ArCH: improving the performance of clonal hematopoiesis variant calling and interpretation.

MOTIVATION: The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. RESULTS: Here, we developed ArCH, an Artifact filtering Clonal Hematopoiesis variant calling pipeline for detecting single nucleotide variants and short insertions/deletions by combining the output of four variant calling tools and filtering based on variant characteristics and sequencing error rate estimation. ArCH is an end-to-end cloud-based pipeline optimized to accept a variety of inputs with customizable parameters adaptable to multiple sequencing technologies, research questions, and datasets. Using deep targeted sequencing data generated from six acute myeloid leukemia patient tumor: normal dilutions, 31 blood samples with orthogonal validation, and 26 blood samples with technical replicates, we show that ArCH improves the sensitivity and positive predictive value of CH variant detection at low allele frequencies compared to standard application of commonly used variant calling approaches. AVAILABILITY AND IMPLEMENTATION: The code for this workflow is available at: https://github.com/kbolton-lab/ArCH.

Clonal Hematopoiesis in Clinical and Experimental Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Clonal hematopoiesis (CH), which results from an array of nonmalignant driver gene mutations, can lead to altered immune cell function and chronic disease, and has been associated with worse outcomes in patients with heart failure (HF) with reduced ejection fraction. However, the role of CH in the prognosis of HF with preserved ejection fraction (HFpEF) has been understudied. This study aimed to characterize CH in patients with HFpEF and elucidate its causal role in a murine model. METHODS: Using a panel of 20 candidate CH driver genes and a variant allele fraction cutoff of 0.5%, ultradeep error-corrected sequencing identified CH in a cohort of 81 patients with HFpEF (mean age, 71±6 years; ejection fraction, 63±5%) and 36 controls without a diagnosis of HFpEF (mean age, 74±7 years; ejection fraction, 61.5±8%). CH was also evaluated in a replication cohort of 59 individuals with HFpEF. RESULTS: Compared with controls, there was an enrichment of TET2-mediated CH in the HFpEF patient cohort (12% versus 0%, respectively; P=0.02). In the HFpEF cohort, patients with CH exhibited exacerbated diastolic dysfunction in terms of E/e' (14.9 versus 11.7, respectively; P=0.0096) and E/A (1.69 versus 0.89, respectively; P=0.0206) compared with those without CH. The association of CH with exacerbated diastolic dysfunction was corroborated in a validation cohort of individuals with HFpEF. In accordance, patients with HFpEF, an age ≥70 years, and CH exhibited worse prognosis in terms of 5-year cardiovascular-related hospitalization rate (hazard ratio, 5.06; P=0.042) compared with patients with HFpEF and an age ≥70 years without CH. To investigate the causal role of CH in HFpEF, nonconditioned mice underwent adoptive transfer with Tet2-wild-type or Tet2-deficient bone marrow and were subsequently subjected to a high-fat diet/L-NAME (Nω-nitro-l-arginine methyl ester) combination treatment to induce features of HFpEF. This model of Tet2-CH exacerbated cardiac hypertrophy by heart weight/tibia length and cardiomyocyte size, diastolic dysfunction by E/e' and left ventricular end-diastolic pressure, and cardiac fibrosis compared with the Tet2-wild-type condition. CONCLUSIONS: CH is associated with worse heart function and prognosis in patients with HFpEF, and a murine experimental model of Tet2-mediated CH displays greater features of HFpEF.

Pilot Study of Donor-Engrafted Clonal Hematopoiesis Evolution and Clinical Outcomes in Allogeneic Hematopoietic Cell Transplantation Recipients Using a National Registry.

