Molecular Investigation of Iranian Patients Suspected to Hereditary Spherocytosis

Research Article

J Blood Disord. 2021; 8(1): 1063.

Molecular Investigation of Iranian Patients Suspected to Hereditary Spherocytosis

Shahab-Movahed Z1, Majd A1, Torbati ES2 and Zeinali S3,4*

1Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Islamic Azad University, North Tehran Branch, Tehran, Iran

2Department of Genetic, Faculty of Science, North Tehran Branch of Islamic Azad University, Tehran, Iran

3Department of Medical Molecular, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

4Kowsar Human Genetic Research Center, Tehran, Iran

*Corresponding author: Sirous Zeinali, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Received: May 31, 2021; Accepted: June 18, 2021; Published: June 25, 2021


Introduction: Hereditary spherocytosis is a heterogeneous disorder with mild to moderate anemia. The aim of this study was to evaluate the inherited spherocytosis gene mutations in patients with RBC cytoplasmic disorders in Iranian population.

Materials and Methods: In this study, Whole Exome Sequencing (WES) was performed for patients suspected to hereditary spherocytosis and their relatives.

Results: Sequence analysis of the probands and their parents identified variations in ANK1 gene (NM_001142446.1:c.127-2A>G), SPTB (c. 14delC, p.Thr5LysfsTer41), SPTA1 (c.466C>T), SLC4A1 (c.2494C>T) and SLC25A38 gene (c.683G>T, NP_060345.2:p.Gly228Val that could be related to the patients clinical manifestation.

Conclusion: Findings are in line with the appropriate diagnostic yield of WES in determining the causative variant especially in those disorders that many genes are involved like anemia. This is the first report of a cohort of Iranian patients with anemia suspected to that were investigated using WES technology. Further studies are needed to investigate the distribution of gene mutations in patients with RBC membrane disorders in Iran

Keywords: Hereditary spherocytosis; Whole-exome sequencing; Hemolytic anemia; Erythrocyte membrane protein; ANK1


HS: Hereditary Spherocytosis; DRTA: Hereditary Distal Renal Tubular Acidosis; AE1: Anion Exchanger 1; WES: Whole Exome Sequencing; ACMG: American College of Medical Genetics; ANK1: Ankyrin 1; EPB42: Erythrocyte MEMBRANE PROTEIN BAND 4.2; HS: Hereditary Spherocytosis; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; MCV: Mean Corpuscular Volume; PCR: Polymerase Chain Reaction; RBCs: Red Blood Cells; SLC4A1: Solute Carrier Family 4 Member 1; SPTA1: Spectrin a Erythrocytic 1; SPTB: Spectrin β;Variants of Unknown Significance (VUS)


Red cell membrane disorders that affect its membrane stability results in irregular red cell shape and disorders including Hereditary Spherocytosis (HS), Hereditary Elliptocytosis (HE), Hereditary Pyropoikilocytosis (HPP) and Hereditary Stomatocytosis (HSt) [1,2]. Hereditary spherocytosis (HS) refers to a common heterogeneous group of congenital hemolytic anemia due to red blood cell structural proteins disorder resulting in spherical-shaped erythrocytes on the peripheral blood smear [1] with wide clinical heterogeneity ranging from asymptomatic cases to severe hemolytic anemia [3] besides reticulocytosis, jaundice, splenomegaly, increased indirect bilirubin and red blood cell osmotic fragility. The worldwide incidence is about one in 2,000 to 5,000 Caucasians [4] and few cases were reported in the black population [2,5] with dominant inheritance in most cases (75%) [6]. Five genes correlated with the pathogenesis of HS so far including Ankyrin (ANK1) (most common (7)), a-spectrin (SPTA1), β-spectrin (SPTB), band 3 (SLC4A1) and protein 4.2 (EPB42) that interplay between the erythrocyte membrane and the lipid bilayer [8]. Defects in protein product of these genes, result in loss of vertical junction between cell membrane and lipid bilayer [1]. High-throughput Next Generation Sequencing (NGS) technology has promoted rapid and cost-effective genetic diagnosis of various genes, especially when routine laboratory testing fails [9,10]. Here, we present clinical and molecular genetics of Iranian cases with HS investigated with whole exome analysis to find the causative mutations to provide prenatal diagnosis in the future pregnancies.

