Increased frequency of the single nucleotide polymorphism of the <i> <scp>DARC</scp> / <scp>ACKR1</scp> </i> gene associated with ethnic neutropenia in a cohort of European patients with chronic idiopathic neutropenia
Authors: Irene Fragiadaki, Stavros Papadakis, Georgia Sevastaki, Katerina Sfyridaki, Irene Mavroudi, George N. Goulielmos, Peggy Kanellou, Anette Mörtberg, Petter Höglund, Katerina Gemenetzi, Kostas Stamatopoulos, Anastasia Chatzidimitriou, Jan Palmblad, Helen A. Papadaki
Journal: American Journal of Hematology (2020)

Chronic neutropenia is defined as the reduction of peripheral blood (PB) absolute neutrophil counts (ANC) below the lower limit of the normal distribution for a period exceeding 3 months.1, 2 The investigation of chronic neutropenia in adults comprises rare congenital conditions that involve germline mutations of a variety of genes including ELANE and HAX1. Also involved are common acquired disorders associated with reduced or ineffective neutrophil production in the bone marrow (BM), increased neutrophil margination to the vascular endothelium, neutrophil sequestration in the spleen, or accelerated neutrophil destruction by immune mechanisms.1, 2 Approximately 30% of patients with chronic neutropenia do not have an apparent underlying cause; the term chronic idiopathic neutropenia (CIN) is used for these cases.2 Ιndividuals with African and Near and Middle East ancestry present lower ANCs than individuals of European ancestry, a situation known as ethnic neutropenia (ENP) commonly associated with absence of Duffy antigens from red blood cells.3 Duffy antigens are encoded by the Duffy antigen receptor for chemokines (DARC), also known as Atypical Chemokine Receptor-1 (ACKR1), gene.4 The Duffy blood group has two major codominant alleles, FY*A and FY*B, differentiated by a single nucleotide polymorphism (SNP) (G125A, rs12075) resulting in the Fya and Fyb variants, respectively.4 The most common genetic determinant associated with ENP is a second SNP (rs2814778) at the GATA promoter region of the DARC/ACKR1 gene associated with a T to C substitution (−46 T > C, rs2814778). The homozygous C/C polymorphism prevents the Duffy antigens' expression on erythroid cells resulting in the Duffy-null Fy(a-b-) (also known as erythroid silent, ES) phenotype.4 In brief, the main SNPs associated with ENP are the combination of the rs12075 (A > G; FY*B > FY*A) and rs2814778 (−46 T > C; FY*B > FY*B ES). The correlation between the C/C rs2814778 genotype (FY*B ES/FY*B ES) and ENP has been associated with a natural selection process of the C allele genotype (FY*B ES) in malaria-endemic regions. On the contrary, the C/C rs2814778 (FY*B ES/FY*B ES) genotype is rare or absent among white European (WE) individuals.5 Therefore, the investigation of unexplained neutropenia in European patients does not usually include Duffy polymorphisms genotyping and thus, data on Duffy phenotype/genotype/allele frequencies in neutropenic compared to normal individuals are missing. In the present study we evaluated the presence of the DARC/ACKR1 SNP -46 T > C rs2814778 associated with the Fy(a-b-) phenotype in 124 WE patients with unexplained neutropenia provisionally characterized as CIN.2 We also assessed the frequency of the Duffy alleles and genotypes among these patients compared with 500 age-matched and sex-matched WE healthy individuals from the same region. Patient characteristics and diagnostic criteria are shown in the Table S1. All subjects were living and originated from the island of Crete/Greece except for one CIN patient and four healthy controls born in and originated from the United Kingdom, and four CIN patients and eight healthy controls born in and originated from Bulgaria. The study was approved by the Ethics Committee of the University Hospital of Heraklion. Genotyping of the CIN patients and controls for the rs12075 and rs2814778 SNPs was performed using a real-time PCR assay as previously described.5 Duffy phenotyping of red blood cells was determined by a direct antiglobulin test using commercially available anti-Fya, anti-Fyb reagents and the LISS/Coombs ID-cards. The T-cell receptor beta (TRB) chain (TRBV-TRBD-TRBJ) gene rearrangements were studied by next generation sequencing (NGS) as previously described.6 Detailed description of the methods (including representative allelic discrimination plots of the SNPs rs12075 and rs2814778) is given in the supplementary file. Statistical analysis (GraphPad Software; San Diego, CA, USA) was performed using the chi-square test with Yates' correction when necessary, the one-way analysis of variance test and the Student t test. The TRB gene repertoires were further analyzed by the Kruskal test followed by the Benjamini-Hochberg correction. The frequencies of the rs12075 A/A (FY*B/FY*B), A/G (FY*B/FY*A) and G/G (FY*A/FY*A) genotypes did not differ significantly between CIN patients and controls (Figure 1A). As anticipated for European populations, the FY*B was the most frequent allele in both groups.5 No statistically significant difference was identified in the frequency of the alleles FY*A (rs12075, 125G) and FY*B (rs12075, 125A) between patients and controls (Figure 1B). Interestingly, a statistically significant difference was identified in the frequency of the rs2814778 T/T (FY*B/FY*B), T/C (FY*B/FY*B ES) and C/C (FY*B ES/FY*B ES) genotypes between CIN patients (108/124 that is, 87.10%, 10/124 that is, 8.06% and 6/124 that is, 4.84%, respectively) and controls (482/500 that is, 96.40%, 18/500 that is, 3.60%, 0/500 that is, 0%, respectively) (P < .001) (Figure 1C). A statistically significant difference was also identified in the frequency of the alleles C (FY*B ES) and T (FY*B) between CIN patients (8.87% and 91.13%, respectively) and controls (1.80% and 98.20%, respectively) (P < .001) (Figure 1D). These data suggest that the FY*B ES allele, associated with the Duffy-null phenotype, is found at higher frequency among CIN patients than among healthy individuals. The presence of the FY*B ES allele gives a relative risk 2.94 (95% confidence interval [CI] 2.169-3.984) for