DIAPH2 gene polymorphisms and laryngeal cancer risk in men

Background The DIAPH2 gene is one of the genes commonly associated with laryngeal squamous cell carcinoma (LSCC). In our study, we considered the four polymorphisms of this gene, i.e. rs5920828, rs4322175, rs12851931 and rs5921830 as potential genetic risk factors for LSCC. Methods We determined the genotyping of the genetic variants of DIAPH2 in 230 male patients with histologically confirmed LSCC compared to the European population. Demographic and environmental exposure data of each subject were examined. To conduct the genetic tests, extraction of total DNA was performed. We genotyped all four variants in each patient and determined their frequencies. Results In the case of the rs12851931 polymorphism in the DIAPH2 gene, a significant difference was observed in the distribution of the T stage depending on the polymorphism. Heterozygotes were more often associated with T2 stage, while homozygotes were more likely to have higher tumor stages. The rs12851931 homozygotes of DIAPH2 were statistically significantly more prevalent in smokers. The results suggested that rs12851931 polymorphism in DIAPH2 could increase the onset risk of LSCC. Conclusions Our results provide further information on the role of the DIAPH2 gene in the pathogenesis of LSCC. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-024-05820-4.


Introduction
Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignancies of the head and neck.It is a significant clinical problem because of its high incidence and the lack of effective treatment.Due to higher rates of tobacco and alcohol use among males, LSCC is more prevalent in men than in women (the male-to-female ratio ranging from 5:1 to 10:1) (Talamini et al. 2002;Zhang et al. 2021).The incidence of LSCC varies across different countries in Europe.The highest rates are reported in Central and Eastern European countries, while the lowest rates are observed in Northern European countries (Igissin et al. 2023;Wierzbicka et al. 2016).
The cause and etiopathogenesis of LSCC are still not fully understood.Epidemiological studies have indicated environmental factors as significant risk factors for developing LSCC.Among the crucial risk factors, the following should be mentioned: tobacco and alcohol use, human papillomavirus (HPV) infection and prolonged exposure to certain chemicals (asbestos fibers, wood dust, chemicals in the metalworking industry and exhaust fumes) (Kreimer et al. 2005).
The incidence of LSCC is strongly associated with age and gender.It typically occurs in male patients over the age of 40, with the peak incidence in the sixth and seventh decades of life.The prognosis for advanced-stage LSCC is poorer, with 5-year survival rates typically below 50%.
In the era of the growing importance of personalized cancer therapy, including LSCC, it is essential to search for genetic markers that increase the risk of disease development and use them for diagnosis, prognosis and personalized treatment strategies for LSCC patients.
Several genetic markers for LSCC have already been identified, including TP53 mutations (Szczepny et al. 2018), mutations in the Ras family genes (H-K-N-Ras) (Bos 1988), EGFR (epidermal growth factor receptor) gene amplification or overexpression (Politi et al. 2019), Cyclin D1 (CCND1) gene amplification (Kyrodimos et al. 2020), FGFR3 (fibroblast growth factor receptor) gene mutations (Manterola et al. 2018), loss of heterozygosity (LOH) (Grbesa et al. 2008), DNA (deoxyribonucleic acid) methylation, SLC22A23 gene (Ekizoglu et al. 2018), protein tyrosine phosphatase receptor Type D (PTPRD) gene and the rs3765142 polymorphism in PTPRD (Szaumkessel et al. 2017;Śnit et al. 2021), hypermethylated-in-cancer-1 (HIC1) gene (Ekizoglu et al. 2018) and the rs9901806 polymorphism in the HIC1 gene (Śnit et al. 2021;Dubuissez et al. 2020).Diaphanous-related formin 2 (DIAPH2) is a member of the diaphanous-related formin family, which encodes proteins involved in cytoskeletal organization and cellular processes, such as cell division, migration and adhesion.The DIAPH2 gene is located on the long arm (q) of chromosome X at position 21, 23.Several studies have highlighted the involvement of DIAPH2 gene abnormalities in LSCC (Śnit et al. 2021;Kostrzewska-Poczekaj et al. 2019).Some studies have identified specific mutations in the DIAPH2 gene in LSCC patients.DIAPH2 mutations were present in approximately 4% of LSCCs, which was mainly related to DIAPH2 gene expression.High expression of DIAPH2 was associated with advanced tumor stage, lymph node metastasis and poor overall survival in LSCC patients.Alterations in DIAPH2 increase cell motility and may contribute to the metastatic potential of LSCC (Kostrzewska-Poczekaj et al. 2019).Upregulation of DIAPH2 in LSCC may act as an oncogene by inhibiting apoptosis through the ART/p53/caspase-3 pathway (Yang et al. 2019).So far, one of the DIAPH2 gene polymorphisms has been associated with LSCC.
