Diagnosis of gene dosage alterations at the PMP22 gene using MAPH.

G enomic rearrangements due to submicroscopic duplications or deletions are responsible for many inherited disorders, and a gene (or genes) sensitive to dosage alterations may be involved in these structural rearrangements. The segment involved is generally smaller than 2 Mb and cannot be detected by conventional karyotyping. Charcot-Marie-Tooth disease type 1A (CMT1A) [OMIM #118220] and hereditary neuropathy with liability to pressure palsies (HNPP) [OMIM #162500] are well characterised as genomic disorders. By a gene dosage mechanism, CMT1A or HNPP respectively result from duplication or deletion of a 1.4 Mb DNA fragment in 17p12 containing the PMP22 gene. Amplification of the PMP22 gene from two copies in normal people to three and even (in severe cases) four copies has been detected in CMT1A cases, and deletion of the gene to one copy results in HNPP. CMT1A/HNPP are caused by a reciprocal unequal crossover event arising from the misalignment of homologous repeat sequences, CMT1AREPs, which flank the 1.4 Mb region. These 24 kb duplicons are 98.7% identical and are thought to mediate this misalignment. 3 In a minority of cases, point mutations in the same gene can also result in CMT1A or HNPP. These two distinct conditions are autosomal dominant traits in a clinically and genetically heterogeneous group of peripheral neuropathies termed hereditary motor and sensory neuropathies (HMSN). The overall estimated prevalence of HMSN is about 1 in 2500, and CMT1A is the most common inherited peripheral neuropathy, with a prevalence of 10–40/ 100 000. A European collaborative study reported the duplication in 70.7% of unrelated CMT1A patients and the deletion of the same region in 84% of HNPP cases. There is a wide range of approaches for molecular diagnosis of CMT1A and HNPP. These approaches can be classified as (a) methods detecting rearrangements and (b) quantitative methods. In the first group, pulsed field gel electrophoresis (PFGE, the standard approach in the USA) and fluorescence in situ hybridisation (FISH) can detect these duplications and deletions, and PCR methods detect specific junction fragments between CMT1A-REPs. Restriction fragment length polymorphism Southern blot analysis and genotyping of short tandem repeat (STR) markers from the CMT1A/HNPP region are methods belonging to the second group. PFGE depends on high quality DNA from patients’ blood samples. This labour intensive approach can cause an estimated failure rate (noninterpretable results) of about 10%. The fact that only about 70% of the CMT1A duplication carriers demonstrate the junction fragment should be considered when using diagnostic methods that rely on detection of a junction fragment. In interphase FISH for CMT1A, a minimum of 50 nuclei should be counted for relative signals and 10–30 nuclei for HNPP in metaphase FISH. A failure rate of 10–30% was estimated in some laboratories for reasons such as a reduction in the quality of the prepared nuclei. Characteristics of an ideal diagnostic test include technical simplicity, accuracy and reliability—that is, not producing test failure or uninformative results frequently. Routine methods to detect the duplication in CMT1A patients have been compared in UK diagnostic laboratories. The aim of that study was to test the sensitivity and reproducibility of each method. The five approaches compared in the study were: microsatellite analysis; junction fragment detection either by Southern blot or PCR; and STS dosage either with capillary electrophoresis or polyacrylamide gel electrophoresis (PAGE). None of these methods was ideal; methods based on a junction fragment missed 14% of duplications, microsatellite analysis had a high failure rate and several tests were required, and STS dosage methods often needed repeating. Rowland et al concluded that STS dosage analysis was the most sensitive method but is technically more difficult than the other methods. Other gene dosage approaches such as array comparative gene hybridisation or multiplex amplifiable probe hybridisation (MAPH) might preserve the high sensitivity in a more robust assay format. With the currently available technology, molecular genetic diagnosis still remains a labour intensive and costly procedure. We therefore applied MAPH to DNA based