Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays.

BACKGROUND: The recommended genomic DNA input requirements for whole genome single nucleotide polymorphism microarrays can limit the scope of molecular epidemiological studies. We performed a large-scale evaluation of whole genome amplified DNA as input into high-density, whole-genome Illumina® Infinium® SNP microarray. RESULTS: Overall, 6622 DNA samples from 5970 individuals were obtained from three distinct biospecimen sources and genotyped using gDNA and/or wgaDNA inputs. When genotypes from the same individual were compared with standard, native gDNA input amount, we observed 99.94% mean concordance with wgaDNA input. CONCLUSIONS: Our results demonstrate that carefully conducted studies with wgaDNA inputs can yield high-quality genotyping results. These findings should enable investigators to consider expansion of ongoing studies using high-density SNP microarrays, currently challenged by small amounts of available DNA.

Can incorporating molecular testing improve the accuracy of newborn screening for congenital adrenal hyperplasia?

BACKGROUND: Single-tier newborn screening (NBS) for CAH using 17-hydroxyprogesterone (17OHP) measured by fluoroimmunoassay (FIA) in samples collected at 24-48 hours produces a high false-positive rate (FPR). 2nd tier steroid testing can reduce the FPR and has been widely implemented. We investigated the accuracy of an alternative multi-tier CAH NBS protocol that incorporates molecular testing of the CYP21A2 gene and reduces the 1st tier 17OHP cutoff to minimize missed cases. METHODS: Created a Minnesota-specific CYP21A2 pathogenic variants panel; develop a rapid, high-throughput multiplex, allele-specific-primer-extension assay; perform 1-year retrospective analysis of Minnesota NBS results comparing metrics between a conventional steroid-based two-tier protocol and a molecular-based multi-tier NBS protocol, applied post-hoc. RESULTS: CYP21A2 gene sequencing of 103 Minnesota families resulted in a Minnesota-specific panel of 21 pathogenic variants. Centers for Disease Control and Prevention (CDC) created a molecular assay with 100% accuracy and reproducibility. Two-tier steroid-based screening of 68,659 live births during 2015 resulted in 2 false negatives (FNs), 91 FPs, and 1 true positive (TP). A three-tier protocol with a lower 1st-tier steroid cutoff, 2nd-tier 21-variant CYP21A2 panel and 3rd-tier CYP21A2 sequencing would have resulted in 0 FNs, 52 FPs and 3 TPs. CONCLUSIONS: Incorporation of molecular testing could improve the accuracy of CAH NBS, although some distinct challenges of molecular testing may need to be considered before implementation by NBS programs.

Genetic variants modify susceptibility to leukemia in infants: a Children's Oncology Group report.

BACKGROUND: The mixed lineage leukemia (MLL) gene is commonly rearranged in infant leukemia (IL). Genetic determinants of susceptibility to IL are unknown. Recent genome-wide association studies for childhood acute lymphoblastic leukemia (ALL) have identified susceptibility loci at IKZF1, ARID5B, and CEBPE. PROCEDURE: We genotyped these loci in 171 infants with leukemia and 384 controls and evaluated associations overall, by subtype [ALL, acute myeloid leukemia (AML)], and by presence (+) or absence (-) of MLL rearrangements. RESULTS: Homozygosity for a variant IKZF1 allele (rs11978267) increased risk of infant AML [Odds ratio (OR) = 3.9, 95% confidence interval (CI) = 1.8-8.4]; the increased risk was similar for AML/MLL+ and MLL- cases. In contrast, risk of ALL/MLL- was increased in infants homozygous for the IKZF1 variant (OR = 5.1, 95% CI = 1.8-14.5) but the variant did not modify risk of ALL/MLL+. For ARID5B (rs10821936), homozygosity for the variant allele increased risk for the ALL/MLL- subgroup only (OR = 7.2, 95% CI = 2.5-20.6). There was little evidence of an association with the CEBP variant (rs2239633). CONCLUSION: IKZF1 is expressed in early hematopoiesis, including precursor myeloid cells. Our data provide the first evidence that IKZF1 modifies susceptibility to infant AML, irrespective of MLL rearrangements, and could provide important new etiologic insights into this rare and heterogeneous hematopoietic malignancy.

Characteristics of responders to a request for a buccal cell specimen among survivors of childhood cancer and their siblings.