In the previous observation (2021) from our clinical center (Zabrze, Poland), we confirmed for the first time that rs6620138 polymorphism in the DIAPH2 gene was associated with the pathogenesis of LSCC.Significant differences in allele distribution of DIAPH2 rs6620138 in the case and control groups were observed (Śnit et al. 2021).
The rs6620138 polymorphism in the DIAPH2 gene has been implicated in the development and progression of LSCC (Śnit et al. 2021).The rs6620138 polymorphism affects DIAPH2 expression levels.In particular, individuals with specific alleles of rs6620138, such as the TG or GG genotypes, have been found to have lower DIAPH2 expression compared to individuals with the TT genotype.The decreased expression of DIAPH2 is associated with an increased risk of LSCC.Alterations in DIAPH2 expression due to the rs6620138 polymorphism could potentially impact the ability of cells to migrate and invade, contributing to the development and progression of LSCC.Studies have suggested that the rs6620138 polymorphism may influence the response to specific anticancer treatments (Śnit et al. 2021).For example, individuals with the GG genotype of rs6620138 had a better response to chemotherapy compared to those with the TT genotype.This association may be attributed to the influence of DIAPH2 on actin dynamics, which can impact the sensitivity of cancer cells to chemotherapy agents (Śnit et al. 2021).
In this study, we considered the following four polymorphisms of the DIAPH2 gene: rs5920828, rs4322175, rs12851931 and rs5921830 as potential genetic risk factors for LSCC.All of the above polymorphisms have been poorly investigated.
The DIAPH2 rs5920828 is a specific variant of the DIAPH2 gene, which encodes for diaphanousrelated formin-2 protein.As regards the specific variant (rs5920828), its role in disease susceptibility and biology is not well-established.However, genetic variants within the DIAPH2 gene have been associated with several human disorders.For example, mutations in DIAPH2 have been found in individuals with microcephaly and cataract, suggesting its involvement in brain development and eye health (Kostrzewska-Poczekaj et al. 2019).
The DIAPH2 rs4322175 encodes for diaphanous-related formin 2 protein, which is involved in various cellular processes, including cell division, cell migration and cytoskeleton organization.The rs4322175 variant is associated with differences in the expression or activity of the DIAPH2 protein.Studies have shown that individuals carrying the alertative C allele of rs4322175 may have altered DIAPH2 protein levels or activity compared to individuals with the reference allele (T allele).The role of DIAPH2 rs4322175 in different biological processes and diseases is still under investigation.Some research suggests this variant could be associated with susceptibility to some disorders.For example, one study found that individuals with the C allele of rs4322175 showed an increased risk of developing Alzheimer's disease compared to those with the T allele (Labat-de-Hoz and Alonso 2021).
The DIAPH2 rs12851931 is a genetic variant of the DIAPH2 gene.The rs12851931 variant is associated with an increased risk of developing various diseases and conditions.In some studies, this genetic variant was associated with an increased risk of schizophrenia, bipolar disorder, autism spectrum disorder (ASD), an increased risk of ovarian cancer, colorectal cancer and early-onset myocardial infarction (Philippe et al. 1993).
The DIAPH2 rs5921830 is a genetic variant of the DIAPH2 gene that has also been associated with various diseases and conditions.One study found that individuals with the CC genotype of rs5921830 had a higher risk of developing colorectal cancer.Another study found an association with the development of ASD in males.The CC genotype was more prevalent in individuals with ASD compared to controls.The DIAPH2 rs5921830 has been suggested to alter the expression or function of the DIAPH2 protein, leading to dysregulation of cellular processes and potentially increasing disease susceptibility (https:// www.prote inatl as.org/ ENSG0 00001 47202-DIAPH2/ patho logy).

Purpose
The aim of our study was to continue previous research and find a link between the four polymorphisms of the DIAPH2 gene and the risk of LSCC development.We determined the genotyping of the genetic variants of DIAPH2 in 230 male patients with histologically confirmed LSCC compared to the European population.Due to the predominance of men in the incidence of LSCC and the fact that the collected material was obtained from only several women, we decided to standardize the study group and analyze only male patients.

