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TIRAP, a novel familial lymphoma risk gene
Population-based studies reported an increased risk for DLBCL in relatives of individuals with DLBCL, and genome-wide association studies identified several com- mon single nucleotide variants associated with sporadic DLBCL.15-18 Familial clustering provides evidence for Mendelian susceptibility. In very rare cases, familial aggre- gation is associated with hereditary cancer syndromes,18 but as far as other syndromes are concerned, a heritable basis for DLBCL is not fully understood. A germline vari- ant in MLL described in a Finnish family is still the only reported variant linked to familial PMBL.19 Although familial lymphomas account for less than 5% of cases, these pedigrees are a valuable tool to help identify risk genes that might also contribute to a better understanding of more frequent sporadic cases.
Here, we investigate a Swiss/Japanese family in which 2 out of 3 children were diagnosed with aggressive B-cell lymphomas arising in the mediastinum. Whole exome sequencing (WES) on the germline DNA of the affected siblings and healthy family members identified a variant in the TIR-domain-containing adaptor protein (TIRAP). TIRAP engages signals from TLR2 and TLR4 receptors and recruits MyD88 to the plasma membrane mediated through Toll/interleukin-1 receptor (TIR) domain interac- tion.20 Downstream signaling includes activation of IL-1R-associated kinases (IRAK), ultimately culminating in the activation of the transcription factors NF-kB and AP-1. In this family, we identified an inherited TIRAP p.R81C variant in 2 affected siblings. This variant provid- ed B-cells with increased proliferation and survival through enhanced NF-kB activity. Functional studies revealed that TIRAP p.R81C enhanced NF-kB gene signa- ture and reduced stress-triggered cell death. Collectively, we provide evidence that TIRAP p.R81C may act as a novel lymphoma risk variant and our data suggest that TIRAP should be integrated into the complex network of genes contributing to deregulated NF-kB signaling involved in lymphomagenesis.
Methods
Patients
Samples from patients, non-affected family members, and healthy donors were collected after informed consent. This study was approved by the local ethics committee (KEK-BE116/11) and was conducted in accordance with the Declaration of Helsinki. The diagnosis of DLBCL/PMBL was made according to the 2017 World Health Organization classification and pathological review by SD and AT confirmed the diagnosis.1 Genomic DNA was ex- tracted from peripheral blood mononuclear cells (PBMCs) using standard methods. Tumor DNA was isolated from formalin-fixed paraffin embedded tissues (FFPE) using phenol-chloroform. In FFPE samples, tumor cell-rich areas were identified on CD20 stained sections and separated from surrounding tissue by laser microdissection.
Whole exome sequencing
The quality of extracted DNA was assessed by Bioanalyzer (Agilent) and a PCR fragment size-based assay developed at Fasteris (Geneva, Switzerland). Prior to library preparation with TruSeq DNA Sample Preparation Kit (Illumina), DNA samples were treated with PreCR Repair Mix (New England Biolabs). Exome capturing was performed using TruSeq Exome Enrichment Kit (Illumina) and samples were sequenced on an Illumina
HiSeq2500 instrument with 100bp paired-end reads (Fasteris, sequencing performed between 2012 and 2014). As the FFPE lym- phoma sample of sister 2 resulted in a low-yield library of poor quality, the library preparation was modified: after PreCR Repair mix treatment, DNA was split, and four libraries were prepared simultaneously using the Nextera Exome Enrichment Kit (Illumina). Before exome enrichment, libraries were pooled and sequencing was performed as described above. Both exome enrichment kits contained the same set of baits, resulting in iden- tical exome coverage. Whole exome sequencing (WES) data has been deposited at the European Nucleotide Archive (http://www.ebi.ac.uk/ena, accession number PRJEB15254). See Online Supplementary Table S3 for exome data metrics.
Analysis of germline and somatic variants
Raw sequence read quality was assessed using FastQC. Reads were mapped to the human reference genome hg19 using Bowtie- 2 v.2.2.1, and duplicated reads were removed by Pi-card-tools v.1.80. Germline variant calling was performed using the Genome Analysis Toolkit (GATK v.3.3.0) best practices workflow using Haplotype Caller and limiting the analysis to enriched targets ±100bp. We used GATK v.3.3.0 to recalibrate the variant quality and refine the genotypes using population (1000 Genomes Project, phase 1 data) and pedigree information. Variants in low complex- ity regions were removed.21 Germline variants were prioritized as following: 1) good quality genotype in both sisters (Phred quality ≥20); 2) moderate/high impact based on SnpEff v.3.2 prediction; 3) novel/known variant at frequency of less than 1% (not polymor- phisms) according to the 1000 Genomes Database and the Exome Variant Server; and 4) the presence of at least one copy of the puta- tively harmful allele in both siblings. Only variants not present as homozygote in healthy family members were selected. Possible links between genes with germline variants and terms related to cancer and malignant lymphomas were assessed by Ariadne Genomics Pathway Studio v.9 (Elsevier). Alterations with a pre- dicted link to the disease were annotated with PolyPhen-2, SIFT, MutationTaster and GERP++ effect prediction scores using dbNSFP v.2.1, and Combined Annotation Dependent Depletion (CADD) scores v.1.1 (available from https://cadd. gs.washington.edu/). Pathway Studio was used to identify gene net- works and canonical pathway enriched for genes containing puta- tively deleterious variants. The enrichment-scores were calculated using c2 test comparing genes with putatively deleterious muta- tions to the proportion of background genes in the Gene Ontology group. An enrichment-score ≥3 corresponded to a significant link (P<0.05). See also Online Supplementary Appendix.
Results
Clinical and histopathological characterization of the mediastinal B-cell lymphomas
The Swiss/Japanese family investigated includes 2 female siblings with lymphomas (Figure 1). The older sis- ter 1 developed a PMBL at 30 years of age and died with primary progressive disease (Online Supplementary Table S1). At 25 years of age, sister 2 was diagnosed with a stage IIA non-germinal center (GC) DLBCL, not otherwise spec- ified (NOS) with a mediastinal mass and cervical lym- phadenopathy. Chemo-immunotherapy achieved an ongoing remission. Smoldering myelomas IgGλ were detected in the father and his monozygotic twin at 65 years of age. Other family members are currently healthy, and there are no other hematologic malignancies in the extended family.
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