Exploring Genetic Testing Requests, Genetic Alterations and Clinical Associations in a Cohort of Children with Autism Spectrum Disorder

Several studies show great heterogeneity in the type of genetic study requested and in the clinicopathological characteristics of patients with ASD. Objective: The following study aims, �rstly, to explore the factors that might in�uence professionals' decisions about the appropriateness of requesting genetic testing for their patients with ASD and, secondly, to determine the prevalence of genetic alterations in a representative sample of children with a diagnosis of ASD. Methods: We studied the clinical factors associated with the request for genetic testing in a sample of 440 children with ASD and the clinical factors of present genetic alterations. Results: Even though the main guidelines recommend genetic testing all children with an ASD diagnosis, only 56% of children with an ASD diagnosis were genetically tested. The prevalence of genetic alterations was 17.5%. These alterations were more often associated with intellectual disability and dysmorphic features. There are no objective data to explicitly justify the request for genetic testing, nor are there objective data to justify requesting one genetic study versus multiple studies. Remarkably, only 28% of males were genetically tested with the recommended tests (fragile X and CMA). As expected, children with dysmorphic features and organic comorbidities were more likely to be genetic tested than those without. Previous diagnosis of ASD (family history of ASD) and attendance at specialist services were also associated with Autism Spectrum Disorder Genetically tested GTASD. Conclusions: Our results re�ect the need to develop algorithms that could make it possible to offer genetic testing not only to children with intellectual disability and dysmorphic features, but also to the whole ASD population.


Introduction
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder (NDD) that is characterised by di culties in communication and social interaction, and the presence of repetitive behaviours and interests.ASDs share many clinical features with other NDDs, and most also have de cits or delays in other areas of neurodevelopment.In recent years, the prevalence of patients with ASD has increased drastically and also varies depending on the series, but is estimated at 1-1.5% (Lai et al., 2014).The causes of this increase are to some extent justi ed by a better knowledge of its pathophysiology and increasingly better established diagnostic criteria (Zeidan et al., 2022), currently using by consensus the criteria established by the DSM-5 (American Psychiatric Association & American Psychiatric Association, 2013) and ICD-11.Families receiving a diagnosis of autism are typically offered a range of recommendations, which may include educational, behavioural and medical community resources, including genetic testing.
There have also been major advances in the aetiological and, in particular, genetic diagnosis of ASD in recent years.In families with a child with autism, the risk of recurrence increases 8. 4-fold for siblings and 2-fold for cousins (Jin et al., 2020).This fact re ects the key role of genetics in the aetiology of ASD, with heritability estimated at 65-90% (Sandin et al., 2017;Tick et al., 2016).Autism is associated with a number of genetic disorders, but relatively few have a monogenic cause; for example, fragile X syndrome and tuberous sclerosis together are estimated to account for < 10% of autism cases, unless 50-70% of FXS patients show ASD symptoms.More commonly, heterozygous de novo single nucleotide polymorphisms (SNPs) and/or abnormalities in submicroscopic segments of DNA called copy number variations (CNVs) are found, with over 100 genes associated with autism having been identi ed and described in multiple studies over the last decade (Bacchelli et al., 2020;Leppa et al., 2016).Recent studies have found that using chromosomal microarray (CMA), approximately 10% of autistic individuals receive a molecular diagnosis (Shen et al., 2010;Tammimies et al., 2016).CMA for copy number changes has been recommended as the rst clinical diagnostic test for children with NDDs (Manning & Hudgins, 2010;Miller et al., 2010;Waggoner et al., 2018).
The American College of Medical Genetics and Genomics (ACMGG) guidelines also recommend CMA for all children with ASD, fragile X testing for males and additional genetic sequencing, including PTEN gene in children with macrocephaly and MECP2 gene in girls with psychomotor regression.However, there is clear variation in the request for genetic testing within the aetiological algorithm of ASD diagnosis.Several studies show great heterogeneity in the type of testing requested and the clinicopathological characteristics of the patients (Hendel et al., 2021;Moreno-De-Luca et al., 2020;Smith et al., 2021).It is known that genetic alteration is not necessarily associated with dysmorphic features and organic comorbidities (Harris et al., 2020).However, these patients are more likely to undergo genetic testing than those without dysmorphic features.Similarly, patients with complex diagnoses and those referred earlier to specialist autism services are more likely to receive genetic testing than those with more obvious clinical criteria for ASD diagnosed in primary care.A recent study suggests that 50 per cent of requests for genetic testing from patients with autism were refused because the test was not considered essential for medical management (Smith et al., 2021).Other studies have shown that genetic testing was irrelevant to families due to unclear medical recommendations (Hendel et al., 2021).In Spain, there is a noticeable underutilization of genetic services (Codina-Solà et al., 2017).The present study aims to explore the factors that may in uence clinicians' decisions about the appropriateness of requesting genetic testing in their patients with ASD, and to determine the prevalence of genetic alterations in a representative sample of 440 children diagnosed with ASD.

