Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3

Abstract

With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations.

Fig 1. Identification and confirmation of a 78 kb chromosome 8q24.3 insertion in patients with CMTX3.

(A) Whole genome sequencing depth of coverage for affected (red) and normal (black) males across the 8q24.3 insertion and flanking sequence. (B) Depiction of wild type chromosome X (top) and mutant chromosome X (bottom). The location of primers and amplicon sizes for the multiplex PCR genotyping assay are shown. Dotted red lines represent insertion breakpoints. (C) Size fractionation of multiplex PCR genotyping assay for a subset of family members from CMT623. Individual genotypes are depicted above the gel lane. Expected band sizes for the various primer combinations are listed to the right. Unaffected hemizygous males and homozygous females generate a single 340 bp amplicon; affected hemizygous males generate 595 bp and 235 bp amplicons crossing the proximal and distal breakpoints, respectively; carrier females amplify all three amplicons.

Fig 2. Characterization of the CMTX3 insertion.

Sequence analysis of the proximal (A) and distal (B) breakpoints. Reference sequence for chromosome X and chromosome 8 are indicated in blue and orange, respectively. The distal breakpoint includes additional sequence from chromosome 12 (in green) and small rearrangements of the chromosome X sequence including an inversion of 12 bp, and a base pair substitution and a base pair deletion. (C) The 78 kb 8q24.3 sequence (in orange) contains the partial 5’ARHGAP39 transcript which has been inserted 330 kb downstream and 84 kb upstream of the genes LOC389895 and SOX3, respectively (in blue). The direction of transcripts are indicated by the arrow. (D) Location of the 78kb 8q24.3 insertion sequence (in orange) relative to the whole of the 5.7 Mb CMTX3 locus (in blue).

Fig 3. FGF13 mRNA expression is elevated in patient lymphoblasts.

Quantitative RT-PCR showing mRNA levels for ARHGAP39 (A) and FGF13 (B) from patient lymphoblasts relative to three normal controls. Bars show the mean mRNA levels (± SD; error bars) relative to Control 1, which has been set to +1. A student t-test was performed comparing each value to Control 1 (*, p < 0.05).

Fig 4. The breakpoints of disease-associated interchromosomal insertions at Xq27.1 localize near the center of 180 bp palindrome sequence.

Cartoon depicts a portion of the palindrome sequence (chrX:139,502,939–139,502,970) with the positive strand folded upon itself in a hairpin loop (black). The four non-palindromic bases in the middle of the 180 bp sequence (TATC, bolded black) are predicted to form the head of the hairpin loop. The locations of the breakpoints on chromosome Xq27.1 for CMTX3 (orange); hypertrichosis¹ (red, [37]); hypertrichosis² (blue, [37]); hypertrichosis³ (green,[38]); ptosis (pink; Bunyan [39]); and XX sex reversal (purple, Haines [40]) are marked out on the hairpin structure. Single breakpoints are depicted by a solid line. Multiple breakpoints are indicated by broken lines.