Genetic and epigenetic disease modifiers in an Italian C9orf72 family expressing ALS, FTD or PD clinical phenotypes

Abstract

Objective: The presence of the hexanucleotide repeat expansion (HRE) in C9orf72 gene is associated to the ALS/FTD spectrum, but also to parkinsonisms. We here describe an Italian family with the father diagnosed with Parkinson disease (PD) at the age of 67 and the two daughters developing FTD and ALS at 45 years of age. We searched for C9orf72 HRE with possible genetic and epigenetic modifiers to account for the intrafamilial phenotypic variability. Methods: C9orf72 mutational analysis was performed by fragment length analysis, Repeat-primed PCR and Southern blot. Targeted next generation sequencing was used to analyze 48 genes associated to neurodegenerative diseases. Promoter methylation was analyzed by bisulfite sequencing. Results: Genetic analysis identified C9orf72 HRE in all the affected members with a similar repeat expansion size. Both the father and the FTD daughter also carried the heterozygous p.Ile946Phe variant in ATP13A2 gene, associated to PD. In addition, the father also showed a heterozygous EIF4G1 variant (p.Ala13Pro), that might increase his susceptibility to develop PD. The DNA methylation analysis showed that all the 26 CpG sites within C9orf72 promoter were unmethylated in all family members. Conclusions: Neither C9orf72 HRE size nor promoter methylation act as disease modifiers within this family, at least in blood, not excluding HRE mosaicism and a different methylation pattern in the brain. However, the presence of rare genetic variants in PD genes suggests that they may influence the clinical manifestation in the father. Other genetic and/or epigenetic modifiers must be responsible for disease variability in this C9orf72 family case.

Motor, cognitive and behavioural profiles of C9orf72 expansion-related amyotrophic lateral sclerosis

“…Some studies describe significantly larger pathogenic HRE size in ALS patients compared to FTD cases [ 31 , 32 ], which was not confirmed by other reports [ 33 ]. We too did not find any association with HRE length and disease manifestation in an Italian C9Pos pedigree presenting high intrafamilial variability [ 10 ]. In contrast to other repeat expansion disorders, the phenomenon of anticipation is still disputable since both expansions and contractions of the G 4 C 2 repeat number have been described transgenerationally in C9Pos families [ 11 , 34 – 36 ].…”

“…Genetic analysis of C9orf72 was performed on DNA extracted from peripheral blood using a two-step PCR protocol, including a first fluorescent amplicon-length analysis with primers designed in the unique sequence flanking the hexanucleotide repeat (FAM-labelled forward 5′-TGTAAAACGACGGCCAGTCAAGGAGGGAAACAACCGCAGCC-3′ and reverse 5′-GCAGGCACCGCAACCGCAG-3′) and, only for samples showing one fluorescent peak, the Repeat-PCR (RP-PCR) was carried out, as previously described [ 10 ]. For both amplicon length analysis and RP-PCR, amplicons were run on ABI Prism 3500 Genetic Analyzer (Applied Biosystems) and visualized using Gene Mapper v.4 software (Applied Biosystems).…”

“…The behavioural profile was evaluated considering the symptoms detected at the ECAS Carer Interview (disinhibition, apathy/inertia, loss of sympathy/empathy, perseverative/stereotyped/compulsive/ritualistic behaviour and hyperorality/altered food preferences), as well as the scores at FBI-A (negative behaviours) and FBI-B (positive/disinhibited behaviours). Psychological status was explored with the Beck Depression Inventory-II (BDI-II; cognitive-affective and somatic symptoms) [24] and the State-Trait Anxiety Inventory (STAI; Y1 state anxiety, Y2 trait anxiety) [25] C9orf72 genetic analysis Genetic analysis of C9orf72 was performed on DNA extracted from peripheral blood using a two-step PCR protocol, including a first fluorescent amplicon-length analysis with primers designed in the unique sequence flanking the hexanucleotide repeat (FAM-labelled forward 5′-TGT AAA ACG ACG GCC AGT CAA GGA GGG AAA CAA CCG CAGCC-3′ and reverse 5′-GCA GGC ACC GCA ACC GCA G-3′) and, only for samples showing one fluorescent peak, the Repeat-PCR (RP-PCR) was carried out, as previously described [10]. For both amplicon length analysis and RP-PCR, amplicons were run on ABI Prism 3500 Genetic Analyzer (Applied Biosystems) and visualized using Gene Mapper v.4 software (Applied Biosystems).…”

“…Southern blot was performed using a unique sequence probe, mapping within C9orf72 first intron and obtained by PCR amplification with the following primers: forward 5′-CTT TCT CCA GAT CCA GCA GCC TCC -3′ and reverse 5′-CTG AGT TCC AGA GCT TGC TACAG-3′. Upon probe labelling with dCTP 32 , hybridized membranes were resolved by autoradiography as previously described [10]. For each sample, the HRE size was calculated as mode of the observed smear range [26].…”

“…C9Pos cases also have an increased frequency of comorbid frontotemporal dementia (ALS-FTD) and a higher prevalence of a family history of ALS and/or other neurodegenerative diseases [ 2 , 5 , 8 , 9 ]. Intriguingly, the same HRE length may cause ALS, FTD or mixed ALS-FTD manifestations with no clear genotype–phenotype correlations [ 10 , 11 ]. However, the current molecular diagnosis of C9orf72 HRE by repeat-primed PCR (RP-PCR) gives no information on HRE length above a certain threshold (50–150 repeats), and the difficulty in performing and including Southern blot assay in a clinical setting has made accurate genotype–phenotype correlations inconclusive.…”