Study design and setting
We conducted a retrospective chart review of 440 participants recruited between 1 January 2020 and 30 December 2021.The study included children with a diagnosis of autism spectrum disorder who were being followed at the Mental Health Unit of the Hospital Virgen del Rocío in Seville, as part of the evaluation prior to the implementation of a protocol to improve the clinical care of children with ASD in 2020 and 2021.We looked at the number of children with a diagnosis of GTASD compared with those with NGTASD.This programme was approved by the local institutional review board (the Clinical Research Ethics Committee of Andalusia) in accordance with international research ethics standards.

Clinical assessment
The diagnosis of ASD was made according to the DSM-5 criteria.In cases of diagnostic complexity, the ADOS-2 test, which has been internationally validated for the diagnosis of patients with ASD, was used.The ADOS-2 is a semi-structured, 45-60 minute session of observation and interaction between a clinician and the child, used to support the diagnosis of ASD (Lord et al., 2012).Data were collected via the patient's digital health record.We collected demographic, medical and neuropsychological information for all subjects.We also recorded the presence of a rst-and second-degree family history of neurodevelopmental disorders.The presence of dysmorphic features or organic co-morbidities was also recorded.According to key clinical guidelines, MRI was requested in patients with dysmorphic features and/or organic comorbidities.(More details in supplementary material)

Genetic Testing
All genetic testing was prescribed by consultant paediatric neurologists, with indications noted in their medical records.No genetic sequencing was requested, for example, of PTEN in children with macrocephaly or MECP2 in girls with psychomotor regression.The following are descriptions of the different genetic tests performed: i) The CMA test employs comparative genomic hybridisation technology (Agilent CGX™ v1.1 v1.1 8-plex array platform with 60K oligonucleotide probes) on a commercial same-sex diploid DNA sample to search for insertions and/or deletions (indels, CNVs) throughout the genome.The identi ed variants were compared to the databases DECIPHER, ClinVar, SFARI, GenoGlyphix (Perki-Elmer), DGV, Dosage Sensitivity ClinGen, ISCA, HGMD, Autism Chromsoome Rearrangement Database, Gnomad and OMIM (de Leeuw et al., 2012;Hanemaaijer et al., 2012;Kearney et al., 2011) The variants were then classi ed as either pathogenic, benign, or of uncertain signi cance (Biesecker & Harrison, 2018) ii) The Fragile X test is designed to detect FMR1 disorders.The main cause of the syndrome is a CGG trinucleotide repeat expansion in the 5'UTR region of the FMR1 gene.Normal and mutated categories of FMR1 alleles were determined in accordance with ACMG guidelines (Kearney et al., 2011).The normal repeat size is from 5 to 44 repeats, the grey zone ranges from 45 to 54, premutation is from 55 to 200, and full mutation is greater than 200.iii) Karyotype analysis is conducted to evaluate the number and structural aspects of chromosomes.Standard methods were utilised for cell culture and subsequent analysis using GTG banding and/or uorescence in situ hybridisation (FISH) (Gardner et al., 2012).iv) We also identi ed a subset of patients for whom a clinical exome was performed, which is not a routine test.Exome sequencing is conducted to obtain quantitative and qualitative extraction and evaluation of the DNA sample.This involves capturing and enriching the exonic regions and anking intronic areas of the genes contained in the SureSelect Exome v6 sequencing panel from Agilent, which comprises of over 20,000 genes.Variants of interest located within the exonic and intronic regions, up to +/-10 nucleotides of the studied genes, were identi ed with respect to the reference genome (hg19).The variants were ltered based on speci c quality criteria, including call quality > 20, coverage > 10x, genotype quality > 20, and allele fraction > 20.All ndings were classi ed according ACMG recommendations (Richards et al., 2015).Any nding was classi ed as variant with a frequency above 1%.All ndings were interpreted based on the consultation of different databases.(See supplmentary material)

