Role of Duffy-Negative Phenotype in Pediatric Sickle Cell Population

Introduction

Neutrophils are well known for their pathogen-killing mechanisms and play an important role in body defense. Impaired neutrophil count and function are associated with recurrent infections. ¹ It has long been recognized that certain individuals, who are otherwise healthy and asymptomatic, have low leukocyte and neutrophil counts. ² In studies focusing on African Americans, it was found that a low white blood cell (WBC) count was predominantly due to low neutrophil counts. This phenomenon has been called “benign ethnic neutropenia” (BEN) as opposed to leukopenia. ³

BEN is one of the most prevalent causes of chronic neutropenia observed in individuals of African, Middle Eastern, and West Indian descent, affecting many people worldwide, without predisposing them for infections. ⁴

The Duffy Antigen Receptor for Chemokines (DARC) is a glycosylated cell membrane receptor, encoded by the DARC gene that is located on chromosome 1. A single nucleotide polymorphism in the DARC promoter region abolishes the gene expression and results in selective loss of DARC expression on red blood cells causing the DARC-null genotype or Duffy-negative trait. The (DARC)-null genotype is known to be predominant in people of African descent and is considered to be the major genetic determinant of benign ethnic neutropenia. However, the mechanism through which the DARC-null genotype induces neutropenia is not well understood. ¹

Sickle cell disease (SCD) is a hemolytic anemia that is inherited in an autosomal recessive fashion, caused by a point mutation in the β-globin chain of hemoglobin (Hb). ⁵ It has been estimated that more than 2 million U.S. residents are either heterozygous or homozygous for the genetic substitution. Most of those affected are of African ancestry and a minority are of Hispanic or southern European, Middle Eastern, or Asian Indian descent. ⁶

While many African American children with SCD are found to be Duffy antigen negative, they are shown to have elevated white blood counts. This may be explained by the fact that SCD is associated with a pro-inflammatory state, which accounts for the elevated baseline leukocyte count and inflammatory cytokines in this subset of population. ⁷

The Duffy antigen on red blood cells has been known to cause clearance of inflammatory cytokines, which may play a role in the pathogenesis of vaso-occlusion in SCD. Persons who are Duffy negative may less efficiently clear inflammatory cytokines. Therefore, SCD patients with Duffy-negative genotype may present with more severe disease. ⁸

Though there have been many studies related to Duffy antigen on adult population, there seems to be very limited literature in pediatric age group.

Our primary aim was to assess the variation in absolute neutrophil count among Duffy-negative children with and without SCD. We hypothesized that Duffy-negative patients with SCD can have an elevated WBC and ANC compared with Duffy-negative children without SCD.

Methods

This is a retrospective study where Duffy-negative children with SCD were compared with Duffy-negative children without SCD.

The study data were collected from the hospital’s electronic medical records. The study protocol was approved by the Institutional Review Board at Brookdale University Hospital Medical Center.

All study participants were ≤18 years of age who were followed up in the Division of Pediatric Hematology and Oncology at One Brooklyn Health, Brookdale Hospital Medical Center between January 2019 to June 2021. Cases included sickle cell pediatric patients with 1 of 4 SCD genotypes (SS, SC, S-beta+-thalassemia, and S-beta0-thalassemia) and being Duffy negative. Steady state data were obtained for them during their routine clinic visit. The control group consisted of asymptomatic pediatric patients without SCD who were referred by our primary care pediatricians to the hematology clinic for assessment of persistent neutropenia that was detected during regular annual physical examination. Later, they were found to be Duffy negative and diagnosed with BEN.

Children with immunodeficiency, any infection, recent steroid use, and SCD patients on hyper transfusion were excluded. Demographic data studied included age and sex of patient, race and ethnicity, and type of SCD. Sickle cell group was characterized according to the genotype. Primary outcome studied was the absolute neutrophil count (ANC). Secondary outcomes included WBC, hemoglobin, platelet count, and C-reactive protein (CRP).

To describe data of demographics, the mean and standard deviation was computed for continuous variables, and the frequency and percentages for categorical variables. To test the differences between Duffy-negative patients with SCD vs without SCD, Mann-Whitney U test was used for continuous outcome variables after checking for normal distribution and equal variances, and chi-square test for categorical outcome variables. The major outcome, absolute neutrophil count, was analyzed both as a continuous scale and an ordinal scale. To study the association with SCD, a linear regression model was fitted to the continuous scale and an ordinal regression model to the ordinal scale absolute neutrophil count. Statistical significance was evaluated with α = 0.05. IBM SPSS Statistics Version 26 was used for all statistical analyses.

Results

A total of 62 children were included in the analysis (30 with SCD and 32 without SCD). The median age of the cohort was 12 years; 69.4% were males and 80.6% were African Americans (Table 1).

Table 1.

Descriptive Statistics and Comparison of Outcome Variable by sickle cell disease Status.