PURPOSE: Analysis of biological samples in large cohort studies may provide insight into the mechanism of, and risk factors for, disease onset and progression. METHODS: This study describes the methods used to collect biological samples from a large multi-center cohort of childhood cancer survivors and siblings of childhood cancer survivors and evaluates the predictors of a positive response among these individuals. RESULTS: Among survivors, female sex, white race/ethnicity, college graduation, never smoking, accessing the healthcare system in the past 2 years, and having a second malignant neoplasm were the strongest predictors of returning a sample. Among siblings, a similar demographic profile defined those likely to submit the requested sample. CONCLUSION: To reduce selection bias and increase the value of these samples for future analysis, additional phone calls and reminders targeting non-responders are needed to improve response rates among those least likely to respond to a single mailed request.

MDM2 polymorphism increases susceptibility to childhood acute myeloid leukemia: a report from the Children's Oncology Group.

BACKGROUND: The variant polymorphism in the gene MDM2, SNP309, leads to increased level of mdm2 protein and subsequent downregulation of p53 tumor suppressor pathway. Presence of this single nucleotide polymorphism (SNP) has been associated with earlier tumorigenesis in patients with Li-Fraumeni syndrome, as well as decreased survival in patients with CLL. In addition, cells homozygous (G/G) for SNP 309 were found to have 10-fold increase resistance to topoisomerase II inhibitors in vitro. PROCEDURE: We genotyped children (n = 575) with de novo acute myeloid leukemia (AML) treated on three Children's Oncology Group protocols (CCG 2941/2961/AAML 03P1) for the presence of SNP309. Healthy blood donors were genotyped as control population. RESULTS: The variant G/G genotype was associated with an increased susceptibility to AML (OR 1.5; P = 0.049). However, the presence of the variant allele at SNP309 did not modify disease response or toxicity in children treated on CCG protocols 2941/2961. CONCLUSIONS: The variant SNP 309 influences susceptibility to pediatric AML, but does not impact overall response to therapy.

Genomic Variants of Cytarabine Sensitivity Associated with Treatment-Related Mortality in Pediatric AML: A Report from the Children's Oncology Group.

PURPOSE: Cytarabine is an effective treatment for AML with associated toxicities including treatment related mortality (TRM). The purpose is to determine the clinical relevance of SNPs identified through the use of HapMap lymphoblastoid cell-based models, in predicting cytarabine response and toxicity in AML. EXPERIMENTAL DESIGN: We tested clinical significance of SNPs associated with cytarabine sensitivity in children with AML treated on Children's Oncology Group regimens (CCG 2941/2961). Endpoints included overall survival (OS), event-free survival (EFS), and TRM. Patients who received bone marrow transplant were excluded. We tested 124 SNPs associated with cytarabine sensitivity in HapMap cell lines in 348 children to determine whether any associated with treatment outcomes. In addition, we tested five SNPs previously associated with TRM in children with AML in our independent dataset of 385 children. RESULTS: Homozygous variant genotypes of rs2025501 and rs6661575 had increased in vitro cellular sensitivity to cytarabine and were associated with increased TRM. TRM was particularly increased in children with variant genotype randomized to high-dose cytarabine (rs2025501: P = 0.0024 and rs6661575 P = 0.0188). In analysis of previously reported SNPs, only the variant genotype rs17202778 C/C was significantly associated with TRM (P < 0.0001). CONCLUSIONS: We report clinical importance of two SNPs not previously associated with cytarabine toxicity. Moreover, we confirm that SNP rs17202778 significantly impacts TRM in pediatric AML. Cytarabine sensitivity genotypes may predict TRM and could be used to stratify to standard versus high-dose cytarabine regimens, warranting further study in prospective AML trials.

Genome-Wide Association Study to Identify Susceptibility Loci That Modify Radiation-Related Risk for Breast Cancer After Childhood Cancer.

