Genetic alterations in the alpha‐synuclein (SNCA) gene have been implicated in Parkinson Disease (PD), including point mutations, gene multiplications, and sequence variations within the promoter. Such alterations may be involved in pathology through structural changes or overexpression of the protein leading to protein aggregation, as well as through impaired gene expression. It is, therefore, of importance to specify the parameters that regulate SNCA expression in its normal and mutated state. We studied the expression of SNCA alleles in a lymphoblastoid cell line and in the blood cells of a patient heterozygous for p.Ala53Thr, the first mutation to be implicated in PD pathogenesis. Here, we provide evidence that: (1) SNCA shows monoallelic expression in this patient, (2) epigenetic silencing of the mutated allele involves histone modifications but not DNA methylation, and (3) steady‐state mRNA levels deriving from the normal SNCA allele in this patient exceed those of the two normal SNCA alleles combined, in matching, control individuals. An imbalanced SNCA expression in this patient is thus documented, with silencing of the p.Ala53Thr allele and upregulation of the wild‐type‐allele. This phenomenon is demonstrated for a first time in the SNCA gene, and may have important implications for PD pathogenesis. Hum Mutat 31:1–7, 2010.

Summary: The mechanisms by which mutations within the 5′ untranslated region (UTR) of the human β‐globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four ‘silent’ mutations in the human β‐globin 5′UTR, namely: +10(−T), +22(G → A), +33(C → G) and +(40–43)(−AAAC), which are present in patients with β‐thalassaemia intermedia. Expression of these genes under the control of the β‐globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61·6%, 68%, 85·2% and 70·6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3′ end processing or mRNA stability. Nuclear run‐on experiments demonstrated that mutations +10(−T) and +33(C → G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G → A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post‐transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.

Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34⁺ haematopoietic cells, but only transiently. To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR. All vectors supported stable transfections in K562 cells. Transfections of CD34⁺ cells from peripheral blood of healthy donors reached 30% efficiency. Upon evaluation of CD34⁺/eGFP⁺ cells in colony-forming cell (CFC) assays, vector pEP-IR showed superior performance after 14 days, by fluorescent microscopy: 100% eGFP⁺-colonies against 0% for pEPI-eGFP, 56.9% for pEPI-SFFV and 49.8% for pEPI-EF1/HTLV; 50% more plasmid copies per cell and 3-fold eGFP expression compared to the latter two constructs, by quantitative (q)PCR and RT-qPCR, respectively. Importantly, the establishment rate in CFC assays was 15% for pEP-IR against 5.5% for pEPI-SFFV and 5% for pEPI-EF1/HTLV. Vector pEP-IR shows extremely low delivery rate but supports eGFP expression in thalassaemic mouse haematopoietic progenitor cells. The IR is a novel human control element for improved episomal gene transfer into progenitor cells.