Family Trio-Based Whole Genome Optical Mapping Identifies Candidate Structural Variations Predisposing Children to Acute Lymphoblastic Leukemia

Germline mutations account for a substantial proportion of childhood cancer and may critically affect disease characteristics, therapy efficacy, severity of treatment side effects and patient outcome. To date, only 8-10% of childhood cancer cases can be explained by germline mutations identified in known cancer predisposing genes. This is in part due to the technical limitation of next generation short read sequencing, which detects single nucleotide variants, small deletions/insertions or simple copy number variations, but is not a reliable tool to identify larger structural variations (SVs, >500 bp) which are frequent in the human genome and may impact on disease predisposition.

Using whole genome optical mapping (WGOM) we aimed at identification of de novo and inherited germline SVs in a cohort of patients with clinically suspected cancer predisposition but without informative findings in short read sequencing analyses.

After informed consent we performed family trio based short read (2x 100 bp) whole exome sequencing (WES) on a HiSeq2500 (Illumina) and collected clinical and demographic data for a cohort of >100 families with children affected by cancer who were treated in our hospital. About 25% of the patients either (1) had a family history indicative of cancer susceptibility, or (2) had accompanying clinical findings (e.g. developmental delay, congenital anomalies) or (3) experienced excessive toxicity during chemotherapy. From this subgroup we selected four patients with acute lymphoblastic leukemia whose sequencing data and routine genetic workup were not informative of a known cancer predisposing syndrome and employed family trio-based next generation WGOM on a Saphyr instrument equipped with Access software (Bionano Genomics) to identify genomic SVs. To this end, we extracted and labeled high molecular weight DNA molecules at specific hexamer sequence motifs (average distance: 5 kb) using a DNA methyltransferase-based direct labeling reaction. Imaging was carried out on single-molecule level and each sample genome was de novo assembled from molecule data. Consensus genome maps were clustered into two alleles and diploid assemblies created. Genomes of patients were compared to parental genomes and the GRCh38 reference genome. SVs were inferred from de novo assemblies and genome comparisons with respect to quality scores, overall molecule coverage, fraction of molecules displaying the SV event, and chimeric DNA fragment mapping. Specific SV calls were compared to a set of > 160 human control samples (provided by Bionano Genomics) to filter against common SVs and potential artifacts. Filtered SVs were annotated using structural variant and gene databases.

Employing WGOM we analyzed DNA molecules 300.000 bp long on average and achieved genomic coverage ranging from 90-132x corresponding to 330-480 Gbp. For instance, for one patient, we obtained 1751 insertions, 624 deletions, 77 inversions, 21 duplications, 1 intra- and 2 inter-chromosomal translocations before filtering. The majority of these events (78%) were inherited from both parents. 20% were inherited from either father or mother and 2% were generated de novo. As the family history of this patient was inconspicuous for tumor diseases, we removed all inherited events and filtered against common variants. This resulted in only two candidate de novo lesions: a heterozygous 129,495 bp deletion framed by inversions (chr9: 66,156,733-66,622,623) in a gene-less region and a heterozygous inverted 352,667 bp duplication (chr22: 15,522,454-15.875,120) that spanned the genes OR11H, POTEH, POTEH-AS1, LINC01297, DUXAP8, and BMS1P22. Of these genes DUXAP8 is an oncogenic non-coding RNA of the homeobox gene family that has been associated with increased tumor growth and poorer prognosis in a wide variety of somatic cancers. It functions as a regulator of transcription by binding to key components of the developmental regulator epigenetic polycomb repressive complex 2 and may thus account for additional presentations of the child (dwarfism, accelerated skeletal age, linguistic developmental delay, morphological traits).

Our results indicate that WGOM is a useful technology to identify candidate SVs in children predisposed to cancer and developmental syndromes. Several candidates are currently being tested and the results will be presented.