Multi-Omics Genetic Investigation Strategies to Enhance the Diagnostic Workflow for Rare Diseases

Neurodevelopmental disorders (NDDs) represent a major group of rare diseases and are frequently associated with substantial morbidity due to underlying genetic alterations. Despite advances in genetic testing, chromosomal microarray analysis (CMA) and exome sequencing (ES) identify a causal genetic diagnosis in only approximately 30-40% of affected individuals. The remaining diagnostic gap may reflect both the limitations of current methods in detecting certain types of genetic alterations and the difficulty of determining the clinical significance of identified variants.

The LEOPARD project aims to improve diagnostic yield in patients with NDDs of unknown genetic etiology through a two-phase approach.

Phase 1: Reanalysis of existing genetic data. Genetic data from 500 patients previously investigated by ES or CMA will be reanalyzed using updated bioinformatics algorithms and current biomedical knowledge. Some patients have already provided consent for the use of their genomic and clinical data in previous studies conducted at the participating clinical centers. The remaining patients will be contacted to obtain informed consent for the use of their data. Approximately one-third of these patients are expected to receive a genetic diagnosis through data reanalysis alone.

Phase 2: Additional molecular investigations. Up to 200 patients who remain undiagnosed after data reanalysis will undergo further molecular investigations, selected according to the clinical and technical eligibility criteria for each test. These investigations will include functional studies to assess the clinical significance of variants of uncertain significance, RNA sequencing to evaluate gene expression and transcript integrity, and DNA methylation profiling to identify disease-associated methylation signatures (episignatures).

If the number of eligible patients remaining undiagnosed after Phase 1 is insufficient, additional patients will be recruited through the participating clinical centers. This phase will require the collection of new biological samples using minimally invasive procedures, including peripheral blood sampling and the collection of buccal and urinary epithelial cells. Biological samples will be sent to the appropriate laboratories according to the analyses required and stored in the study biobanks.

Genetic alterations and identified variants will be evaluated according to applicable national and international scientific guidelines, including recommendations from the Italian Society of Human Genetics (SIGU) and the American College of Medical Genetics and Genomics (ACMG).

The diagnostic contribution of each additional molecular investigation will be assessed using statistical methods to estimate the improvement in diagnostic sensitivity and overall diagnostic yield.

The project will run for 24 months and will involve three clinical and laboratory units and one bioinformatics unit. The participating centers have the clinical expertise and patient population required to support recruitment in accordance with the study objectives.

Primary outcomes

• Proportion of Participants Receiving a Genetic Diagnosis [Up to 24 months]: Proportion of participants in whom a specific genetic diagnosis is established following reanalysis of existing genetic data and, where applicable, additional molecular investigations. The diagnostic yield will be assessed by identifying the proportion of participants receiving a genetic diagnosis among those evaluated.

Sex: All

Inclusion Criteria:

* Diagnosis of autism spectrum disorder and/or another neurodevelopmental disorder.
* Moderate-to-severe intellectual disability, documented by psychometric assessment, with an intelligence quotient (IQ) below 50.
* Previous genetic testing by chromosomal microarray analysis (CMA) and/or exome sequencing (ES).
* Availability and willingness to provide biological samples, including peripheral blood, saliva, and urine, as required for the planned molecular investigations.
* Availability of parental biological samples, whenever feasible.

Exclusion Criteria:

* An established clinical diagnosis that already explains the participant's clinical presentation.
* Intellectual disability of a known non-genetic etiology, such as perinatal hypoxia.