Down syndrome (DS) is the most prevalent chromosomal disorder in the population and associated with alterations in development in a range of organ systems. The Centers for Disease Control and Prevention estimates DS to occur in approximately 1 in 733 newborns in the United States. Children with DS may be variably afflicted with a range of conditions including cognitive challenges, congenital heart defects, endocrinopathies, autoimmunity, orthopedic problems, and hematologic disorders. Children with DS therefore represent a unique population in which to study acute leukemia. These children are ten to twenty times more likely than age-matched peers to develop leukemia, making them an excellent model for the study of leukemogenesis. In addition to their propensity to develop leukemia, DS children have other underlying metabolic abnormalities that may contribute to their development of leukemia including their decreased intrinsic antioxidant activity and inability to process free radicals.
Dr. Pope's work on analysis of multiple genes in key pathways influencing the ability to repair DNA damage and metabolize free radicals. They are studying genes with known functional polymorphisms that are common in the population, many of which are associated with increased risk of cancer or of heart disease, often an endpoint for free radical damage.
Preliminary Data: We do understand some of the pathogenesis of leukemia in DS patients with transient myeloproliferative disease (TMD), a known precursor event to leukemia, and acute megakaryoblastic leukemia (AMKL) through work in our lab and others.
The AMKL of children with DS is uniquely characterized by acquired somatic mutations at exon 2 of the transcription factor gene GATA1. GATA1 is essential for normal erythropoiesis and plays a critical role in megakaryocytic development. In the vast majority of patients with DS with TMD and/or AMKL, the blast populations possess acquired somatic mutations affecting exon 2 of the GATA1 transcription factor, resulting in a shortened version of the protein (GATA1s). The reason for the particular propensity of GATA1s mutations in DS AMKL/TMD is unknown, but this mutation appears to cause accumulation and a block of differentiation in megakaryocytic precursors. Notably, mutations in GATA1 may be necessary but not sufficient for leukemogenesis in DS children, as analysis of cord blood specimens have revealed GATA1 mutations in a small number of unaffected DS infants, as well as multiple separate GATA1 mutant clones in some DS AMKL patients. It appears likely that additional cancer-causing mutations, or genetic “hits,” are also necessary for the full development of AMKL.
Overexpression of genes located on chromosome 21 regulating oxidant and single carbon metabolism may represent a “first hit” in the pathogenesis of TMD and AMKL in children with Down syndrome. Human chromosome 21 contains over 300 genes and other regulatory transcripts, including the Down syndrome critical region at 21q22, which is associated with the predominance of phenotypic characteristics of Down syndrome. Multiple lines of evidence implicate additional copies of this region of chromosome 21 as at least partially involved in DS leukemogenesis. Notably, in individuals who are mosaic for Down syndrome, if a leukemia or transient myeloproliferative disorder occurs, it is always found to harbor additional copies of chromosome 21. Though the chromosome 21q22 region includes a variety of genes implicated in leukemia, examination of oncogenes found on chromosome 21 have, as of yet, not identified consistent frequent patterns of mutation leading to pathologically constitutive activation. These observations have led to the search for other associated genetic mutations that may be responsible for or contribute to the evolution of leukemia in patients with DS. Multiple studies have suggested that cells from patients with DS exhibit high oxidative stress and potentially increased endogenous DNA damage. Notably, key cellular enzymes located on chromosome 21 including cystathionine-ß-synthase (CBS) and zinc-copper superoxide dismutase (SOD1) participate in the normal metabolism of endogenous oxidants and have been postulated to contribute to the “oxidant stress phenotype”. A recent analysis from the Taub laboratory of the mutational spectrum of GATA1 in leukemic blast samples from in children with DS identified patterns of mutations, suggesting that increased oxidative stress may represent a key pathway in leukemogenesis.
Cellular enzymes not located on chromosome 21 participate in the normal metabolism of endogenous oxidants as well as in DNA repair; many of these enzymes and proteins exhibit polymorphisms that influence their function. We hypothesized that background polymorphic variations in the ability to metabolize oxidants or repair associated DNA damage may identify the subgroup of children with Down syndrome who carry an increased risk of TMD and AMKL.
Work in the Perentesis laboratory has focused on studying oxidant metabolism and DNA repair genes that cause similar diseases to TMD and AMKL in mice, and are implicated in the causation of other forms of leukemia. The gene NADPH:Quinone Oxidoreductase 1 (NQO1) directly participates in the metabolism of benzene, chemotherapy drugs, intracellular free radicals, and other carcinogens. Mice with loss of NQO1 develop spontaneous and radiation-induced myeloproliferative disease. In humans, NQO1 has a polymorphic variant (NQO1 Pro187Ser) that is associated with lower function, and an increased risk of leukemia and development of other cancers. We sought to examine if the NQO1 slow metabolism variant was associated with an increased risk of TMD and/or AMKL in children with Down syndrome. Our study population included 200 children with Down syndrome and leukemia enrolled on a national Down syndrome AMKL and TMD treatment study between 1997 and 2004 (Children’s Oncology Group Study A2971).
