Analysis of the CMT1A-REP repeat: mapping crossover breakpoints in CMT1A and HNPP

The CMT1A-REP repeat sequence flanks a 1.5 megabase pair (Mb)segment of chromosome 17p11.2–12 which is duplicated inCharcot—Marie—Tooth neuropathy type 1A (CMT1A) anddeleted in hereditary neuropathy with liability to pressurepalsies (HNPP). The CMT1A-REP repeat is proposed to mediatemisalignment and unequal crossover resulting in reciprocal chromosomalrearrangements in CMT1A and HNPP. We have constructed a physicalmap of the proximal and distal CMT1A-REP repeats. Cloned fragmentsfrom CMT1A-REP repeat regions were used to determine the sizeof the repeats and to assess regions of homology. The crossoverbreakpoints were mapped in a series of 30 unrelated CMT1A patientsand 22 unrelated HNPP patients. The CMT1A-REP repeat spans approximately27 kilobase pairs and appears to be continuous. Locations ofrestriction enzyme sites are highly conserved for the proximaland distal CMT1A-REP repeats. All crossovers mapped within theCMT1A-REP repeat sequence and heterogeneity for breakpoint locationwas demonstrated. Seventy-seven percent (40 of 52) of CMT1Aand HNPP chromosomes contained breakpoints which mapped withina 7.9 kb interval, suggesting the presence of a possible ‘hotspot’for recombination in CMT1A-REP. DNA sequence analysis for 4kb of the interval containing the majority of crossovers revealedover 98% sequence identity between proximal and distal CMT1A-REPrepeat sequences. Probes useful for molecular-based diagnosisof CMT1A and HNPP are described.     

Fine mapping of de novo CMT1A and HNPP rearrangements within CMT1A-REPs evidences two distinct sex-dependent mechanisms and candidate sequences involved in recombination

The molecular mechanism resulting in the duplication or deletion of a 1.5 Mb region of 17p11.2-p12, associated, respectively, with Charcot- Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), has been proposed to be an unequal crossing- over during meiosis between the two chromosome 17 homologues generated by misalignment of the proximal and distal CMT1A-REP repeats, two homologous sequences flanking the 1.5 Mb CMT1A/HNPP monomer unit. In a recent study of a large series of de novo cases of CMT1A and HNPP, two distinct sex-dependent mechanisms were identified. Rearrangements of paternal origin, essentially duplications, were indeed generated by unequal meiotic crossing-over between the two chromosome 17 homologues, but duplications and deletions of maternal origin resulted from an intrachromosomal process, either unequal sister chromatid exchange or, in the case of deletion, excision of an intrachromatidal loop. In order to determine how these recombinations occur, 24 de novo crossover breakpoints were localized within the 1.7 kb rearrangement hot spot by comparing the sequences of the parental CMT1A-REPs with the chimeric copy in affected offspring. Nineteen out of 21 paternal crossovers were found in a 741 bp hot spot. All the breakpoints of maternal origin (n = 3), however, were located outside this interval, but in closely flanking sequences, supporting the hypothesis that two distinct sex- dependent mechanisms are involved. Several putative recombination promoting sequences in the hot spot, which are rare or absent in the surrounding 7.8 kb, were identified.      

Prenatal detection of a 17p11.2 duplication resulting from a rare recombination event and novel PCR-based strategy for molecular identification of Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth disease, type 1A (CMT1A) is caused in most cases by a 1.5 Mb duplication on chromosome 17p11.2 arising after unequal crossing-over between repeated sequences called CMT1A-REPs, flanking the 1.5 Mb unit. A 3.2 kb recombination hot spot has been defined, resulting in a junction fragment between EcoRI (distal CMT1A-REP) and SacI (proximal CMT1A-REP). This was further reduced to a 1.7kb EcoRI-NsiI fragment, and recently to a 731 bp hot spot region within this fragment. We describe the CMT1A-REPs-based PCR method used to identify CMT1A duplications and report on a family case in which a 29-year-old pregnant woman requested prenatal diagnosis for two successive pregnancies because her husband was affected with CMT1A. Our method enabled us to characterise the duplication in both foetuses and demonstrate that it arose from a rare recombination event taking place outside the 1.7 kb region. Since our approach is simple and enables the entire set of duplications occurring after recombination in the enlarged 3.2kb region including the hot spot to be detected, we suggest it might be considered for use in primary screening for pre- and postnatal diagnosis of CMT1A.     

