Identification of a novel splice variant in SEC23B gene in a patient with concomitant presence of congenital dyserythropoietic anemia II and Gilbert’s syndrome

ABSTRACT

Background:

Congenital dyserythropoietic anemia Ⅱ (CDA Ⅱ) is a rare inherited disorder of defective erythropoiesis caused by SEC23B gene mutation. CDA Ⅱ is often misdiagnosed as a more common type of clinically related anemia, or it remains undiagnosed due to phenotypic variability caused by the coexistence of inherited liver diseases, including Gilbert’s syndrome (GS) and hereditary hemochromatosis.

Methods:

We describe the case of a boy with genetically undetermined severe hemolytic anemia, hepatosplenomegaly, and gallstones whose diagnosis was achieved by targeted next generation sequencing.

Results:

Molecular analysis revealed a maternally inherited novel intronic variant and a paternally inherited missense variant, c.[994-3C > T];[1831C > T] in the SEC23B gene, confirming diagnosis of CDA Ⅱ. cDNA analysis verified that the splice acceptor site variant results in two mutant transcripts, one with an exon 9 skip and one in which exons 9 and 10 are deleted. SEC23B mRNA levels in the patient were lower than those in healthy controls. The patient was also homozygous for the UGT1A1*6 allele, consistent with GS.

Conclusion:

Identification of the novel splice variant in this study further expands the spectrum of known SEC23B gene mutations. Molecular genetic approaches can lead to accurate diagnosis and management of CDA Ⅱ patients, particularly for those with GS coexisting.

KEYWORDS:

• Congenital dyserythropoietic anemia II
• next generation sequencing
• SEC23B
• splicing defects
• Gilbert’s syndrome

Introduction

Congenital dyserythropoietic anemias (CDAs) comprise a heterogeneous group of genetic disorders of red cell production characterized by ineffective erythropoiesis and dyserythropoiesis. Four major types of CDA, including CDA I (OMIM # 224120 for CDA 1A; # 615631 for CDA 1B), CDA II (# 224100), CDA III (# 105600), and CDA IV (# 613673) have been designated based on morphological findings in bone marrow and genetic variations [1]. CDA Ⅱ (the most common form of CDA) is a rare autosomal recessive disorder with major clinical manifestations involving mild to transfusion-demanding hemolytic anemia, fatigue, jaundice, and hepatosplenomegaly.

Due to the rarity of the disease and its clinical overlap with other inherited hemolytic anemias, it is difficult to predict which of the known CDA-related genes is causative in any given affected individual [2]. Furthermore, coexistence with more frequent autosomal recessive genetic disorders such as hereditary hemochromatosis (HHC) or Gilbert’s syndrome (GS), can result in phenotypic variability in CDA Ⅱ patients. Iron overload may be exacerbated when HHC and CDA Ⅱ are concomitant, whereas coexistence of GS with CDA Ⅱ is associated with an unusually high level of hyperbilirubinemia.

Overall, the difficulty in diagnosing this disorder leads to treatment delays and increased morbidity and mortality. Genetic testing can improve the speed and accuracy of diagnosis, and the vast majority of patients with a confirmed diagnosis of CDA II harbor biallelic SEC23B gene mutations. Indeed, over 100 pathogenic or likely pathogenic variants have been reported to date in patients of different origins [3]. However, clinical and genotypic data for CDA II in Asian populations are insufficient. To the best of our knowledge, mutations underlying CDA II have not been discovered in Korean patients. We herein report the first genetically confirmed case of coexisting CDA II and GS in Korea resulting from a novel splicing SEC23B variant associated with exon skipping.

Methods

Next-generation panel sequencing

This study was approved by the Institute Review Board of Inha University of Korea (IRB No. 2021-05-030). The requirement for written informed consent was waived by the IRB because of the anonymous and retrospective nature of the study. Genomic DNA was enriched by using TruSight One sequencing panels (Illumina, San Diego, CA, U.S.A.) and sequenced using the MiSeqDx platform (Illumina). This panel targets exonic regions that harbor disease-causing variants in up to approximately 4,800 genes associated with known clinical phenotypes according to Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php), GeneTests (www.genetests.org), and Online Mendelian Inheritance in Man (OMIM, www.omim.org). Extracted sequencing data were processed using MiSeq Reporter (Illumina), which includes Burrows–Wheeler Aligner (BWA) for alignment to a reference genome (GRCh37/hg19) and Genome Analysis Toolkit (GATK) for variant calling. The Variant Call Format (VCF) file was analyzed by applying BaseSpace Variant Interpreter Software (Illumina). Clinically relevant variants were validated by Sanger sequencing.

