Sequencing of Charcot-Marie-Tooth disease genes in a toxic polyneuropathy

Abstract

Objective: Mutations in Charcot-Marie-Tooth disease (CMT) genes are the cause of rare familial forms of polyneuropathy. Whether allelic variability in CMT genes is also associated with common forms of polyneuropathy-considered "acquired" in medical parlance-is unknown. Chemotherapy-induced peripheral neuropathy (CIPN) occurs commonly in cancer patients and is individually unpredictable. We used CIPN as a clinical model to investigate the association of non-CMT polyneuropathy with CMT genes.

Methods: A total of 269 neurologically asymptomatic cancer patients were enrolled in the clinical trial Alliance N08C1 to receive the neurotoxic drug paclitaxel, while undergoing prospective assessments for polyneuropathy. Forty-nine CMT genes were analyzed by targeted massively parallel sequencing of genomic DNA from patient blood.

Results: A total of 119 (of 269) patients were identified from the 2 ends of the polyneuropathy phenotype distribution: patients that were most and least susceptible to paclitaxel polyneuropathy. The CMT gene PRX was found to be deleteriously mutated in patients who were susceptible to CIPN but not in controls (p = 8 × 10(-3)). Genetic variation in another CMT gene, ARHGEF10, was highly significantly associated with CIPN (p = 5 × 10(-4)). Three nonsynonymous recurrent single nucleotide variants contributed to the ARHGEF10 signal: rs9657362, rs2294039, and rs17683288. Of these, rs9657362 had the strongest effect (odds ratio = 4.8, p = 4 × 10(-4)).

Interpretation: The results reveal an association of CMT gene allelic variability with susceptibility to CIPN. The findings raise the possibility that other acquired polyneuropathies may also be codetermined by genetic etiological factors, of which some may be related to genes already known to cause the phenotypically related Mendelian disorders of CMT.

Figure 1. Neuropathy phenotyping by serial symptom assessment in paclitaxel-treated patients to score CIPN susceptibility

A. A CIPN susceptibility score was determined for each of the n=248 patients in N08C1, for whom three (or more) serial CIPN20 assessments were available. All patients were treated with paclitaxel for up to twelve weeks. Patients with scores that were significantly different from the study median of 1.0 were chosen for exome sequencing, n=119. Patients with a significantly higher score constituted CIPN-susceptible cases and patients with a significantly lower score were the controls. Patients with intermediate susceptibility scores (close to 1), or few or missing data points, or unreliable responses (indicated by large standard errors), n=129, remained uncategorized. Thereby the approach yielded two “extreme phenotype” groups for genetic comparison. Data points shown are the CIPN susceptibility scores with standard errors for all patients normalized to the median slope for the specific paclitaxel regimen received: paclitaxel single agent (top); paclitaxel with carboplatin (bottom); weekly administration (circles); every-three-week administration (squares). B. Raw CIPN20 scores are shown for two illustrative patients, a control- and a CIPN subject over a period of up to twelve weekly administrations of paclitaxel. The heat map depicts responses to the CIPN20 questionnaire items in rows and time points (weekly) in columns. Both patients were free of neuropathy symptoms at study outset as confirmed by their response “not at all” (a score of 1) to all questions at the earliest time point. Subsequently one of the patients continued to answer “not at all” to almost all CIPN20 items in the twelve repeat assessments indicating lack of susceptibility to neuropathy (or “resistance” to neuropathy); as a consequence, this patient was assigned to the control group. The other depicted patient responded with scores rising from week to week (yellow, red), indicating early onset of neuropathy and progressive worsening over time; treatment was terminated after fewer than twelve treatments. This patient was classified as belonging to the group of cases, susceptible to CIPN, because the slope of worsening of CIPN symptoms was steep.

Figure 2. CMT gene categorization and -testing

A. Sequencing read coverage. The genomic target region consisting of 235 kB nucleotides covering the 801 exons of 49 canonical CMT genes was sequenced at a multiplicity (“depth”) exceeding the requirement of >20-fold unique read coverage at >80% of base pair positions. Shown is the average and 95% CI for all study samples. B. SNV discovery and grouping. SNV were categorized according to whether they occurred only in one patient in the study (“singleton”) or were seen in several patients (“recurrent”). SNV counts across the target region are shown for the entire study (left panel) and as average per patient (right panel). Distinct singleton SNV were nearly as common as distinct recurrent SNV. However, because the same recurrent SNV occurs in several patients, recurrent SNV are in aggregate far more commonly found than singleton SNV as shown on the right. SNV were further sub-grouped into synonymous SNV, expected to be biologically silent in most cases, and non-synonymous SNV, i.e., missense or non-sense SNV, which are predicted to alter the encoded protein. All subsequent analyses were based on non-synonymous SNV. C. Categorization of CMT genes according to non-synonymous SNV. CMT genes were categorized according to whether they harbored singleton and/or recurrent SNV. Only non-synonymous SNV were considered. D. Burden testing of CMT genes for singleton SNV association with CIPN-susceptibility. A p-p plot shows the results of Burden tests performed on genes harboring ≥5 singleton SNV. The gene PRX was found to be associated with CIPN-susceptibility. E. C-alpha testing of CMT genes for recurrent SNV association with CIPN-susceptibility. A p-p plot shows the results of C-alpha tests performed on genes harboring ≥1 recurrent SNV. The gene ARHGEF10 was found to be associated with CIPN-susceptibility.

Figure 3. Allelic variants in CIPN-susceptibility associated CMT genes

A. PRX singleton SNV. Of the CIPN-susceptible patients, 8 carried a non-synonymous SNV in PRX. The protein encoded by PRX is shown with the positions of SNV found in CIPN susceptible patients (blue) and sites of known CMT mutations (grey). B. Validation by Sanger sequencing. Individual SNV in PRX were confirmed by Sanger sequencing. Shown are stacks of next generation sequencing reads from the present study and confirmatory electrophoretic tracings obtained by capillary sequencing for the heterozygous variant at chr19:40909664 in a CIPN-susceptible patient and the reference genotype (in another patient). C. AHRGEF10 recurrent SNV. Three recurrent non-synonymous SNV were detected. The observed MAF was similar to the MAF reported in the 1000 Genomes Project and NHLBI_6500 exome sequencing reference datasets. The three SNV were not correlated (occur independently) in reference cohorts as shown in a linkage disequilibrium (LD) plot generated from 1000 Genomes Project data. D. Observed frequency of hetero- and homozygous genotypes and validation by TaqMan PCR. For each SNV the relative frequencies of the three possible genotypes observed in the study (homozygous for the major allele, or heterozygous, or homozygous for the minor allele) were consistent with the Hardy Weinberg equilibrium. The accuracy of SNV was confirmed by orthogonal laboratory techniques. E. AHRGEF10 alleles in CIPN-susceptible patients and controls. The association of ARHGEF10 alleles with CIPN-susceptibility was dominated by rs9657362. Absence of its minor allele was strongly associated with CIPN-susceptibility with an OR=4.8; the same result can also be expressed as the inverse, i.e., OR=0.21, to mean that the minor allele was associated with control group status, i.e. it was protective. The association of rs9657362 was statistically highly significant with p=4×10⁻⁴. The SNV rs17683288 and rs2294039 showed a trend toward a protective and a risk-effect respectively. A Venn diagram shows the frequency of all possible AHRGEF10 genotypes (all 8 possible combinations of carrying a minor allele at rs9657362, rs17683288 and rs2294039) among CIPN-susceptible and control patients.