The human COX10 gene is disrupted during homologous recombination between the 24 kb proximal and distal CMT1A-REPs

The CMT1A-REPs are two large directly repeating DNA sequences located on chromosome 17p11.2-p12 flanking the region duplicated in patients with Charcot-Marie-Tooth disease type 1A (CMT1A) and deleted in patients with hereditary neuropathy with liability to pressure palsies (HNPP). We have sequenced two cosmids, c74F4 and c15H12, which contain the entire proximal and distal CMT1A-REPs and determined that these repeats are approximately 99% identical across a 24,011 bp region. In addition, both contain an exon of the human heme A:farnesyltransferase gene (COX10). Hybridization studies revealed that COX10 spans the distal CMT1A-REP, while the proximal CMT1A-REP contains an isolated COX10 'pseudo-exon'. There is also a COX10 hybridization signal on chromosome 10 which appears to represent a processed pseudogene. We propose that the distal CMT1A-REP represents the progenitor copy of COX10 exon VI which was duplicated with surrounding intronic sequences during mammalian genome evolution and that the HNPP deletion results in a COX10 null allele.      

Asian hereditary neuropathy patients with peripheral myelin protein-22 gene aneuploidy

Japanese hereditary neuropathy with liability to pressure palsy (HNPP) patients have a deletion of one peripheral myelin protein-22 (PMP22) gene region in distal chromosome band 17p11.2 as do Caucasian patients. Japanese and Asiatic Indian CMT1A patients have a PMP22 gene duplication that results in Charcot-Marie-Tooth disease type IA (CMT1A; HMSNIA) in patients of European and Middle Eastern ancestry. About 70% of Japanese CMT1 patients have a PMP22 duplication as do Caucasians, while Japanese CMT1B, CMT2 and Dejerine-Sottas patients do not have PMP22 gene region aneuploidy. Although HNPP and CMT1A genotypes are generated simultaneously by unequal recombination that results in PMP22 gene aneuploidy in each daughter cell, only 3 Japanese HNPP probands with PMP22 deletion from a large patient population were referred to a single center compared to 18 referred CMT1A probands with PMP22 duplication. This lower HNPP frequency more likely reflects lower HNPP reproductive fitness than patient ascertainment bias because disease severity and variation in severity is about the same in CMT1A and HNPP patients and because all patients of both types were referred regardless of disease severity. These results, along with an apparently high de novo CMT1A mutation rate, suggest that common ancestors of Japanese, Asian Indians, and Caucasians carried PMP22 geneflanking sequences that enhance unequal crossing over. © 1995 Wiley-Liss, Inc.     

Detection of the CMT1A/HNPP recombination hotspot in unrelated patients of European descent.

Charcot-Marie-Tooth type 1 disease (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP) are common inherited disorders of the peripheral nervous system. The majority of CMT1 patients have a 1.5Mb tandem duplication (CMT1A) in chromosome 17p11.2 while most HNPP patients have a deletion of the same 1.5 Mb region. The CMT1A duplication and HNPP deletion are the reciprocal products of an unequal crossing over event between misaligned flanking CMT1A-REP elements. We analysed 162 unrelated CMT1A duplication patients and HNPP deletion patients from 11 different countries for the presence of a recombination hotspot in the CMT1A-REP sequences. A hotspot for unequal crossing over between the misaligned flanking CMT1A-REP elements was observed through the detection of novel junction fragments in 76.9% of 130 unrelated CMT1A patients and in 71.9% of 32 unrelated HNPP patients. This recombination hotspot was also detected in eight out of 10 de novo CMT1A duplication and in two de novo HNPP deletion patients. These data indicate that the hotspot of unequal crossing over occurs in several populations independently of ethnic background and is directly involved in the pathogenesis of CMT1A and HNPP. We conclude that the detection of junction fragments from the CMT1A-REP element on Southern blot analysis is a simple and reliable DNA diagnostic tool for the identification of the CMT1A duplication and HNPP deletion in most patients.     