Reverse transcription (RT)-PCR analysis of the SEC23B gene

Reverse transcription (RT)-PCR analysis was performed to determine whether the c.994-3C > T mutation produces different SEC23B transcripts as a splice site mutation. Total RNA was extracted from the peripheral blood lymphocytes using High Pure RNA Isolation Kit (Roche Applied Sciences, Germany), followed by cDNA synthesis using Transcriptor First Strand cDNA Synthesis Kit (Roche). A region encompassing exons 8–12 was amplified using one set of primer pairs developed by the authors. The cDNA amplification products were analyzed by agarose gel electrophoresis and sequencing. To quantify SEC23B mRNA expression, RT–qPCR amplification was performed using the TaqMan® gene expression assay (Applied Biosystems, Foster City, CA, U.S.A.) and ABI 7500 Real Time PCR system (Applied Biosystems). TaqMan probes were employed for the SEC23B gene (Hs00197211_m1) and the endogenous control (GAPDH; Hs99999905_m1). Relative levels of SEC23B gene expression were analyzed using the 2^−ΔCt method after normalization to levels of GAPDH gene transcripts. The mRNA expression analysis was performed independently three times.

Case presentation

A newborn (Proband, II-2 in Figure 1A) without antenatal or perinatal problems was born to healthy nonconsanguineous Korean parents. His older sister had been diagnosed with hereditary spherocytosis (HS); she received several blood transfusions and underwent laparoscopic cholecystectomy and splenectomy but died of sepsis at the age of 13 years. The proband was referred for icteric skin and abdominal distension on his second day of life. Blood testing revealed total bilirubin level of 14.73 mg/dL, direct bilirubin level of 0.83 mg/dL, lactate dehydrogenase level of 272 IU/L, hemoglobin 10.7 g/dL, hematocrit 30.3%, and reticulocyte 10.14%, indicating hemolytic anemia, but his Coombs reaction test was negative. Liver function testing also showed increases, including aspartate aminotransferase (AST) 248 IU/L and alanine aminotransferase (ALT) 49 IU/L. The iron balance test revealed ferritin level of 139.09 ng/mL, iron level of 296 ug/dL, and total iron binding capacity (TIBC) level of 311 ug/dL (Table 1). Peripheral blood smears indicated the presence of anisocytosis, poikilocytosis, and occasional spherocytosis. The osmotic fragility test was normal. Subsequently, bone marrow aspirates revealed the presence of increased bi – and multinucleated erythroblasts with erythroid hyperplasia (Figure 1B). During follow-up observations at an outpatient clinic, severe anemia (hemoglobin, 4.0–6.5 g/dL) requiring red blood cell transfusions every 3 months persisted. Eventually, he underwent laparoscopic splenectomy at 4 years of age. However, his anemia persisted, and subsequent spleen scans confirmed the presence of an accessory spleen, which was removed. After abdominal ultrasonography revealed several GB stones with diameters less than 0.4 cm, which were associated with abdominal pain accompanied by Murphy's sign, the patient underwent laparoscopic cholecystectomy at the age of 8 years.

Figure 1. (A) Pedigree of the proband with congenital dyserythropoietic anemia II. (B) Bone marrow smear of the proband revealed erythroid hyperplasia, binucleation of erythroblasts (green arrows), dysplastic, and multilobulated (red arrows) erythroid precursors (Wright-Giemsa stain, x 400). (C) Integrative Genomics Viewer (IGV) snapshot showing the likely pathogenic variants of SEC23B gene in the proband. Sanger sequencing confirmed compound heterozygous variants (NM_006363.6:c.994-3C > T and c.1831C > T) in the gene. (D) IGV snapshot showing the homozygous UGT1A1*6 allele in the proband. Sanger sequencing of family members showing UGT1A*6 heterozygosity and homozygosity in his father and mother, respectively.

Table 1. Laboratory results of the proband.

