Current Status of Molecular Diagnosis of Hereditary Hemolytic Anemia in Korea

Epidemiology of HHA in Korea

In 1991 a nationwide multi-center epidemiological study of HHA in Korea covered the period from 1981 to 1990. This study covered 315 patients, including 162 pediatric patients aged 0–15 years.36 Since 1997, epidemiological studies on HHA have been conducted in 10-year cycles by the RBC Disorder WP of the KSH. The first 10-year national epidemiological study of HHA, covering the period from 1997 to 2006, was published in 2007.4 This study covered 431 subjects, including 333 pediatric patients from 35 hospitals across Korea. The second 10-year nationwide Korean epidemiological study, covering the period from 2007 to 2016, was reported in 2020.6 This study included 369 pediatric patients with HHA diagnosed using the Korean SOP for HHA.4, 5 These epidemiological studies between 1997 and 2016, HS is the most common type of HHA, accounting for over 70% of all nonimmune hemolytic anemia. From 2007 to 2016, 253 pediatric patients were diagnosed with HS, whereas 10 of the 369 patients with nonimmune hemolytic anemia were diagnosed with HE.

Thalassemia major and other hemoglobinopathies were previously uncommon. However, recent increases have occurred4, 6 primarily because of increased thalassemia minor (carriers of thalassemia) attributed to an increase in international marriage contacts.37 Additionally, clinicians may have increased their knowledge of hemoglobinopathy in Korean children with anemia.2

RBC enzymopathies are rare in Korea, with G6PD deficiency and PKD the most common.38, 39 From 2007 to 2016, 23 pediatric patients with RBC enzymopathy were recorded, including 12 with PKD and nine patients with G6PD deficiency. More recently, the incidence of PKD has increased markedly compared with the period 2007 to 2016.6 One factor may be external immigration, especially from South-East Asia, because of international marriages.40, 41, 42, 43 The increase in the diagnostic confirmation of RBC enzymopathy may be attributed to the increased availability of genetic diagnostic techniques, including NGS.16, 44, 45 Fig. 1 illustrates the shifts in the epidemiology of HHA in Korea. Furthermore, Table 1 provides a summary of HAA diagnosis in Korea, encompassing all HAA patients recruited in the three preceding nationwide studies.4, 6, 36

Fig. 1
Changes in the epidemiology of hereditary hemolytic anemia in Korea over three decades. Although membranopathy is predominant, there has been a marked increase in the detection rates of hemoglobinopathy and red blood cell enzymopathy as well.

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Table 1
Trend of studies on the epidemiology of hereditary hemolytic anemia in Korea

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Clinical and laboratory features of HHA in Korea

HHA is suspected with symptoms such as anemia, jaundice, and splenomegaly.2 The most common symptom/sign of HHA is neonatal jaundice, which may require medical intervention.46

With RBC membranopathy, most patients with autosomal dominant-type HS have a mild phenotype. whereas those with autosomal recessive type HS may have severe anemia requiring transfusions.46 Most patients with HS may also show splenomegaly.12 Those with well-compensated anemia in HS may receive a diagnosis later in life because of cholelithiasis.47 For example, in one study on HHA, jaundice and splenomegaly were the most common symptoms among patients with RBC membranopathy.6

Patients with hemoglobinopathy may show facial changes, characterized by enlarged maxillary sinuses resulting from blood cell production. These facial changes can lead to ear, nose, and throat infections in patients, especially those with thalassemia major, to infections of the ears, nose, and throat. Adults with thalassemia major with chronic transfusion-dependent anemia and iron overload may develop complications such as cardiomyopathy, diabetes mellitus, hypopituitarism, hypoparathyroidism, hypothyroidism, or testicular/ovarian failure.20 The most common presentation in Korea is thalassemia minor, which is usually asymptomatic. In tests conducted on Koreans with HHA, the median value of Hb was the highest, whereas those of the RBC profiles were the lowest. The median value of corrected reticulocytes was also low, as was that of lactate dehydrogenase. In contrast, the median value of haptoglobin was high in Korean individuals with hemoglobinopathy, suggesting that hemolysis is not severe among patients with hemoglobinopathy in Korea.6

