Somatic CAG repeat instability in intermediate alleles of the HTT gene and its potential association with a clinical phenotype

Abstract

Huntington disease (HD) is a neurodegenerative disorder caused by ≥36 CAGs in the HTT gene. Intermediate alleles (IAs) (27-35 CAGs) are not considered HD-causing, but their potential association with neurocognitive symptoms remains controversial. As HTT somatic CAG expansion influences HD onset, we hypothesised that IAs are somatically unstable, and that somatic CAG expansion may drive phenotypic presentation in some IA carriers. We quantified HTT somatic CAG expansions by MiSeq sequencing in the blood DNA of 164 HD subjects and 191 IA (symptomatic and control) carriers, and in the brain DNA of a symptomatic 33 CAG carrier. We also performed genotype-phenotype analysis. The phenotype of symptomatic IA carriers was characterised by motor (85%), cognitive (27%) and/or behavioural (29%) signs, with a late (58.7 ± 18.6 years), but not CAG-dependent, age at onset. IAs displayed somatic expansion that were CAG and age-dependent in blood DNA, with 0.4% and 0.01% of DNA molecules expanding by CAG and year, respectively. Somatic expansions of +1 and +2 CAGs were detected in the brain of the individual with 33 CAGs, with the highest expansion frequency in the putamen (10.3%) and the lowest in the cerebellum (4.8%). Somatic expansion in blood DNA was not different in symptomatic vs. control IA carriers. In conclusion, we show that HTT IAs are somatically unstable, but we found no association with HD-like phenotypes. It is plausible, however, that some IAs, close to the HD pathological threshold and with a predisposing genetic background, could manifest with neurocognitive symptoms.

Fig. 1. Somatic expansions in HTT alleles by CAG length.

The ratio of CAG expansions (number of somatic expansion sequenced reads/number of inherited allele sequenced reads) relative to the number of CAGs from the inherited allele in peripheral blood is depicted for (a) allele sizes ranging from 10 to 66 CAGs (n = 337), and (b) a closer view for allele sizes ranging from 10 to 40 CAGs.

Fig. 2. The effect of CAG length and age in the somatic expansions of HTT alleles.

The graphs show the ratio of somatic expansions (number of somatic expansion sequenced reads/number of inherited allele sequenced reads) in peripheral blood plotted against the age of the carriers of (a) 47 normal, (b) 135 intermediate, (c) 37 reduced-penetrance and (d) 74 full penetrance alleles. Each data point is coloured with respect to the CAG length of the inherited allele. The scatterplots also present the linear regression lines for each CAG repeat length, which include the interaction between age at sampling and CAG repeat length.

Fig. 3. Neurocognitive phenotype of HTT intermediate alleles carriers.

The graphs summarize the clinical characteristics of symptomatic cases represented as, (a) Onset of symptoms in intermediate (green, n = 49), reduced-penetrance (orange, n = 13) and full-penetrance (blue, n = 229) alleles carriers by the number of CAG repeats. The purple lines indicate the prediction of age at onset by Langbehn et al. [50], average (solid line) ± SD (dotted lines). (b) Venn diagram depicting the breakdown of neurocognitive signs into motor, cognitive and behavioural in 82 HTT intermediate allele carriers.

Fig. 4. Somatic instability of the CAG repeats of a 33 CAG allele carrier.

Somatic expansion ratio in single 33 CAG molecule data, showing the proportion of expansions generated by PCR (only +1 CAG) (blue), and in peripheral blood (orange) and brain tissues (a). The proportion of reads relative to the inherited CAG length in the cerebellum (b) and putamen (c). Caud N caudate nucleus, Occip L occipital lobe, Temp L temporal lobe, Front L frontal lobe, Subst N substantia nigra, HPC hippocampus.