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1.  Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways 
BMC Medical Genomics  2013;6:22.
Background
SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age.
Methods
Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR.
Results
We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways.
Conclusions
Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.
doi:10.1186/1755-8794-6-22
PMCID: PMC3689607  PMID: 23777634
Autosomal dominant cerebellar ataxia; Spinocerebellar ataxia; SCA28; AFG3L2; Genome-wide expression; LCLs
2.  Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes 
In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene.
We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were clearly distinguishable from clones with two or more NEO copies.
This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.
doi:10.1186/1480-9222-13-10
PMCID: PMC3226651  PMID: 22035318
3.  Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele-Specific Expression 
Human Mutation  2013;34(8):1160-1171.
ABSTRACT
Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels.
doi:10.1002/humu.22348
PMCID: PMC3714349  PMID: 23649844
Lamin B1; leukodystrophy; ADLD; duplication Alu; NHEJ; FoSTeS; MMBIR

Results 1-3 (3)