For RBC enzymopathy, patients with G6PD deficiency typically develop jaundice a few days after birth.46 However, beyond infancy, most G6PD-deficient patients have no symptoms under normal conditions. Hemolysis in these individuals is triggered by oxidative stressors such as infections or some medications.26, 46 When exposed to oxidative stressors, G6PD-deficient patients can experience abrupt hemolytic anemia, with pallor, jaundice, and the presence of red-brown urine. Patients with PKD present a wide spectrum of clinical manifestations.48 The severity of hemolysis in PKD can be exaggerated during infections or pregnancy.49 Some patients with PKD have lifelong, fully compensated, or mild anemia, whereas others experience severe anemia requiring repetitive RBC transfusions.50 In severe cases of PKD, patients may develop transfusion-dependent iron overload or cholelithiasis.48, 49 In a Korean epidemiological study for HHA, patients with RBC enzymopathy exhibited the most severe degree of anemia among individuals with HHA. Additionally, the diagnostic age of individuals with RBC enzymopathy was the lowest among the different HHA groups.

Genetic characteristics of HHA in Korea

Studies utilizing confirmation tests for patients with HHA in Korea are scarce because of the limited availability of certain confirmation test facilities. Lee et al.51 conducted a study on 27 patients with HS and identified various types of HS, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): spectrin deficiency in 7.4% of cases (2/27), ankyrin deficiency in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27), and protein 4.2 deficiency in 14.8% (4/27). These findings indicated that ankyrin deficiency alone and its combination with spectrum deficiency accounted for 33.3% of cases (9/27), as the most common biochemical defects. Notably, protein 4.2 deficiency was more prevalent among Koreans than Caucasians. Protein defects were not observed in nine out of the 27 patients (33.3%).51

Park et al.52 reported 42 suspected patients with HS that were screened via OFT and confirmed using the molecular tic test of Sanger sequencing. Of these, 25 patients carried one heterozygous mutation of ANK1 (n = 13) or SPTB (n = 12).52 Deleterious mutations, including frameshift, nonsense, and splice site mutations were found in 91% (21/23) of cases, with non-hotspot mutations dispersed across multiple exons. This was the first large-scale single-center study to examine ANK1 or SPTB mutational characteristics and genotype-phenotype correlation. Anemia was most severe in patients with HS who carried mutations on the ANK1 spectrin-binding domain, whereas SPTB mutations in HS patients spared the tetramerization domain, where HE and pyropoikilocytosis mutations are typically located. The study also found a high frequency of splenectomy in ANK1-mutant HS (32%), as compared to that in SPTB-mutant HS (10%) (P = 0.028).

Choi et al.53 conducted the first multi-center genetic study of Korean patients with HS. Genetic variations in RBC membrane protein-encoding genes were examined using multi-gene targeted NGS (t-NGS) in patients with HS diagnosed by Korean HHA guidelines. These findings were compared with clinical features. A total of 43 genes were included, comprising 17 RBC membrane protein and 20 RBC enzyme-encoding genes, along with six additional candidate genes, for differential diagnoses (thalassemia, congenital dyserythropoietic anemia [CDA], paroxysmal nocturnal hemoglobinuria, and Gilbert syndrome). Of the 59 patients with HS, 50 (84.7%) had one or more critical variants in the RBC membrane protein-encoding genes. A total of 54 critical variants, including 46 novel mutations, were detected in six RBC membrane protein-encoding genes. The highest number of variants were found in SPTB (n = 28), followed by those in ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations in genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were also detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). The positive rate of OFT was 86.8% among patients harboring HS-related gene mutations. As a result, many single-gene confirmatory tests that utilized Sanger sequencing have transitioned to NGS, particularly t-NGS for HHA.45, 53, 54, 55 Following these studies, cases with novel mutations have continued to be reported using Sanger sequencing, t-NGS, or whole-exome sequencing (WES).56, 57, 58, 59, 60

