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. 2008 Sep-Oct;95(1-2):31-8.
doi: 10.1016/j.ymgme.2008.06.006. Epub 2008 Aug 3.

The molecular basis of pyruvate carboxylase deficiency: mosaicism correlates with prolonged survival

Dong Wang  1 , Hong Yang, Kevin C De Braganca, Jiesheng Lu, Ling Yu Shih, Paz Briones, Tim Lang, Darryl C De Vivo
Affiliations

The molecular basis of pyruvate carboxylase deficiency: mosaicism correlates with prolonged survival

Dong Wang et al. Mol Genet Metab. 2008 Sep-Oct.

Abstract

Pyruvate carboxylase (PC) deficiency (OMIM, 266150) is a rare autosomal recessive disease. The revised PC gene structure described in this report consists of 20 coding exons and four non-coding exons at the 5'-untranslated region (5'-UTR). The gene codes for three transcripts due to alternative splicing: variant 1 (NM_000920.3), variant 2 (NM_022172.2) and variant 3 (BC011617.2). PC deficiency is manifested by three clinical phenotypes-an infantile form (Type A), a neonatal form (Type B), and a benign form (Type C). We report the molecular basis for eight cases (one Type A, five Type B and two Type C) of PC deficiency. Eight novel complex mutations were identified representing different combinations of missense mutations, deletions, a splice site substitution and a nonsense mutation. The classical phenotypes (A, B and C) correlated poorly with clinical outcomes. Mosaicism was found in five cases (one Type A, three Type B and one Type C) and four of these cases had prolonged survival. Death in the fifth case resulted from unrelated medical complications. The discrepancy between the current findings and the existing classification system should be addressed to accommodate these new observations.

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Figures

Fig. 1
Fig. 1. PC gene structure, sequence variance distribution and three transcript variants
The coding exons of PC gene are represented by rectangles with different symbols and Arabic numbers on the top. The four non-coding exons are labeled with UE1-4 on the top. The arrows before UE1, UE2 and UE4 represent the transcription initiation sites. Primers used for the RT-PCR F0, F1, L3, M3, R1 and R2 are shown with an arrow head to designate the direction. All previously reported mutations in the literature are shown above the gene structure(3, 24, 33). All the novel complex mutations identified in this study are shown under the gene structure with arrow heads to show the locations of alterations in the gene. Three transcript variants are shown with the same coding region and different non-coding exons in the 5’-UTR. BC: Biotin carboxylation domain. TC: Transcarboxylation domain. BIO: Biotin carboxyl carrier domain
Fig. 2
Fig. 2. Splice donor site destroyed in patient 3
Partial genomic DNA sequence from exons 2 and 3 and intron 2 around the splice donor and acceptor sites are shown. The amino acids (aa) and their locations are shown under the DNA sequence. Part of the mutant mRNA is shown with the deletion of nine nucleotides resulting in the deletion of amino acids V105, A106 and K107.
Fig. 3
Fig. 3. Quantitative PCR-RFLP
Quantitative PCR-RFLP was done as described in the Materials and Methods section. Molecular weight markers and patients’ codes are indicated on the top part of the figure. The marker ladder is spaced 100bp apart. 5m and 5f are the mother and father of patient 5. C is a carrier control and Ctr is the undigested control. A. The PCR mutant fragment (Mt) NE3-NE4 (401bp) with substitution c. [1892G>A] could not be digested by Msp I, whereas the wild-type DNA (Wt) NE3-NE4 fragment could be cut into two fragments (189bp and 212bp) as shown by an arrow head on the right side. B. The CO1-CO2 (398bp) mutant fragment (Mt) containing c. [2540C>T] could not be cut by Fnu4H I, whereas the wild-type fragment(Wt) could be cut into two fragments (239 bp and 159bp) as shown by the arrow head on the right side.
Fig. 4
Fig. 4. Western blot analysis
Western blot was done as described in the Materials and Methods section. The membrane was then probed with streptavidin-HRP conjugate. PC, MCC and PCC were visualized using the horseradish peroxidase ECL system (Amersham, Piscataway, NJ) following the protocol. C is a carrier control and Ctr is normal control. A. molecular weight markers and patient codes are shown on the top. 5m is the mother of patient 5. On the left side of the figure the molecular weight of each band in the marker are labeled with arrow heads (in KDa). The location of PC, MCC, and PCC are shown by arrow heads. B. the western blot was analyzed using Image J. The density of PC protein was normalized by the total density of MCC and PCC, further normalized to the control (100%). The data are shown as mean ± SD, n =3.
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