the genetic causes of TSA will allow the granting of appropriate genetic counseling and the risk of recurrence of the disease in the family, prevention being an important goal in this pathology.
The project will generate social and economic effects, deliverables that will result in the assessment and monitoring protocols in TSA, adding a touch of classical control techniques and enhancing the quality of treatment and the evident benefit of patients.

Materials and Methods

Patients with a clinical diagnosis of ASD made by board-certificated child psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders-V (DSM-V), were recruited from the Clinic of Psychiatry and Neurology for Children and Adolescents, Timisoara, from „Casa Faenza” Timisoara and from the Children’s Mental Health Center from Timisoara [11]. Patients with a clinical diagnosis of PWS and AS will be recruited from Prader Willi Asssociation, Rare Diseases Association and from Genetic Department of Children’s Emergency Hospital “Louis Turcanu” Timişoara.
The clinical diagnosis is confirmed by the use of standardized diagnostic tools considered the “gold standard” – SCQ association tests, ADI, ADOS for clinical diagnosis.
Autism Diagnostic Interview-Revised (ADI-R) is a detailed interview of the parent (or caregiver) of an individual suspected of having autism spectrum disorder [1]. The interview can be used to assess adults and children, provided that their mental age is over 2 years. ADOS-G (Autism Diagnostic Observation Schedule) is a standardized semi-structured instrument for assessing social interaction, communication, play and imaginative use of certain materials by patients suspected of autism spectrum disorders.
Social Communication Questionnaire (SCQ, Social Communication Questionnaire) – helps to assess skills and social functioning of children who may suffer from autism or autism spectrum disorders [14]. Evaluation of patients with ASD, PWS and AS will be in the present and in collaboration with parents / foster parents, and data recording will be made with the coefficient D / intellect level for the 2 categories: TSA and syndromes caused by genetic 11-13- 15q region PWS and AS. Genetic study of autism spectrum disorder include genetic markers and determination CNV 15q region by qPCR. Genotyping by RT-PCR involves the use of kits and reagents compatible with the machine 7900HT Applied Biosystem. For GABRB3 genotype analysis will be used allelic discrimination method using TaqMan SNP genotyping kits Assay Cat. No. 4351379 (Life Technologies, Carlsbad, CA) and probes Locked Nucleic Acids (LNA) with specific design (Sigma Aldrich) according to the manufacturer’s instructions. Determination CNV 15q region by qPCR amplification oligonucleotides involves the generation of this region using Primer3 software and check the instrument COUNTER TOP specificity using UCSC Human Genome Browser browser. The working protocol for qPCR is based on the use of SYBR Green I dye (available under the iQ ™ SYBR ® Green Supermix produced by BioRad, Cat. No.170-8880) containing Itaqui DNA polymerase, a hot-start polymerase, that minimizes the amplification of PCR products nonspecific.
Regarding the collection of biological samples will be both genotyping samples (EDTA peripheral blood of which will be extracted DNA). Statistical analysis methodology includes assessing the role of chance, bias and the confounding factors with alternative explanations for the results achieved. No topics included in a study group is not omitted from the analysis. The statistical processing of survey results will be used for correlation analysis SPSS – relationships between two quantitative variables.
It shall establish: degree of dependence, linear correlation, Regression, Correlation for ordinal variables.

Results and Discussions

No doubt today PCR is themost important technique used in molecular biology. What can be done by PCR in technical terms can be described very simply enables fast and unlimited amplification of specific sequences of nucleic acids independent of concentration in which they are present in the solution / sample source. Motivation widespread use of PCR in the research, diagnosis and monitoring therapeutic progress is given by the sensitivity of the method – single molecules of DNA can be detected and analyzed; specificitatea- all the polymorphisms of a single nucleotide change in large rearrangements may be identified by appropriate design of the PCR test. The third argument is rapiditatea- can generate a response to a research or diagnostic hypothesis in a period of one to several days. Moreover, recent years have added another quality metodei- democracy: when certain pairs of nucleotide sequences used to query a specific PCR reaction is published, anyone can use these sequences to reproduce the same reaction.

