Stefanie Huhn

Genetic Polymorphisms in Host Innate Immune Sensor Genes and the Risk of Nasopharyngeal Carcinoma in North Africa

Nasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world. It is an Epstein-Barr virus−associated malignancy with an unusual racial and geographical distribution. The host innate immune sensor genes play an important role in infection recognition and immune response against viruses. Therefore, we examined the association between polymorphisms in genes within a group of pattern recognition receptors (including families of Toll-like receptors, C-type lectin receptors, and retinoic acid−inducible gene I−like receptors) and NPC susceptibility. Twenty-six single-nucleotide polymorphisms (SNPs) in five pattern-recognition genes were genotyped in 492 North African NPC cases and 373 frequency-matched controls. TLR3_rs3775291 was the most significantly associated SNP (odds ratio [OR] 1.49; 95% confidence interval [95% CI] 1.11−2.00; P = 0.008; dominant model). The analysis showed also that CD209_rs7248637 (OR 0.69; 95% CI 0.52−0.93; P = 0.02; dominant model) and DDX58_rs56309110 (OR 0.70; 95% CI 0.51−0.98; P = 0.04) were associated with the risk of NPC. An 18% increased risk per allele was observed for the five most significantly associated SNPs, TLR3_rs3775291, CD209_rs7248637, DDX58_rs56309110, CD209_rs4804800, and MBL2_rs10824792, (ptrend = 8.2 × 10−4). Our results suggest that genetic variation in pattern-recognition genes is associated with the risk of NPC. These preliminary findings require replication in larger studies.

Single Nucleotide Polymorphisms within Interferon Signaling Pathway Genes Are Associated with Colorectal Cancer Susceptibility and Survival

Interferon (IFN) signaling has been suggested to play an important role in colorectal carcinogenesis. Our study aimed to examine potentially functional genetic variants in interferon regulatory factor 3 (IRF3), IRF5, IRF7, type I and type II IFN and their receptor genes with respect to colorectal cancer (CRC) risk and clinical outcome. Altogether 74 single nucleotide polymorphisms (SNPs) were covered by the 34 SNPs genotyped in a hospital-based case-control study of 1327 CRC cases and 758 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall survival and event-free survival in a subgroup of 483 patients. Seven SNPs in IFNA1, IFNA13, IFNA21, IFNK, IFNAR1 and IFNGR1 were associated with CRC risk. After multiple testing correction, the associations with the SNPs rs2856968 (IFNAR1) and rs2234711 (IFNGR1) remained formally significant (P = 0.0015 and P<0.0001, respectively). Multivariable survival analyses showed that the SNP rs6475526 (IFNA7/IFNA14) was associated with overall survival of the patients (P = 0.041 and event-free survival among patients without distant metastasis at the time of diagnosis, P = 0.034). The hazard ratios (HRs) for rs6475526 remained statistically significant even after adjustment for age, gender, grade and stage (P = 0.029 and P = 0.036, respectively), suggesting that rs6475526 is an independent prognostic marker for CRC. Our data suggest that genetic variation in the IFN signaling pathway genes may play a role in the etiology and survival of CRC and further studies are warranted.

Genetic variants in C-type lectin genes are associated with colorectal cancer susceptibility and clinical outcome

Inflammatory responses play a vital role at different stages of colorectal carcinogenesis. C‐type lectins mediate inflammatory/immune responses and participate in immune escape of pathogens and tumors. Our study aimed to evaluate the correlation between polymorphisms in three C‐type lectin genes, CD209, MBL2 and REG4, and colorectal cancer (CRC) risk and clinical outcome. We genotyped 15 potentially functional single nucleotide polymorphisms (SNPs) and assessed their associations with CRC risk in a case‐control study of 1353 CRC cases and 767 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall and event‐free survival in 414 patients. Two CD209 SNPs were associated with CRC risk after adjustment for multiple comparison. Minor allele carriers of the promoter SNP rs2287886 had an increased risk of CRC (OR 1.30, 95% CI 1.08–1.56), while minor allele carriers of the 3′UTR SNP, rs7248637, had a decreased risk (OR 0.74, 95% CI 0.60–0.91). Multivariate survival analyses, including age, gender, TNM stage and grade, showed that patients without distant metastasis at the time of diagnosis and carrying the rs2994809 T allele had a decreased overall and event‐free survival (HR 2.11, 95% CI 1.20–3.72 and HR 2.00, 95% CI 1.18–3.39, respectively). We show that SNPs in CD209 may affect CRC risk, while a SNP in REG4 may be a useful marker for CRC progression.

A coding IRAK2 protein variant compromises Toll-like receptor (TLR) signaling and is associated with colorectal cancer survival.

