NEW YORK (GenomeWeb) – Through exome sequencing, researchers from Baylor College of Medicine and elsewhere identified apparent causal mutations and an increased burden of rare variants for patients with Charcot-Marie-Tooth-like peripheral neuropathies that had eluded diagnosis.
CMT is a hereditary peripheral neuropathy with two major clinical types as well as other less-common forms that have been linked to a range of gene mutations, including 
ones in CMT1A, CMTX1, RAB7, and PMP22, among others. As it is a heterogeneous disease both genetically and clinically, the researchers noted that molecular diagnosis using single-gene or gene panel testing has been challenging.
As Baylor’s James Lupski and his colleagues noted in Cell Reports today, they sequenced the exomes of 40 people from 37 unrelated families to zero in on the molecular cause of peripheral neuropathies in 17 of the 37 families.
From this, they proposed three candidate disease genes — PMP2, SPTLC3, and DNAJB5 — but also noticed that affected patients had a significant increase of rare variants in neuropathy-linked genes, as compared to controls.
“Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity,” Lupski and his colleagues wrote in their paper.
The researchers performed whole-exome sequencing on 40 patients with peripheral neuropathies from 37 families. In six of these patients, Lupski and his colleagues uncovered known disease-causing alleles, including mutations in MFN2, MED25, and ABHD12, among others. They also identified novel alleles in known neuropathy-related genes, such as a compound heterozygous truncating mutation in SURF1.
They also found variants in three new candidate neuropathy genes in three different families.
In one family, they homed in on a missense variant in PMP2, a stabilizing component of the myelin sheath that insulates axons in the peripheral nervous system.
This variant, the researchers noted, was present in the patient and his affected father, but not in his unaffected mother or sister. Further, morpholino-mediated knockdown of the zebrafish orthologs of this gene led to a motor neuron phenotype that could be rescued by injection of the morpholino and wild-type human PMP2.
In another patient, the researchers noted a variant affecting a highly conserved residue of SPTLC3, which encodes a subunit of the serine palmitoyltransferase enzyme that’s involved in the biosynthesis of sphingolipids. Again, zebrafish-based studies indicated that knocking this gene down led to motor neuron axon defects.
Finally, in three affected individuals from a large family, they also found a variant in DNAJB5 that affects a highly conserved amino acid in the DnaJ domain of the protein. That protein, the researchers added, encodes part of HSP40/DNAJ family of molecular co-chaperones. Variants in related genes, they noted, have been linked to similar phenotypes.
At the same time, Lupski and his colleagues noticed that patients often had more than one rare variant in a neuropathy gene, and suspected that this could influence the presentation and severity of the disease.
For instance, they observed a higher-than-expected heterozygous carrier frequency of the MED25 mutation in their cohort as compared to control samples, a minor allele frequency of 5 percent versus one of 0.5 percent.
In addition, they calculated an overall average of 2.3 nonsynonymous rare variants in each individual in 58 known neuropathy-related genes among their cohort, versus an average of 1.3 such variants among controls.
To confirm this observation, the researchers turned to whole-exome data from cohort of 32 patients from 30 families from Turkey with clinical Charcot-Marie-Tooth diagnoses. This cohort, too, exhibited an increased number of rare variants, as compared to controls.
In a zebrafish-based functional assay, the researchers suppressed various neuropathy-associated genes on their own and in combination. From this, they noted increased phenotypic severity with pairwise combinations of these genes.
“This mutation burden may well influence the phenotype, contributing to the clinical heterogeneity and the spectrum of severity observed in the disease,” Lupski and his colleagues said.