Data Analysis
Initially, we determined the percentage of genetic exams requested (karyotype, CMA array, fragile X, exome sequencing) in the overall sample and compared the ndings with the primary clinical guidelines (Kearney et al., 2011).Subsequently, we separated the sample into two subsets: genetically tested ASD GTASD and non-genetically tested NGTASD individuals were compared based on demographic and clinical assessment data, including age at diagnosis, sex, urban or rural area, family history of neurodevelopmental disorders, diagnostic complexity, dysmorphic features, and organic comorbidities.Non-clinical factors such as the time from referral to the child and adolescent mental health unit and age at enrollment were also evaluated.We aimed to identify any signi cant differences between the two groups.We calculated descriptive statistics for all variables and determined correlations and statistical signi cance using chi-squared and t-tests for independent groups.We identi ed and compared the language and intellectual functioning characteristics of individuals with genetic changes and those without.To provide a more detailed qualitative description, we created tables of ndings for individuals with genetic alterations.Statistical signi cance was established at a 2-sided P value of less than .05.The Statistical Package for Social Sciences (IBM SPSS, version 28.0, Armonk, NY: IBM Corp) was utilised for the analysis.

Demographic and clinical factors
The age of the four hundred forty participants with a con rmed diagnosis of ASD ranged from 2 to 18 years, and the male/female sex ratio was 369/71.One hundred eighty-four children (41.8%) were from rural areas.The mean age at diagnosis was 37 months, 110 children (25.0%) had a family history of NDD, 132 (30.0%) of the patients had dysmorphic features and organic comorbidities, 159 (36.1%) were referred to a complex diagnostic service, and the mean referral time to specialist services was 22 months.

Genetic alterations
From the 246 ASD genetically tested individuals, 17.5% (n=43) had genetic alterations, variants of unknown signi cance or pathogenic ndings.Of those 43 patients, 46% ( n =20) had alterations on CMA, 39% (n=17) had alterations on exome, 9.3 % (n=4) had chromosome abnormalities and 9.3% (n=4) had pathogenic fragile X ndings.Medical.recommendations in response to the genetic nding such referral to medical subspecialties (e.g.nephrology because of associated renal disorders in a case of kidney dyspasia) and screening for associated comorbidities were made for 43% of patients with pathogenic genetic ndings.

Genetic testing yield
The genetic testing yield including CMA, fragile X (males), karyotype, and exome was 17.47%.However, when we calculated the genetic testing yield excluding exome, the yield decreased to 11%.It is important to clarify that exome sequencing was only performed on a very selected sample of patients who presented normal CMA but additional dysmorphic features or comorbidities besides autism.The speci c CMA yield was 10% (20/186).As for the alterations found in CMA, 10 were classi ed as pathological,  2).The most common CNVs in this sample were deletions or duplications on 15q(n=5), 16p (n=2) and 17q (n = 2).With respect to the karyotype, the yield was 4% (4/127) (Table 3).Only 1 alteration, 46,XX,del(10)(q26.13-q26.3) was reported as uncertain signi cance.Three inversions were observed, a normal variant inv(2) and two apparently balanced chromosome inversion.The fragile X yield in the male population was 2% (3/142 performed).Among the 4 subjects with FMR1 diseases ndings, there were 3 full mutations (FRAXA), and 1 premutation (fragile X-associated tremor/ataxia syndrome (FXTAS) (Table 4).The speci c exome yield was 45% (17/37).Twenty seven mutations were found in 17 different patients, from those 27 only 5 were reported as pathogenic mutations and 7 were reported as probably pathogenic mutations.No mutated gene appeared in two different subjects (Table 5)   ,2,3,4,5,6 The variants with the same number were found in the same individual respectively