Background: Interleukin-1 receptor-associated kinases (IRAKs) play a critical role in TLR signaling and thus innate immunity. Results: A coding IRAK2 variant, rs35060588, affects TLR signaling and colorectal cancer survival. Conclusion: IRAK2 rs35060588 is a functional, disease-relevant variant. Significance: IRAK2 and its variant rs35060588 may serve as a point of therapeutic intervention and predictive biomarker, respectively. Within innate immune signaling pathways, interleukin-1 receptor-associated kinases (IRAKs) fulfill key roles downstream of multiple Toll-like receptors and the interleukin-1 receptor. Although human IRAK4 deficiency was shown to lead to severe immunodeficiency in response to pyogenic bacterial infection during childhood, little is known about the role of human IRAK2. We here identified a non-synonymous IRAK2 variant, rs35060588 (coding R214G), as hypofunctional in terms of NF-κB signaling and Toll-like receptor-mediated cytokine induction. This was due to reduced ubiquitination of TRAF6, a key step in signal transduction. IRAK2 rs35060588 occurs in 3–9% of individuals in different ethnic groups, and our studies suggested a genetic association of rs35060588 with colorectal cancer survival. This for the first time implicates human IRAK2 in a human disease and highlights the R214G IRAK2 variant as a potential novel and broadly applicable biomarker for disease or as a therapeutic intervention point.

Genome-wide association study of immunoglobulin light chain amyloidosis in three patient cohorts: comparison with myeloma

Immunoglobulin light chain (AL) amyloidosis is characterized by tissue deposition of amyloid fibers derived from immunoglobulin light chain. AL amyloidosis and multiple myeloma (MM) originate from monoclonal gammopathy of undetermined significance. We wanted to characterize germline susceptibility to AL amyloidosis using a genome-wide association study (GWAS) on 1229 AL amyloidosis patients from Germany, UK and Italy, and 7526 healthy local controls. For comparison with MM, recent GWAS data on 3790 cases were used. For AL amyloidosis, single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at P<10−5 with homogeneity of results from the 3 sample sets; some of these were previously documented to influence MM risk, including the SNP at the IRF4 binding site. In AL amyloidosis, rs9344 at the splice site of cyclin D1, promoting translocation (11;14), reached the highest significance, P=7.80 × 10−11; the SNP was only marginally significant in MM. SNP rs79419269 close to gene SMARCD3 involved in chromatin remodeling was also significant (P=5.2 × 10−8). These data provide evidence for common genetic susceptibility to AL amyloidosis and MM. Cyclin D1 is a more prominent driver in AL amyloidosis than in MM, but the links to aggregation of light chains need to be demonstrated.

Genome-wide association study of clinical parameters in immunoglobulin light chain amyloidosis in three patient cohorts

Immunoglobulin light chain (AL) amyloidosis is a progressive plasma cell dyscrasia which is characterized by the deposition of amyloid fibers derived from immunoglobulin light chain or their fragments systemically in many organs. The characteristics of amyloids relate to disease severity and sequelae, including the target organs where amyloids accumulate, such as the heart, kidney, liver, gut and peripheral nerves. Heart failure is usually the critical life-threatening condition; the median survival time may range from months to some years. We have recently characterized ten putative genetic risk loci (at a significance level of <10) for AL amyloidosis using a genome-wide association study (GWAS) approach on a total of 1229 German, UK and Italian patients. In the study herein we carried out a systematic GWAS-based association study on clinical data, including the affected organs and the isotype of serum immunoglobulins (Ig). We hypothesized that clinical profiles may be able to define distinct molecular subtypes. AL amyloidosis and multiple myeloma (MM) patient populations are described in Table 1; more details can be found in the study by da Silva Filho and colleagues. A total of nine clinical profiles were selected based on patient numbers, amyloid organ involvement (heart, kidney, heart + kidney and liver, irrespective of whether other organs were involved) and Ig profiles (intact IgG with l or k, l any, k any, l/k light chain only (LCO), and l LCO). Baseline assessments and procedures included physical examination, amyloid organ involvement and standard laboratory values in addition to serum monoclonal (M)-protein, free light chains, N-terminal pro btype natriuretic peptide (NT-proBNP) and cardiac troponin T (cTNT)/ high-sensitive (hs)TNT analyses. Organ involvement was uniformly assessed according to the consensus criteria agreed on by the three centers involved. The collection of patient samples and associated clinical information was approved by the relevant ethical review boards in accordance with the tenets of the Declaration of Helsinki. Analysis of the GWAS data was performed using imputed data as described. Single-nucleotide polymorphisms (SNPs) possessing a minor allele frequency (MAF) of <1% were excluded. Associations based on imputed SNPs alone were not considered. The association test between SNPs and AL amyloidosis was performed in SNPTEST v2.5. The three data sets were combined in meta-analysis and heterogeneity was assessed by the I statistic (interpreted as low <0.25, moderate 0.50 and high >0.75). For genome-wide significance, a limit of P<5x10 was used. Details of the bioinformatic analyses can be found in the study conducted by da Silva Filho et al. and in the Online Supplementary Material. Z-scores were calculated for SNPs as log odds ratio (OR) divided

Inbreeding and homozygosity in breast cancer survival.