Facilitated diagnosis of CMT1A duplication in chromosome 17p11.2-12: Analysis with a CMT1A-REP repeat probe and photostimulated luminescence imaging

Charcot-Marie-Tooth disease type 1A (CMT1A) is a common autosomal dominant demyelinating peripheral neuropathy. Most patients with CMT1A have been found to have a 1.5 megabase tandem DNA duplication in chromosome 17p11.2-12. Meiotic unequal crossover mediated by the CMT1A-REP repeat is a proposed mechanism for generation of the duplication in CMT1A and a reciprocal deletion seen in hereditary neuropathy with liability to pressure palsies. Testing for the CMT1A duplication is frequently the first step in the molecular diagnosis of patients with suspected inherited demyelinating neuropathy. We used a 1.0 kb EcoRI-PstI DNA fragment (pHK1.OP) from the proximal CMT1A-REP repeat as a probe for Southern blot analysis and detected increased gene dosage in CMT1A by determining measuring radioactivity ratios with a photostimulated luminescence imaging plate. We found that this method is useful for rapid diagnosis of the DNA duplication associated with CMT1A. Hum. Mutat. 9:563–566, 1997. © 1997 Wiley-Liss, Inc.     

Unequal exchange at the Charcot-Marie-Tooth disease type 1A recombination hot-spot is not elevated above the genome average rate

An increasing number of human diseases and syndromes are being found to result from micro-duplications or microdeletions arising from meiotic recombination between homologous repeats on the same chromosome. The first microduplication syndrome delineated, Charcot-Marie-Tooth disease type 1A (CMT1A), results from unequal crossing over between two >98% identical 24 kb repeats (CMT1A-REPs) on chromosome 17. In addition to its medical significance, the CMT1A region has features that make it a unique resource for detailed analysis of human unequal recombination. Previous studies of CMT1A patients showed that the majority of unequal crossovers occurred within a small region (<1 kb) of the REPs suggesting the presence of a recombination hot-spot. We directly measured the frequency of unequal recombination in the hot-spot region using sperm from four normal individuals. Surprisingly, unequal recombination between the REPs occurs at a rate no greater than the average rate for the male genome (approximately 1 cM/Mb) and is the same as that expected for equally aligned REPs. This conclusion extends to humans the findings in yeast that recombination between repeated sequences far apart on the same chromosome may occur at similar frequencies to allelic recombination. Finally, the CMT1A hot-spot stands in sharp contrast to the human MS32 mini-satellite-associated hot-spot that exhibits highly enhanced recombination initiation in addition to positional specificity. One possibility is that the CMT1A hot-spot may consist of a region with genome average recombination potential embedded within a recombination cold-spot.     

Copy number variation upstream of PMP22 in Charcot�CMarie�CTooth disease

In several individuals with a Charcot–Marie–Tooth (CMT) phenotype, we found a copy number variation (CNV) on chromosome 17p12 in the direct vicinity of the peripheral myelin protein 22 (PMP22) gene. The exact borders and size of this CNV were determined by Southern blot analysis, MLPA, vectorette PCR, and microarray hybridization analyses. All patients from six apparently unrelated families carried an identical 186-kb duplication different from the commonly reported 1.5-Mb duplication associated with CMT1A. This ancestral mutation that was not reported in the human structural variation database was only detected in affected individuals and family members. It was absent in 2124 control chromosomes and 40 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP) and therefore should be regarded as causative for the disease. This variant escapes most routine diagnostic screens for CMT1A, because copy numbers of PMP22 probes were all normal. No indications were found for the involvement of the genes that are located within this duplication. A possible association of this duplication with a mutation in the PMP22 coding regions was also excluded. We suggest that this CNV proximal of the PMP22 gene leads to CMT through an unknown mechanism affecting PMP22 expression.     

