Fig. 1. Nucleotide sequence of the simple sequence repeat RM11-GT. Autoradiogram of a DNA sequencing gel showing the repeat, which lies at the basis of the polymorphic DNA marker RM11-GT. Initial evidence for the Charcot-Marie-Tooth type 1A (CMT1A) duplication was revealed by this marker that showed three alleles (i.e., triallelic) in fully informative CMT patients.
made an interesting observation for one of the SSR markers, termed RM11-GT with SSR (TA)5(GT)17(AT)8 (see Fig. 1) (14). When primers were used to type the CMT1A families for this marker, one often found three alleles rather than the usual two expected with one inherited from each parent. Three alleles were observed in many individuals with CMT1A, but not in all—the marker was not always fully informative. Three alleles were not observed in unaffected family members with the exception of three individuals who were asymptomatic; however, these latter three seemingly unaffected individuals had not had nerve conduction studies. Subsequent NCV studies revealed decreased motor NCVs consistent with CMT1 and confirmed our suspicion that these individuals had subclinical, not yet penetrant disease.
Roberto examined some of the Southern blots that utilized an RFLP marker from the same locus and noted that often when there were three alleles revealed by RM11-GT (14), a dosage difference could be observed between the two alleles if the affected individual was heterozygous for that RFLP. These initial observations suggested that there may be three copies of the genomic region that was being assayed, potentially reflecting genomic duplication at the CMT1A locus. The entire laboratory now focused on the "duplication hypothesis" and, to keep our hypothesis quiet, it was referred to as the "D" word within the laboratory because we all focused on gathering data to support or refute the duplication hypothesis using multiple independent molecular approaches. When now correcting for diagnosis (i.e., making sure that all apparent unaffected individuals did not have subclinical disease by performing NCVs and systematically examining CMT1A families), the putative duplication appeared to cosegregate with the CMT1A phenotype as determined by objective NCV measurements (15).
To reconcile dosage differences of heterozygous RFLP alleles with the three RM11-GT alleles observed in CMT1A duplication patients, Pragna performed an important experiment. For one of the RFLP markers revealing dosage differences, and from which the PCR-typeable RM11-GT marker was derived, the MspI alleles were separated on preparative agarose gels and used as templates for PCR amplifications of RM11-GT. As anticipated, from the RFLP allele showing increased dosage she could amplify 2 RM11-GT alleles, whereas only one was found from the PCR of the other RFLP allele that displayed normal dosage (15). Similar types of experiments, to examine the molecular basis for the dosage differences of alleles, were performed by first physically separating the two chromosome homologs in rodent somatic cell hybrids (15).
The allele dosage differences revealed by RFLP analyses could also be observed, although it was much more difficult to see and less informative, for two other CMT1A-linked markers that by genetic mapping studies were adjacent to the initial marker revealing duplication. This suggested the putative duplication might be large and we, thus, attempted to obtain further, physical evidence for its existence. Pentao Liu applied pulsed-field gel electrophoresis (PFGE) as a means to try to resolve a potentially large genomic change. Indeed, he was able to identify a 500-kb apparent junction fragment in CMT1A patients that was not observed in controls. Furthermore, he showed that this junction fragment cosegregated with CMT1A (15). Interestingly, this junction fragment was increased in dosage in a patient, whom we presumed was homozygous for the CMT mutation given the severe clinical picture, where both parents had CMT1A. In collaboration with Barbara Trask (Seattle, WA), we attempted to resolve the duplication by fluorescence in situ hybridization of metaphase spreads from lymphoblastoid cell lines constructed from CMT1A patients and controls. The metaphase analysis failed, but on interphase spreads she could identify duplication on one of the two chromosome 17 homologs (15). Moreover, she identified an apparent duplication on both homologous chromosomes in the patient presumed to be homozygous on clinical grounds. Because these were interphase cells, it was important to distinguish duplication from replication and this was done by comparison to a nearby control probe. To our knowledge, this was the first time that a common autosomal dominant human disease trait was diagnosed using a microscopic technique. Although it was 55 years later, I think we were probably as excited as Calvin Bridges was when he initially applied the then new technique of polytene chromosomes to the study of fruit fly traits and found that the Bar gene was a duplication (16).
We had accumulated very strong physical evidence thus far; 3 GT alleles, dosage differences of heterozygous RFLP alleles, a PFGE junction fragment, and interphase fluorescence in situ hybridization revealing a duplicated signal, for the CMT1A duplication. However, what remained was reconciliation with the genetic data. The marker VAW409, from which RM11-GT was derived and which physically revealed the duplication by virtue of dosage differences of heterozygous RFLP alleles in CMT1A patients, also appeared to reveal recombinants in the genetic analysis. We thought it would be hard to publish our CMT1A duplication findings without reconciling the physical and genetic data. Through conversations I was having with Markus Grompe (Portland, OR), a then clinical genetics fellow with me at Baylor, it became clear that the duplication could have consequences for the interpretation of marker genotypes and thus linkage analyses. The failure to account for the duplication in linkage analyses produces false recombinants (Fig. 2A). Linkage programs score f &
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