Megan H. Brewer

Distal enhancers upstream of the Charcot-Marie-Tooth type 1A disease gene PMP22

Myelin insulates axons in the peripheral nervous system to allow rapid propagation of action potentials, and proper myelination requires the precise regulation of genes encoding myelin proteins, including PMP22. The correct gene dosage of PMP22 is critical; a duplication of PMP22 is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth Disease (CMT) (classified as type 1A), while a deletion of PMP22 leads to another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies. Recently, duplications upstream of PMP22, but not containing the gene itself, were reported in patients with CMT1A like symptoms, suggesting that this region contains regulators of PMP22. Using chromatin immunoprecipitation analysis of two transcription factors known to upregulate PMP22-EGR2 and SOX10-we found several enhancers in this upstream region that contain open chromatin and direct reporter gene expression in tissue culture and in vivo in zebrafish. These studies provide a novel means to identify critical regulatory elements in genes that are required for myelination, and elucidate the functional significance of non-coding genomic rearrangements.

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.

Haplotype-specific modulation of a SOX10/CREB response element at the Charcot–Marie–Tooth disease type 4C locus SH3TC2

Loss-of-function mutations in the Src homology 3 (SH3) domain and tetratricopeptide repeats 2 (SH3TC2) gene cause autosomal recessive demyelinating Charcot-Marie-Tooth neuropathy. The SH3TC2 protein has been implicated in promyelination signaling through axonal neuregulin-1 and the ERBB2 Schwann cell receptor. However, little is known about the transcriptional regulation of the SH3TC2 gene. We performed computational and functional analyses that revealed two cis-acting regulatory elements at SH3TC2-one at the promoter and one ∼150 kb downstream of the transcription start site. Both elements direct reporter gene expression in Schwann cells and are responsive to the transcription factor SOX10, which is essential for peripheral nervous system myelination. The downstream enhancer harbors a single-nucleotide polymorphism (SNP) that causes an ∼80% reduction in enhancer activity. The SNP resides directly within a predicted binding site for the transcription factor cAMP response element binding protein (CREB), and we demonstrate that this regulatory element binds to CREB and is activated by CREB expression. Finally, forskolin induces Sh3tc2 expression in rat primary Schwann cells, indicating that SH3TC2 is a CREB target gene. These findings prompted us to determine if SNP genotypes at SH3TC2 are associated with differential phenotypes in the most common demyelinating peripheral neuropathy, CMT1A. Interestingly, this revealed several associations between SNP alleles and disease severity. In summary, our data indicate that SH3TC2 is regulated by the transcription factors CREB and SOX10, define a regulatory SNP at this disease-associated locus and reveal SH3TC2 as a candidate modifier locus of CMT disease phenotypes.

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

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.

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

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.

Evidence of a founder haplotype refines the X-linked Charcot–Marie-Tooth (CMTX3) locus to a 2.5 Mb region

X-linked Charcot–Marie-Tooth (CMTX) disease is a common inherited degenerative disorder of the peripheral nerve. Previously, our laboratory identified a large New Zealand/United Kingdom (NZ/UK) family mapping to the CMTX3 locus (Xq26.3–27.1). We have now identified a second large, Australian X-linked CMT family that links to the CMTX3 locus. This new family has the same phenotype as our previously described CMTX3 family, with slightly milder disease in males than CMTX1 and asymptomatic carrier females. This study also includes the re-analysis of one of the original US pedigrees reporting the CMTX3 locus. The large Australian family shared the complete disease haplotype with our original NZ/UK family, while the American family shared only the distal portion of the disease haplotype. Comparison of the frequency of the CMTX3 haplotype to the normal population showed strong statistical evidence (p < 0.0001) indicating that the smaller shared haplotype is identical by descent. This suggests that the new CMTX3 family, our previously reported family, and the original American CMTX3 family have a common ancestor, and the disease in these families is caused by a founder mutation. The ancestral recombination observed in the American family refines the CMTX3 interval to a 2.5 Mb region between DXS984 and DXS8106. In this region, 11 out of the 15 annotated genes have been excluded for pathogenic mutations.

X-linked CMT: genes and gene loci in an Australian cohort.

Dear Sirs, Charcot-Marie-Tooth (CMT) disease encompasses a clinically and genetically heterogeneous group of inherited peripheral neuropathies affecting bothmotor and sensory neurons. AnXlinked form of CMT (CMTX) accounts for up to 15% of all CMT cases [1, 2]. OMIM reports five X-linked loci associated with CMT disease: CMTX1 (OMIM 302800), CMTX2 (OMIM 302801), CMTX3 (OMIM 302802), Cowchock syndrome (CMTX4, OMIM 310490) and CMTX5 (Rosenberg-Chutorian syndrome, OMIM 311070). Clinically, families linked to the CMTX1 and CMTX3 loci generally present pure CMT peripheral neuropathies. The families reported for CMTX2, Cowchock syndrome and CMTX5, on the other hand, are perhaps better described as syndromic peripheral neuropathies rather than CMT as the CMTX2 locus is associated with mental retardation [3], Cowchock syndrome is associated with mental retardation and deafness [4] and CMTX5 is associated with deafness and optic neuropathy [5]. Genetic mutations in the gap junction beta 1/ connexin 32 (GJB1/Cx32) and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) genes cause CMTX1 and CMTX5, respectively [6, 7]. CMTX1 accounts for up to 90% of all CMTX cases. The prevalence of the remaining Xlinked CMT loci is unknown as there is little information reported. Previously, we reported an Australian cohort of 77 probable X-linked CMT families defined by no male-tomale inheritance and a CMT phenotype with females showing either a normal or mildly affected phenotype indicated by normal or mildly reduced nerve conduction velocities. These families were also negative for the PMP22 duplication or mutations in PMP22 and P0. Only 51% (N=39) were caused by a Cx32 mutation [8]. This report examines known CMTX loci in this family cohort using a combination of mutation scanning and haplotype analysis. A total of 70 probable X-linked CMT families from our original cohort of 77 were studied. The cohort number has decreased as three families are caused by other CMT gene mutations, and the genetic material for four families is no longer available for study. Individuals examined gave informed consent in accordance with protocols approved by the Sydney South West Area Health Service Ethics Review Committee (Sydney, Australia). Figure 1 gives a breakdown of CMTX loci in our cohort. Thirty-nine families were shown to have a mutation in Cx32. An affected individual from each of the 31 Cx32negative families was analysed for mutations in the coding exons of PRPS1 using high-resolution melt analysis as previously described [9]. All the families were negative for PRPS1 mutations. No families tested had clinical symptoms M. H. Brewer (*) :R. Chaudhry : S. Chu : B. Kowalski : G. Nicholson :M. Kennerson Northcott Neuroscience Laboratory, ANZAC Research Institute, Concord, New South Wales, Australia 2139 e-mail: mbrewer@med.usyd.edu.au