the development of CIN (odds ratio [OR] 5.311; 95% CI 2.801-10.07) in the population of our study. The allele and genotype frequencies of the SNP rs2814778 in our control population corresponds to the anticipated frequencies in individuals of European ancestry.5 Duffy phenotyping of red blood cells was performed in 62/124 CIN patients and in all 500 normal controls (Table S2). An absolute correlation was identified between the Duffy phenotype and genotype in both, the CIN and control groups. A statistically significant difference was observed in the frequency of the Fy(a-b+), Fy(a + b+), Fy(a + b-), Fy(a-b-) phenotypes between CIN patients and controls (P < .001) that was mainly due the identification of the Duffy-null phenotype in 3/62 CIN patients and in none of the controls (P < .001). These patients with the Duffy-null phenotype displayed the anticipated FY*B ES/FY*B ES genotype. We also investigated whether the different SNP rs12075 and rs2814778 genotypes have an impact in the total white blood cell (WBC) and subtype absolute counts in CIN patients and controls (Table S3). We only identified statistically significant differences in ANC between CIN patients with different rs2814778 genotypes (T/T, T/C, C/C). Specifically, CIN patients with the C/C genotype had significantly lower ANCs compared to those with T/T (P < .001) or T/C (P = .007) genotypes; no significant difference was identified in ANCs between patients with T/T and T/C genotypes. These data suggest that the rs2814778 C/C (FY* ES/FY*B ES) genotype is not only related with increased risk for the development of neutropenia in WE individuals according to the estimated relative risk, but also with more pronounced neutropenia among neutropenic patients. We have previously shown that CIN patients display lower absolute lymphocyte counts compared to healthy individuals.2 In the current study we found that CIN patients with the C/C genotype represent a distinct group with higher absolute lymphocyte counts compared to patients with the T/T genotype (P = .0006) and T/C genotype (although not statistically significant) (Table S3). The absolute lymphocyte counts in the group of patients with the C/C genotype were similar to that of normal controls, whereas patients with the T/T and T/C genotypes displayed significantly lower lymphocyte counts compared to the controls (P < .001 and P = .001, respectively) in accordance with previously reported data on CIN-related lymphopenia.2 We have also previously shown that CIN patients display remarkable TRB repertoire skewing and oligoclonality with shared clonotypes.6 In the current study we performed immunogenetic analysis in the six CIN patients with the rs2814778 C/C (FY*B ES/FY*B ES), and in four random CIN patients with T/T (FY*B/FY*B) genotypes (Table S4). All C/C CIN patients showed a T-cell polyclonal profile (Figure 1E). Among T/T CIN patients, the three displayed an oligoclonal profile (47.8% median T-cell clonality) while the remaining was polyclonal (10.2% median T-cell clonality) (Figure 1F). The TRBV repertoire was characterized by significant restrictions in both the C/C and T/T patients (Figure 1G,H). The biases were distinct between C/C vs T/T CIN patients and reached statistical significance in the case of 12 TRBV genes (P < .05). Two out of the 12 TRBV genes, namely TRBV20-1 and TRBV6-6, were overrepresented in the repertoire of C/C patients, while TRBV6-5 predominated in the T/T group (Figure 1I). In the C/C group, 660 distinct clonotypes were shared between 2-3 patients, while in the T/T group, 2373 distinct clonotypes were shared between 2-4 cases. Finally, 151 clonotypes were present in both patient groups; these clonotypes did not include the immunodominant clonotypes of the T/T patients. The data support the notion that the C/C CIN patients represent a group with distinct characteristics compared to the T/T CIN patients. In conclusion, we provide for the first time evidence that the DARC/ACKR1 SNP -46 T > C rs2814778 (FY*B ES/FY*B ES) genotype occurs at a substantial frequency among WE individuals with unexplained neutropenia. Homozygous individuals represent a group with different hematological/immunogenetic characteristics compared to CIN patients. It is unknown if these individuals have very distant roots in Africa. Possible admixture of African and other genetic material, representing millenia of migration patterns all over the Mediterranean basin into today's Europe, needs further evaluation. Further studies are needed to evaluate the frequency of the SNP rs2814778 and Duffy-null genotype and phenotype among patients with unexplained neutropenia originating from various European regions. Until such studies become available, we recommend the incorporation of the genetic test for the rs2814778 SNPs (or the Duffy antigen red blood cell phenotype if the genetic test is not available) in the diagnostic algorithm of European patients with unexplained neutropenia. If the test is positive and the individual is asymptomatic, further hematological evaluations can be stopped and the subject can be informed about the benign basis of the disorder, wheres the rest CIN patients need further investigation and follow-up. This study indicates further the heterogeneity of CIN and highlights the importance of future genome-wide analysis studies in different populations for the better understanding of the diverse pathogenetic mechanism underlying the disorder. This article is based upon work from COST Action CA18233 "European Network for Innovative Diagnosis and treatment of Chronic Neutropenias, EuNet-INNOCHRON" supported by European Cooperation in Science and Technology (COST). Appendix S1. Supporting information. Table S1. Clinical and laboratory data of the CIN# patients studied Table S2. Duffy blood group phenotypes in CIN patients and healthy controls Table S3. Absolute white blood cell counts in CIN patients and normal controls grouped by rs12075 and rs2814778 genotypes Table S4. TRB NGS analysis of CIN patients with rs2814778 C/C and T/T genotype Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.