Discussion
After analyzing the factors related to clinicians' decisions regarding genetic testing requests in a representative sample of children with ASD, we found that: i) even though the primary guidelines (Kearney et al., 2011) recommend testing for all children diagnosed with ASD, only 56% of them were genetically tested.Furthermore, the prevalence of genetic alterations was 17.5% among those who were tested.These changes were more commonly linked to intellectual disability and physical abnormalities.ii) We couldn't nd explicit objective evidence to support or oppose genetic testing, nor to justify requesting one genetic assessment over multiple.iii) Notably, only 28% of males were tested using the suggested tests (Fragile X and CMA).iv) As anticipated, children exhibiting physical abnormalities and organic comorbidities were more likely to be genetically studied than those without.v) Prior diagnosis of ASD and enrollment in specialist services were also linked to ASD genetic testing.
At the moment, there is no clinical or demographic data available to help us identify patients or phenotypes suitable for genetic studies, and the request is driven solely by medical decision.However, referring to other international studies (Ho et al., 2016), it is advisable to recommend genetic testing for all children diagnosed with ASD.In fact, exome sequencing has been suggested as a primary test before or at the same time as CMA, due to its higher molecular diagnostic yield for neurodevelopmental disorders compared to CMA (Degenhardt et  In terms of clinical factors, our ndings are consistent with the scienti c literature, as we found that intellectual disability and dysmorphic features were associated with the presence of genetic alterations, but it should be noted that, as we also found, these patients are more likely to be genetically tested than patients without dysmorphic features or intellectual disability.Among the identi ed mutations related to intellectual disability, we found, for example, the CIC c.The speci c detection rate of CMA in our study was 10%, which is similar to previous studies reporting also 10% (Kalsner et al., 2018;Tammimies et al., 2015).However, the wide variation in the utility of the test depending on the study population is evident, for example, current studies report higher performance until 25% (Ho et al., 2016).This may be because the detection rate and pathogenic yield of CMA varies signi cantly depending on the primary indications for testing, the age of the individuals tested, and the specialty of the ordering physician.
There could be many reasons that could explain why genetic studies are not requested for all ASD patients, such as the complexity of a patient's phenotypic pro le or insu cient consultation time to obtain a complete diagnosis.Recent studies (Moreno-De-Luca et al., 2020) have found that dissonance between professional recommendations and clinical practice may be explained by limitations in clinicians' knowledge and comfort with genetic testing, a lower frequency of genetic testing in patients diagnosed with ASD by psychiatrists and psychologists than by pediatricians, changes in genetic testing practices over time, and a reduced likelihood of testing being offered to adolescents with ASD.
On the other hand, there are subjective or observer-dependent assessments of the changes found in CMA.
Duplications in the 15q13.3region have been associated with intellectual disability, global developmental delay, speech and language delay and, to a lesser extent, autism spectrum disorder and epilepsy.The 15q13.3 duplication presents greater clinical variability and lower penetrance than the corresponding deletion of the region.It has not been possible to associate a de nitive phenotype to this duplication.
Deletions at 15q11.2 BP1-BP2 have been shown to confer a slightly increased risk of ASD with low penetrance (10.4%) (Chaste et al., 2014;Rosenfeld et al., 2013).We observed discrepancies in the way laboratories reported some results, for example, we found 5 patients with 15q duplications.The 15q11.2 BP1-BP2 duplications were reported by the genetics department on one occasion as uncertain signi cance and on another occasion as pathogenic, and to date there is strong evidence that the 15q11.2BP1-BP2 duplication is associated with a modestly increased risk of ASD and with a speci c pattern of non-syndromic ASD (Wilson et al., 2020).This discrepancy in interpretation of a nding between different laboratories is also found between different databases and even within the same database over time.In addition, 9 CNVs of uncertain signi cance have been reported.Although the impact of individual CNVs remains uncertain, their contribution to ASD risk cannot be excluded due to ongoing reclassi cations and new lines of research.There are several studies showing the conversion of uncertain CNVs into pathological ones (Kalsner et al., 2018).
One of the key strengths of this study is the large sample.The study's results have practical implications for clinical practitioners, suggesting that implementing protocols and algorithms could be a valuable strategy for improving the detection of genetic alterations in children with ASD.Some limitations of this study should be noted, including the absence of standardized vocabulary, such as the Human Phenotype Ontology (HPO) (Kohler et al., 2021), to detail phenotypic pro les.Additionally, the information collected on the rationale for genetic study requests is based on clinical subjectivity.The lack of parental data for many of the CNVs and variants identi ed precludes clear categorization as pathogenic or uncertain.In our study, there are several mutations with unknown heritability due to the lack of familial segregation, which could be limitations in classifying these mutations as benign or pathogenic (Manrai et al., 2016).
It is not only the phenotypic heterogeneity but also the genetic heterogeneity (Alvarez-Mora et al., 2016) and the low penetrance of same ndings that adds to the complexity of autism and there is still a long way to go in genomic sequencing research to improve understanding of the impact of individual variants on the pathogenesis and severity of autism.While the primary cause of ASD is attributed to genetics, future research should also explore the potential role of epigenetic mechanisms, as they involve regulatory molecules like miRNAs, which may impact the risk of autism through genetic regulation.
and 10 of uncertain signi cance (VUS) after their study by geneticists in multiple databases(Abrahams et al., 2013;Amberger et al., 2015;Landrum et al., 2016)(Table 6746A > C; p.(Lys2249Thr) an autosomal dominant mutation in the CIC gene associated with intellectual disability type 45 (Lu et al., 2017) and the recurrent deletion/duplication of the 15q13.3region associated with phenotypes with different degrees of intellectual disability (van Bon et al., 1993).

