Mechanism, prevalence, and more severe neuropathy phenotype of the Charcot-Marie-Tooth, Type 1A triplication. V. Gelowani1, P. Liu1, F. Zhang2, V. Drory3,6, S. B. Shachar4, E. Roney1, W. B. Burnette5, J. Li5, A. Orr-Urtreger4,6, J. R. Lupski1,7,8 1) Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai 200433, China; 3) Department of Neurology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel;; 4) Genetic Institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 5) Department of Neurology, Center for Human Genetics Research, Vanderbilt University; 6) Sacker Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel;; 7) Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; 8) Texas Childrens Hospital, Houston, TX, USA.

   Early studies in the 1930s on the Bar locus in drosophila raised questions about molecular origin, mechanism, and frequency of triplications. To address these questions experimentally in humans we chose as our genetic model Charcot-Marie-Tooth, type 1 (CMT1A, which is one of the most common dominant disease traits and due to duplication. We hypothesized that a) triplications convey a more severe neuropathy phenotype compared to duplications, b) triplications arise from duplications through nonallelic homologous recombination (NAHR), and c) that the frequency of triplication arising from a pre-existing duplication is higher compared to that of duplication arising from a wild type allele.
   We identified families with one member having the disease at the very extreme end of severity. Array CGH and interphase FISH identified individuals with either neutral copy number (unaffected), duplication (trait manifestation), or triplication (severe disease) within the CMT1A families. These data support the gene dosage hypothesis as the patients carrying triplications were more severe by both clinical examination and objective electrophysiological measurements compared to patients with duplications. To investigate the mechanism of crossover, we designed microsatellite genotyping assays to phase haplotypes. In both families, the de novo triplication arose from a pre-existing duplication during maternal meiosis. The triplication arose through one interchromosomal NAHR event in one family, and involved two steps in the other family. Theoretically, there are more ways for a chromosome to have copy number gain through NAHR when there is a duplication at the locus than when there is not. Data extracted from de-identified CMT samples analyzed by MLPA (courtesy A. Medeiros, R. Moore, Dr. J. Higgins, Quest/Athena Diagnostics) showed that the frequency of triplication among CNV gains is ~1/500. Thus, the frequency of triplication arising from a pre-existing duplication is higher, by about 200-fold, compared to the frequency of de novo duplication (1.73x10-5) arising from a wildtype allele. We conclude that CMT1A triplication causes a severe clinical phenotype and its prevalence may have been substantially underestimated. Patients with severe neuropathy and with a CMT1A duplication family history should be evaluated for triplication using adequate molecular assays that can distinguish triplication from duplication.

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