Once the alternative alleles were sequenced and a single nucleotide change was found between the two alleles becomes possible design of a PCR protocol allowing segregation follow one of them. Initially are identified the primers that allow specific amplification of the region of interest containing the site where it was nominated variant nucleotide (for this application sequence length is not critical, and can be spread from 150 to 400 nucleotides). Then two allele-specific oligonucleotides are produced for each variant, trying to locate as close to the center variant sequence. Ideal it is used a sequence of 19-21 nucleotides, quite short to allow differential hybridization based on the presence of deference just a nitrogenous base and long enough to allow locus specificity.
Q-PCR is a quantitative PCR, used to measure the amount of specific nucleic acids (DNA / RNA) in the sample by measuring the amplification in the exponential growth phase but the amount of product measured reflects the initial amount of the target sample. Methods for Q-PCR using SYBR Green fluorescent dye and the sample containing or fluorophores, such as TaqMan, to measure the amount of product amplified as the process of amplification are made.
GABAergic system plays a crucial role in the initial development of the brain. Both genetic association studies and the expression suggests a key role of the GABAergic system in autism. More specifically, GABRB3 gene variations have been previously implicated in empathy and TSA studies in humans. The first study GABRB3 gene variations associated with TSA has identified a statistically significant association between marker 155CA-2 and individuals with ASD using multiple transmission disequilibrium test to 140 families [5]. The result was subsequently replicated by another study that included 80 families with autism [3]. Variations of the GABRB3 have also been associated with the tactile sensitivity which is unusual for some individuals with ASD [16]. Moreover, GABRB3 knockout murine model showed deficits in social networking and comportementul can be regarded as a potential animal model for autism [6].
B3 has GABRB3 encoding the GABAA receptor subunit. GABAA receptor is a ligand-dependent ionotropic receptors, which is part of the adult brain inhibitory synapses and selective lead ions Cl. Throughout development is very important GABRB3 bags for growth and differentiation of neural and signaling mediates excitatory [10]. Warrier et al. tested importance GABRB3 gene as candidate gene in TSA and potential measurement endofenotipică 6 autistic spectrum: 3 self-reporting measures (trasăuri autistic, empathy, systematization) and 3 measures the performance (recognition of emotions, attention to detail, spatial processing) [18]. Self-reporting measurements used are as follows; Autism Spectrum Quotient (AQ) is a measure of autistic traits. Interpretation – AQ score greater than 32 is predictive of TSA and AQ scores show a significant heritability general population sample. Empathy was measured using the Empathy Quotient (EQ). On average, individuals diagnosed with ASD will have significantly lower scores than the control groups EQ.
The attention to detail was measured using the Embedded Figures Test (EFT) in which participants were asked to locate the form enclosed in a complex pattern. Individuals with ASD have scores above average. Spatial processing was measured using the Mental Rotation Test (MRT) in which individuals were asked to match part of a letter and identify whether part of the letter are identical or mirror images. In this study, individuals with ASD had better outcomes than the control group at MRT.
Three SNPs (rs7180158, rs7165604, rs12593579) were significantly associated with Asperger’s syndrome, while the other two SNPs (rs9806546, rs11636966) were significantly associated with EQ. Two pairs of SNPs, rs12438141, rs1035751 and rs12438141, rs7179514 showed significant association with variation EFT scores. Another pair of SNPs, rs7174437-rs1863455 was significantly associated with scores varying MRT. Furthermore, several haplotypes, including a block of 19 kb in this study showed linkage to linkage disequilibrium, that contained SNPs significant association was found in Asperger’s syndrome.
Chromosome 15q11-q13 is an chromosomal region increased incidence of deletions and duplications that are commonly associated with developmental disorders including TSA. Deletion 15q11.2-q12 paternal segment is associated with Prader-Willi syndrome, while the maternal 15q11-13 deletion segment is associated with Angelman syndrome, genetic disorder affecting both global development and related phenotypic features as language, visuo – spatial processing and interpersonal relationship with a significant degree of overlap with pervasive pathology. Moreover, 15q11-q13 mother of duplicate was found in about 1-3% of patients with TSA. Therefore, the genes located in this region are considered as candidate genes for TSA.
