Data supporting the co-expression of PDHA1 gene and of its paralogue PDHA2 in somatic cells of a family

This article presents a dataset proving the simultaneous presence of a 5′UTR-truncated PDHA1 mRNA and a full-length PDHA2 mRNA in the somatic cells of a PDC-deficient female patient and all members of her immediate family (parents and brother). We have designed a large set of primer pairs in order to perform detailed RT-PCR assays allowing the clear identification of both PDHA1 and PDHA2 mRNA species in somatic cells. In addition, two different experimental approaches were used to elucidate the copy number of PDHA1 gene in the patient and her mother. The interpretation and discussion of these data, along with further extensive experiments concerning the origin of this altered gene expression and its potential therapeutic consequences, can be found in “Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells” (A. Pinheiro, M.J. Silva, C. Florindo, et al., 2016) [1].


a b s t r a c t
This article presents a dataset proving the simultaneous presence of a 5 0 UTR-truncated PDHA1 mRNA and a full-length PDHA2 mRNA in the somatic cells of a PDC-deficient female patient and all members of her immediate family (parents and brother).
We have designed a large set of primer pairs in order to perform detailed RT-PCR assays allowing the clear identification of both PDHA1 and PDHA2 mRNA species in somatic cells. In addition, two different experimental approaches were used to elucidate the copy number of PDHA1 gene in the patient and her mother.
The interpretation and discussion of these data, along with further extensive experiments concerning the origin of this altered gene expression and its potential therapeutic consequences, can be found in "Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: null mutations in the PDHX gene associated with unusual expression Value of the data These data, reporting on PDHA2 gene expression in somatic cells, may trigger new research related to the activation of a paralogue gene as a therapeutic target to loss-of-function mutations.
Data revealing the co-existence of both PDHA1 and PDHA2 mRNAs in somatic cells will be useful for future experiments addressing the impact between both isoforms in the assembly of a fully functional PDC.
Data concerning gene copy number may assist the choice of the underlying methodology. These dataset may contribute for designing further experiments aiming the development of alternative therapies for metabolic disorders.

Data
The E1 rate-limiting enzyme of pyruvate dehydrogenase complex (PDC) is a heterotetramer (α 2 β 2 ) and its α subunit is encoded by PDHA1 gene, located in X chromosome and presenting ubiquitous expression in somatic tissues. Nevertheless a paralogue gene exists, PDHA2, which is located in chromosome 4 and expressed only in spermatocytes and spermatids [2]. Table 1 shows the primers used for the amplification of the analyzed genes, according to the used methodology. Fig. 1 presents the results of PDHA1 and PDHA2 gene expression in somatic cells of the individuals under study and in controls. Fig. 2 displays the alignment of PDHA1 and PDHA2 mRNAs Table 1 List of primers used in this study.

Primer
Sequence Position cDNA amplification showing that the specific primers were designed to anneal to regions with null or very low homology between the two genes, thus proving the simultaneous presence of both transcripts. Fig. 3 depicts the scheme of PDHA1 mRNA with the localization of all the primers used to prove the presence of the 5 0 UTR truncated PDHA1 mRNA detected in the family samples, and to localize the truncation point. Table 2 and Fig. 4 show the results of the two different methodologies used to evaluate PDHA1 gene copy number: quantitative real time PCR (Table 2) and microarray analyses (Fig. 4).

Sample preparation
Lymphocytes were isolated from three independent peripheral blood samples obtained from the index case and her parents and brother, as well as from control individuals.
Patient's fibroblast cultures were established from a diagnostic skin biopsy and grown under standard conditions.
Positive controls for PDHA2 gene expression were obtained from two different sources; a commercially available human testis total RNA sample (Clontech Laboratories Inc., Mountain View, CA, USA) and human testis specimens from eight cases requiring open testicular biopsy for the retrieval of testicular sperm for intracytoplasmic sperm injection [3].

Nucleic acids preparation
Genomic DNA, total RNA and cDNA were prepared according to standard methods and described in [1].  Amplification of the 11 individual exons of the PDHA1 gene and related intron-exon boundaries were amplified using primers already published [4]. PDHA1 and PDHA2 cDNAs were amplified under conditions previously described [5] and using primers listed in Table 1, which were designed to annealing to regions displaying no homology between transcripts [6].

Evaluation of PDHA1 and PDHA2 expression and PDHA1 gene dosage
PDHA1 and PDHA2 transcriptional levels were evaluated by quantitative real time RT-PCR under conditions previously described [1].
The copy number of PDHA1 gene was evaluated by two methods, quantitative real time PCR and microarray analysis, as previously described [1].