Traditional laboratory diagnosis

Molecular diagnosis of HHA

The molecular testing strategies for HHA have developed with the adoption of NGS in many clinical laboratories. Many causative genes are associated with a single HHA entity, and these may overlap between diseases with different phenotypes.64 In addition, the clinical presentation of HHA can overlap and lack specificity, requiring complex analyses and consequent complicate the diagnosis.47, 70 In one study, multi-gene analysis modified the original clinical diagnosis in 45.8% of patients with congenital anemia, including HHA. For example, 45.5% of the probands initially classified as CDA were diagnosed with chronic anemia because of enzymatic defects, primarily caused by mutations in the PKLR gene.54 In another study, t-NGS enabled molecular diagnosis in 38.6% of patients, which accounted for only 13.6% of the known cases, leading to altered treatment approaches.71 Co-occurrence of multiple disorders can also complicate phenotypic assessment, necessitating molecular testing. Recent studies report co-inheritance of G6PD in 15% of patients with HS,72 as well as that of G6PD and dehydrated stomatocytosis,54 and combined metabolic deficiencies with RBC membrane defects.73 Accurate diagnosis is crucial to avoid complications or unnecessary interventions for HHA. In cases of hereditary stomatocytosis, splenectomy is contraindicated, whereas in PKD, a new oral activator of PK may offer therapeutic potential.50, 74

The British Society of Hematology and the European Hematology Association recently published a practice paper on the use of NGS for inherited anemias, including HHA, and recommended NGS after characterization of the phenotype, including gene panels for Diamond-Blackfan anemia, CDA, congenital sideroblastic anemia, RBC enzymopathy, and RBC membranopathy.55 Patients suspected of a specific type of HHA achieved higher diagnostic yields than those with an unspecified HHA subtype.73 However, in Korea, because of the widespread availability of t-NGS testing and limited access to tests characterizing the phenotype, such as RBC membrane analysis, ektacytometry, or RBC enzyme testing, molecular diagnosis is recommended as the initial step in the diagnostic workup, with phenotypic correlation performed later in the process.2 Several reports of t-NGS results for HHA in Korea have been published, especially for HS, followed by genetic profiling to confirm the HHA diagnoses.53, 57, 58, 75

Several steps are required before implementing t-NGS for HHA diagnosis.76 Target genes should be carefully selected to include potential causative genes for HHA and diseases that mimic or overlap with HHA. The clinical t-NGS panels reported in the Genetic Testing Registry for HHA typically include 28–40 genes, and a recent Korean clinical practice guideline for HHA diagnosis recommends specific genes for testing.2

Regarding the type of genetic variants, all possible pathogenic variants, including single nucleotide variants, indels, or copy number variations, should be considered and evaluated. If necessary, valid ancillary assays should be performed to complement the analysis.55 As copy number variations cannot be fully analyzed in some t-NGS panels, additional molecular testing is required in conjunction with t-NGS, especially for RBC hemoglobinopathy. Additionally, common variants detected in the population can be considered for the screening test.77, 78 In HS, common pathogenic modifiers (e.g., alpha-LeLY/LePRA) are present in over 1% of the general population and should be checked in patients suspected of HS or HE, along with other SPTA1 variants. Although alpha-LeLY is usually included in most t-NGS panels, alpha-LePRA (c.4339-99C>T) may not be covered within ± 10 base pairs of the exons in many panels, or might be lost during data processing; thus, it should be considered. However, alpha-LePRA is often co-inherited with the α-Bughill allele (missense variant p.[Ala970Asp]), which is covered by standard t-NGS panels, allowing confirmation of α-LEPRA using alternate methods.79, 80 For thalassemia, common globin gene deletions are analyzed using gap-PCR or MLPA,81 and t-NGS simultaneously tests for HBA1, HBA2, and HBB. Depending on the chosen method, the laboratory should be aware of any limitations and either reduce them or clearly state what has not been tested in the report. In cases where no causative variants are identified, WES or whole-genome sequencing (WGS) may be considered, although this remains under research.16, 71, 82 Although t-NGS may offer testing for a limited number of genes and variable phenotypes, comprehensive genetic analyses using WES have allowed the diagnosis of G6PD deficiency in patients initially suspected of CDA and the identification of genetic variants in non-HHA genes that explained part of the phenotypes. Some laboratories have opted for WES over t-NGS (because of its increasing availability) and have explored the identification of causative genes via trio-analysis of WES.83, 84, 85 Various methods for diagnosing HHA have been succinctly summarized in Table 2, including their respective strengths, weaknesses, sensitivities, and specificities.

Table 2
Comparison of different methods to differentiate and diagnose HHAs

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However, despite advances in sequencing techniques, genetic causes may remain unexplained in some patients with HHA.86 In addition, the clinical importance of some detected mutations may be unclear, often classifying them as a ‘variant of uncertain importance.’ Even if a genetic abnormality is found, the genotype may not align with the patient’s phenotype. Therefore, characterizing a patient's phenotype using traditional laboratory tests remains essential for individual patient diagnosis, severity assessment, and medical management planning. Several traditional diagnostic laboratory methods for HHA have undergone improvements in Korea.

