A recurrent stop-codon mutation in succinate dehydrogenase subunit B gene in normal peripheral blood and childhood T-cell acute leukemia

Abstract

Background: Somatic cytidine mutations in normal mammalian nuclear genes occur during antibody diversification in B lymphocytes and generate an isoform of apolipoprotein B in intestinal cells by RNA editing. Here, I describe that succinate dehydrogenase (SDH; mitochondrial complex II) subunit B gene (SDHB) is somatically mutated at a cytidine residue in normal peripheral blood mononuclear cells (PBMCs) and T-cell acute leukemia. Germ line mutations in the SDHB, SDHC or SDHD genes cause hereditary paraganglioma (PGL) tumors which show constitutive activation of homeostatic mechanisms induced by oxygen deprivation (hypoxia).

Principal findings: To determine the prevalence of a mutation identified in the SDHB mRNA, 180 samples are tested. An SDHB stop-codon mutation c.136C>T (R46X) is present in a significant fraction (average = 5.8%, range = less than 1 to 30%, n = 52) of the mRNAs obtained from PBMCs. In contrast, the R46X mutation is present in the genomic DNA of PBMCs at very low levels. Examination of the PBMC cell-type subsets identifies monocytes and natural killer (NK) cells as primary sources of the mutant transcript, although lesser contributions also come from B and T lymphocytes. Transcript sequence analyses in leukemic cell lines derived from monocyte, NK, T and B cells indicate that the mutational mechanism targeting SDHB is operational in T-cell acute leukemia. Accordingly, substantial levels (more than 3%) of the mutant SDHB transcripts are detected in five of 20 primary childhood T-cell acute lymphoblastic leukemia (T-ALL) bone marrow samples, but in none of 20 B-ALL samples. In addition, distinct heterozygous SDHB missense DNA mutations are identified in Jurkat and TALL-104 cell lines which are derived from T-ALLs.

Conclusions: The identification of a recurrent, inactivating stop-codon mutation in the SDHB gene in normal blood cells suggests that SDHB is targeted by a cytidine deaminase enzyme. The SDHB mutations in normal PBMCs and leukemic T cells might play a role in cellular pre-adaptation to hypoxia.

Figure 1. Analysis of SDHB R46X mRNA mutation.

A. The SDHB gene has 843 bp coding nucleotides spread to 8 exons within ∼35 kb at chromosome band 1p36.13. The 5′- portion of the gene shows the position of the R46X mutation and the mitochondrial signal peptide cleavage site (filled circle). B. Sequence chromatograms of RT-PCR products show samples that have high (top), low (middle), or undetectable (bottom) amounts of mutant R46X sequences. C. Quantification of the mutant fraction involved TaqI RE digestion of fluorescently-labeled RT-PCR products and capillary gel electrophoresis. The red peaks denote the molecular weight marker. D. The agarose gel electrophoresis shows variable fractions of mutant RT-PCR products from normal PBMCs detected by TaqI RE digestion, which releases two bands 159 and 126 bp in size from the wild-type sequence (also see Fig. S1A). E. Fractions of TaqI RE resistant transcripts in the purified PBMC cell types are shown in an agarose gel. The negative image is presented to enhance the visibility of mutant transcripts.

Figure 2. The steady-state fraction of mutant SDHB cDNAs resistant to TaqI RE digestion.

Each circle represents a sample from a different subject (total indicated by n) except for those in the PHA stimulated PBMCs which were obtained from two normal donors, stimulated by 2.5 µg/ml and 5.0 µg/ml concentrations of PHA and tested at days 2, 5 and 8 for a total of 12 samples. The value corresponding to each circle was derived from three RT-PCR reactions. Two outlier PBMC values were shown at the top with their mutant transcript fractions in parentheses. Boxes and the vertical lines denote the means and their 95% confidence intervals of samples sets. “PBMC mutation carrier” group contains 5 SDHC and 24 SDHD mutation carriers. The leukemic cell lines were derived from B cells (n = 3), T cells (n = 9), NK cells (n = 1) and monocytes (n = 2).

Figure 3. SDHB mutations in genomic DNAs (gDNAs).

A. TaqI RE digestion of PCR-amplified exon 2 shows no evidence of undigested mutant DNAs at 260 bp size (pointed by an arrow head). TaqI RE digestion products of the wild-type DNA co-migrate at sizes of 128 and 132 bps (denoted by a star). B. Gel electrophoresis of TaqI RE-digested (mutation) enrichment PCR (ePCR) products shows variable amounts of mutant gDNAs at 233 bp size (pointed by an arrow head), which are confirmed to be primarily composed of c.136C>T by sequencing (Table 1). TaqI digestion of wild-type ePCR products gives two bands at sizes, 101 and 132 bp (shown by a star). C. ePCR analyses of leukemic cell lines demonstrate the presence of mutant gDNA in the MOLT-4 cell line (pointed by an arrow head) but not in other leukemic cell lines in this experiment. D. Direct sequence analysis of a gDNA ePCR product shows selective enrichment of the R46X mutation. E, F. Sequence chromatograms of the heterozygous DNA mutations detected in two T-ALL cell lines are shown.

Figure 4. SDH genetic downregulation as a mechanism for early detection of hypoxia.

Data from PGL research suggest that SDH normally inactivates a messenger(s) critical for downstream hypoxia-signaling. Consequently, heterozygous SDH germ line mutations over-activate the hypoxia signaling pathways in PGL. Loss of the wild-type allele by loss-of-heterozygosity (LOH) leads to constitutive hypoxic signaling and tumorigenesis in PGL. Transcript mutations, genomic imprinting, balancing selection or germ line mutations are postulated to reduce SDH activity and change the hypoxia-detection threshold from a lower oxygen concentration (a) to higher oxygen concentrations (b, c), thus leading to earlier detection of impending hypoxia.