	Initial workup	Reference range
Complete blood count
WBC (×10^3/μL)	13.6	4.0–10.0
RBC (×10^6/μL)	3.04	4.14–5.81
Hb (g/dL)	10.7	13.1–17.5
Hct (%)	30.3	39.0–52.0
MCV (fL)	100	83.1–100.3
MCH (pg)	35.3	27.6–34.1
MCHC (g/dL)	35.0	31.8–35.4
RDW (%)	26.9	11.9–14.1
Ret (%)	10.14	0.50–1.50
PLT (/μL)	235,000	140,000–400,000
Biochemical, laboratory data and iron balance
Total bilirubin (mg/dL)	14.73	0.10–1.30
Direct bilirubin (mg/dL)	0.83	0.10–0.40
Ferritin (ng/mL)	139.09	15–332
Iron (ug/dL)	296	65–157
Total iron binding capacity (ug/dL)	311	256–426
ALT (IU/L)	49	4–43
AST (IU/L)	248	7–38
LDH (IU/L)	272	106–211

WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood cell distribution width; Ret, reticulocytes; PLT, platelets; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase.

Molecular analysis

Considering the family history of HS, mutation screening of HS-associated genes (ANK1, SPTB, SLC4A1, SPTA1, and EPB41) was initially performed, but no mutations were identified. We did detect two heterozygous variants in the SEC23B gene, c.994-3C > T and c.1831C > T. c.1831C > T (p.Arg611Trp) is classified as a variant of uncertain significance (VUS) by ACMG guidelines [4]. p.Arg611Trp was described in the Genome Aggregation Database (gnomAD) at an extremely low frequency of 0.00003182 (0.0001088 in East Asian) but was absent from the Korean Reference Genome Database (KRGDB). In silico analyses performed using PolyPhen-2 (score, 1.0), SIFT (0), MutationTaster (1.0), and REVEL (0.856) predicted that p.Arg611Trp has a damaging effect on the protein. This variant was previously detected in a single family with CDA II [5], but the c.994-3C > T mutation in intron 8 is not reported in disease or general population databases. In silico analysis using Human Splicing Finder Matrices and SpliceAI predicted no significant impact on splicing signals [6, 7]. A family study conducted using targeted mutation analyses revealed that c.994-3C > T was inherited from his mother (I-2 in Figure 1A) and c.1831C > T from his father (I-1 in Figure 1A), which confirmed autosomal recessive inheritance of CDA II (Figure 1C). In addition, we assessed the consequence of the defects on SEC23B gene transcription and RNA splicing. RNA analysis revealed expression of two aberrant transcripts along with the wild-type transcript (567 bp) in the proband and the mother but not in the father (Figure 2A). Transcript 1 (451 bp) corresponds to a transcript with exon 9 deleted; exon 9 and 10 are deleted in smaller transcript 2 (327 bp) (Figure 2A). Additionally, the patient exhibited lower SEC23B mRNA levels than healthy controls (Figure 2B). This splice variant leading to exon skipping supports a deleterious effect of this VUS of the SEC23B as PVS1 based on ACMG criteria for classifying pathogenic variants. We were able to upgrade the classification of these two variants from VUS to likely pathogenic variants and confirmed the diagnosis of CDA II.

Figure 2. Results of mRNA analysis. (A) RT-PCR analysis extending from exon 7–12 of the SEC23B gene was conducted for the proband, his parents, and healthy controls. Three different products were present in the proband and his mother, who harbored SEC23B c.994-3C > T. The upper band corresponds to the wild-type product, the middle band to the exon 9-deleted product, and the lower band to the exon 9 and exon 10-deleted product. Sequencing analysis of RT-PCR products from the proband confirmed the generation of multiple mutant transcripts. (B) SEC23B mRNA levels in blood samples from the proband and healthy controls.

Furthermore, coinheritance of GS was determined according to the presence of the c.211G > A (p.Gly71Arg) homozygous mutation in the UGT1A gene (UGT1A1*6). The father was shown to carry the heterozygous p.Gly71Arg mutation, and the mother also carried this mutation in the homozygous state (Figure 1D).