Improved treatment options, such as reduced-intensity conditioning (RIC), enable older patients to receive potentially curative allogeneic hematopoietic cell transplantation (HCT). This progress has led to increased use of older HLA-matched sibling donors. An unintended potential risk associated with older donors is transplantation of donor cells with clonal hematopoiesis (CH) into patients. We aimed to determine the prevalence of CH in older HLA-matched sibling donors pretransplantation and to assess the clinical impact of donor-engrafted CH on HCT outcomes. This was an observational study using donor peripheral blood samples from the Center for International Blood and Marrow Transplant Research repository, linked with corresponding recipient outcomes. To explore engraftment efficiency and evolution of CH mutations following HCT, recipient follow-up samples available through the Bone Marrow Transplant Clinical Trials Network (Protocol 1202) were included. Older donors and patients (both ≥55 years) receiving first RIC HCT for myeloid malignancies were eligible. DNA from archived donor blood samples was used for targeted deep sequencing to identify CH. The associations between donor CH status and recipient outcomes, including acute graft-versus-host disease (aGVHD), chronic GVHD (cGVHD), overall survival, relapse, nonrelapse mortality, disease-free survival, composite GVHD-free and relapse-free survival, and cGVHD-free and relapse-free survival, were analyzed. A total of 299 donors were successfully sequenced to detect CH. At a variant allele frequency (VAF) ≥2%, there were 44 CH mutations in 13.7% (41 of 299) of HLA-matched sibling donors. CH mostly involved DNMT3A (n = 27; 61.4%) and TET2 (n= 9; 20.5%). Post-HCT samples from 13 recipients were also sequenced, of whom 7 had CH+ donors. All of the donor CH mutations (n = 7/7; 100%) were detected in recipients at day 56 or day 90 post-HCT. Overall, mutation VAFs remained relatively constant up to day 90 post-HCT (median change, .005; range, -.008 to .024). Doubling time analysis of recipient day 56 and day 90 data showed that donor-engrafted CH mutations initially expand then decrease to a stable VAF; germline mutations had longer doubling times than CH mutations. The cumulative incidence of grade II-IV aGVHD at day 100 was higher in HCT recipients with CH+ donors (37.5% versus 25.1%); however, the risk for aGVHD by donor CH status did not reach statistical significance (hazard ratio, 1.35; 95% confidence interval, .61 to 3.01; P = .47). There were no statistically significant differences in the cumulative incidence of cGVHD or any secondary outcomes by donor CH status. In subset analysis, the incidence of cGVHD was lower in recipients of grafts from DNMT3A CH+ donors versus donors without DNMT3A CH (34.4% versus 57%; P = .035). Donor cell leukemia was not reported in any donor-recipient pairs. CH in older HLA-matched sibling donors is relatively common and successfully engrafts and persists in recipients. In a homogenous population (myeloid malignancies, older donors and recipients, RICr, non-cyclophosphamide-containing GVHD prophylaxis), we did not detect a difference in cGVHD risk or other secondary outcomes by donor CH status. Subgroup analyses suggest potential differential effects by clinical characteristics and CH mutations. Larger prospective studies are needed to robustly determine which subsets of patients and CH mutations elicit meaningful impacts on clinical outcomes.

KMT2A-D pathogenicity, prevalence, and variation according to a population database.

INTRODUCTION: The KMT2 family of genes is essential epigenetic regulators promoting gene expression. The gene family contains three subgroups, each with two paralogues: KMT2A and KMT2B; KMT2C and KMT2D; KMT2F and KMT2G. KMT2A-D are among the most frequent somatically altered genes in several different cancer types. Somatic KMT2A rearrangements are well-characterized in infant leukemia (IL), and growing evidence supports the role of additional family members (KMT2B, KMT2C, and KMT2D) in leukemogenesis. Enrichment of rare heterozygous frameshift variants in KMT2A and C has been reported in acute myeloid leukemia (AML), IL, and solid tumors. Currently, the non-synonymous variation, prevalence, and penetrance of these four genes are unknown. METHODS: This study determined the prevalence of pathogenic/likely pathogenic (P/LP) germline KMT2A-D variants in a cancer-free adult population from the Genome Aggregation Database (gnomAD). Two methods of variant interpretation were utilized: a manual genomic variant interpretation and an automated ACMG pipeline. RESULTS: The ACMG pipeline identified considerably fewer P/LP variants (n = 89) compared to the manual method (n = 660) in all 4 genes. Consequently, the total P/LP prevalence and allele frequency (AF) were higher in the manual method (1:112, AF = 4.46E-03) than in ACMG (1:832, AF = 6.01E-04). Multiple ancestry-exclusive P/LP variants were identified along with an increased frequency in males compared to females. Many of these variants identified in this population database are also associated with severe juvenile conditions. CONCLUSION: These data demonstrate that putatively functional germline variation in these developmentally important genes is more common than previously appreciated and identification in cancer-free adults may indicate incomplete penetrance for many of these variants. Future research should examine a genetic predisposing role in IL and other pediatric cancers.

Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification.

Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.

Germline Sequencing Identifies Rare Variants in Finnish Subjects with Familial Germ Cell Tumors.

