The spectrum of C9orf72 repeat lengths in Portuguese frontotemporal dementia and amyotrophic lateral sclerosis patients: from pathogenic expansions to intermediate alleles.

INTRODUCTION

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically distinct disorders that share a common genetic etiology, with pathogenic hexanucleotide repeat expansions in the C9orf72 gene representing the most frequent genetic cause of both conditions. In this study we aim to determine the frequency of C9orf72 repeat expansions in patients with FTD and/or ALS and their at-risk relatives from central Portugal, and to analyze the distribution and repeat size of intermediate alleles within this cohort investigating their potential pathogenicity.

METHODS

Between January 2016 and January 2026, C9orf72 repeat expansions were assessed in 610 patients with the clinical presentation within the spectrum of FTD/ALS and in 63 asymptomatic relatives. C9orf72 repeat expansions were identified using a two-step PCR protocol comprising STR-PCR and RP-PCR, followed by repeat-length sizing with a commercial kit. Capillary electrophoresis traces were subsequently analyzed using AmplideX® PCR/CE Reporter software. Furthermore, carriers of intermediate alleles underwent comprehensive clinical, fluid-biomarker, and neuropsychological characterization to investigate the potential pathogenicity of these alleles.

RESULTS

Pathogenic C9orf72 expansions were identified in 11% of patients. The vast majority carried long expansions (>145 repeats), whereas three patients exhibited shorter expansions ranging from 38 to 78 repeats. Intermediate alleles were found in seven subjects with heterogeneous clinical presentations. Clinically, these individuals were indistinguishable from carriers of longer expansions.

DISCUSSION: C9orf72 hexanucleotide repeat expansions are a major genetic cause of FTD, ALS, and FTD-ALS in this cohort of patients, highlighting the importance of genetic testing for accurate counselling and clinical management. These findings support routine C9orf72 testing in all patients with these disorders, particularly those of European ancestry. Our findings also support the emerging view that intermediate C9orf72 repeat expansions may act as a genetic risk factors contributing to a broad spectrum of clinical phenotypes.