Background: Fragile X syndrome (FXS) is a genetic disorder caused by loss of function of the FMR1 gene, located on the X chromosome. It represents one of the most common monogenic causes of intellectual disability (ID) and autism spectrum disorder (ASD), and is traditionally included in first-line etiological investigations of cognitive impairment. However, recent evidence has raised questions regarding the diagnostic yield of FMR1 testing, which appears lower than expected, considering the worldwide estimated prevalence. This study aimed to assess the diagnostic performance of FMR1 analysis within the Neurodevelopment Unit of a tertiary paediatric hospital in Portugal.Method: We conducted a retrospective review of all FMR1 gene analyses requested by the Neurodevelopment Unit between January 2014 and May 2025 for patients with ID and/or ASD. Results were categorized according to CGG trinucleotide repeat size: normal, intermediate (45–54 repeats), premutation (55–200 repeats), and full mutation (>200 repeats). Diagnostic yield was calculated as the ratio of confirmed FXS cases among all tested individuals. Data analysis was performed using Excel®.Results: A total of 1095 patients were tested during the study period (73.9% male). Only one case of FXS was identified: a female patient carrying one normal allele (29 CGG repeats) and one allele with a pathogenic expansion, exhibiting mosaicism for a premutation (~120 CGG repeats) and an inactive full mutation (>200 CGG repeats). This case presents ethical concerns, as it involves in vitro fertilization with donor sperm. No FXS cases were detected in the male cohort, resulting in a diagnostic yield of 0.1% (1/286 females; 0/809 males). Beyond confirmed cases of FXS, 18 patients (4 females and 14 males) were identified with an intermediate number of repeats, and 2 patients (1 male and 1 female) with premutations.Conclusion: While previous reports have estimated the diagnostic yield of FMR1 testing in ID and/or ASD to range from 1–3%, more recent studies suggest lower rates, consistent with our findings. These results suggest that FMR1 testing may not be optimal as a routine first-line diagnostic tool and should instead be prioritized in patients with specific clinical features suggestive of FXS. Nonetheless, identification of intermediate alleles and premutations carries important clinical implications, as these variants confer increased risk for Fragile X-associated tremor/ataxia syndrome (FXTAS) and primary ovarian insufficiency. It also allows genetic counseling by highlighting the potential for trinucleotide expansion in future generations.Multicentre and nationwide studies are needed to more accurately determine the diagnostic yield of FMR1 testing across the country and support evidence-based adjustments to current etiological investigation protocols.