PURPOSE: Pediatric germ cell tumors are rare, representing about 3% of childhood malignancies in children less than 15 years of age, presenting in neonates or adolescents with a greater incidence noted in older adolescents. Aberrations in primordial germ cell proliferation/differentiation can lead to a variety of neoplasms, including teratomas, embryonal carcinoma, choriocarcinoma, and yolk sac tumors. PATIENTS AND METHODS: Three Finnish families with varying familial germ cell tumors were identified, and whole-genome sequencing was performed using an Illumina sequencing platform. In total, 22 unique subjects across the three families were sequenced. Family 1 proband (female) was affected by malignant ovarian teratoma, Family 2 proband (female) was affected by sacrococcygeal teratoma with yolk sac tumor in the setting of Cornelia de Lange syndrome, and Family 3 proband (male) was affected by malignant testicular teratoma. Rare variants were identified using an autosomal recessive or de novo model of inheritance. RESULTS: For family 1 proband (female), an autosomal recessive or de novo model of inheritance identified variants of interest in the following genes: CD109, IKBKB, and CTNNA3, SUPT6H, MUC5AC, and FRG1. Family 2 proband (female) analysis identified gene variants of interest in the following genes: LONRF2, ANO7, HS6ST1, PRB2, and DNM2. Family 3 proband (male) analysis identified the following potential genes: CRIPAK, KRTAP5-7, and CACNA1B. CONCLUSION: Leveraging deep pedigrees and next-generation sequencing, rare germline variants were identified that were enriched in three families from Finland with a history of familial germ cell tumors. The data presented support the importance of germline mutations when analyzing complex cancers with a low somatic mutation landscape.

Strong concordance between RNA structural and single nucleotide variants identified via next generation sequencing techniques in primary pediatric leukemia and patient-derived xenograft samples.

Acute leukemia represents the most common pediatric malignancy comprising diverse subtypes with varying prognosis and treatment outcomes. New and targeted treatment options are warranted for this disease. Patient-derived xenograft (PDX) models are increasingly being used for preclinical testing of novel treatment modalities. A novel approach involving targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was employed to compare 25 primary pediatric acute leukemia samples and their corresponding PDX samples. A comparison of the primary samples and PDX samples revealed a high concordance between single nucleotide variants and gene fusions whereas other complex structural variants were not as consistent. The presence of gene fusions representing the major driver mutations at similar allelic frequencies in PDX samples compared to primary samples and over multiple passages confirms the utility of PDX models for preclinical drug testing. Characterization and tracking of these novel cryptic fusions and exonal variants in PDX models is critical in assessing response to potential new therapies.

The evolutionary dynamics and fitness landscape of clonal hematopoiesis.

Somatic mutations acquired in healthy tissues as we age are major determinants of cancer risk. Whether variants confer a fitness advantage or rise to detectable frequencies by chance remains largely unknown. Blood sequencing data from ~50,000 individuals reveal how mutation, genetic drift, and fitness shape the genetic diversity of healthy blood (clonal hematopoiesis). We show that positive selection, not drift, is the major force shaping clonal hematopoiesis, provide bounds on the number of hematopoietic stem cells, and quantify the fitness advantages of key pathogenic variants, at single-nucleotide resolution, as well as the distribution of fitness effects (fitness landscape) within commonly mutated driver genes. These data are consistent with clonal hematopoiesis being driven by a continuing risk of mutations and clonal expansions that become increasingly detectable with age.

Error-corrected sequencing strategies enable comprehensive detection of leukemic mutations relevant for diagnosis and minimal residual disease monitoring.

BACKGROUND: Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. METHODS: Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4-11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. RESULTS: Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. CONCLUSIONS: Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

Engraftment of rare, pathogenic donor hematopoietic mutations in unrelated hematopoietic stem cell transplantation.

Clonal hematopoiesis is associated with various age-related morbidities. Error-corrected sequencing (ECS) of human blood samples, with a limit of detection of ≥0.0001, has demonstrated that nearly every healthy individual >50 years old harbors rare hematopoietic clones below the detection limit of standard high-throughput sequencing. If these rare mutations confer survival or proliferation advantages, then the clone(s) could expand after a selective pressure such as chemotherapy, radiotherapy, or chronic immunosuppression. Given these observations and the lack of quantitative data regarding clonal hematopoiesis in adolescents and young adults, who are more likely to serve as unrelated hematopoietic stem cell donors, we completed this pilot study to determine whether younger adults harbored hematopoietic clones with pathogenic mutations, how often those clones were transferred to recipients, and what happened to these clones over time after transplantation. We performed ECS on 125 blood and marrow samples from 25 matched unrelated donors and recipients. Clonal mutations, with a median variant allele frequency of 0.00247, were found in 11 donors (44%; median, 36 years old). Of the mutated clones, 84.2% of mutations were predicted to be molecularly pathogenic and 100% engrafted in recipients. Recipients also demonstrated de novo clonal expansion within the first 100 days after hematopoietic stem cell transplant (HSCT). Given this pilot demonstration that rare, pathogenic clonal mutations are far more prevalent in younger adults than previously appreciated, and they engraft in recipients and persist over time, larger studies with longer follow-up are necessary to correlate clonal engraftment with post-HSCT morbidity.