Structural variations causing inherited peripheral neuropathies: A paradigm for understanding genomic organization, chromatin interactions, and gene dysregulation

Inherited peripheral neuropathies (IPNs) are a clinically and genetically heterogeneous group of diseases affecting the motor and sensory peripheral nerves. IPNs have benefited from gene discovery and genetic diagnosis using next‐generation sequencing with over 80 causative genes available for testing. Despite this success, up to 50% of cases remain genetically unsolved. In the absence of protein coding mutations, noncoding DNA or structural variation (SV) mutations are a possible explanation. The most common IPN, Charcot‐Marie‐Tooth neuropathy type 1A (CMT1A), is caused by a 1.5 Mb duplication causing trisomy of the dosage sensitive gene PMP22. Using genome sequencing, we recently identified two large genomic rearrangements causing IPN subtypes X‐linked CMT (CMTX3) and distal hereditary motor neuropathy (DHMN1), thereby expanding the spectrum of SV mutations causing IPN. Understanding how newly discovered SVs can cause IPN may serve as a useful paradigm to examine the role of topologically associated domains (TADs), chromatin interactions, and gene dysregulation in disease. This review will describe the growing role of SV in the pathogenesis of IPN and the importance of considering this type of mutation in Mendelian diseases where protein coding mutations cannot be identified.

SOX10 Regulates an Alternative Promoter at the Charcot-Marie-Tooth Disease Locus MTMR2

Schwann cells are the myelinating glia of the peripheral nervous system and dysfunction of these cells causes motor and sensory peripheral neuropathy. The transcription factor SOX10 is critical for Schwann cell development and maintenance, and many SOX10 target genes encode proteins required for Schwann cell function. Loss-of-function mutations in the gene encoding myotubularin-related protein 2 (MTMR2) cause Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a severe demyelinating peripheral neuropathy characterized by myelin outfoldings along peripheral nerves. Previous reports indicate that MTMR2 is ubiquitously expressed making it unclear how loss of this gene causes a Schwann cell-specific phenotype. To address this, we performed computational and functional analyses at MTMR2 to identify transcriptional regulatory elements important for Schwann cell expression. Through these efforts, we identified an alternative, SOX10-responsive promoter at MTMR2 that displays strong regulatory activity in immortalized rat Schwann (S16) cells. This promoter directs transcription of a previously unidentified MTMR2 transcript that is enriched in mouse Schwann cells compared to immortalized mouse motor neurons (MN-1), and is predicted to encode an N-terminally truncated protein isoform. The expression of the endogenous transcript is induced in a heterologous cell line by ectopically expressing SOX10, and is nearly ablated in Schwann cells by impairing SOX10 function. Intriguingly, overexpressing the two MTMR2 protein isoforms in HeLa cells revealed that both localize to nuclear puncta and the shorter isoform displays higher nuclear localization compared to the longer isoform. Combined, our data warrant further investigation of the truncated MTMR2 protein isoform in Schwann cells and in CMT4B1 pathogenesis.

Unique clinical and neurophysiologic profile of a cohort of children with CMTX3

Objective To describe in detail the clinical profile of Charcot-Marie-Tooth disease subtype 3 (CMTX3) to aid appropriate genetic testing and rehabilitative therapy. Methods We reviewed the clinical and neurophysiologic profile and CMT Pediatric Scale (CMTPedS) assessments of 11 children with CMTX3. Results Compared with the more common forms of CMT, CMT1A and CMTX, CMTX3 was characterized by early onset with early and progressive hand weakness. Most affected children were symptomatic within the first 2 years of life. The most common presentation was foot deformity in the first year of life. CMTPedS analysis in these children revealed that CMTX3 progressed more rapidly (4.3 ± 4.1 points over 2 years, n = 7) than CMT1A and CMTX1. Grip strength in affected boys was 2 SDs below age- and sex-matched normative reference values (z score −2.05 ± 1.32) in the second decade of life. The most severely affected individual was wheelchair bound at 14 years of age, and 2 individuals had no movement in the small muscles of the hand in the second decade of life. Nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity ≤23 m/s. Conclusions CMTX3 had an earlier onset, severe hand weakness, and more rapidly progressive disability compared to the more common forms of CMT. Understanding the unique phenotype of CMTX3 is essential for directing genetic testing because the CMTX3 insertion will not be seen on a routine microarray or neuromuscular gene panel. Early diagnosis will enable rehabilitation to be started early in this rapidly progressive neuropathy.