Study population
The study protocol was approved by the Bioethics Committee of the Medical University of Silesia (Poland).The study population consisted of 230 subjects with histologically confirmed LSCC treated in the Department of Otorhinolaryngology and Laryngological Oncology, Medical University of Silesia (Zabrze, Poland), Department of Laryngology and Laryngological Oncology, Upper Silesian Medical Centre of Silesian Medical University, (Katowice, Poland) and Department of Otolaryngology, Head and Neck Surgery, Holy-Cross Cancer Centre (Kielce, Poland).All patients underwent laryngectomy and had no distant metastases (M0 according to the TNM classification).Demographic and environmental exposure data of each subject were examined, including sex, age, smoking, alcohol consumption, a history of diseases and a family history of cancer.Information on smoking and alcohol consumption was obtained from the medical history.For smokers, the number of packyears smoked was calculated.Non-smokers were defined as patients who had stopped smoking at least 10 years before cancer diagnosis.To assess alcohol consumption, patients declared the amount of alcohol consumed per week.Increased exposure to alcohol was considered when the alcohol intake was more than 280 mg of pure alcohol per week (approximately 13 0.5-L bottles of beer, 3 0.75-L bottles of wine and 0.8 L of vodka).To assess the genetic predisposition to cancer, we took into account the occurrence of cancer, particularly in the first-and second-degree relatives.
The study material was obtained in cooperation with the Department of Pathomorphology at the Medical University of Silesia (Zabrze, Poland).
For the European population, we used the most frequently selected database, i.e.NCBI.Unfortunately, there is no division into gender or age in the database, only the final size group, ethnicity and allele distribution for the selected SNPs.The European population used to compare the distribution of the polymorphisms (identifier: BioSample: SAMN10492695) was taken from the NCBI database on 5th January 2024.The largest population was selected for comparison.

Genetic analysis
To conduct the genetic tests, extraction of total DNA was performed with the MagCore Genomic DNA FFPE (formalin-fixed paraffin-embedded) One-Step kit (IVD) using MagCore®Compact.We used a NanoDrop spectrophotometer (Thermo Scientific, Waltham, USA) to measure the concentration and purification of the DNA.The genetic material was then used to genotype four variants of the DIAPH2 gene.The SNPs identifiers were as follows: rs4322175, rs5920828, rs5921830 and rs12851931.We used TaqMan SNP genotyping assays (ThermoFisher, USA) to discriminate alleles (Table S1).For PCR, we used FastStart Essential DNA Probes Master (Roche, Switzerland) for real-time PCR using the LifeCycler 96 thermocycler (Roche, Switzerland).
We genotyped all four variants in each patient and determined their frequencies.

Statistical analysis
Data were presented as the number of cases with the percentage for qualitative variables.To assess the normality of the variables, we used a histogram and a quantile-quantile (QQ) plot.Quantitative variables with a normal distribution were presented as means with standard deviation and variables with deviations from the normal distribution were presented as the median with the first and third quartiles.For statistical analyses of quantitative variables with the normal distribution, the Student's t test was used.The Wilcoxon test was applied for quantitative variables with the deviation from the normal distribution.The results for smokers and drinkers were presented in boxplots.The comparison of qualitative variables was based on the Pearson's chi-squared test.The GLM method was used to assess the correlations between the polymorphisms and other variables.The comparison of allele frequencies in the study population with the general population was made using the test for proportions.
The analysis was performed using the R language in the RStudio environment (Wickham et al. 2019) and Tidyverse and Janitor packages (Zhang et al. 2022).p values <0.05 were considered statistically significant.1).The median pack-year history of smoking was 25 years.84.30% of patients were smokers, and 79.10% were drinkers.68.70% of patients were both smokers and drinkers (Table 2).As shown in Table 3, the mean age of smokers was 63.7 years, and 63.4 years in non-smokers (p = 0.83, NS).The mean age of drinkers was 63.2 years compared to 65.3 years for non-drinkers (p = 0.099, NS).The mean age of drinkers and smokers was 63.4 years compared to 64.2 years for both non-smokers and non-drinkers (p = 0.52, NS).Allele distribution in smokers, drinkers and both smokers and drinkers is given in Table 4. Homozygotes of rs12851931 polymorphism in DIAPH2 were statistically significantly more prevalent in smokers than in non-smokers (Table 4).The distribution of particular alleles in the study population is given in Table 5.The allele distribution of rs5920828 and rs5921830 polymorphisms of DIAPH2 differed statistically from the distribution reported in the European population (Table 6).The rs4322175 and rs12851931 polymorphisms in DIAPH2 "0"-no-smokers, no-drinkers, no-smokers and no-drinkers "1"-smokers, drinkers, smokers and drinkers  showed a trend to statistical significance (p = 0.08 and p = 0.07, respectively).The assessment of the relationship between the polymorphisms and the TNM stages showed no difference in three polymorphisms (rs5920828, rs4322175 and rs5921830).
In the case of the rs12851931 polymorphism in the DIAPH2 gene, a significant difference was observed in the distribution of the T stage depending on the polymorphism.Heterozygotes were more often associated with T2 stage, while homozygotes were more likely to have higher tumor stages (Table 7).GLM analysis for the polymorphisms and cancer history, survival and cancer recurrence is given in Tables 8, 9, and 10.No statistically significant differences were found between cancer history, survival, risk of cancer recurrence and the analyzed polymorphisms.