The Zebrafish Xenograft Platform-A Novel Tool for Modeling KSHV-Associated Diseases.

Kaposi's sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.

Novel mutation in AIRE gene with autoimmune polyendocrine syndrome type 1.

PURPOSE: Autoimmune polyendocrine type 1 (APS-1) is a complex inherited autosomal recessive disorder. Classically, it appears within the first decade of life followed by adrenocortical insufficiency, mucocutaneous candidiasis, Addison's disease, and hypoparathyroidism. The clinical phenotype of APS-1 varies depending upon mutations in the autoimmune regulator gene (AIRE) on chromosome 21q22.3. METHODS: In this study, we performed Sanger sequencing ofAIRE in Iranian patients to identify different variants and probable new mutations corresponding to a clinical diagnosis of APS-1. RESULTS: After analyzing 14AIRE exons, we detected a novel insertion mutation in exon 2 in a patient who presented with severe APS-1, Lys50AsnfsX168. Furthermore, the known mutations in AIRE, including Arg139X, Arg257X, and Leu323SerfsX51, were detected in enrolled patients. DISCUSSION: According to our results, sequencing analysis ofAIRE provides a useful screening method to diagnose patients with incomplete or unusual clinical presentations of APS-1.

Clonal hematopoiesis and risk of acute myeloid leukemia.

Nearly all adults harbor acute myeloid leukemia (AML)-related clonal hematopoietic mutations at a variant allele fraction (VAF) of ≥0.0001, yet relatively few develop hematologic malignancies. We conducted a nested analysis in the Nurses' Health Study and Health Professionals Follow-Up Study blood subcohorts, with up to 22 years of follow up to investigate associations of clonal mutations of ≥0.0001 allele frequency with future risk of AML. We identified 35 cases with AML that had pre-diagnosis peripheral blood samples and matched two controls without history of cancer per case by sex, age, and ethnicity. We conducted blinded error-corrected sequencing on all study samples and assessed variant-associated risk using conditional logistic regression. We detected AML-associated mutations in 97% of all participants (598 mutations, 5.8/person). Individuals with mutations ≥0.01 variant allele fraction had a significantly increased AML risk (OR 5.4, 95%CI: 1.8-16.6), as did individuals with higher-frequency clones and those with DNMT3A R882H/C mutations. The risk of lower-frequency clones was less clear. In the 11 case-control sets with samples banked ten years apart, clonal mutations rarely expanded over time. Our findings are consistent with published evidence that detection of clonal mutations ≥0.01 VAF identifies individuals at increased risk for AML. Further study of larger populations, mutations co-occurring within the same pre-leukemic clone and other risk factors (lifestyle, epigenetics, etc.), are still needed to fully elucidate the risk conferred by low-frequency clonal hematopoiesis in asymptomatic adults.

Distal chromosome 16p11.2 duplications containing SH2B1 in patients with scoliosis.

INTRODUCTION: Adolescent idiopathic scoliosis (AIS) is a common musculoskeletal disorder with strong evidence for a genetic contribution. CNVs play an important role in congenital scoliosis, but their role in idiopathic scoliosis has been largely unexplored. METHODS: Exome sequence data from 1197 AIS cases and 1664 in-house controls was analysed using coverage data to identify rare CNVs. CNV calls were filtered to include only highly confident CNVs with >10 average reads per region and mean log-ratio of coverage consistent with single-copy duplication or deletion. The frequency of 55 common recurrent CNVs was determined and correlated with clinical characteristics. RESULTS: Distal chromosome 16p11.2 microduplications containing the gene SH2B1 were found in 0.7% of AIS cases (8/1197). We replicated this finding in two additional AIS cohorts (8/1097 and 2/433), resulting in 0.7% (18/2727) of all AIS cases harbouring a chromosome 16p11.2 microduplication, compared with 0.06% of local controls (1/1664) and 0.04% of published controls (8/19584) (p=2.28×10-11, OR=16.15). Furthermore, examination of electronic health records of 92 455 patients from the Geisinger health system showed scoliosis in 30% (20/66) patients with chromosome 16p11.2 microduplications containing SH2B1 compared with 7.6% (10/132) of controls (p=5.6×10-4, OR=3.9). CONCLUSIONS: Recurrent distal chromosome 16p11.2 duplications explain nearly 1% of AIS. Distal chromosome 16p11.2 duplications may contribute to scoliosis pathogenesis by directly impairing growth or by altering expression of nearby genes, such as TBX6. Individuals with distal chromosome 16p11.2 microduplications should be screened for scoliosis to facilitate early treatment.