General guidelines on diagnosis of HHA in other countries

Diagnosis of HHA is often challenging because of the potential overlap of clinical symptoms and laboratory test results. In some cases, identifying the causative gene may be necessary to establish a clear diagnosis, and to avoid unnecessary investigations and ensure timely and appropriate treatment. Therefore, many countries have developed their own practice guidelines and recommendations tailored to their unique epidemiological and medical environments. However, the core approach to diagnosing HHA remains similar across these guidelines. When suspecting HHA in a patient, a comprehensive diagnostic process is essential, which includes a thorough physical examination, as well as a detailed review of the patient’s medical and family history. The laboratory evaluation process may vary depending on the specific type of HHA suspected or the patient’s epidemiological/ethnic background.

Guidelines for diagnosing HS are abundant because of its high incidence, whereas those for diagnosing other types of HHA are relatively limited. In most cases of RBC membranopathy and hemoglobinopathy, the key lies in interpreting the peripheral blood smear findings, which then guide the subsequent evaluation. When HS is suspected, many countries recommend the OFT as a screening test, whether the classical method or the EMA-binding test. However, it is important to note that OFT can yield both false positives and false negatives, especially in neonates or younger patients with conditions such as CDA type II (CDA-II) or other membranopathies. To address these challenges, guidelines in the UK and from the International Council for Standardization in Haematology recommended membrane protein analysis as a screening or diagnosis test.12, 17 More recent guidelines advocate for molecular genetic testing such as PCR or NGS, especially in neonates or individuals who do not have documented family histories.87 A recent Chinese protocol also suggests NGS as the next step after screening tests such as the OFT, assessment of G6PD levels, and acidified glycerol lysis test, instead of the SDS-PAGE test.88

However, a recent meta-analysis on the diagnostic methods for RBC disorders in Asia revealed varying proportions of using genetic analysis to identify mutations, with Sanger sequencing, NGS, and WES being used in 33.3%, 22.2%, and 13.8% of cases, respectively.35 Surprisingly, proteomics was employed in only 5.5% of the total studies, which is a relatively low proportion given the existing recommendations and guidelines. In contrast, guidelines for diagnosing enzymopathy are relatively scarce, with the most recent (2019) recommendations for PKD provided by EuroBloodNet, the European Reference Network in Rare Hematological Diseases representing one of the latest guidelines for enzymopathy.89

Current SOP of HHA in Korea

The RBC Disorder WP of KSH has introduced an SOP for the diagnosis of HHA, as illustrated in Fig. 2.5, 15 When dealing with patients suspected of having HHA, it is crucial to conduct a thorough review of their medical history, including familial and ethnic backgrounds, in addition to appropriate laboratory tests for HHA. The primary focus should initially be on complete blood count, with RBC indices and peripheral blood smear findings. If RBC membranopathy is suspected, especially HS, OFT, flow cytometric OFT, or EMA-binding test are recommended. Although the EMA-binding test is highly predictive for HS, the fluorescence intensity of RBCs in Southeast Asian ovalocytosis, cryohydrocytosis, and some cases of CDA-II can fall within the same range as that expected for HS. Additionally, patients with HS who have isolated ankyrin deficiency may exhibit normal fluorescence results. Clinicians should, therefore, correlate these findings with family studies and other test results, for comprehensive diagnosis and accurate assessment.90

Fig. 2
SOP for the diagnosis of HHA by the RBC Disorder Working Party of the KSH.5, 15
HHA = hereditary hemolytic anemi, CBC = complete blood count, RBC = red blood cell, TIBC = total iron binding capacity, LDH = lactate dehydrogenase, EP = electrophoresis, PB = peripheral blood, OFT = osmotic fragility test, EMA = eosin 5-maleimide, HPLC = high-performance liquid chromatography, NGS = next-generation sequencing, SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis, WES = whole-exome sequencing, WGS = whole-genome sequencing, SOP = standard operating procedure, KSH = Korean Society of Hematology.

^*Prioritize enzyme analysis before transfusion in suspected patients; if unclear, perform NGS testing afterwards.

