Molecular Genetic Analysis of the PLP1 Gene in 38 Families with PLP1-related disorders: Identification and Functional Characterization of 11 Novel PLP1 Mutations

<p>Abstract</p> <p>Background</p> <p>The breadth of the clinical spectrum underlying Pelizaeus-Merzbacher disease and spastic paraplegia type 2 is due to the extensive allelic heterogeneity in the X-linked <it>PLP1 </it>gene encoding myelin proteolipid protein (PLP). <it>PLP1 </it>mutations range from gene duplications of variable size found in 60-70% of patients to intragenic lesions present in 15-20% of patients.</p> <p>Methods</p> <p>Forty-eight male patients from 38 unrelated families with a PLP1-related disorder were studied. All DNA samples were screened for <it>PLP1 </it>gene duplications using real-time PCR. <it>PLP1 </it>gene sequencing analysis was performed on patients negative for the duplication. The mutational status of all 14 potential carrier mothers of the familial <it>PLP1 </it>gene mutation was determined as well as 15/24 potential carrier mothers of the <it>PLP1 </it>duplication.</p> <p>Results and Conclusions</p> <p><it>PLP1 </it>gene duplications were identified in 24 of the unrelated patients whereas a variety of intragenic <it>PLP1 </it>mutations were found in the remaining 14 patients. Of the 14 different intragenic lesions, 11 were novel; these included one nonsense and 7 missense mutations, a 657-bp deletion, a microdeletion and a microduplication. The functional significance of the novel <it>PLP1 </it>missense mutations, all occurring at evolutionarily conserved residues, was analysed by the <it>MutPred </it>tool whereas their potential effect on splicing was ascertained using the <it>Skippy </it>algorithm and a neural network. Although <it>MutPred </it>predicted that all 7 novel missense mutations would be likely to be deleterious, <it>in silico </it>analysis indicated that four of them (p.Leu146Val, p.Leu159Pro, p.Thr230Ile, p.Ala247Asp) might cause exon skipping by altering exonic splicing elements. These predictions were then investigated <it>in vitro </it>for both p.Leu146Val and p.Thr230Ile by means of RNA or minigene studies and were subsequently confirmed in the case of p.Leu146Val. Peripheral neuropathy was noted in four patients harbouring intragenic mutations that altered RNA processing, but was absent from all <it>PLP1</it>-duplication patients. Unprecedentedly, family studies revealed the <it>de novo </it>occurrence of the <it>PLP1 </it>duplication at a frequency of 20%.</p