Discussion

CDA II is traditionally diagnosed based on the acidified serum test (Ham test), SDS polyacrylamide gel electrophoresis (SDS-PAGE), bone marrow examination, and electron microscopy findings. However, diagnostic delays and misdiagnoses are common, as these more complicated tests are not readily available in many diagnostic laboratories. The most common erroneous diagnosis is HS [1, 8], which can be diagnosed without additional tests if a patient has a family history of HS as well as typical clinical manifestations and blood parameters [9], but this could increase the risk of misdiagnosis, as in our case. Clinical application of next generation sequencing enables rapid testing of multiple genes and provides a means of clarifying diagnosis in cases of highly heterogeneous, both clinically and genetically, diseases such as CDA [10, 11].

In this report, we describe the case of a patient with concomitant CDA II and GS that was genetically confirmed. He carried a compound heterozygous mutation in the SEC23B gene, with one allele of the same variant described in a single Chinese family [5], and another allele containing a new mutation. Pedigree analysis revealed that the proband inherited the mutations from separate alleles, which strongly suggests that the deceased sibling (DNA unavailable), who had similar clinical manifestation, harbored the same mutations.

To date, approximately 15 splice-site variants in the SEC23B gene have been reported in patients diagnosed with CDA II [2, 12]. However, the majority of these variants were not experimentally validated in in vitro functional assays but rather were assessed using in silico tools to predict pathogenicity [8, 13]. Our novel intronic variant is not located at a canonical splice site. Although multiple in silico tools are available, they were insufficient to accurately predict the effects of this variant on mRNA splicing. We experimentally validated a novel SEC23B acceptor-splice site mutation, c.994-3C > T, that generates two mutant transcripts, one with an exon 9 skip and one with a two-exon skip of exon 9 and downstream exon 10. Splice mutations can lead to skipping of several exons and production of multiple mRNA isoforms in rare genetic disorders [14]. In general, the order of intron change may play an important role in two-exon skipping [15, 16]. Two mechanisms have been proposed to explain the genesis of double exon skipping due to a splicing mutation: (1) the formation of a large multiexon structure called spliced exon clusters in RNA intermediates (SECRI) and (2) splicing paralysis. Further study is needed to reveal in detail the outcome and to better understand the splicing processes resulting from splice site mutations in CDA II patients.

Many founder and private mutations have been identified in different ethnic groups. Although CDA II cases confirmed by the molecular investigation are predominant in Mediterranean areas, Southeast Asia, and Oceania, occurrence in East Asia is very low [17, 18]. Of the 17 recurrent mutations described by Russo et al. [2], p.Arg550Ter was the only variant observed in the general Korean population (KRGDB, MAF, 0000294). Our patient harbored one private mutation, whereas the other was reported in a Chinese patient. These findings suggest that certain potential founder mutations are probably present in East Asians.

Previous studies on genotype-phenotype correlations show that patients with compound heterozygosity for missense and nonsense/hypomorphic alleles exhibit more severe clinical phenotypes than other genotypic groups (2 missense alleles, 2 hypomorphic alleles, or compound heterozygosity nonsense and hypomorphic alleles) [2, 19]. Our patient carried a missense allele in compound heterozygosity with a hypomorphic allele, and the disease followed a severe clinical course requiring regular blood transfusion. Additionally, coincidental coinheritance of GS was confirmed by the presence of the homozygous UGT1A1*6 genotype. GS is considered a benign inherited disorder characterized by increased bilirubin levels due to UGT1A1 gene mutation. However, risk of gallstones and hyperbilirubinemia is increased in CDA II patients with GS [20]. The gallstone formation in early childhood in our patient can be explained by his homozygous UGT1A1*6 genotype. In fact, UGT1A1*6 is a common among Asian populations, with allele frequencies in Japanese, Korean and Chinese populations of 0.13, 0.23 and 0.23, respectively. Therefore, genetic testing should be considered for UGT1A1 in patients with CDA II to evaluate the influence of coinherited GS on clinical manifestation.

In summary, this is the first report of a Korean patient with CDA II and GS confirmed by molecular genetic analysis. We identified a novel SEC23B acceptor-splice site variant through NGS and RNA analyses. Genetic diagnostic testing can improve understanding of the molecular characteristics and increase the diagnostic yield of CDA II, which is probably underestimated due to clinical heterogeneity and diagnostic difficulties. We further recommend screening for UGT1A1 genetic variants in patients with confirmed CDA II.

Acknowledgments

We are grateful to the patient and his parents.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by INHA UNIVERSITY Research Grant.