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For diagnosis of RBC membranopathy, the International Council for Standardization in Haematology guidelines recommends traditional methods for quantifying RBC membrane proteins, using SDS-PAGE. Although more advanced techniques have emerged, including matrix-assisted laser desorption/ionization-time of flight mass spectrometry or LC-MS/MS, these are currently not available in Korea.54, 91, 92 Therefore, in cases of RBC membranopathy in Korea, the recommendation is to confirm the diagnosis using molecular genetic methods, which can provide accurate results.

For RBC enzymopathy, the classical spectrophotometric methods used to measure RBC enzyme activity are typically labor-intensive and time-consuming. To address the limitations, a recommended approach is to use the ion-pairing UPLC-MS/MS method. It is important to note that the current UPLC-MS/MS RBC enzyme activity assay covers only six enzymes. Thus, clinicians should be aware of which specific enzymes are tested and avoid ruling out untested diagnoses. Additionally, when interpreting enzyme assay results, factors such as previous RBC transfusions, which can affect accuracy, must also be considered.

Hemoglobinopathies in Korea are typically screened using either high-performance liquid chromatography or hemoglobin electrophoresis. Abnormal results obtained from these methods are usually confirmed via genetic testing, primarily employing Sanger sequencing and/or MLPA.93

Molecular diagnosis of HHA in Korea

RBC membrane disorders are usually caused by membrane structural defects or altered membrane transport function. As further evaluations for membrane proteins are not available in Korea, molecular genetic tests can be considered. Genetic variations reported in Korea include those related to cytoskeleton and membrane protein quantitative deficiencies including ankyrin, erythrocytic α- and β-spectrin chains, band 3 and protein 4.2.53 ^, 80 Although genetic testing is a powerful tool in the diagnosis of these disorders, approximately 50% of patients remain undiagnosed despite NGS testing.80

The diagnosis of enzymopathy in Korea is currently dependent on UPLC-MS/MS RBC enzyme activity assay, which has a limitation of covering six enzymes. Therefore, molecular genetic tests are recommended as the next step for patients who are suspected of enzymopathy.

For every patient suspected of having HHA, a genetic test is strongly recommended as a confirmation test. t-NGS is now routinely used in undiagnosed patients and family members with HHA. t-NGS is preferred over WES or WGS because of its lower cost, quicker turnaround time, simpler data analysis, better coverage of relevant regions of interest, and reduced risk of incidental findings. The RBC Disorder WP of the KSH has created a t-NGS panel that encompasses genes associated with RBC membranopathy, enzymopathy, and other diseases that share overlapping features with HHA (Table 3), including ADA, AK1, ALDOA, ANK1, CYB5R3, EPB41, EPB42, G6PD, GCLC, GPI, GSR, GSS, HBA1, HBA2, HBB, HK1, NT5C3A, PFKM, PGK1, PIEZO1, PKLR, SLC4A1, SPTA1, SPTB, TPI1, and UGT1A1.2 It is worth noting that in Korea, t-NGS is often considered before RBC enzyme or membrane protein analysis because of its greater availability. Of note, specific considerations may be required for the detection of large deletions/duplications, variants in introns, and copy number variations related to hemoglobinopathy.

Table 3
Genes recommended for hereditary hemolytic anemia gene panel testing

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In the case of hemoglobinopathy, especially thalassemia, it is recommended to perform Sanger sequencing and MLPA as the initial approach, rather than t-NGS. A retrospective epidemiological study of HHA conducted in Korea revealed that most of the hemoglobinopathies were β-thalassemia minor (83.1%) or α-thalassemia minor (15.3%). In the case of β-thalassemia, the primary mutations involve single nucleotide variants in the HBB gene, which can be detected using sequence analysis. However, this may also encompass variants in non-coding regions, and rarely, large deletions. If HBB sequencing reveals only one pathogenic variant or none at all, deletion/duplication analysis is typically conducted. In contrast, α-thalassemia cases in Korea are primarily caused by a deletion in the HBA1 or HBA2 genes, accounting for up to 85% of all causative mutations. As mentioned above, such large deletions can be missed in sequencing or t-NGS. It is worth noting that, despite the importance of MLPA in diagnosing α-thalassemia, it is not routinely performed in Korea because of it not being covered by the health insurance, potentially leading to an underestimation of α-thalassemia in the country.