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].

Specifications Table

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 (α2β2) 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 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′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).

Table 1

List of primers used in this study.

Primer	Sequence		Position
cDNA amplification
PDHA1messenger
PDHA1-F	5′–AGCATCCCGTAATTTTGC–3′		+75 to +92
PDHA1-R	5′–CTTTAGTTCTTCCACACTGG–3′		+989 to +1008
PDHA1-5’-F	5′–GGGCACCTGAAGGAGACTT–3′		−85 to −66
PDS1	5′–TGTGAGGAGTCGCCGCTGCC–3′		−37 to −18
PDSTr-F	5′–GCCACTGCCTGTGCTTCAT–3′		−17 to +2
PDSTr-R	5′–ACTCCATTCGGCGTACAGTCT–3′		+207 to +226
PDHA2messenger
PDHA2-F	5′–TGCCATCTACAGCACTCCGT–3′	−27 to −8
PDHA2-R	5′–CCTCCTTGAGTTGAGAACAC–3′	+1235 to +1254
PDHXmessenger
PXF2	5′–CTGCTGCGTTATCTTGTGGGCT–3′	+37 to +58
PXW2	5′–TGAGTGAATGTGCCCACTGCATTG–3′	+812 to +835
PXP2	5′–CAATGCAGTGGGCACATTCACTGA–3′	+812 to +835
PXR2	5′–TAACAACTACTGAATCAACTAAGC–3′	+2060 to +2083
Genomic DNA amplification
PDHA1gene
PDHA1-P1-F	5′–CCCTTGTTGCTTTGGTGTTT–3′	4383 to 4403
PDHA1-P1-R	5′–AGATTGCTCTGCTGACTACCG–3′	4762 to 4784
PDHA1-P2-F	5′–TGAGCATGCTGCTAATCTTCA–3′	4642 to 4682
PDHA1-P2-R	5′–CGGCGTGACAGAGTCGTAAT–3′	5114 to 5133
PDHA1-P3-F	5′–CTGGACGCCGTTCTGGTT–3′	4966 to 2983
PDHA1-P3-R	5′–GCGGAGGCGAAGTAAAGG–3′	4323 to 4340
PDHA1-P4-F	5′–TGCTTCATGAGGAAGATGCT–3′	5140 to 5159
PDHA1-P4-R	5′–AGGGTGCTGTTTGAACGAAG–3′	5526 to 5645
PDHA2gene
PDHA2-A-F	5′–GAGTAAGGAAAAGTGGAATGTCA–3′	−841 to −819
PDHA2-A-R	5′–ATCCTGCTCCATAATGTGCC–3′	−200 to −181
PDHA2-B-F	5′–GCCATCAGGATAAATGTGGC–3′	−657 to −638
PDHA2-B-R	5′–CCCTTTTCCCTGTTAAACCC–3′	−322 to −303
PDHA2-C-F	5′–AACTCTCAGAACTCTCATGTGCC–3′	−415 to −393
PDHA2-C-R	5′–ACGGAGTGCTGTAGATGGCA–3′	−27 to −8
PDHA2-D-F	5′–CAGGACCTGCCTCTATCACC–3′	−142 to +123
PDHA2-D-R	5′–AAACCGCGAATGAATTTCTG–3′	+244 to +263
PDHA2-F-F	5′–GCATGGAATTGAAGGCAGAT–3′	+212 to +231
PDHA2-F-R	5′–CCTCCTTGAGTTGAGAACAC–3′	+1298 to+1317
PDHXgene
PX1F	5′–AGAGACCTAAAGGCACCGCT–3′	+5414 to +5433
PX1R	5′–AAGCAGGCCCTCAATCATAA–3′	+5751 to +5770
PX2F	5′–TGGGAATCTTTTAGACTTTGGA–3′	+20,144 to + 20,165
PX2R	5′–TGCTGAACCCAGAAAACCTT–3′	+20,531 to + 20,550
PX3F	5′–CAACCCAGAAATAGCTACGGA–3′	+36,259 to + 36,279
PX3R	5′–CACATTAAAAATAAGGAGGCAAAA–3′	+36,557 to + 36,581
PX4F	5′–TGCAGTCATGGGGTTTTACTT–3′	+46,205 to + 46,225
PX4R	5′–ACAGCAACTTCCTACGTGATG–3′	+46,549 to +46,570
PX5F	5′–GTGACCATCTGTGGGAGTCA–3′	+49,159 to +49,173
PX5R	5′–TTATTCAGAAAACAACTCTTGCAT–3′	+49,549 to +49,573
PX6F	5′–TCACCTGCGTTTTCTGAAAGT–3′	+55,435 to + 55,456
PX6R	5′–GTGAGCCAAGATTGTGCCAT–3′	+55,779 to +55,798
PX7F	5′–TTCCACTTGTGGTTTAACGGA–3′	+58,968 to +58,988
PX7R	5′–TTTCCTCTAGCACAAATATACCCA–3′	+59,294 to +59,318
PX8F	5′–ACAAGTTTGAAGTTGTAATGGTCA–3′	+66,918 to +66,941
PX8R	5′–GAGGGAGATCAAACGATAGGA–3′	+67,178 to +67,198
PX9F	5′–TTTTTCTGTAACCGCCTTGG–3′	+73,376 to +73,395
PX9R	5′–TCTCCCCTTCACACACACAA–3′	+73,700 to +73,719
PX10F	5′–GGTAACAAAATCAAATCAAGGCA–3′	+81,064 to +81,085
PX10R	5′–TTCAGATAAATGAAAGGCTGACA–3′	+81,315 to +81,337
PX11F	5′–ACGGAAAGGGGACTTTGATT–3′	+83,725 to +83,744
PX11R	5′–TTGAGGACTAGGCAAGTCGG–3′	+84,031 to +84,050
PDHA2gene methylation analysis
CpGI-M-F	5′–ATAAATTAGTTAGTTTAGGTTGCGT–3′	−188 to −164
CpGI-M-R	5′–ATAACGTCATTTAAAAAATTACGAA–3′	+74 to +98
CpGI-U-F	5′–ATAAATTAGTTAGTTTAGGTTGTGT–3′	−188 to −64
CpGI-U-R	5′–ATAACATCATTTAAAAAATTACAAA–3′	+74 to +98
CpGII-F	5′–TGGAATTGAAGGTAGATTAGTTGTATAAAT–3′	+205 to+234
CpGII-R	5′–ATACCATTACCCCCATAAAAATTCT–3′	+406 to +431
Gene dosage analysis
PDHA1gene
PDHA1-exon7F	5′–AGGAGGCCTTTCTGTGCTTT–3′	11,341 to 11,359
PDHA1-exon7R	5′–CGGCCCCACCACAGGGTTCCT–3′	11,616 to 11,636
PAHgene
PAH-exon1F	5′–GCTTTACTGTGCGGAGATCACCAC–3′	5315 to 5339
PAH-exon1R	5′–CTTATGAAACCAGGAAGCAC–3′	5606 to 5625

