Sequence variations in C9orf72 downstream of the hexanucleotide repeat region and its effect on repeat-primed PCR interpretation: a large multinational screening study

Abstract

A large GGGGCC-repeat expansion mutation (HREM) in C9orf72 is the most common known cause of ALS and FTD in European populations. Sequence variations immediately downstream of the HREM region have previously been observed and have been suggested to be one reason for difficulties in interpreting RP-PCR data. Our objective was to determine the properties of these sequence variations with regard to prevalence, the range of variation, and effect on disease prognosis. We screened a multi-national cohort (n = 6981) for the HREM and samples with deviant RP-PCR curves were identified. The deviant samples were subsequently sequenced to determine sequence alteration. Our results show that in the USA and European cohorts (n = 6508) 10.7% carried the HREM and 3% had a sequence variant, while no HREM or sequence variants were observed in the Japanese cohort (n = 473). Sequence variations were more common on HREM alleles; however, certain population specific variants were associated with a non-expanded allele.In conclusion, we identified 38 different sequence variants, most located within the first 50 bp downstream of the HREM region. Furthermore, the presence of an HREM was found to be coupled to a lower age of onset and a shorter disease survival, while sequence variation did not have any correlation with these parameters.

Keywords:

• C9orf72
• ALS
• FTD
• variants
• RP-PCR interpretation

Acknowledgements

We thank the patients and their relatives for participating in this study. We also wish to thank Ann-Charloth Nilsson, Anna Birve, Anna-Karin Rikardsson, and Ulla-Stina Spetz for technical assistance and the many neurologists who have provided samples from their patients. We are also grateful to Jonathan Gilthorpe proof reading the manuscript.

Funding

More than 60 grants over a 20 years period has funded this project. This project was funded by the Swedish Science Council, the Brain Research Foundation, B. Hållsten’s Brain Research Foundation, Neuroförbundet, the Ulla-Carin Lindquist’s Foundation for ALS Research, the Knut and Alice Wallenberg Foundation, Swedish Brain Power, the German Federal Ministry of Education and Research (STRENGTH consortium and BMBF; 01GI0704, German network for ALS research (MND-NET)), the German Research Foundation (SYNERGY excellence cluster), the Charcot Foundation for ALS Research (ACL, JHW), the virtual Helmholtz Institute ‘RNA-Dysmetabolismus in ALS and FTD’ and the DFG-funded Swabian ALS Registry, Grant-in-Aid for the Research Committee of CNS Degenerative Diseases and Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labour and Welfare in Japan.

Declaration of interest

The authors declare no conflict of interest.