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. A group of genes including GABAA receptor subunit GABRB3, encoding subunits GABRG3 GABRA5 and β3, α5, γ3, was mapped to chromosome 15q12. Recent studies have shown that GABA-ergic path change is associated with the pathogenesis of autism. For example, reducing GABA_A receptor expression, including GABRB3 subunit enzymes synthesized GABA, glutamic acid decarboxylase (GAP) 65 and 67 were found in several regions of the brain of patients with autism.
Several linkage and association studies have investigated the association of three receptor subunit genes on chromosome 15q11-13 GABA_A with ASD. Cook et al., Reported the first time the existence of an association between autism imbalance and genetic marker GABRB3 [5, 8, 16].
Menold et al., Found two genetic markers associated with autism in GABR3 and Mc Canley et al. conducted an analysis of genetic markers linked to the imbalance that stretch of chromosome 15q12 1MB; they found six markers between GABRB3 and GABRA5 associated with autism [12].
In the view of the clinical heterogeneity of patients with ASD, several groups have studied the genetic association of these receptors subunit gene sets GABA_A patients with ASD phenotypes based on particular. Warrier et al. examined the association between SNPs and 45 GABRB3 and Asperger syndrome, finding a significant association of three SNPs with Asperger syndrome and multiple endofenotipuri in connection with ASD.
Atypical sensory sensitivity is one of the characteristic core of patients with autism and Tavassoli et al., Found an association between genetic markers of GABRB3 and tactile sensitivity in typical development in children. These converging evidence from these studies support the idea that GABRB3 can be an important candidate gene for ASD.
Acrocentric proximal regions chromosome specifically 15q11.2, are frequently involved in structural rearrangement. Between the region 15q rearrangements, duplications are more common interstitial few patients analyzed by molecular techniques. Christofolini et al., Described a rearrangement of chromosome 15 consists of interstitial triplication of -6.9Mb between 15q11.2 to 15q13.2 followed by duplicating -2.4Mb between 15q13.2 to 15q13.3 [4, 7, 17, 18]. These patients have different clinical manifestations including developmental delays and intellectual disabilities, minor facial dysmorphism, epilepsy and autistic behavior.
Genome screening by changes in the DNA copy number was achieved either by classical cytogenetic techniques, such as comparative genomic hybridization of metaphase and fluorescence in situ hybridization (FISH), or by arrayCGH or quantitative PCR. Although arrayCGH and FISH techniques have been widely used for the detection of modified DNA copy number, these approaches have their own limitations. FISH analysis is applied when the region of interest was previously defined; its use does not provide sufficient resolution to detect microdeliţii and requires high technical expertise and a prolonged period of time to obtain results.
ArrayCGH is utillised to detect small changes in DNA copy number simultaneously across the entire genome. Have shown that increasing the specificity, sensitivity and transfer compared to other methods used for analyzing copy number changes. Recent research studies in developing arrayCGH for microdeletions / microduplicaţii indicates that validation is required to make significant phenotype-genotype interpretation. So far this procedure is not widely available for screenarea diagnosis of a large number of patients and limited costs. In this study we chose to validate the PCR using quantitative (qPCR) for the detection of microdeletions and microduplicaţii 15q region using genomic DNA. qPCR was previously used to quantify the gene expression but not used for routine screenning children with autism for CNV of 15q.
The study Van Esch et al. used DNA to confirm qPCR children arrayCGH 4 patients with MECP2 duplications [7, 17]. Taking into consideration our design and technical advantages biobăncii I propose to develop qPCR performed in a single tube using the DNA of patients for the detection of changes in the number of children in the GABRB3 gene and the rest of the region 15q. qPCR site offers great adaptability and flexibility, while requiring less extensive than technical framework FISH, available in most laboratories. This technique offers the advantage of high resolution mapping of the region of interest and a high adaptability design based on different pairs of primers and PCR conditions optimization for a specific project. The proposed method will be validated using DNA from patients already diagnosed by array CGH. Even if the condition occurs spontaneously in most affected individuals, the risk of recurrence of the second child is less than 1%, it is recommended to offer prenatal diagnosis in these couples, even if the genetic modification or not the parents identified.