Genome-wide association studies (GWASs) help to understand the effects of single nucleotide polymorphisms (SNPs) on breast cancer (BC) progression and survival. We performed multiple analyses on data from a previously conducted GWAS for the influence of individual SNPs, runs of homozygosity (ROHs) and inbreeding on BC survival. (I.) The association of individual SNPs indicated no differences in the proportions of homozygous individuals among short-time survivors (STSs) and long-time survivors (LTSs). (II.) The analysis revealed differences among the populations for the number of ROHs per person and the total and average length of ROHs per person and among LTSs and STSs for the number of ROHs per person. (III.) Common ROHs at particular genomic positions were nominally more frequent among LTSs than in STSs. Common ROHs showed significant evidence for natural selection (iHS, Tajima’s D, Fay-Wu’s H). Most regions could be linked to genes related to BC progression or treatment. (IV.) Results were supported by a higher level of inbreeding among LTSs. Our results showed that an increased level of homozygosity may result in a preference of individuals during BC treatment. Although common ROHs were short, variants within ROHs might favor survival of BC and may function in a recessive manner.

Shared ancestral susceptibility to colorectal cancer and other nutrition related diseases

BackgroundThe majority of non-syndromic colorectal cancers (CRCs) can be described as a complex disease. A two-stage case–control study on CRC susceptibility was conducted to assess the influence of the ancestral alleles in the polymorphisms previously associated with nutrition-related complex diseases.MethodsIn stage I, 28 single nucleotide polymorphisms (SNPs) were genotyped in a hospital-based Czech population (1025 CRC cases, 787 controls) using an allele-specific PCR-based genotyping system (KASPar®). In stage II, replication was carried out for the five SNPs with the lowest p values. The replication set consisted of 1798 CRC cases and 1810 controls from a population-based German study (DACHS). Odds ratios (ORs) and 95% confidence intervals (CIs) for associations between genotypes and CRC risk were estimated using logistic regression. To identify signatures of selection, Fay-Wu’s H and Integrated Haplotype Score (iHS) were estimated.ResultsIn the Czech population, carriers of the ancestral alleles of AGT rs699 and CYP3A7 rs10211 showed an increased risk of CRC (OR 1.26 and 1.38, respectively; two-sided p≤0.05), whereas carriers of the ancestral allele of ENPP1 rs1044498 had a decreased risk (OR 0.79; p≤0.05). For rs1044498, the strongest association was detected in the Czech male subpopulation (OR 0.61; p=0.0015). The associations were not replicated in the German population. Signatures of selection were found for all three analyzed genes.ConclusionsOur study showed evidence of association for the ancestral alleles of polymorphisms in AGT and CYP3A7 and for the derived allele of a polymorphism in ENPP1 with an increased risk of CRC in Czechs, but not in Germans. The ancestral alleles of these SNPs have previously been associated with nutrition-related diseases hypertension (AGT and CYP3A7) and insulin resistance (ENPP1). Future studies may shed light on the complex genetic and environmental interactions between different types of nutrition-related diseases.

Coding variants in NOD-like receptors: An association study on risk and survival of colorectal cancer

Nod-like receptors (NLRs) are important innate pattern recognition receptors and regulators of inflammation or play a role during development. We systematically analysed 41 non-synonymous single nucleotide polymorphisms (SNPs) in 21 NLR genes in a Czech discovery cohort of sporadic colorectal cancer (CRC) (1237 cases, 787 controls) for their association with CRC risk and survival. Five SNPs were found to be associated with CRC risk and eight with survival at 5% significance level. In a replication analysis using data of two large genome-wide association studies (GWASs) from Germany (DACHS: 1798 cases and 1810 controls) and Scotland (2210 cases and 9350 controls) the associations found in the Czech discovery set were not confirmed. However, expression analysis in human gut-related tissues and immune cells revealed that the NLRs associated with CRC risk or survival in the discovery set were expressed in primary human colon or rectum cells, CRC tissue and/or cell lines, providing preliminary evidence for a potential involvement of NLRs in general in CRC development and/or progression. Most interesting was the finding that the enigmatic development-related NLRP5 (also known as MATER) was not expressed in normal colon tissue but in colon cancer tissue and cell lines. Future studies may show whether regulatory variants instead of coding variants might affect the expression of NLRs and contribute to CRC risk and survival.

ELDA qASO-PCR for High Sensitivity Detection of Tumor Cells in Bone Marrow and Peripheral Blood

A quantitative allele-specific polymerase chain reaction in combination with an extreme limiting dilution approach (ELDA qASO-PCR) enables the detection of tumor cells in patients with multiple myeloma (MM) in bone marrow (BM) samples and in peripheral blood (PB) with a sensitivity of <10-6. The two-step procedure of patient-specific tumor cell identification via the immunoglobulin heavy chain (IgH) and kappa/lambda light chain (k/λ LC) locus, followed by tumor cells quantification by ELDA qASO-PCR allows for the application of this method to the majority of MM patients, including those with Bence Jones proteinuria.