Discovery of novel predisposing coding and noncoding variants in familial Hodgkin lymphoma.

Familial aggregation of Hodgkin lymphoma (HL) has been demonstrated in large population studies, pointing to genetic predisposition to this hematological malignancy. To understand the genetic variants associated with the development of HL, we performed whole genome sequencing on 234 individuals with and without HL from 36 pedigrees that had 2 or more first-degree relatives with HL. Our pedigree selection criteria also required at least 1 affected individual aged <21 years, with the median age at diagnosis of 21.98 years (3-55 years). Family-based segregation analysis was performed for the identification of coding and noncoding variants using linkage and filtering approaches. Using our tiered variant prioritization algorithm, we identified 44 HL-risk variants in 28 pedigrees, of which 33 are coding and 11 are noncoding. The top 4 recurrent risk variants are a coding variant in KDR (rs56302315), a 5' untranslated region variant in KLHDC8B (rs387906223), a noncoding variant in an intron of PAX5 (rs147081110), and another noncoding variant in an intron of GATA3 (rs3824666). A newly identified splice variant in KDR (c.3849-2A>C) was observed for 1 pedigree, and high-confidence stop-gain variants affecting IRF7 (p.W238∗) and EEF2KMT (p.K116∗) were also observed. Multiple truncating variants in POLR1E were found in 3 independent pedigrees as well. Whereas KDR and KLHDC8B have previously been reported, PAX5, GATA3, IRF7, EEF2KMT, and POLR1E represent novel observations. Although there may be environmental factors influencing lymphomagenesis, we observed segregation of candidate germline variants likely to predispose HL in most of the pedigrees studied.

Genome-wide DNA methylation profiling in chronic lymphocytic leukaemia.

Background: DNA methylation aberrations are widespread among the malignant B lymphocytes of patients with chronic lymphocytic leukaemia (CLL), suggesting that DNA methylation might contribute to the pathogenesis of CLL. Aim: We aimed to explore the differentially methylated positions (DMPs) associated with CLL and screen the differentially methylated and expressed genes (DMEGs) by combining public databases. We aimed to observe the direction of each DMEG in CLL based on the DMPs in the promoter and the body region respectively to narrow down DMEGs. We also aimed to explore the methylation heterogeneity of CLL subgroups and the effect of B cells maturation on CLL. Methods: In this population-based case control study, we reported a genome-wide DNA methylation association study using the Infinium HumanMethylation450 BeadChip, profiling the DNA methylation of CD19+ B Cells from 48 CLL cases and 28 healthy controls. By integrating methylation data and expression data from public databases, gene sets were jointly screened, and then the relationship between methylation sites in promoter and body region and expression of each gene was explored. In addition, support vector machine (SVM) classification algorithm was used to identify subgroups of CLL cases based on methylation pattern, and the effect of B-cell differentiation related methylation sites on CLL-related sites was observed. Results: We identified 34,797 DMPs related to CLL across the genome, most of which were hypomethylated; the majority were located in gene body regions. By combining these DMPs with published DNA methylation and RNA sequencing data, we detected 26,244 replicated DMPs associated with 1,130 genes whose expression were significantly different in CLL cases. Among these DMEGs, nine low expressed DMEGs were selected with hypermethylated in promoter and hypomethylated in body region, and 83 high expressed DMEGs were selected with both hypomethylated in promoter and body region. The 48 CLL cases were divided into 3 subgroups based on methylation site by SVM algorithm. Over 92% of CpGs associated with B cell subtypes were found in CLL-related DMPs. Conclusion: The DNA methylation pattern was altered across the genome in CLL patients. The methylation of ZAP70, FMOD, and ADAMTS17 was significantly different between CLL cases and controls. Further studies are warranted to confirm our findings and identify the underlying mechanisms through which these methylation markers are associated with CLL.

Association of elevated serumfree light chains with chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis.

