Autosomal dominant leukodystrophy (ADLD) is a slowly progressive demyelinating disease, with onset in the 4th to 6th decade of life. It is characterized by autonomic dysregulation, pyramidal signs, cerebellar dysfunctions, and symmetrical primary myelin loss in the central nervous system. Duplication of the lamin B1 gene (LMNB1) has been implicated as the cause of disease. Eleven ADLD families of different ethnic origins have been published so far, but the duplication breakpoint has been characterized at the nucleotide level in only three. Here, we characterize the LMNB1 duplication in eight patients with ADLD originating from Italy (n=3), France (n=3), Brasil (n=1) and North America (n=1). A custom array-CGH allowed us to define the extent of the duplications, which ranged from 128 to 474 Kb. Seven duplications were unique, proving their origin from separate mutational events. Two French families, coming from the same geographic region, shared the same duplication, and are therefore likely to have a common ancestor. The largest duplication boundaries extend centromerically up toGRAMD3(also involving ALDH7A1 and PHAX), and telomerically up to MARCH3. Using long-range PCR and sequencing, the duplication breakpoints were characterized in six patients; a microhomology region was found in two, an insertion in two, and a microduplication in one, suggesting microhomology-mediated, breakinduced replication (MMBIR) as a possible mechanism of origin. We studied the expression of the LMNB1 alleles using a primer extension assay of the SNP rs1051644 in the 3’UTR (c.*239C>T); lymphoblastoid cell lines of three different patients showed a 2:1 ratio of the duplicated allele vs. the nonduplicated allele on both genomic DNA and cDNA. This proves that the duplication increases LMNB1 expression by only 50%, whereas published real-time PCR data show several fold increases in LMNB1 expression. In cell models, lamin B1 overexpression alters the transcription of many genes, and we suggest this may affect the real-time experimental results. In summary, we define seven novel LMNB1 duplications in ADLD patients. Expression analysis experiments using primer extension suggest that in lamin B1 duplication carriers the gene and protein levels are increased approximately 50%. (Work supported by Telethon grant GGP10184 and S.Paolo Neuroscience grant to A. Brussino, ELA foundation 2009-007I4 grant to O Boespflug- Tanguy).
Characterization of lamin B1 duplication breakpoints and expression analysis in ADLD patients
BRUSSINO, Alessandro;DI GREGORIO, ELEONORA;GIORGIO, ELISA;LACERENZA, Daniela;BRUSCO, Alfredo
2011-01-01
Abstract
Autosomal dominant leukodystrophy (ADLD) is a slowly progressive demyelinating disease, with onset in the 4th to 6th decade of life. It is characterized by autonomic dysregulation, pyramidal signs, cerebellar dysfunctions, and symmetrical primary myelin loss in the central nervous system. Duplication of the lamin B1 gene (LMNB1) has been implicated as the cause of disease. Eleven ADLD families of different ethnic origins have been published so far, but the duplication breakpoint has been characterized at the nucleotide level in only three. Here, we characterize the LMNB1 duplication in eight patients with ADLD originating from Italy (n=3), France (n=3), Brasil (n=1) and North America (n=1). A custom array-CGH allowed us to define the extent of the duplications, which ranged from 128 to 474 Kb. Seven duplications were unique, proving their origin from separate mutational events. Two French families, coming from the same geographic region, shared the same duplication, and are therefore likely to have a common ancestor. The largest duplication boundaries extend centromerically up toGRAMD3(also involving ALDH7A1 and PHAX), and telomerically up to MARCH3. Using long-range PCR and sequencing, the duplication breakpoints were characterized in six patients; a microhomology region was found in two, an insertion in two, and a microduplication in one, suggesting microhomology-mediated, breakinduced replication (MMBIR) as a possible mechanism of origin. We studied the expression of the LMNB1 alleles using a primer extension assay of the SNP rs1051644 in the 3’UTR (c.*239C>T); lymphoblastoid cell lines of three different patients showed a 2:1 ratio of the duplicated allele vs. the nonduplicated allele on both genomic DNA and cDNA. This proves that the duplication increases LMNB1 expression by only 50%, whereas published real-time PCR data show several fold increases in LMNB1 expression. In cell models, lamin B1 overexpression alters the transcription of many genes, and we suggest this may affect the real-time experimental results. In summary, we define seven novel LMNB1 duplications in ADLD patients. Expression analysis experiments using primer extension suggest that in lamin B1 duplication carriers the gene and protein levels are increased approximately 50%. (Work supported by Telethon grant GGP10184 and S.Paolo Neuroscience grant to A. Brussino, ELA foundation 2009-007I4 grant to O Boespflug- Tanguy).
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