Received on February 1, 2006
Accepted on April 10, 2006

Molecular Diagnostics and Genetics

Multiplex Ligation-Dependent Probe Amplification for Rapid Detection of Proteolipid Protein 1 Gene Duplications and Deletions in Affected Males and Carrier Females with Pelizaeus-Merzbacher Disease

¹ Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH
² Brain Tumor Institute, Cleveland Clinic Foundation, Cleveland, OH
³ Institute of Human Genetics, University Hospital Eppendorf, Hamburg, Germany
⁴ Department of Human Genetics, Medical University of Vienna, Vienna, Austria
⁵ Department of Pediatrics and Pediatric Neurology, Georg August University, Göttingen, Germany
⁶ Department of Clinical Pathology, and Department of Pediatrics, and Department of Neurology, and Genomic Medicine Institute, Cleveland Clinic Foundation, Cleveland, OH

^* To whom correspondence should be addressed. E-mail: warshai{at}ccf.org.

Background: Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for 50%-75% of cases and point variations for 15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions.

Methods: We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex.

Results: All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%-9.8%.

Conclusions: MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.