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The Peripheral Neuropathy Solution

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I started my PhD in October 1988 at the University of Antwerp, Belgium in the laboratory of Christine Van Broeckhoven, currently the scientific director of the Molecular Genetics Department affiliated to the Flanders Interuniversity Institute for Biotechnology. I became interested in her molecular genetic research of neurological disorders, after reading a paper in Nature on Alzheimer's disease (21). I selected this paper as a topic for a course in the frame of my master studies in biotechnology (applied in agriculture) at the University of Leuven, Belgium. When I joined the Antwerp team, Peter Raeymaekers was the only other PHD student, performing molecular genetics on a multi-generation Belgian CMT family with autosomal dominant transmission. In fact, Peter initially started his PHD on genetics of Alzheimer disease, but because the families were still being sampled, he initially spent a lot of effort in developing protocols for isolating human DNA, and in cloning probes that recognized RFLPs. When Peter De Jonghe and Jan Gheuens, clinical neurologists at the Neurology Department of the University Hospital Antwerp, presented to him the large pedigree of a CMT family, he decided to switch to research into genetics of CMT. It became apparent that the pedigree of this CMT family was huge, with more than 350 family members in five generations. In total we sampled 51 affected and 60 healthy relatives for linkage studies. Because we were convinced that CMT was a very rare disorder at that time, we used alphabetical letters in the acronyms of the CMT families we ascertained in Belgium. Still, we had not changed our opinion of the disease frequency when we reached the letter Z, and considered starting again with A-A, in retrospect it is fortunate that we decided to switch to numbers. At this moment we have nearly 2000 CMT families under investigation, either sampled in Belgium or obtained through international collaboration, particularly within the European CMT consortium founded in 1991. However, looking back it seems like the alphabetical letters had some magic value: the CMT-A family turned out to belong to the CMT1A subtype (22), family CMT-B belongs to the CMT1B subtype (23), and CMT-M has a pure motor phenotype (24).

Fortunately, Belgium is a small country (you can hardly drive 2 hours by car without ending up in a neighboring country), in which people tend to continue living in the village where they were born. Every week Peter De Jonghe and his wife Gisèle Smeyers, at that time the research nurse on the project, made many trips visiting family members of family CMT-A at their homes to collect blood samples. Gisèle made the first contact with the patients and relatives to explain the aims of the study and to ask whether she could visit again, but now with the neurologist Peter De Jonghe, who was leading the project. What she did not tell was that the neurologist was in fact her husband, because she wanted the family members to feel free to criticize doctors because of lack of attention for the problems of a CMT patient. However, there soon came a moment when she had to disclose the husband-wife relationship. When visiting a CMT patient whose husband was a forester, she was offered a rabbit to take home. The next visit, the man offered her again a rabbit but said "and here is one for the doctor too." Not to look greedy, she disclosed that the doctor was her husband Peter De Jonghe, but still received the two rabbits.

In the Belgian CMT-A family, Peter Raeymaekers used RFLPs to exclude the first CMT locus on chromosome 1-designated CMT1B in 1982 (25), and initiated a genome search using some of his in-house developed RFLPs. However, shortly before Peter Raeymaekers' PhD thesis defense June 1989, Jeffery Vance (Durham, NC) reported linkage with two chromosome 17p markers (D17S58 and D17S71) in CMT1A families (6). We confirmed the linkage with the two 17p DNA markers in the Belgian CMT-A family, and obtained a log of the odd (LOD) score of 10.67 (significant linkage is obtained when the LOD > 3) (22). We proceeded with the genetic analysis of eight additional chromosome 17 markers, and showed that the CMT1A mutation was mapped in the 17p11.2-p12 region between the marker D17S71 and the gene coding for myosin heavy polypeptide 2 (MYH2) (26).

At that time only partial genetic maps were available for linkage studies (27,28). To fine-map the CMT1A locus, we genotyped additional RFLPs and detected informative recombinants in family CMT-A. However, the genotypes obtained for two DNA markers (pVAW409R1 and pVAW409R3), representing the same locus D17S122, were hard to interpret on RFLP analysis. For one marker we obtained a significant LOD score of 16.20, but it recombined with the second marker at D17S122. These results were hard to believe, and our first reaction was that we had misinterpreted the genotypes. The autoradiograms of the Southern blots were messy, with high backgrounds owing to the presence of repetitive sequences that made it

Asel Sacll

Fig. 3. Hybridization signals obtained with probe pVAW409R3a (D17S122). (A) Southern blot of genomic DNA digested with MspI of Charcot-Marie-Tooth neuropathy type 1A (CMT1A) duplication patients (C) and healthy relatives (N) of three different CMT families. Dosage differences between the alleles are seen in each patient, either in the upper allele (2.8 kb) or lower allele (2.7 kb). (B,C) Southern patterns of genomic DNA digested with rare cutter restriction enzymes AscI and Sacll. DNA fragments were separated by pulsed-field gel electrophoresis. The 500-kb junction fragment (arrow) is only present in the CMT1A duplication patients belonging to a small branch of the Belgian family CMT-A.