The 1.4-Mb CMT1A duplication/HNPP deletion genomic region reveals unique genome architectural features and provides insights into the recent evolution of new genes

Duplication and deletion of the 1.4-Mb region in 17p12 that is delimited by two 24-kb low copy number repeats (CMT1A-REPs) represent frequent genomic rearrangements resulting in two common inherited peripheral neuropathies, Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsy (HNPP). CMT1A and HNPP exemplify a paradigm for genomic disorders wherein unique genome architectural features result in susceptibility to DNA rearrangements that cause disease. A gene within the 1.4-Mb region, PMP22, is responsible for these disorders through a gene-dosage effect in the heterozygous duplication or deletion. However, the genomic structure of the 1.4-Mb region, including other genes contained within the rearranged genomic segment, remains essentially uncharacterized. To delineate genomic structural features, investigate higher-order genomic architecture, and identify genes in this region, we constructed PAC and BAC contigs and determined the complete nucleotide sequence. This CMT1A/HNPP genomic segment contains 1,421,129 bp of DNA. A low copy number repeat (LCR) was identified, with one copy inside and two copies outside of the 1.4-Mb region. Comparison between physical and genetic maps revealed a striking difference in recombination rates between the sexes with a lower recombination frequency in males (0.67 cM/Mb) versus females (5.5 cM/Mb). Hypothetically, this low recombination frequency in males may enable a chromosomal misalignment at proximal and distal CMT1A-REPs and promote unequal crossing over, which occurs 10 times more frequently in male meiosis. In addition to three previously described genes, five new genes (TEKT3, HS3ST3B1, NPD008/CGI-148, CDRT1, and CDRT15) and 13 predicted genes were identified. Most of these predicted genes are expressed only in embryonic stages. Analyses of the genomic region adjacent to proximal CMT1A-REP indicated an evolutionary mechanism for the formation of proximal CMT1A-REP and the creation of novel genes by DNA rearrangement during primate speciation.     

Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17

Charcot — Marie — Tooth disease type 1A (CMT1A)is a common autosomal dominant demyellnating neuropathy thatis associated with a 1.5 megabase (Mb) tandem DNA duplicationin chromosome 17p11.2-p12. Hereditary neuropathy with liabilityto pressure palsies (HNPP, tomaculous neuropathy) is anotherless frequently diagnosed autosomal dominant neuropathy andis associated with a 1.5 Mb deletion in chromosome 17p11.2-12.Meiotic unequal crossover is a proposed mechanism for the generationof both the duplication In CMT1A and the deletion in HNPP. CMT1A-REPis a repeat that flanks the region which is duplicated/deletedIn CMT1A/HNPP. The CMT1A-REP repeat sequence may mediate unequalcrossover through misalignment of the homologous, repeated sequences.Three copies of the CMT1A-REP repeat are present on stably InheritedCMT1A duplication chromosomes. In this report, molecular analysisin multiple patients detected three copies of the CMT1A-REPsequence on both inherited and de novo CMT1A duplication chromosomes,and one copy of the CMT1A-REP repeat on the deleted chromosomein both inherited and de novo HNPP. These observations supportthe hypothesis that a reciprocal recombination mechanism involvingthe CMT1A-REP is responsible for the generation of both theduplicated and deleted chromosomes, and document the first examplesin humans of Mendellan syndromes resulting from the reciprocalproducts of unequal exchange Involving large Intra-chromosomalsegments.     

Origin of the de novo duplication in Charcot -- Marie -- Tooth disease type 1A: unequal nonsister chromatid exchange during spermatogenesis

A 1.5 Mb duplication within 17p11.2 is the major mutation causingboth autosomal dominant and sporadic Charcot - Marie - Toothdisease type 1A (CMT1A). An Independent origin for the mutationIn each family has been postulated. The proposed genetic mechanismcausing the CMT1A duplication is unequal nonsister chromatldexchange at melosis (unequal crossing-over). We studied theparental origin of the duplication In nine genetically sporadicCMT1A patients and demonstrated that in all cases the mutationwas the product of an unequal nonsister chromatld exchange duringspermatogenesis. The fact that only paternal de novo duplicationswere observed In the sporadic CMT1A patients suggests that malespecific factors may be operating during spermatogenesis thateither help forming the duplication and/or stabilize the duplicatedchromosome.     