We found that children with DS and the NQO1 Pro187Ser allele had a significantly increased risk of AMKL (Odd Ratio (OR) = 1.52, p = 0.11) and TMD (OR = 1.70, p = 0.04). We next studied the Fanconi anemia complementation group A protein gene (FANCA) and the x-ray repair cross-complementing 1 gene (XRCC1). FANCA is a critical DNA damage repair gene, abnormalities in these genes have been associated with the development of malignancy in otherwise healthy individuals. We found that DS children and a FANCA 1501 variant allele had a significantly increased risk of AMKL (OR = 2.04, p = 0.01) and TMD (OR = 1.55, p = 0.07). The related XRCC1 DNA repair pathway includes a polymorphic variant (XRCC1 Arg399Gln) that is associated with a protective effect against the development of leukemia. This enzyme is important in the repair of single-stranded DNA breaks, which are the most common forms of DNA damage and also result from oxidative stress. We found that the XRCC1 Arg399Gln variant allele was associated with a markedly decreased risk of AMKL (OR = 0.43, p = 0.001) and TMD (OR = 0.66, p = 0.11), similar to the protective effect described in non-DS adult acute myeloid leukemia.
These results fit a model in which additional copies of chromosome 21 produce increased oxidative stress and diminished DNA repair capacity in general, but do not directly cause leukemia in all individuals with DS because additional “hits” are required. However, DS patients possessing low functioning polymorphisms in the NQO1 oxidant metabolizing gene and/or low-functioning polymorphisms in the FANCA DNA repair gene and/or polymorphisms in the XRCC1 gene have a markedly increased risk of development of TMD and AMKL because they have limited capacity to metabolize mutagens. Notably, these observations are very similar to the NQO1 low function mouse model of myeloproliferative disorder.
Study Design: My work on analysis of multiple genes in key pathways influencing the ability to repair DNA damage and metabolize free radicals. We are studying genes with known functional polymorphisms that are common in the population, many of which are associated with increased risk of cancer or of heart disease, often an endpoint for free radical damage. Examples of genes in the DNA repair pathway associated with risk for cancer include ERCC2 and XRCC3. ERCC2, an excision repair cross-complementing protein rodent repair deficiency gene, is an integral part of the nucleotide excision pathway which recognizes and repairs DNA structural damage. XRCC3, the x-ray repair cross-complementing protein group 3 gene, participates in DNA repair by initiating homologous recombination at sites of double-stranded breaks. The genes identified that play an important role in free radical metabolism include p22phox, SOD2 and PON1. The p22phox gene encodes a NADPH oxidase that when activated is responsible superoxide generation. SOD2 encodes superoxide dismutase, a key enzyme in the antioxidant pathway and scavenger of reactive oxygen species. PON1, paraoxonase -1, plays an essential role in the detoxification of organophosphates. The table below outlines key SNPs that are being examined individually as well as those included on the Affymetrix DMET platform.
The population that will undergo analysis includes 200 patients diagnosed with TMD or AML that were enrolled on the Children’s Oncology Group study A2971, 492 patients with leukemia but without DS enrolled on recent COG leukemia clinical trials, and 400 controls comprised of healthy blood donors. An additional separate cohort of 205 samples from patients with DS AML will be analyzed from the separate and on-going COG-AAML0431 national treatment study for children with DS and AML. We are also collecting buccal swab DNA from children with DS without leukemia or a history of TMD followed in our program with a projected accrual of 400 individuals. These investigations and sample procurement are included in the above referenced clinical trials, and have current Human Subject/IRB approval.
The single nucleotide polymorphisms (SNPs) that will be analyzed in this study were chosen after an extensive review of the literature to identify those involved in DNA repair and oxidant metabolism (see above). The SNPs that will be analyzed include: ERCC2 (rs1799793), p22phox (rs4673), PON1 (rs662), PON1 (854560), and SOD2 (rs 4880). SNP analysis will be carried out using TaqMan Universal PCR Master Mix (Applied Biosystems, USA) and TaqMan SNP Genotyping Assays (Applied Biosystems, USA). We will also employ the Affymetrics DMET large-scale drug metabolizing enzyme polymorphism analysis platform, and will also study 1,936 drug metabolism markers in 225 genes, or 90% of the current ADME Core markers as defined by the PharmaADME group. Whole genome single nucleotide polymorphism (SNP)-based copy number variation (CNV) analysis will be performed on purified genomic DNA from paired samples of TMD, AMKL and normal cells from the same patient in order to distinguish acquired CNVs in AMKL and TMD from polymorphic CNVs in the human genome. We use the Illumina human660w-quad beadchip, a SNP-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV.
The prevalence of polymorphisms in the children with DS and TMD or AMKL will be estimated as binomial proportions and corresponding confidence intervals will be calculated. The half-width of corresponding 95% confidence intervals will be provided for various observed prevalences assuming all patients and only 80% of patients have genotype data available. On-study characteristics of patients with different genotypes will be compared. The significance of observed differences in proportions will be tested using the Chi-squared test and Fisher’s exact test when data are sparse. For continuous data, the Mann-Whitney test will be used to compare the medians of distributions. CNV data will be analyzed using CNV partition (Illumina Genome Studio software).