Background: Childhood cancer survivors treated with chest-directed radiotherapy have substantially elevated risk for developing breast cancer. Although genetic susceptibility to breast cancer in the general population is well studied, large-scale evaluation of breast cancer susceptibility after chest-directed radiotherapy for childhood cancer is lacking. Methods: We conducted a genome-wide association study of breast cancer in female survivors of childhood cancer, pooling two cohorts with detailed treatment data and systematic, long-term follow-up: the Childhood Cancer Survivor Study and St. Jude Lifetime Cohort. The study population comprised 207 survivors who developed breast cancer and 2774 who had not developed any subsequent neoplasm as of last follow-up. Genotyping and subsequent imputation yielded 16 958 466 high-quality variants for analysis. We tested associations in the overall population and in subgroups stratified by receipt of lower than 10 and 10 or higher gray breast radiation exposure. We report P values and pooled per-allele risk estimates from Cox proportional hazards regression models. All statistical tests were two-sided. Results: Among survivors who received 10 or higher gray breast radiation exposure, a locus on 1q41 was associated with subsequent breast cancer risk (rs4342822, nearest gene PROX1 , risk allele frequency in control subjects [RAF controls ] = 0.46, hazard ratio = 1.92, 95% confidence interval = 1.49 to 2.44, P = 7.09 × 10 -9 ). Two rare variants also showed potentially promising associations (breast radiation ≥10 gray: rs74949440, 11q23, TAGLN , RAF controls = 0.02, P = 5.84 × 10 -8 ; <10 gray: rs17020562, 1q32.3, RPS6KC1 , RAF controls = 0.0005, P = 6.68 × 10 -8 ). Associations were restricted to these dose subgroups, with consistent findings in the two survivor cohorts. Conclusions: Our study provides strong evidence that germline genetics outside high-risk syndromes could modify the effect of radiation exposure on breast cancer risk after childhood cancer.

High-producer interleukin-2 genotype increases risk for acute graft-versus-host disease after unrelated donor bone marrow transplantation.

BACKGROUND: Cytokine polymorphisms may modulate immunologic reactivity, including graft-versus-host disease (GVHD). A single nucleotide polymorphism resulting in a thymine-to-guanine transition in the interleukin (IL)-2 gene promoter region occurs at position -330. In vitro studies have shown that the G allele is associated with early and sustained enhancement of IL-2 production, a so-called high-producer genotype. Because IL-2 is a proinflammatory cytokine, we hypothesized that recipients with high-producer genotypes would have increased frequency of GVHD after allogeneic bone marrow transplantation (BMT). METHODS: We studied 95 consecutive donor and recipient pairs who received an unrelated donor BMT at the University of Minnesota. The median age at time of BMT was 14.1 years (range 0.9-54.8 years). Stem cells were human leukocyte antigen-A, B, and DRB1 matched in 70 cases (74%) and single-antigen mismatched in 25 cases (26%). GVHD prophylaxis consisted of cyclosporine-containing regimens (53%), T-cell depletion by elutriation (42%), and others (2%). RESULTS: The probability of grade II-IV acute GVHD at day 100 was 36% (95% confidence interval 26%-46%) and was significantly affected by the presence of recipient IL-2 G allele. The probability of acute GVHD was 49% in 49 patients (52%) with at least one G allele compared with 24% in 42 patients (44%) with no G allele (P<0.01). In the Cox regression analysis, the presence of at least one IL-2 G allele was associated with a twofold increased risk of acute GVHD. CONCLUSIONS: If confirmed by others, our results indicate that more intensive GVHD prophylaxis is needed for patients with at least one IL-2 G allele, possibly directed toward blunting early host cell production of IL-2.

GST genotype may modify clinical phenotype in patients with Fanconi anaemia.

In the search for genetic modifiers of the Fanconi anaemia (FA) phenotype, we examined the role of polymorphism in three glutathione s-transferase genes (GSTT1, GSTM1 and GSTP1) in 356 FA patients enrolled in the International Fanconi Anaemia Registry (IFAR). Cellular sensitivity to 1,2:3,4 diepoxybutane was significantly increased in GSTT1 deleted compared with GSTT1 positive cases (median chromosomal breaks 11.1 vs. 8.3, P < 0.01) but there was no effect on clinical manifestations of FA. GSTM1 genotype significantly influenced time to bone marrow failure in complementation group FA-C, (median age 3.0 years vs. 7.0 years, P < 0.01). GSTP1 genotype did not influence FA phenotype.

Interleukin-1 genotype and outcome of unrelated donor bone marrow transplantation.