Clustering of CMT1A duplication breakpoints in a 700 bp interval of the CMT1A-REP repeat

The CMT1A-REP repeat is proposed to mediate unequal crossover leading to a 1.5 Mb duplication in chromosome 17p11.2-12 associated with Charcot-Marie-Tooth neuropathy type 1A (CMT1A). There is an apparent recombinational “hotspot” in the CMT1A-REP repeat since the majority of crossover breakpoints for CMT1A are located within a 1.7 kb interval. Further to characterize the crossover breakpoint region, we constructed PCR primers that specifically amplify the duplication breakpoint junctions in a series of Japanese and Caucasian CMT1A patients. We mapped the breakpoints in 89% of patients within a 700 bp interval of the CMT1A-REP repeat. This 700 bp region is 1.3 kb telomeric to a previously described mariner-like transposable element. Our observations further define the location of crossovers for CMT1A and provide additional evidence that this region is a recombinational “hotspot” within the CMT1A-REP repeat. Hum Mutat 11:109–113, 1998. © 1998 Wiley-Liss, Inc.     

Proximal microdeletions and microduplications of 1q21.1 contribute to variable abnormal phenotypes

Chromosomal band 1q21.1 can be divided into two distinct regions, proximal and distal, based on segmental duplications that mediate recurrent rearrangements. Microdeletions and microduplications of the distal region within 1q21.1, which are susceptibility factors for a variety of neurodevelopmental phenotypes, have been more extensively studied than proximal microdeletions and microduplications. Proximal microdeletions are known as a susceptibility factor for thrombocytopenia-absent radius (TAR) syndrome, but it is unclear if these proximal microdeletions have other phenotypic consequences. Therefore, to elucidate the clinical significance of rearrangements of the proximal 1q21.1 region, we evaluated the phenotypes in patients identified with 1q21.1 rearrangements after referral for clinical microarray testing. We report clinical information for 55 probands with copy number variations (CNVs) involving proximal 1q21.1: 22 microdeletions and 20 reciprocal microduplications limited to proximal 1q21.1 and 13 microdeletions that include both the proximal and distal regions. Six individuals with proximal microdeletions have TAR syndrome. Three individuals with proximal microdeletions and two individuals with larger microdeletions of proximal and distal 1q21.1 have a 'partial' TAR phenotype. Furthermore, one subject with TAR syndrome has a smaller, atypical deletion, narrowing the critical deletion region for the syndrome. Otherwise, phenotypic features varied among individuals with these microdeletions and microduplications. The recurrent, proximal 1q21.1 microduplications are enriched in our population undergoing genetic testing compared with control populations. Therefore, CNVs in proximal 1q21.1 can be a contributing factor for the development of abnormal phenotypes in some carriers.     

A de novo case of hereditary neuropathy with liability to pressure palsies (HNPP) of maternal origin: a new mechanism for deletion in 17p11.2?

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant neuropathy, most often associated with a deletion of the 17p11.2 region, which is duplicated in 70% of patients with Charcot- Marie-Tooth type 1 (CMT1A). Most de novo CMT1A and HNPP cases have been of paternal origin. A rare case of de novo HNPP of maternal origin was analysed to determine the underlying mechanism. Affected individuals in the family carried a deletion corresponding to the CMT1A/HNPP monomer unit associated with a rearrangement of the CMT1A-REP sequences. Segregation analysis of 17p11-p12 markers in the family indicated that the deletion was not generated by unequal crossing over between homologous 17 chromosomes, as in de novo cases from paternal origin, but rather by an intrachromosomal rearrangement. Two distinct mechanisms can therefore lead to the same 17p11.2 deletion. This result suggests that intrachromosomal rearrangement may be specific to maternal transmissions.      

Sex vesicle "entrapment": translocation or nonhomologous recombination of misaligned Yp and Xp as alternative mechanisms for abnormal inheritance of the sex-determining region

Abnormal inheritance of the sex determining region, normally located on Yp, results in about 1 in 20,000 phenotypic males with a 46,XX genotype. Studies to date indicate that many 46,XX males apparently arise due to a balanced, yet abnormal, nonhomologous interchange of Xp and Yp termini. However, 2 of the 5 XX males we report here have 3 copies of the pseudoautosomal locus, MIC2. Thus, they appear to have inherited the sex determining region as a result of Yp sequences being added onto the X pseudoautosomal region. Such an unequal, extremely nonhomologous interchange could alternatively be considered to arise from an unbalanced translocation of Yp to Xp. Our results suggest that very unequal interchange or translocation of Yp sequences onto the X pseudoautosomal region is not as rare a mechanism for XX males as originally thought. We also suggest that sex vesicle "entrapment" favors the association of a Yp fragment to the X pseudoautosomal region over a translocation to either Xq or an autosome.     

