Repeat length in spinocerebellar ataxia type 4 (SCA4) predicts age at onset and disease severity

Abstract

Background: Recently, an exonic GGC repeat expansion (RE) was identified by long-read genome sequencing in the ZFHX3 gen, causing spinocerebellar ataxia type 4 (SCA4), a dominant form of ataxia with sensory neuropathy. However, the analysis of larger cohorts of patients remained demanding, resulting in a challenge to diagnose patients and leaving the question of anticipation in SCA4 unanswered.

Objectives: We aimed to develop a GGC repeat test for clinical SCA4 screening and to apply this test to screen two large German SCA pedigrees and samples of unrelated patients collected over the last 25 years.

Methods: We modulated a commercial GGC-RE kit (Bio-Techne AmplideX^® Asuragen^® PCR/CE FMR1 Reagents) with ZFHX3-specific primers and adapted PCR conditions. The test was applied to patients and 50 healthy controls to determine the exact repeat number. Clinical data were revised and correlated with the expanded allele sizes and an exploratory analysis of structural MRI was performed.

Results: Repeat size, determined by our protocol for (GGC)_n RE analysis shows a strong inverse correlation between repeat length and age at onset and anticipation in subsequent generations. The phenotype also appears to be more strongly expressed in carriers of longer RE. Clinical red flags were slowed saccades, sensory neuropathy and autonomic dysfunction.

Conclusion: Our protocol enables cost-effective and robust screening for the causative SCA4 RE within ZFHX3. Furthermore, detailed clinical data of our patients gives a more precise view on SCA4, which seems to be more common among patients with ataxia than expected.

Keywords: Anticipation; Repeat expansion; SCA4; Spinocerebellar ataxia.

Fig. 1

Family I and II; Standard symbols were used. Black symbols represent individuals with cerebellar ataxia with sensory and autonomic neuropathy. Individual indices, allele lengths and age of onset (AOO) are given beneath pedigree symbols. Red expanded allele sizes

Fig. 2

a 2% Agarose gel: PCR products of different patients (Family I); lane 1 50 bp-Ladder (NEB), lane 2 100 bp-Ladder (Invitrogen); lane 3 and 4 control samples, lane 5 patient IV:10; lane 6 patient V:17; lane 7 patient V:8; lane 8 patient III:7; lane 9 patient VI:3; lane 10 negative PCR control; b Capillary gel electrophoresis of (i) smallest, (ii) intermediate and (iii) longest pathological allele in our cohort. Numbers indicate allele repeat size: (21 = common normal allele); c Capillary peaks with repeat sizes of three control samples. Upper lane homozygous 21 repeats, middle lane heterozygous 19 and 21 repeats, bottom lane heterozygous 18 and 21 repeats. d Sequences of heterozygous controls with 21, 19 and 18 repeat alleles, respectively

Fig. 3

Anticipation in SCA4 in subsequent generations of families I and II and inverse correlation between age of onset (AOO) and repeat length (RL) of 26 SCA4 patients

Fig. 4

Homology models of region 3452–3560 of the human ZFHX3 protein (GenBank: AAC14462.1) and a variant containing 47 additional glycine residues