Katia Margiotti^1*, Marco Fabiani¹, Alvaro Mesoraca¹, Claudio Giorlandino^1,2

¹Human Genetics Lab, Altamedica Main Centre, Viale Liegi 45, 00198 Rome, Italy

²Department of Prenatal Diagnosis, Altamedica, Fetal-Maternal Medical Centre, Viale Liegi 45, 00198 Rome, Italy

^*Corresponding Author: Katia Margiotti, Human Genetics Lab Altamedica Main Centre Viale Liegi 45 00198 Rome, Italy.

Abstract
Approximately half of patients with rare genetic diseases remain undiagnosed after clinical exome sequencing (CES). We performed a genetic re-evaluation of undiagnosed patients more than 4 years after their initial negative CES report to determine the efficiency of reanalysis strategies. A total of 28 clinical exomes from patients with autism spectrum disorder (ASD) were genetically re-evaluated after several years from negative test results. The clinical phenotypes were categorized as ASD-Simplex (no dysmorphic features) and ASD-Complex (with dysmorphic features) by a specialist. No pathogenic or likely pathogenic mutations were identified in all patients through chromosomal microarray (CMA) and clinical exome sequencing panels at the initial diagnosis. We found that 14 % (4/28) of the patient’s diagnosis could be resolved after re-evaluating clinical exome data. Demonstrating that reanalysis after several years is a necessary action to find causative gene disorder.

Keywords: Autism Spectrum Disorder; ASD; clinical exome sequencing; CES.

Introduction
Autism is a group of genetically distinct neurodevelopmental disorders characterized by impaired early social interaction and repetitive behaviors and interests [1]. Autism is four times more prevalent in males than in females, and it frequently co-occurs with epilepsy, melancholy, anxiety, attention deficit hyperactivity disorder, and challenging behaviors such as sleep and self-harm [2]. Autism can be classified as simplex or essential (when there are no physical abnormalities or microcephaly present), complicated (when there are dysmorphic traits and microcephaly present), or syndromic (when autism is part of a genetic condition that has already been described) [3]. Currently, several specific genetic variants are known to be associated with ASD. As of May 2023, the Simons Foundation Autism Research Initiative (SFARI) included a list of approximately 1195 risk genes and more than 20 recurrent copy number variant (CNV) loci that are relevant to both syndromic and non-syndromic autism (https://gene.sfari.org/). The American Academy of Pediatrics and the American College of Medical Genetics and Genomics both recommend chromosomal microarray (CMA), which is a technique that detects large duplications or deletions, as part of the first-tier evaluation for children with either a developmental disability or ASD [4]. Current guidelines recommend for ASD next-generation sequencing (NGS) panel testing if CMA-based evaluations yield a negative result.

On the other hand, the majority of these patients will have normal results on both tests. Recent research indicates that periodic negative Clinical Exome data reanalysis using improved bioinformatic tools and current gene–disease databases can identify additional candidate variants. Estimates suggest that the diagnostic yield by Clinical Exome could be increased by ~15 % when using up-to-date software, literature, and phenotypic information for reinterpretation [5-10]. Thus, there is a need to re-analyze negative Clinical Exome to give a better possibility for those who do not receive a molecular diagnosis in the first evaluation. In this study, using a clinical exome cohort enrolled in the Altamedica Center, we present a retrospective reanalysis of 28 negative clinical exome tests.

Results and Discussion
To emphasize the significance of CES reanalysis, we revisited a cohort of 28 patients with complex (n=9) and simplex ASD (n=19), originally enrolled at Altamedica Medical Center. We selected all negative Array Comparative Genomic Hybridization (Array-CGH) and negative clinical exome analyzed over more than 4 years to gain insight into the relative contribution of reanalysis strategies. Array CGH was performed to exclude microdeletion/microduplication copy number variants (CNV) as possible causes of ASD. We performed a systematic reanalysis, with an advanced clinical exome pipeline (i.e., including low-quality variants, copy number variant (CNV) analysis, and up-to-date disease-gene panels), of all patients in this cohort. Systematic follow-up of all patients without a conclusive diagnosis after diagnostic testing (n=28) revealed 4 new definitive genetic diagnoses in simplex ASD patients, resulting from updated bioinformatic information tools (Table 1). In total, 14 % (4/28) of the patients in this study required reanalysis for conclusive diagnoses based on variants not identified in the initial analysis. For these 4 patients, the definitive genetic diagnosis was based on variants not prioritized in the initial CES analysis (Table 1). Variants were detected after updated bioinformatic analyses and interpretation. Publications of the 4 disease-variants associations appeared after our initial research since they were not seen or prioritized during the initial CES analysis. This emphasizes the necessity of routinely reviewing CES data since novel genotype-phenotype relationships can be discovered at almost any moment.

Table 1. Novel genetic diagnoses after more than 4 years from the initial analysis.

Author Contributions: Conceptualization and writing the original draft, KM; data analysis KM, FM; visualization and supervision, AM and CG. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding

Institutional Review Board Statement: The study was approved by the local ethical committee of Artemisia SPA.

Informed Consent Statement: Informed consent was obtained from subjects involved in the study.

Data Availability Statement: Not applicable.

Conflicts of Interest: The authors declare no conflict of interest.

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