Asel Sacll

Fig. 3. Hybridization signals obtained with probe pVAW409R3a (D17S122). (A) Southern blot of genomic DNA digested with MspI of Charcot-Marie-Tooth neuropathy type 1A (CMT1A) duplication patients (C) and healthy relatives (N) of three different CMT families. Dosage differences between the alleles are seen in each patient, either in the upper allele (2.8 kb) or lower allele (2.7 kb). (B,C) Southern patterns of genomic DNA digested with rare cutter restriction enzymes AscI and Sacll. DNA fragments were separated by pulsed-field gel electrophoresis. The 500-kb junction fragment (arrow) is only present in the CMT1A duplication patients belonging to a small branch of the Belgian family CMT-A.

difficult to "read" the MspI alleles. To avoid these "dirty blots," we decided to "clean up" the pVAW409R1 and pVAW409R3 clones, by recloning the non-repetitive restriction fragments into derivative probes designated pVAW409R1b and pVAW409R3a. The hybridization results were squeaky clean; however, much to my amazement, in each genotype one allele had the double density of the other. There was also no consistency because in one patient it was the upper band and in another patient the lower band revealing the increased dosage intensity. After checking and rechecking, I realized that the data could only be explained if one of the alleles was duplicated. I reinterpreted the genotypes, and yes, the recombinants had disappeared.

I can still feel the excitement that went through my body that summer in 1990. Although I was convinced that the data were true, I refrained from telling Peter Raeymaekers and my supervisor Christine. Could it still be that I mixed up samples? I redid the entire experiment, but no, the same results appeared (Fig. 3A). Now it was time to share my findings! Suddenly, the project became the hottest one in the group, we worked hard and step-by-step discovered that the duplication had to be >1 Mb in size based on other duplicated markers in the region (pVAW412R3 [D17S125] and pEW401 [D17S61]) and PFGE data. We wrote the paper and submitted it to Nature. While under editorial review, we continued the work and found the real genetic proof that it was the duplication that caused the disease, namely in one family we observed the duplication appearing de novo together with the disease. In this family (CMT-G), the grandparents were unaffected, and among their six children there was only one patient, who transmitted CMT to his son.

Peter Raeymaekers, Peter De Jonghe and I attended the seventh International Congress on Neuromuscular Diseases in Munich, September 1990. During this meeting our excitement about the finding of the CMT1A duplication gradually turned into sheer paranoia. Jeffery Vance was giving the plenary lecture on CMT; did he not know about the duplication, or did he and would not tell? Who was chatting to whom during the poster session and what about? Could it be that the CMT1A duplication would be revealed in the "surprise box," the last presentation of the meeting? To us shareholders of the CMT1A duplication an extraordinary event took place at this meeting. During the poster session, authors had to present their poster in three slides in a session chaired by Peter James Dyck (Rochester, NY) one of the forefathers of the entire field of peripheral neuropathies. At some point, there was a heated discussion regarding controversial data presented by Victor Ionascescu (Iowa City, IA). To change the subject and calm the audience, P. J. Dyck suddenly asked, "in all these fancy molecular genetic studies, has someone of you ever seen something special like a duplication?" Peter Raeymaekers turned pale and almost fainted. However, nobody noticed the remark, and later it became apparent that P. J. Dyck had absolutely no knowledge of the CMT1A duplication at that time.

Nature did not send our paper for review. Christine, my supervisor, had many hours of discussion with the associate editor Kevin Davies handling the paper; she added new data to the paper (e.g., the de novo duplication data, the size of the duplication estimated at around 1 Mb, additional CMT1A duplication families), but nothing helped. The final verdict was "since we were so close to the gene we might consider coming back when we found it." This illustrated the disbelief in the scientific community that an autosomal dominant disease could result from a gene dosage defect, a genetic mechanism that now is commonly accepted! Also, in 1990 we did not have available the technology we have today, and cloning a gene from a larger than 1 Mb region was still a major challenge. Later, Kevin Davies became editor of Nature Genetics, and invited Christine to tell the story of the CMTA duplication at the first International Conference of Nature Genetics on "Human Genetics: Mapping the Future," Washington, April 1993.

We sent the paper to Science and Lancet and neither were prepared to send it for review and comments ranged from "not interesting for the larger public" to "the duplication does not provide insight in the identity of the genetic defect causing CMT1A." By now it was spring 1991, we were desperate and terribly disappointed, while running into another problem.