Special Therapy and Psychosocial Needs Identified in a Multidisciplinary Cancer Predisposition Syndrome Clinic.

Identification of patients with cancer predisposition syndromes (CPSs) can provide vital information to guide care of an existing cancer, survey for future malignancy, and counsel families. The same underlying mutation responsible for a CPS may also result in other phenotypic abnormalities amenable to therapeutic intervention. The purpose of this study was to examine patients followed in our multidisciplinary CPS clinic to determine the prevalence and scope of medical and psychosocial needs. Data from a baseline evaluation of a single-center patient registry was reviewed. Eligible patients included those with a known or suspected CPS. Over 3 years, 73 patients consented and had successful follow-up. Utilization rate of special therapies, defined as speech therapy, occupational therapy, and/or physical therapy, in the CPS population was 50.7%, significantly higher than a representative sample of children with special needs. Prevalence of 504/IEP (Individualized Education Program) utilization was 20.5%. Patients with CPSs have a high prevalence of medical and psychosocial needs beyond their risk for cancer, for which early screening for necessary interventions should be offered to maximize the patient's developmental potential. Future research is needed to further define the developmental and cognitive phenotypes of these syndromes, and to evaluate the effectiveness of subsequent interventions.

Age-specific changes in genome-wide methylation enrich for Foxa2 and estrogen receptor alpha binding sites.

The role of DNA methylation patterns in complex phenotypes remains unclear. To explore this question, we adapted our methods for rare variant analysis to characterize genome-wide murine DNA hybridization array to investigate methylation at CpG islands, shores, and regulatory elements. We have applied this platform to compare age and tissue- specific methylation differences in the brain and spleen of young and aged mice. As expected from prior studies, there are clear global differences in organ-specific, but not age-specific, methylation due mostly to changes at repetitive elements. Surprisingly, out of 200,000 loci there were only 946 differentially methylated cytosines (DMCs) between young and old samples (529 hypermethylated, 417 hypomethylated in aged mice) compared to thousands of tissue-specific DMCs. Hypermethylated loci were clustered around the promoter region of Sfi1, exon 2 of Slc11a2, Drg1, Esr1 and Foxa2 transcription factor binding sites. In particular, there were 75 hypermethylated Foxa2 binding sites across a 2.7 Mb region of chromosome 11. Hypomethylated loci were clustered around Mid1, Isoc2b and genome-wide loci with binding sites for Foxa2 and Esr1, which are known to play important roles in development and aging. These data suggest discreet tissue-independent methylation changes associated with aging processes such as cell division (Sfi1, Mid1), energy production (Drg1, Isoc2b) and cell death (Foxa2, Esr1).

Rare Event Detection Using Error-corrected DNA and RNA Sequencing.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5-2.0%. Thus, standard NGS has a limit of detection for mutations that are >0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is <0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Parental Age and Risk of Infant Leukaemia: A Pooled Analysis.

BACKGROUND: Infant leukaemia (IL) is extremely rare with fewer than 150 cases occurring each year in the United States. Little is known about its causes. However, recent evidence supports a role of de novo mutations in IL aetiology. Parental age has been associated with several adverse outcomes in offspring, including childhood cancers. Given the role of older parental age in de novo mutations in offspring, we carried out an analysis of parental age and IL. METHODS: We evaluated the relationship between parental age and IL in a case-control study using registry data from New York, Minnesota, California, Texas, and Washington. Records from 402 cases [219 acute lymphoblastic leukaemia (ALL), 131 acute myeloid leukaemia (AML), and 52 other] and 45 392 controls born during 1981-2004 were analysed. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression. Estimates were adjusted for infant sex, birth year category, maternal race, state, and mutually adjusted for paternal or maternal age, respectively. RESULTS: Infants with mothers' age ≥40 years had an increased risk of developing AML (OR 4.80, 95% CI 1.80, 12.76). In contrast, paternal age <20 was associated with increased risk of ALL (OR 3.69, 95% CI 1.62, 8.41). CONCLUSION: This study demonstrates increased risk of infant ALL in relation to young paternal age. Given record linkage, there is little concern with recall or selection bias, although data are lacking on MLL gene status and other potentially important variables. Parent of origin effects, de novo mutations, and/or carcinogenic exposures may be involved in IL aetiology.