The interleukin 1 (IL-1) gene family includes three members (IL-1-alpha, IL-1-beta and IL-1Ra) that mediate immune and inflammatory responses through two specific cell surface receptors. Cytosine to thymine transitions at codons -889 and -511 in the IL-1-alpha and IL-1-beta genes, respectively, and an 86-base pair repeat in the IL-1Ra are believed to influence gene transcription. We have genotyped these three polymorphisms in 90 donor/recipient pairs undergoing unrelated donor bone marrow transplantation (BMT) at the University of Minnesota. We found no association between the occurrence of acute GVHD and donor and/or recipient polymorphisms of any of the three IL-1 genes. The presence of at least one IL-1alpha- 889 T allele in the donor was associated with significantly improved survival in univariate analysis (survival at 1 year 40% C/C donor, 68% T/C donor, 75% T/T donor, P < 0.01). Multiple regression analysis showed that if the donor and recipient each possessed the IL-1alpha T allele there was significantly improved survival [relative risk (RR) 0.2, P < 0.01] and decreased treatment-related mortality (TRM; RR 0.2, P = 0.01). The presence of the IL-1beta T allele in donor and recipient was also associated with improved survival (RR 0.2, P < 0.01) and decreased TRM (RR 0.1, P < 0.01). These data suggest that donor polymorphism in IL-1alpha and IL-1beta might influence survival after unrelated donor BMT, but does not alter risk of GVHD.

Glutathione S-transferase genotypes, genetic susceptibility, and outcome of therapy in childhood acute lymphoblastic leukemia.

The glutathione S-transferase (GST) genes are involved in the metabolism of environmental carcinogens and of some classes of chemotherapy drugs. GSTM1 and GSTT1 genotypes are polymorphic in humans, and the phenotypic absence of enzyme activity is caused by a homozygous inherited deletion of the gene. Previous, smaller studies of childhood acute lymphoblastic leukemia (ALL) provided contrasting data on the role of the GST genotype in susceptibility and treatment outcomes. We analyzed GST genotypes in 710 children with ALL treated by the Children's Cancer Group. Frequencies were compared with those of normal controls, and outcomes were analyzed according to genotype. Comparisons of gene frequencies in ALL case and control patients showed similar frequencies (54% vs 53% GSTM1 null in whites, P =.9; 40% versus 32% in blacks, P =.45; 16% versus 15% GSTT1 null in whites, P =.8; 17% versus 28% in blacks, P =.3). ALL was not associated with the GSTM1-null genotype or the double-null genotype in blacks or whites, in contrast to previous reports. Stratification of cases by age at diagnosis, sex, white blood cell count at diagnosis, B or T lineage, or cytogenetics revealed no differences in genotype frequencies. Analysis of treatment outcomes showed no differences in outcome according to GST genotype; in particular, there were no differences in frequencies of relapse at any site. These data, representing a larger series than any reported previously, suggest that GST genotype does not affect etiology or outcome of childhood ALL.

XRCC1 and glutathione-S-transferase gene polymorphisms and susceptibility to radiotherapy-related malignancies in survivors of Hodgkin disease.

BACKGROUND: One of the most serious late effects of treatment for childhood cancer is the occurrence of subsequent malignancy. Survivors of Hodgkin disease (HD), in particular, have been shown to be at high risk of subsequent malignancy, the occurrence of which has been associated strongly with exposure to radiotherapy. METHODS: In the current study, the authors investigated the association between polymorphisms in 3 genes--glutathione-S-transferase M1 (GSTM1), glutathione-S-transferase T1 (GSTT1), and XRCC1, with roles in protection from a variety of DNA-damaging agents-and the risk of subsequent malignancy in 650 survivors of HD enrolled in the Childhood Cancer Survivor Study who had received radiotherapy. RESULTS: Individuals lacking GSTM1 but not GSTT1 were at increased risk of any subsequent malignancy (odds ratio [OR], 1.5; 95% confidence interval [CI], 1.0-2.3), and for subsequent cancer within the radiation field (OR, 1.4; 95% CI, 0.9-2.1). A nonsignificant increased risk of thyroid carcinoma was observed in individuals lacking either GSTM1 (OR, 2.9; 95% CI, 0.8-10.9) or GSTT1 (OR, 3.7; 95% CI, 0.6-23.5). Individuals having the genotype of the arginine/glutamine polymorphism at codon 399 in the XRCC1 gene (R399) showed a nonsignificant increased risk of breast carcinoma compared with those without (OR, 1.4; 95% CI, 0.7-2.7), and a nonsignificant decreased risk against a subsequent thyroid carcinoma (OR, 0.6; 95% CI, 0.2-1.6). No differences in genotype frequencies were observed between survivors with basal cell carcinoma when compared with survivors without a subsequent cancer. CONCLUSIONS: These data illustrated the potential value of incorporating the collection of genomic DNA in longitudinal cohort studies of populations with well defined, potentially carcinogenic exposures. Evaluation of additional genetic polymorphisms in this cohort may help define genes that influence individual sensitivity to radiotherapy.