Re-analysis of an original CMTX3 family using exome sequencing identifies a known BSCL2 mutation

Introduction: Charcot-Marie-Tooth (CMT) disease is a group of peripheral neuropathies affecting both motor and sensory nerves. CMTX3 is an X-linked CMT locus, which maps to chromosome Xq26.3-q27.3. Initially, CMTX3 was mapped to a 31.2-Mb region in 2 American families. We have reexamined 1 of the original families (US-PED2) by next generation sequencing. Methods: Three members of the family underwent exome sequencing. Candidate variants were validated by PCR and Sanger sequencing analysis. Conclusion: No pathogenic coding variants localizing to the CMTX3 region were identified. However, exome sequencing identified a known BSCL2 mutation (N88S). This study demonstrates the power of exome sequencing as a tool to identify gene mutations for a small family in the absence of statistically significant linkage data.3 page(s

Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing

Inherited peripheral neuropathies (IPNs) are a group of related diseases primarily affecting the peripheral motor and sensory neurons. They include the hereditary sensory neuropathies (HSN), hereditary motor neuropathies (HMN), and Charcot-Marie-Tooth disease (CMT). Using whole-exome sequencing (WES) to achieve a genetic diagnosis is particularly suited to IPNs, where over 80 genes are involved with weak genotype–phenotype correlations beyond the most common genes. We performed WES for 110 index patients with IPN where the genetic cause was undetermined after previous screening for mutations in common genes selected by phenotype and mode of inheritance. We identified 41 missense sequence variants in the known IPN genes in our cohort of 110 index patients. Nine variants (8%), identified in the genes MFN2, GJB1, BSCL2, and SETX, are previously reported mutations and considered to be pathogenic in these families. Twelve novel variants (11%) in the genes NEFL, TRPV4, KIF1B, BICD2, and SETX are implicated in the disease but require further evidence of pathogenicity. The remaining 20 variants were confirmed as polymorphisms (not causing the disease) and are detailed here to help interpret sequence variants identified in other family studies. Validation using segregation, normal controls, and bioinformatics tools was valuable as supporting evidence for sequence variants implicated in disease. In addition, we identified one SETX sequence variant (c.7640T>C), previously reported as a putative mutation, which we have confirmed as a nonpathogenic rare polymorphism. This study highlights the advantage of using WES for genetic diagnosis in highly heterogeneous diseases such as IPNs and has been particularly powerful in this cohort where genetic diagnosis could not be achieved due to phenotype and mode of inheritance not being previously obvious. However, first tier testing for common genes in clinically well-defined cases remains important and will account for most positive results.12 page(s

A New locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.13 page(s

Characterizing the molecular phenotype of an Atp7a(T985I) conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX)

ATP7A is a P-type ATPase essential for cellular copper (Cu) transport and homeostasis. Loss-of-function ATP7A mutations causing systemic Cu deficiency are associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome. We previously identified two rare ATP7A missense mutations (P1386S and T994I) leading to a non-fatal form of motor neuron disorder, X-linked distal hereditary motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency. Recent investigations using a tissue specific Atp7a knock out model have demonstrated that Cu plays an essential role in motor neuron maintenance and function, however the underlying pathogenic mechanisms of ATP7A mutations causing axonal degeneration remain unknown. We have generated an Atp7a conditional knock in mouse model of dHMNX expressing Atp7a(T985I), the orthologue of the human ATP7A(T994I) identified in dHMNX patients. Although a degenerative motor phenotype is not observed, the knock in Atp7a(T985I/Y) mice show altered Cu levels within the peripheral and central nervous systems, an increased diameter of the muscle fibres and altered myogenin and myostatin gene expression. Atp7a(T985I/Y) mice have reduced Atp7a protein levels and recapitulate the defective trafficking and altered post-translational regulatory mechanisms observed in the human ATP7A(T994I) patient fibroblasts. Our model provides a unique opportunity to characterise the molecular phenotype of dHMNX and the time course of cellular events leading to the process of axonal degeneration in this disease

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

<p>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, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref037" target="_blank">37</a>]); hypertrichosis² (blue, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref037" target="_blank">37</a>]); hypertrichosis³ (green,[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref038" target="_blank">38</a>]); ptosis (pink; Bunyan [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref039" target="_blank">39</a>]); and XX sex reversal (purple, Haines [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref040" target="_blank">40</a>]) are marked out on the hairpin structure. Single breakpoints are depicted by a solid line. Multiple breakpoints are indicated by broken lines.</p

Average depth ± SD of sequence coverage across 8q24.3.

<p>Average depth ± SD of sequence coverage across 8q24.3.</p

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

<p>(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.</p

Characterization of the CMTX3 insertion.

<p>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’<i>ARHGAP39</i> transcript which has been inserted 330 kb downstream and 84 kb upstream of the genes <i>LOC389895</i> and <i>SOX3</i>, 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).</p

Split-reads and discordant paired ends mapping to Xq27.1 and 8q24.3 in whole genome sequencing data from affected males.

<p>Split-reads and discordant paired ends mapping to Xq27.1 and 8q24.3 in whole genome sequencing data from affected males.</p