Sex vesicle “entrapment”: Translocation or nonhomologous recombination of misaligned Yp and Xp as alternative mechanisms for abnormal inheritance of the sex-determining region

Abnormal inheritance of the sex determining region, normally located on Yp, results in about 1 in 20,000 phenotypic males with a 46,XX genotype. Studies to date indicate that many 46,XX males apparently arise due to a balanced, yet abnormal, nonhomologous interchange of Xp and Yp termini. However, 2 of the 5 XX males we report here have 3 copies of the pseudoautosomal locus, MIC2. Thus, they appear to have inherited the sex determining region as a result of Yp sequences being added onto the X pseudoautosomal region. Such an unequal, extremely nonhomologous interchange could alternatively be considered to arise from an unbalanced translocation of Yp to Xp. Our results suggest that very unequal interchange or translocation of Yp sequences onto the X pseudoautosomal region is not as rare a mechanism for XX males as originally thought. We also suggest that sex vesicle “entrapment” favors the association of a Yp fragment to the X pseudoautosomal region over a translocation to either Xq or an autosome.     

Relationship between Charcot - Marie-Tooth 1A and Smith - Magenis regions. snU3 may be a candidate gene for the Smith - Magenis syndrome

The juxtacentromeric region of the human chromosome 17 shortarm (17p11.2-p12) contains genes Involved in the Charcot - Marie- Tooth type 1A disease (CMT1A) and the Smith-Magenis syndrome(SMS). CMT1A Is associated with a duplication of a short segmentwhereas SMS is linked to microdeletions, extending toward thecentromere. We describe the construction and analysis of a 5Mb YAC contig spanning the CMT1A duplicated segment and thedistal part of four SMS microdeletions. We concluded that theYAC contig contains about 1Mb of genomic DNA which is deletedin the four SMS patients analysed. Moreover two YACs containboth STS deleted in SMS (U3) and STS duplicated in CMT1A (5H5),but the proximal breakpoint associated with the CMT1A duplicationis not the same as the distal SMS breakpoint we studied. Finallywe located five new STS In SMS deletion. Two of them, a microsatellite(D17S805(23)) and the gene coding for small nuclear RNA U3,have been localized In the contig we described. We may alsonote that snU3 Is the first expressed sequence localized Inan SMS deletion so far. The possible participation of this genein the SMS phenotype is discussed.     

Homologous DNA exchanges in humans can be explained by the yeast double-strand break repair model: a study of 17p11.2 rearrangements associated with CMT1A and HNPP.

Rearrangements in 17p11.2, responsible for the 1.5 Mb duplications and deletions associated, respectively, with autosomal dominant Charcot-Marie-Tooth type 1A disease (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are a suitable model for studying human recombination. Rearrangements in 17p11.2 are caused by unequal crossing-over between two homologous 24 kb sequences, the CMT1A-REPs, that flank the disease locus and occur in most cases within a 1.7 kb hotspot. We sequenced this hotspot in 28 de novo patients (25 CMT1A and three HNPP), in order to localize precisely, at the DNA sequence level, the crossing-overs. We show that some chimeric CMT1A-REPs in de novo patients (10/28) present conversion of DNA segments associated with the crossing-over. These rearrangements can be explained by the double-strand break (DSB) repair model described in yeast. Fine mapping of the de novo rearrangements provided evidence that the successive steps of this model, heteroduplex DNA formation, mismatch correction and gene conversion, occurred in patients. Furthermore, the model explains 17p11.2 recombinations between chromosome homologues as well as between sister chromatids. In addition, defective mismatch repair of the heteroduplex DNA, observed in two patients, resulted in two heterozygous chimeric CMT1A-REPs which can be explained, as in yeast, by post-meiotic segregation. This work supports the hypothesis that the DSB repair model of DNA exchange may apply universally from yeasts to humans.