Christine was organizing and chairing the eighth workshop on "The Genetics of Hereditary Motor and Sensory Neuropathies" sponsored by the European NeuroMuscular Center (ENMC), in May 1991, in The Netherlands. The paper was not yet resubmitted, and thus we were confronted with a major dilemma: should we be quiet while the workshop aimed at defining criteria for sampling CMT families using strict diagnostic criteria of CMT1 for mapping and cloning of the CMT1A defect, although we knew about the duplication? We decided not, what could we lose at this point. Also, if it became later known to the participants that we as organizers had this information at the time of the workshop, all European CMT researchers would feel deceived. We decided to share the exciting, although still unpublished data, having it presented by Peter Raeymaekers.

While Peter was gradually building the story towards the discovery of the CMT1A duplication, one could notice the increasing excitement among the participants who became silent, stopped taking notes and started whispering "they found it." After the applause, Alan Emery, former research director of the ENMC stood up, congratulated the Antwerp researchers with their important and fascinating finding that they were sharing prepublication. He asked all participants to keep all the presented data confidential, to remember they heard about this unpublished data at the ENMC workshop, and refrain from publishing their data on the duplication obtained without crediting the Antwerp group. To us, he suggested to publish our manuscript in Neuromuscular Disorders, a new journal of his good friend Victor Dubowitz (20). This explains why our most cited paper was published in the second issue of a journal that had not yet had an impact factor in 1991. Later on in June 1991, my supervisor Christine Van Broeckhoven attended the MDA-organized workshop on CMT in Tucson, AZ, chaired by Kurt Fischbeck (Bethesda, MD). Here, again there was the dilemma, but now all European researchers that were present at the workshop had brought data from their families confirming that the duplication was the major CMT1A mutation. The evening before the workshop Christine informed Kurt Fischbeck. Also, present in the bar was Garth Nicholson (Sidney, Australia) who bought a bottle of champagne, and made a phone call to his co-workers who, while he was asleep, collected all the data on the Australian families. The next day, Christine presented the Antwerp data and referenced the data of the European groups. Next there was a presentation by Jim Lupski (Houston, TX) who had similar data that was in press in Cell (15). A more extreme difference in journal impact factors, is hard to imagine. Though, we had not published this major finding in a major journal, we did receive substantial recognition thanks to the many European and American colleagues who always cited, and still do, our paper in Neuromuscular Disorders (20). Also, since the MDA workshop in 1991, the Antwerp and Houston labs had a special bond based on mutual respect and friendship, and have been collaborating on the genetics of different inherited peripheral neuropathies ever since.

After the discovery of the CMT1A duplication many labs requested our "clean" RFLP probes for research and DNA-diagnosis of CMT neuropathies. I remember the many tubes we had to prepare to distribute the clones around the world. In the same year we reported our findings to the patients in Belgium. Since then, we organize yearly meetings for the Belgian CMT organization. In some families, such as CMT-G, the disease appeared simultaneously with a de novo duplication originating from an unequal crossover event between two homologous chromosomes (20). These findings indicated that the CMT1A duplication in 17p11.2 was the disease-causing mutation. At that time it was thought that isolated cases of hereditary motor and sensory neuropathies represented autosomal recessive traits. We and others demonstrated that the CMT1A duplication was responsible for most cases of autosomal dominant CMT1, but that de novo mutations occurred in 9 out of10 sporadic patients. This finding became important for genetic counseling of isolated CMT patients (29).

Because the duplicated markers in CMT1A spanned a minimal distance of approx 10 cM on the genetic map of chromosome 17p11.2-p12, we constructed a physical map of the CMT1A region using rare cutter restriction enzymes in combination with PFGE. This was a very laborious undertaking that resulted in determining the size of the CMT1A duplication to about 1.5 Mb. The discrepancy between the genetic and physical map distances suggested that the 17p11.2 region was extremely prone to recombination events, and that the high recombination rate could be a contributing factor to the genetic instability of this chromosomal region. We also determined by PFGE mapping the position of the duplication breakpoints. The discovery of extra restriction fragments or "duplication junction fragments" with the markers in 1.5-Mb region, provided a more accurate DNA-diagnostic tool for the screening of CMT1A patients (Fig. 3B,C). In addition to the unequal crossover resulting into the CMT1A duplication, we also observed in some of our CMT1 families recombination between DNA markers located on the chromosome transmitting the CMT1A duplication, making our research a puzzling event (30).

After the proposed genetic mechanism causing the CMT1A duplication was determined to be owing to unequal crossover during meiosis, we studied the parental origin of the duplication in genetically sporadic CMT1A patients. We demonstrated that in all cases the mutation was the product of an unequal nonsister chromatid exchange during spermatogenesis. The fact that only paternal de novo duplications were observed in the sporadic CMT1A patients, suggested that male-specific factors may be operating during spermatogenesis that either aid in the formation of the duplication and/or stabilize the duplicated chromosome (31). Later, de novo duplications were also described on the maternal chromosome.