Discussion
Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant neoplasms of the respiratory system (Talamini et al. 2002).It is more prevalent in males and often occurs in individuals over the age of 40.Genetic factors also play an essential role in the etiopathogenesis of LSCC.Genetic research on LSCC explores mutations, copy number variations and epigenetic changes.The DIAPH2 gene is one of the potential genetic factors of LSCC.The polymorphisms we have analyzed are still poorly understood.There are only a few studies on their relationship with cancers or other diseases.
We examined the distribution of the studied genotypes and alleles in the whole group of subjects and compared the results to the distribution in the European population.The allele distribution of two polymorphisms (rs5920828 and rs5921830) differed statistically from the distribution found in the European population.The next two polymorphisms (rs4322175 and rs12851931) showed a trend to statistical significance and presumably the difference would be significant in the case of a larger group size.We are aware that trend is not a significant value in statistics but used this statement to highlight the proximity of the p value to 0.05.In our opinion, the described polymorphisms may have the clinical relevance and hence further studies are warranted.
Further analysis showed that three of the studied polymorphisms (rs5920828, rs4322175 and rs5921830) showed no significant difference in relation to the TNM stages.However, a significant difference was observed for rs12851931 in the distribution of the T stage depending on the polymorphism.Heterozygotes of rs12851931 variant of the DIAPH2 gene were more often associated with T2 stage, while homozygotes were more likely to have higher tumor stages.The total number of patients with stage T2 was 11.Therefore, the association of heterozygote of rs12851931 variant of the DIAPH2 should be interpreted with caution, which is a limitation of the study.However, despite the small number of patients, their number was in fact sufficient to obtain the statistical significance.Even more in the case of the other polymorphisms, the total number of patients with T2 was similar (from 10 to 12 patients) and we found no correlation.
As mentioned in Introduction of our manuscript and considering the fact that DIAPH2 gene mutations were present in approximately a few percent of LSCCs and that high expression of DIAPH2 was associated with advanced tumor stage, lymph node metastasis and poor overall survival, it  should be stressed that our results may have the practical aspect and may allow the creation, after confirmation in other studies, of an early marker of LSCC which can be useful in the diagnosis of LSCC at an early stage in some risk groups, such as smokers.The importance of our research (even though it was conducted on such a small group of patients) also increases knowledge related to this very important area in the field of oncology and may be the basis for practical implementation in the future.
In the study population, most patients were homozygous (AA-39.6%,GG-52.8%), which may indicate the relationship between rs12851931 and an increased risk of more advanced LSCC.Furthermore, homozygotes of rs12851931 polymorphism of DIAPH2 were statistically significantly more prevalent in smokers.The rs12851931 variant of the DIAPH2 gene was associated with a higher risk of cancer development (ovarian cancer or colorectal cancer) and could be related to LSCC, as indicated in our study (Zhang et al. 2022;Lin et al. 2014).
It can be suggested that people with the above polymorphism could have a higher risk of developing LSCC than those without them.
However, if our findings are confirmed by other authors, the determination of the rs12851931 variant of the DIAPH2 gene or other variants of the DIAPH2 gene that we have analyzed may have practical significance in predicting the risk of developing LSCC.
Importantly, our study and two previous Polish studies ( Kostrzewska-Poczekaj et al. 2019;Śnit et al. 2021) have indicated an association of the DIAPH2 gene variants with the risk of LSCC.
The study provides insights into the genetic variants of the DIAPH2 gene in relation to LSCC.The findings emphasize the complexity of LSCC etiology and suggest that additional factors may contribute to our clinical outcomes apart from the specific DIAPH2 gene polymorphisms.
Further research is warranted to fully understand the role of DIAPH2 and other genetic markers in LSCC.Studying the functions and potential therapeutic implications of these markers may contribute to the development of targeted therapies for LSCC in the future.
The limitation of our study was a relatively small sample size and only male population.

Conclusions
Our study showed that the rs12851931 polymorphism in the DIAPH2 gene could be associated with the pathogenesis of LSCC in male.Further studies should be conducted on larger study groups, including other races and the female population to confirm the above findings.Our research (even though it was conducted on such a small group of patients) also increases knowledge related to this very important area in the field of oncology and may be the basis for practical implementation in the future.
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Table 2
The age and pack-year history of smoking

Table 4
The allele distribution in smokers, drinkers and both smokers and drinkers

Table 5
The alleles of the polymorphisms in the study population

Table 6
The allele distribution in the study population versus the European population

Table 7
The relationship between the polymorphisms and the TNM stages (Pearson's chi-squared test)

Table 8
GLM analysis for the polymorphisms and cancer history

Table 9
GLM analysis for the polymorphisms and survival