Chronic lymphocytic leukemia (CLL) and its precursor, monoclonal B-cell lymphocytosis (MBL), are heritable. Serumfree light-chain (sFLC) measures are a prognostic factor for CLL, but their role in susceptibility to CLL is not clear. We investigated differences between sFLC measurements in pre-treatment serum from five groups to inform the association of sFLC with familial and sporadic CLL: (1) familial CLL (n = 154), (2) sporadic CLL (n = 302), (3) familial MBL (n = 87), (4) unaffected first-degree relatives from CLL/MBL families (n = 263), and (5) reference population (n = 15,396). The percent of individuals having elevated monoclonal and polyclonal sFLCs was compared using age-stratified and age- and sex-adjusted logistic regression models. In age groups >50 years, monoclonal sFLC elevations were increased in sporadic and familial CLL cases compared to the reference population (p's < 0.05). However, there were no statistically significant differences in sFLC monoclonal or polyclonal elevations between familial and sporadic CLL cases (p's > 0.05). Unaffected relatives and MBL cases from CLL/MBL families, ages >60 years, showed elevated monoclonal sFLC, compared to the reference population (p's < 0.05). This is the first study to demonstrate monoclonal sFLC elevations in CLL cases compared to controls. Monoclonal sFLC levels may provide additional risk information in relatives of CLL probands.

Parental longevity and survival among patients with multiple myeloma and monoclonal gammopathy of undetermined significance: a population-based study.

Parental longevity is associated with an increased life expectancy; results with regard to specific diseases are conflicting. There are limited data focusing on host characteristics and their effect on survival among multiple myeloma (MM) patients and individuals with monoclonal gammopathy of undetermined significance (MGUS). Therefore, our aim was to evaluate the impact of parental longevity on survival of patients with MM and MGUS. A total of 4675 patients with MM, 6812 MGUS patients and 13 398 population-based controls for MM as well as 19 110 controls for MGUS, from 1988 to 2013, were included in the study. Longevity was defined as >90 years of age. Among MM patients, parental longevity was associated with a decreased risk of death [hazard ratio (HR) = 0·92, 95% confidence interval (CI) 0·84-0·99] and the same was true for MGUS patients (HR = 0·87, 95% CI 0·78-0·96). Having one long lived parent significantly decreased the risk of death in both groups, but was not statistically significant when both parents exceeded 90 years of age. In conclusion, parental longevity decreases the risk of death for patients with MM and MGUS which may reflect the importance of the host's genetic and environmental factors in relation to survival.

Sex-related DNA methylation differences in B cell chronic lymphocytic leukemia.

BACKGROUND: Men are at higher risk of developing chronic lymphocytic leukemia (CLL) than women. DNA methylation has been shown to play important roles in a number of cancers. There are differences in the DNA methylation pattern between men and women. In this study, we investigated whether this contributes to the sex-related difference of B cell CLL risk. METHODS: Using the HumanMethylation450 BeadChip, we profiled the genome-wide DNA methylation pattern of CD19+ B cells from 48 CLL patients (29 female patients and 19 male patients) and 28 healthy people (19 women and 9 men). RESULTS: We identified 1043 sex-related differentially methylated positions (DMPs) related to CLL, 56 of which are located on autosomes and 987 on the X chromosome. Using published B cell RNA-sequencing data, we found 18 genes covered by the DMPs also have different expression levels in male and female CLL patients. Among them, TRIB1, an autosome gene, has been shown to promote tumor growth by suppressing apoptosis. CONCLUSIONS: Our study represents the first epigenome-wide association study (EWAS) that investigates the sex-related differences in cancer, and indicated that DNA methylation differences might contribute to the sex-related difference in CLL risk.

Prevalence of pathogenic/likely pathogenic variants in the 24 cancer genes of the ACMG Secondary Findings v2.0 list in a large cancer cohort and ethnicity-matched controls.

BACKGROUND: Prior research has established that the prevalence of pathogenic/likely pathogenic (P/LP) variants across all of the American College of Medical Genetics (ACMG) Secondary Findings (SF) genes is approximately 0.8-5%. We investigated the prevalence of P/LP variants in the 24 ACMG SF v2.0 cancer genes in a family-based cancer research cohort (n = 1173) and in cancer-free ethnicity-matched controls (n = 982). METHODS: We used InterVar to classify variants and subsequently conducted a manual review to further examine variants of unknown significance (VUS). RESULTS: In the 24 genes on the ACMG SF v2.0 list associated with a cancer phenotype, we observed 8 P/LP unique variants (8 individuals; 0.8%) in controls and 11 P/LP unique variants (14 individuals; 1.2%) in cases, a non-significant difference. We reviewed 115 VUS. The median estimated per-variant review time required was 30 min; the first variant within a gene took significantly (p = 0.0009) longer to review (median = 60 min) compared with subsequent variants (median = 30 min). The concordance rate was 83.3% for the variants examined by two reviewers. CONCLUSION: The 115 VUS required database and literature review, a time- and labor-intensive process hampered by the difficulty in interpreting conflicting P/LP determinations. By rigorously investigating the 24 ACMG SF v2.0 cancer genes, our work establishes a benchmark P/LP variant prevalence rate in a familial cancer cohort and controls.