The approach used in this study can be adapted for the molecular diagnosis of any genomic regions may have deletions, or repeats, providing an accurate measurement of DNA copy quantity. Implementing’s qPCR for the number of copies will also produce important data for genotype-phenotype correlation.
Regarding clinical aspects are considered “gold standard” the association of SCQ, ADI, ADOS for clinical diagnosis.
Autism Diagnostic Interview-Revised (ADI-R) is a detailed interview of the parent (or caregiver) an individual suspected of having autism spectrum disorder. The interview can be utilized for de evaluation of adults and children, with the condition that their mental age should be over 2 years. The interview is composed by 93 items, with 3 domains of functioning: language and communication; mutual social interactions; restricted, repetitive, stereotyped behaviours and interests. ADI-R is very effective in differentiating autism from other developmental disorders, and borderline syndromes assessment; were identified as new subgroups of autistic symptomatology could be quantified. In order to make a proper assessment of a person’s behavior, symptoms must be analyzed in different situations which are intentionally structured interaction. To this end, were created psychodiagnostic scales (scales of observation for psychiatric diagnosis).
ADOS-G (Autism Diagnostic Observation Schedule ADOS-G (Autism Diagnostic Observation Schedule) a standardized assessment tool semi-structured social interaction, communication, play and imaginative use of certain materials by suspected autism spectrum disorders. The program consists of four modules observation, each lasting about 40 minutes, depending on the capacity of expressive language and chronological age of the person being tested: 1 – no expressive language; 2 – non-fluent language; 3 – fluent language; 4 – language fluently, adolescents and adults. Play in modules 1 and 2 involves the examination room with toys the child and the examiner can move freely. Modules 3 and 4 can be completed at the table (involving discussion). Filming modules can be subsequently encode. There are cut-off scores for all spectrum disorders – PDD-NOS, atypical autism, autistic spectrum. Encodings ADOS-G set a score that is independent of language ability.
Social Communication Questionnaire (SCQ, Social Communication Questionnaire) is the third component of the triad internationally recognized as the “Golden Standard” for evaluating autism with ADI-R and ADOS. This tool helps concise assessment skills and social functioning of children who may suffer from autism or autism spectrum disorders. Being a questionnaire that can be completed by a parent or guardian by another in less than 10 minutes, the SCQ is an effective way to determine whether a person should or should not be included in a complete diagnostic evaluation. The questionnaire is available in two versions – lifetime – relating to the evaluation of lifetime (Lifetime) and Actual – relating to the evaluation of the current situation (Current). Evaluation version of all life is centered on the history of child development, providing a total score that is interpreted according to certain boundary points. This score can identify people who have autism and who should undergo a full assessment Autism Diagnostic Interview-Revised with (ADIR) Autism Diagnostic Observation Schedule or (ADOS). SCQ has some similarities with the ADI-R and the correlation between ADI-R scores and SCQ is high and is not affected by age, gender, language level and performance IQ. So SCQ is a valid screening tool, providing a reasonable picture of the severity of symptoms. Alternative assessment of the current situation focuses on the child’s behavior in the last 3 months until use. The results provided by this variant may be useful for treatment planning, intervention and educational measure the change in time.

Conclusions

Genetic studies conducted could not identify a single gene responsible for all changes in the autistic spectrum, most studies tracing the importance of genetics in neurodevelopmental direction. The genes involved in brain development (including genes involved in the formation or stabilization of synapses and genes involved in neural circuits neurotansmisia these immature) were consistently associated with autism.
Because the genetic causes of autism are not yet fully decoded, this chapter lists the known genetic syndromes (fragile X syndrome, Joubert and Timothy) that autism can be recognized. It is also recognized contribution to autism several distinct forms of genetic variations (common or rare) and environmental factors.
Highlighting the genetic causes of TSA will allow the granting of appropriate genetic counseling and the risk of recurrence of the disease in the family, prevention is an important goal in this pathology.
The project will generate social and economic effects deliverables that will result in the assessment and monitoring protocols TSA, adding a touch of classical control techniques and enhancing the quality of treatment, the benefit of patients evident.

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