The next step was to identify the gene interrupted by the duplication, or to find a dosage-sensitive gene (three copies instead of two copies), or one in which a position effect on one or more genes is involved. One year after the discovery of the CMT1A duplication, Ueli Suter (Zürich, Switzerland) reported two independent mutations in the transmembrane domain of the mouse peripheral myelin protein 22 (PMP22) gene. These missense mutations occurred spontaneously in the trembler (Tr) and trembler-j (Trj) mouse mutants (32,33). These mice were considered a model for CMT neuropathy owing to weakness and atrophy of distal limb muscles and hypomyelination of peripheral nerves. Interestingly, PMP22 was expressed in the myelin of peripheral nerves and shown to be identical in DNA sequence to the growth arrest specific gene Gas3. The Gas3 gene was mapped to mouse chromosome 11 in a region syntenic to human chromosome 17p11.2. Using our pulsed-field mapping data, we demonstrated that the human PMP22 gene was located in the middle of the duplicated CMT1A region and that this gene was not interrupted by the duplication. Eva Nelis, who joined our small CMT research group as a PhD student, demonstrated that the PMP22 gene showed a dosage-effect because density differences were observed in the hybridization signals on Southern blots. This finding indicated that PMP22 was a good candidate gene for CMT1A. I remember my first trip to the United States, where we presented our physical CMT1A mapping data at a Chromosome 17 workshop in Park City. There it was decided between the participating teams to submit the PMP22 gene data as site-by-site manuscripts.

Finally, the work was published in the first issue of Nature Genetics (34-37). The proof that PMP22 was the disease-causing gene for CMT1A was made after the identification of point mutations in some rare patients (38-40).

After my PhD defense in 1993, Phillip Chance (Seattle, WA) demonstrated that the condition known as hereditary neuropathy with liability to pressure palsies (HNPP) was associated with an interstitial deletion of the same 1.5-Mb region that is duplicated in CMT1A patients (41). The mechanism for unequal crossover was explained by the misalignment at flanking repeat sequences (CMT1A-REPs) leading to a tandem duplication in CMT1A and the reciprocal deletion in HNPP (42,43), and subsequently confirmed by many labs. As a result of another paper by Lupski's team (44), Jim invited me in 1995 as a visiting scientist at the Baylor College of Medicine in Houston, to screen markers located within the CMT1A-REP. Our joint effort allowed analyzing a large group of unrelated CMT1A duplication and HNPP deletion patients from different European countries for the presence of a recombination hotspot in the CMT1A-REP sequences. We confirmed the hotspot for unequal crossover between the misaligned flanking CMT1A-REP elements, and detected novel junction fragments in more than 70% of the unrelated patients. This recombination hotspot was also present in de novo CMT1A duplication and HNPP deletion patients. Our data also indicated that the hotspot of unequal crossover occurred in several populations independent of ethnic background. We concluded that the detection ofjunction fragments from the CMT1A-REP element on Southern blots could be used as a novel and reliable DNA-diagnostic tool in most patients (45). Nowadays, the Southern blot method (Fig. 3A) has been replaced by PCR methods making use of highly informative short tandem repeat markers in the CMT1A region or specific primers located within the CMT1A-REP region.

At the second CMT workshop, sponsored by the ENMC in The Netherlands, researchers from several European countries agreed to contribute to a large study with the aim to estimate the frequency of the CMT1A duplication and HNPP deletion, and to make the first inventory of mutations in the myelin genes causing CMT. I remember the many phone calls (e-mail was not yet available in all 28 centers involved in the study) Eva Nelis made to find out that the CMT1A duplication was present in more than 70% of 800 unrelated CMT1 patients, and the deletion in 84% of more than 150 unrelated HNPP patients. In CMT1 patients negative for the duplication, mutations were identified in PMP22, myelin protein zero (MPZ), and connexin 32 (GJB1/Cx32) (46). These data resulted in the Inherited Peripheral Neuropathy Mutation Database developed and maintained by Eva Nelis (http://www.molgen.ua.ac.be/CMTMutations/).

Without the excellent contacts between our lab and the many CMT patients and their families involved in this research, we could have never detected the CMT1A duplication and the many disease-causing genes currently involved in distinct types of inherited peripheral neuropathies. Professor Alan Emery said at the first European CMT workshop: "This is another step towards discovery of the causes of all these disorders, which will open doors to possible treatments in the future." The CMT1A duplication mechanism is now referred to in many textbooks on Human Molecular Genetics.

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