Germline mutations in Protection of Telomeres 1 in two families with Hodgkin lymphoma.

In a previous whole exome sequencing of patients from 41 families with Hodgkin lymphoma, we identified two families with distinct heterozygous rare coding variants in POT1 (D224N and Y36H), both in a highly conserved region of the gene. POT1 D224N mutant did not bind to a single-stranded telomere oligonucleotide in vitro suggesting the mutation perturbs POT1's ability to bind to the telomeric G-rich overhang. Human HT1080 cells expressing POT1 D224N and lymphoblastoid cells carrying Y36H both showed increased telomere length and fragility in comparison to wild type cells. This strongly suggests that mutant POT1 causes chromosome instability and may play a role in lymphomagenesis in these families.

Association of polygenic risk score with the risk of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis.

Inherited loci have been found to be associated with risk of chronic lymphocytic leukemia (CLL). A combined polygenic risk score (PRS) of representative single nucleotide polymorphisms (SNPs) from these loci may improve risk prediction over individual SNPs. Herein, we evaluated the association of a PRS with CLL risk and its precursor, monoclonal B-cell lymphocytosis (MBL). We assessed its validity and discriminative ability in an independent sample and evaluated effect modification and confounding by family history (FH) of hematological cancers. For discovery, we pooled genotype data on 41 representative SNPs from 1499 CLL and 2459 controls from the InterLymph Consortium. For validation, we used data from 1267 controls from Mayo Clinic and 201 CLL, 95 MBL, and 144 controls with a FH of CLL from the Genetic Epidemiology of CLL Consortium. We used odds ratios (ORs) to estimate disease associations with PRS and c-statistics to assess discriminatory accuracy. In InterLymph, the continuous PRS was strongly associated with CLL risk (OR, 2.49; P = 4.4 × 10-94). We replicated these findings in the Genetic Epidemiology of CLL Consortium and Mayo controls (OR, 3.02; P = 7.8 × 10-30) and observed high discrimination (c-statistic = 0.78). When jointly modeled with FH, PRS retained its significance, along with FH status. Finally, we found a highly significant association of the continuous PRS with MBL risk (OR, 2.81; P = 9.8 × 10-16). In conclusion, our validated PRS was strongly associated with CLL risk, adding information beyond FH. The PRS provides a means of identifying those individuals at greater risk for CLL as well as those at increased risk of MBL, a condition that has potential clinical impact beyond CLL.

Genome-wide association analysis implicates dysregulation of immunity genes in chronic lymphocytic leukaemia.

Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P=5.04 × 10-13), 1q42.13 (rs41271473, P=1.06 × 10-10), 4q24 (rs71597109, P=1.37 × 10-10), 4q35.1 (rs57214277, P=3.69 × 10-8), 6p21.31 (rs3800461, P=1.97 × 10-8), 11q23.2 (rs61904987, P=2.64 × 10-11), 18q21.1 (rs1036935, P=3.27 × 10-8), 19p13.3 (rs7254272, P=4.67 × 10-8) and 22q13.33 (rs140522, P=2.70 × 10-9). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response.

Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene.

Hodgkin lymphoma shows strong familial aggregation but no major susceptibility genes have been identified to date. The goal of this study was to identify high-penetrance variants using whole exome sequencing in 17 Hodgkin lymphoma prone families with three or more affected cases or obligate carriers (69 individuals), followed by targeted sequencing in an additional 48 smaller HL families (80 individuals). Alignment and variant calling were performed using standard methods. Dominantly segregating, rare, coding or potentially functional variants were further prioritized based on predicted deleteriousness, conservation, and potential importance in lymphoid malignancy pathways. We selected 23 genes for targeted sequencing. Only the p.A1065T variant in KDR (kinase insert domain receptor) also known as VEGFR2 (vascular endothelial growth factor receptor 2) was replicated in two independent Hodgkin lymphoma families. KDR is a type III receptor tyrosine kinase, the main mediator of vascular endothelial growth factor induced proliferation, survival, and migration. Its activity is associated with several diseases including lymphoma. Functional experiments have shown that p.A1065T, located in the activation loop, can promote constitutive autophosphorylation on tyrosine in the absence of vascular endothelial growth factor and that the kinase activity was abrogated after exposure to kinase inhibitors. A few other promising mutations were identified but appear to be "private". In conclusion, in the largest sequenced cohort of Hodgkin lymphoma families to date, we identified a causal mutation in the KDR gene. While independent validation is needed, this mutation may increase downstream tumor cell proliferation activity and might be a candidate for targeted therapy.

Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family.

Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia.

Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.

Hodgkin lymphoma risk following infectious and chronic inflammatory diseases: a large population-based case-control study from Sweden.

Patients with Hodgkin lymphoma (HL) have a well-characterized immune deficiency of T cell function, originally identified by increased susceptibility to certain infections. Epidemiological evidence has long pointed to infectious etiologies in younger HL patients. With the aim of expanding our knowledge on the potential role of pre-existing immune deficiency in HL and an infectious/inflammatory etiology, we conducted a comprehensive population-based case-control study in HL patients diagnosed in Sweden in the period 1965-2004, and their matched controls. In a large population-based study including 7,414 HL patients and 29,240 matched controls, we evaluated the subsequent risk of HL in relation to a broad range of infectious and inflammatory conditions, using unconditional logistic regression. A previous history of any reported infection was associated with an 11 % increased risk of HL (P < 0.05). More specifically, we found sinusitis (odds ratio = 1.81; 95 % confidence interval = 1.06-3.07), tuberculosis (1.76; 1.01-3.07), encephalitis (7.88; 1.97-31.5), and herpes zoster (2.20; 1.11-4.35) to be associated with excess HL risk. A personal prior history of chronic inflammatory condition was not associated with an increased risk of HL (0.94; 0.71-1.14). Our results suggest that underlying immune deficiency is a primary phenomenon in HL. Alternatively, certain infectious agents may be potential HL triggers.

Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole-exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin gene POT1 (chromosome 7, g.124493086C>T; p.Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere lengths and numbers of fragile telomeres, suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two separate Italian families, one variant per family, yielding a frequency for POT1 variants comparable to that for CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in US and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Monoclonal gammopathy of undetermined significance and risk of lymphoid and myeloid malignancies: 728 cases followed up to 30 years in Sweden.

In 728 Swedish cases of monoclonal gammopathy of undetermined significance (MGUS), followed up to 30 years (median, 10 years), we estimated the cumulative risk of hematologic disorders originating from lymphoid and myeloid lineages. Using Cox regression models, we examined associations of demographic and laboratory factors with progression and determined the discriminatory power of 3 prediction models for progression. Eighty-four MGUS cases developed a lymphoid disorder, representing a cumulative risk of 15.4%. Multiple myeloma (MM) occurred in 53 patients, and the 30-year cumulative risk was 10.6%; an ∼0.5% annual risk. Three factors were significantly associated with progression: abnormal free light-chain (FLC) ratio (<0.26 or >1.65), M-protein concentration (≥1.5 g/dL), and reduction of 1 or 2 noninvolved immunoglobulin isotype levels (immunoparesis). A prediction model with separate effects for these 3 factors and the M-protein isotype had higher discriminatory power than other models, although the differences were not statistically significant. The 30-year cumulative risk for myeloid malignancies was <2%. Our study confirms that abnormal FLC ratio and M-protein concentration >1.5 g/dL, factors previously considered by Mayo Clinic researchers, are predictors for MM progression and suggests that separate consideration of immunoparesis and the Mayo Clinic risk factors could improve identification of MGUS patients at high risk for progression.

Genetic susceptibility to chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the West and is an incurable malignancy. No firmly established evidence exists for environmental risk factors in the etiology of CLL. However, CLL is estimated to have one of the highest familial risks for a hematologic malignancy; this along with other evidence strongly supports an inherited genetic component. In the past 5 years, genome-wide association studies (GWAS) have provided the foundation for new avenues in the investigation of pathogenesis of this disease with 22 susceptibility loci currently identified. We review here the advances made in identifying these loci, the potential to translate these findings into clinical practice, and future directions needed to advance our understanding of the genetic susceptibility of CLL.