Fig. 1

RT-PCR analyses of PDH E1α transcripts. (a) Using PDHA1 and PDH2 specific primers. PL - patient lymphocytes; PF - patient fibroblasts; T - whole testis tissue; C1 and C2 - control lymphocytes; B1 without PCR control using whole testis total RNA; B2 - PCR control using no biological sample. M - 100 Base Pair Ladder (New England Biolabs). (b) Using forward PDS1 primer and reverse PDHA1 specific primer. PL - patient lymphocytes; PF - patient fibroblasts; C - control lymphocytes; B2 - PCR control using no biological sample. M - 100 Base Pair Ladder (New England Biolabs).

Fig. 2

Alignment of PDHA1 and PDHA2 cDNA sequences and primers’ localization.

Fig. 3

Schematic representation of the PDHA1 mRNA sequence showing the amplified versus non-amplified products in the RT-PCR analysis with the corresponding localization of the forward primers (PDHA1-5′, PDS1, PDSTrF, PDHA1F) and reverse primers (PDHA1R and PDSTrR), as well as the identification of the predicted truncation point.

Table 2

Calculations for determining by qPCR the copy number of PDHA1 gene using as reference the autosomal PAH gene.

PDHA1gene
Sample	Ave ΔCt	ΔΔCt	RQ (2-ΔΔCt)	Copy # (2×RQ)
Patient	0.26	0.91	0.5	1
Control Female 1	−0.65	0	1	2
Control Female 2	−0.33	0.32	0.8	2
Control Female 3	−0.59	0.06	0.9	2
Control Male 1	0.93	1.58	0.3	1
Control Male 2	−0.23	0.42	0.7	1
Control Male 3	−0.01	0.64	0.6	1

Fig. 4

Detailed view of the PDHA1 region on chromosome X. (a) Allele difference and (b) copy number state showing absence of big deletions involving the gene. (c) OMIM genes: PDHA1 (dark green horizontal bar) and MAP3K15 (gray horizontal bar). Intron - horizontal pink lines; Exon - vertical pink bars. (d) Markers present in PDHA1 region. Dark green - non-polymorphic probes; Light green - SNP, single nucleotide polymorphism. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Experimental design, materials and methods

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].

PCR of genomic DNA and cDNA

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].

Subject area	Biology
More specific subject area	Molecular Genetics
Type of data	Tables, figures
How data was acquired	Agarose gel electrophoresis after RT-PCR analyses quantitative real time PCR, microarray analyses, in silico analyses (BLAST software)
Data format	Raw, analyzed
Experimental factors	Genomic DNA and total RNA isolated from whole blood samples and fibroblast cultures
Experimental features	Genomic DNA was amplified by quantitative real time PCR and microarray analyses. Total RNA was reverse transcribed and amplified by semi-quantitative RT-PCR and by quantitative real time PCR using TaqMan assays. Alignment of sequences was performed using the BLAST software.
Data source location	Lisboa, Portugal
Data accessibility	Data provided within the manuscript and available in public databases (NCBI) in case of sequence alignment: GenBank accession numbers GenBank:NM_000284.3(PDHA1) and GenBank:NM_005390.4(PDHA2)