A rapid method for the diagnosis of Charcot Marie Tooth disease Type 1A by FISH

Abstract

▼Charcot Marie Tooth disease Type 1A (CMT1A) is associated with a submicroscopic tandem duplication on the short arm of chromosome 17. The 1.5 Mb duplication contains the peripheral myelin protein 22 (PMP22) gene, the dosage of which plays a crucial role in the aetiology of the disorder. Diagnosis of CMT1A has until recently been limited to studies on Southern blots. Methods derived for fluorescence in situ hybridization (FISH) analysis have relied on traditional 72-h cytogenetic cultures of patient blood samples (Ref. 1) or on the development of more complex blood treatment protocols (Ref. 2). Metaphase chromosomes cannot be used because the duplication is tandem and unresolvable by FISH at metaphase level. We describe a rapid, reliable method producing large numbers of well-conserved nuclei, suitable for FISH, that fits easily into the routine procedures of a busy diagnostic cytogenetic laboratory. The main modification of standard blood culture procedures is the reduction of culture time to overnight, with no colchicine treatment. Stock culture medium can be used, containing phytohemagglutinin (PHA) if this is the way stock is usually prepared, since overnight culture is insufficient to stimulate nuclei past the G0/G1 stage of the cell cycle. This is important, for the replicated chromatids of late S or G2 frequently give double signals, potentially mimicking the CMT1A duplication. This method therefore obviates the need to co-hybridize an alternate-coloured control probe to check the replication status of the 17p segment. Blood cultures for diagnosis are set up the day before any normal harvest day. Any standard chromosome harvesting procedure that includes hypotonic treatment and acetic