Table 1a .
Genetically tested ASD individuals vs non genetically tested ASD individualsOf the total sample, 246 patients (56%) were genetically tested with at least one genetic study.The most commonly requested genetic test was CMA, which was performed in 186 patients (42% of the total sample) with normal results in 167 patients.Of these 167 patients, 37 (20%) underwent a clinical exome and in 17 of these patients an alteration was found.Only 100 of 369 (28%) of men received both recommended tests (Fragile X and CMA).Unfortunately, we did not nd any objective data explicitly justifying when to request of a genetic study and the request of one versus several tests.Regarding the factors related to the decision about the appropriateness of genetic testing, we found statistical differences in the proportion of dysmorphic features and organic comorbidities between GTASD and factors, family history of NDDs, diagnostic complexity or referral time to specialist services between the GTASD and NGTASD groups.
a: P values are for independent sample t tests for continuous dependent variables or χ2 tests whenever both variables were categorical.NGTASD (42.3% vs. 14.4%,F:38.55, p=0.001); we also found that the mean age of ASD diagnosis was lower in the GTASD group than in the NGTASD group (59.5, SD 27 months vs 85.5, SD 41, t student 39 p=0.001) and that the mean age of enrollment in specialist services was lower in the GTASD group than in the NGTASD group (7.5, SD 3.7 years vs 10.5, SD 4.9, t student 26.42 p=0.001).There were no differences in demographic

Table 2
Results of chromosomal microarray analysis CMA NA, Not available; Novo, not observed in blood sample from either parent; U, Unknown.1Thevariants with the same number were found in the same individual respectively

Table 3 .
Results of Karyotype Notes: M indicates male; F, female; inv, inversion of chromosome region; del, deletion of chromosomal material; NA, Not available; de novo, not observed in blood sample from either parent; CMA: chromosomal microarray analysis

Table 4 .
Results of FMR1 gen alterations

Table 5 .
Results of alterations in whole-exome sequencing (Miles et al., 2005;Tammimies et al., 2015), 2013;Tammimies et al., 2015)ple is comparable to the recent report by(Harris et al., 2020), however, it falls short of the recommended standards set by the ACMG and current guidelines which suggest genetic testing for all patients diagnosed with ASD (Cuccaro et al., 2014; Narcisa et al., 2013).Among these GTASD, the diagnostic yield of 11%, apart from exome, is in line with previous studies which have reported an overall diagnostic yield of around 12%(Kalsner et al., 2018;Schaefer & Mendelsohn, 2013;Tammimies et al., 2015).Therefore, our ndings align with previous WHO data(Miles et al., 2005;Tammimies et al., 2015), as there were no disparities observed when examining clinical features between children with or without pathogenic ndings in the genetic test except for those with intellectual disability.