Materials and Methods

Patients and study design

Patients with anemia and primary clinical diagnosis of Hereditary Spherocytosis (HS) were referred from the Blood disorder clinic of Ali-Asghar Children’s Hospital, Iran University of Medical Sciences, Tehran, Iran to Dr. Zeinali’s Medical Genetics Lab, from September 2017 to March 2018 for genetic counseling and genetic diagnosis. Each patient was reviewed to determine clinical features, laboratory data, disease history (age at diagnosis, jaundice, transfusion history and age at first transfusion and family history). Families with clinical evidence of congenital hemolytic anemia suspected to HS were recruited to the study. After genetic counseling, drawing family pedigree, recording the clinical characteristic and obtaining informed consent, peripheral blood sample (10 ml) were taken from patients and other family members on tubes containing EDTA. Hematologic tests including Complete Blood Count and Analysis (CBC), reticulocyte count, NaCl osmotic fragility test (on fresh and incubated blood), red blood cell distribution width (RDW), peripheral blood smear examination, Direct Antiglobulin Test (DAT) and hemoglobin electrophoresis were performed at Ali-Asghar Children’s Hospital laboratory. Genomic DNA was extracted using salting out Proteinase K method (Kawsar Biotech Co., Tehran, Iran) following the manufacturer’s instruction. Quantity and quality of isolated DNA were measured by Nanodrop spectrophotometer (ThermoScientific®, Waltham, USA) and gel electrophoresed on 1% agarose, respectively. This study was approved by the ethics committee of Kawsar Human Genetics Research Center (KHGRC).

Whole genome sequencing was performed using Illumina’s HiSeq X-ten machines (deCODE Genetics, Reykjavik, Iceland) (Supple S1). More than 95% of the targeted sequences being covered adequately for high-confidence variant calling (>30 X coverage). Rare variants with minor allele frequency less than 1% in the population were included in final analysis. We considered coding and splicing variants with high impact (stop, frameshift, and splice site) and moderate impact (missense, in frame and splice region) on protein function and focused on Single-Nucleotide Polymorphisms (SNPs) and small indels (< 20 base pairs) and prioritized the analysis on mutations in phenotypically relevant genes, i.e. genes with a known link to Mendelian disorders according to Online Mendelian Inheritance in Men (OMIM) ( that fit the phenotypes of the index and the inspected inheritance model (especially those relevant to HS including ANK1 (HS type 1, OMIM #182900), SPTB (HS type 2, OMIM #616649), SPTA1 (HS type 3, OMIM #270970), SLC4A1 (HS type 4, OMIM #612653), and EPB42 (HS type 5, OMIM #612690) genes). Vari¬ants in candidate genes that were suspected to be involved in the disease pathogenesis (Variants of unknown significance (VUS), Likely Pathogenic and Pathogenic) were selected for Sanger sequencing in the proband, her/his parents and other family members, if present. Specific forward and reverse primers according to the identified variants were designed with NCBI Primer- Blast Tool ( and synthesized commercially by Metabion Co., Germany. A list of the designed primers and amplification reaction were provided in Supple S2 and Table S1.


Twenty-five samples (9 affected cases and 16 healthy family members from 5 consecutive families (consanguineous marriage in 2 families) were enrolled to the study by anemia and primary clinical diagnosis of Hereditary Spherocytosis (HS). Clinical characteristics and laboratory data in probands at the time of first visit are summarized in Table S1. Following variant filtering of WES results in the probands and their parents, two pathogenic/likely pathogenic variants in ANK1 and SPTB genes were identified in two separate families and confirmed by segregation analysis within the family by direct Sanger sequencing (Table 1, Figure 1 and Figure 2). Furthermore, we identified some candidate variants of uncertain significance in other families that underwent further segregation study within the family to confirm their presence and pathogenicity, as follows.