Variable	Total (N = 62)		Sickle cell disease (N = 30)		No sickle cell disease (N = 32)		P value
	Median(min, max)	Count (%)	Median(min, max)	Count(%)	Median(min, max)	Count(%)
Age (years)	12 (1, 18)		10 (2, 17)		13 (1, 18)		.017
Sex
Male		43 (69.4%)		19 (63.3%)		24 (75.0%)	.32
Female		19 (30.6%)		11 (36.7%)		8 (25.0%)
Race
African American		50 (80.6%)		23 (76.7%)		27 (84.4%)	.74
Hispanic/Latino		7 (11.3%)		4(13.3%)		3 (9.4%)
Other		5 (8.1%)		3 (10.0%)		2 (6.3%)
White blood cell (K/μL)	5.2 (2.2, 15.3)		9.5 (6.2, 15.3)		3.0 (2.2, 5.4)		<.001
Absolute neutrophil count (K/μL)	1.5 (0.5, 11.0)		4.7 (2.6, 11.0)		0.9 (0.5, 1.6)		<.001
Hemoglobin (g/dL)	11.0 (6.8, 15.7)		9.0 (6.8, 13.6)		12.8 (10.2, 15.7)		<.001
Platelet count (K/μL)	289 (122, 645)		363.5 (126, 645)		259 (122, 429)		<.001
C-reactive protein (mg/dL)
0.6 or up		27 (43.5%)		25 (83.3%)		2 (6.3%)	<.001
< 0.5		35 (56.5%)		5 (16.7%)		30 (93.8%)

The ANC which is the primary outcome being analyzed showed a significant increase in Duffy-negative children with SCD (median 9.5 vs 3 in control; p < 0.001). The WBC, platelet count, and CRP all showed a significant increase in the SCD group compared with control group (p < 0.001). A considerable drop in hemoglobin was noted in the SCD group (median 9 vs 12.8; p < 0.001). The elevated CRP in the SCD group may explain the inflammatory state in this group (Table 1).

On further analyzing the data in the SCD group related to Hydroxyurea (HU) use, we found no statistically significant change in the outcome variable (Table 2).

Table 2.

Comparison of Outcome Variable Across Different Type of SCD and Hydroxyurea Use.

Variable	Type of sickle cell disease (N = 30, median)				Hydroxyurea use among SCD subjects (N = 30, median)
	HbSS (n = 23)	HbSC (n = 5)	HbSB0(n = 2)	P value	Yes (n = 24)	No (n = 6)	P value
White blood cell (K/μL)	9.3	11.8	9.1	.33	9.4	11.7	.16
Absolute neutrophil count (K/μL)	4.6	7.2	5.8	.41	4.7	5	.46
Hemoglobin (g/dL)	9	10.5	8.9	.006	8.8	9.9	.018
Platelet count (K/μL)	366	328	231.5	.22	363.5	336.5	.82

Abbreviation: SCD sickle cell disease; HbSS – Hemoglobin SS; HbSC – Hemoglobin SC; HbSB0 – Hemoglobin sickle beta zero thalassemia.

Discussion

Sickle cell disease is common among African American population, and nearly 68% of them are negative for the Duffy antigen. ⁵ , ⁹ Patients who are Duffy negative have been noted to have neutropenia. ³ Therefore, the WBC/ANC baseline in patients with SCD is of interest, particularly because they are prone to develop infections/sepsis. ⁶

Conflicting findings have been reported regarding the clinical significance of RBC Duffy expression in sickle cell anemia. ⁷ In a study by Afenyi-Annan et al, ⁸ it was concluded that the Duffy-negative status was strongly associated with more severe disease among patients with SCD owing to the inefficient clearance of inflammatory cytokines. On the other hand, studies by Schnog et al ¹⁰ and Nebor et al ¹¹ have demonstrated no significant difference in clinical severity between Duffy-positive and Duffy-negative sickle cell patients.

It is a well-known fact that SCD is associated with a chronic inflammatory state. High WBC count is a potential risk factor for severe SCD-related complications. ⁷ Previous studies have shown that WBC levels were considerably higher in Duffy-positive individuals than in Duffy-negative patients. ⁷ , ⁸ Draser et al ¹² found that Duffy-negative individuals with and without SCD had reduced WBC and neutrophil counts.

On the contrary, our research suggests that Duffy antigen-negative children with SCD exhibit higher white blood counts than Duffy-negative children without SCD.

Patients with SCD are given HU to ameliorate the effects of the disease, increase the hemoglobin F, reduce the vaso-occlusive crises, and prevent organ damage. Hydroxyurea’s known side effect is leukopenia/neutropenia, which if present can limit its use. ¹³

The sustained stability of ANC in patients with SCD on HU may be explained due to the persistent inflammatory state and reactive bone marrow that help these otherwise Duffy-negative patients to overcome the potentially lowering of the WBC, which may affect the use of HU.

Our study has some limitations which include a small sample size with predominantly African American population based on a single center.

Further adequately powered studies are needed to determine the variations in WBC counts in SCD patients when they are in a steady state, when they have a vaso-occlusive crises and with HU use based on the Duffy expression. This could be effective in determining the physiologic relevance of this protein in patients with SCD.

Author Contributions

Dr. Jeyamurugan has conceptualized, designed, analyzed, drafted the manuscript and approved the final version of the manuscript. Dr. Jung has contributed to analysis and approved the final version of the manuscript. Dr. Viswanathan has critically revised the manuscript and approved the final version. Dr. Kupferman has contributed to conception and design, critically revised the manuscript and approved the final version of the manuscript. Dr. Peichev has conceptualized, designed, interpreted, critically revised the manuscript and approved the final version of the manuscript.