Charcot-Marie-Tooth 1 (CMT1) and tomaculous neuropathy (HNPP) - Evidence of specific DNA duplications and deletions in the area of 17p11.2-12 by means of a set of dinucleotide markers
Abstract
Hereditary motor-sensory neuropathies (HMSN) are clinically and genetically a heterogeneous group of neuromuscular diseases. In the clinical picture dominate distal muscular weakness, distal muscular atrophy, deformities of the feet in particular pes cavus, areflexia, sensory disorders and in the majority an autosomal dominant heredity of the disorder.
By elektrophysiology type I, with a markedly reduced conduction velocity (NCV) through the peripheral nerve can be readily differentiated from type II with a normal or only slightly reduced NCV. In recent years classification into molecular genetic types I.IV with several sub-types predominated.
At present at least 18 different chromosomal areas responsible for different types of HMSN are known. Direct DNA diagnosis is so far possible only in electrophysiologically confirmed type I Charcot-Marie-Tooth disease (CMT1) which accounts for 70% of HMSN.
Type 1 CMT is however also genetically heterogeneous but 80-90% of the patients are formed by type IA CMT. The latter is autosomally dominant caused by 1,5 Mb submicroscopic duplication in the area 17p11.2-12 in which the gene for the peripheral myelin protein 22 (PMP 22) is situated.
Deletion in the same area is found in patients with so-called tomaculous neuropathy (HNPP). Quantitative PCR (QT-PCR), pulsed gel electrophoresis (PFGE), quantitative Southern blotting and fluorescent in situ hybridization (FISH) are the most frequently used methods for the detection of these duplications/deletions.
These methods are however, very pretentious as regards time, labour, costs and equipment. In our department for detection of these specific reconstructions a set of eight microsatellite (CA)n markers is used (AFM 191xh12, AFM 200yb12, AFM 317ygl, AFM 165, RM11-GT, 142E8, 103B11, 133C4), located inside the mentioned 1,5 Mb CMT/HNPP area.
Specific primer labelled fluorescent PCR products are longitudinally analyzed on an automatic sequantor. Duplication, i.e. the incidence of a total of three copies of the section in the area 17p11.2-12 in affected CMT 1A will be most probably manifested by the finding of three PCR products of different length in at elast one of eight markers used.
In case of deletion in 17p11.2-12 in the affected subject in all 8 markers only 1 allele is found. In segregation analysis in the family with HNPP the only detectable (sound) allele is not transmitted from the affected parent to the affected offspring.
This non-radioactive method is simple, cheap and reliable and thus suitable also for small clinical laboratories and has a great capacity. A certain disadvantage is that it is not possible to rule out duplication completely.
The latter can be only reluiably detected. For quite definit evidence of deletion it is necessary to examine two generations of affected subjects.
However one method makes it possible to detect two different mutations in two frequent diseases of the peripheral nervous system.