A 65-year-old female (Fig. 1A, individual I-2) was referred to the Department of Internal Medicine, Daejeon St. Mary's Hospital (Daejeon, Republic of Korea) for a further evaluation. She had been diagnosed as hemolytic anemia 30 years ago, based on a medical history of persistent anemia and hyperbilirubinemia for several years. She received RBC transfusion several times and a cholecystectomy roughly 20 years ago before without knowing the reason for the operations. The laboratory findings at the time of admission to our hospital were as follows: hemoglobin, 6.6 g/dL; red cell distribution width, 23.9%; reticulocytes, 22.5%; haptoglobin, <20 mg/dL; erythropoietin, 4080 mIU/mL; total bilirubin, 8.7 mg/dL; direct bilirubin, 1.9 mg/dL; and lactate dehydrogenase, 250 IU/L. Autoimmune hemolytic anemia was ruled out because irregular antibody screening, Coombs test, and cold agglutinin test were negative, even though increased osmotic fragility. On the peripheral blood (PB) smear, round, densely staining spherical-shaped RBCs (spherocytes) were frequently found about 10 to 20 cells per high power field, that lack central pallor and have a smaller than normal diameter (Fig. 1B). Pedigree analysis of the proband's family members revealed that her 3rd son (Fig. 1A, individual II-3) showed similar symptoms such as anemia and jaundice, 2 grandchildren (Fig. 1A, individuals III-1 and III-2) experienced neonatal jaundice after birth (Table 1). Numerous spherocytes were frequently found in the PB smears of symptomatic family members. Based on these laboratory findings and clinical manifestations, the proband and symptomatic family members were provisionally diagnosed as HS. At 2 years later, the proband underwent a splenectomy due to transfusion-refractory anemia and splenomegaly. After the splenectomy, her hemoglobin level improved to normal range (14.1 g/dL) and her bilirubin levels decreased dramatically (total bilirubin 1.9 mg/dL; direct bilirubin 0.6 mg/dL).
To confirm the genetic cause of HS, genetic testing using targeted NGS that consists of genes related anemia and bone marrow failure syndromes was performed in the proband. The gene panel included 5 HS-associated genes such as 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). All subjects provided written informed consent for clinical and molecular analyses, and the study protocol was approved by the Institute Review Board of the Catholic University of Korea.
Briefly, genomic DNA was extracted from the PB. Capture of the target regions was performed with reagents from a custom design HaloPlex Target Enrichment kit (Agilent Technologies, Inc., Santa Clara, CA). Massively parallel sequencing was performed on the Illumina HiSeq 2000 platform (Illumina, Inc., San Diego, CA). Average coverage of depth of the entire panel was 101x, and 98.3% of targeted bases were covered by 10x sequence reads. Sequence reads were aligned to hg19 with Burrow-Wheeler Aligner (version 0.7.12, MEM algorithm). Duplicate reads were removed by using Picard-tools1.96. Local realignment and base quality recalibration were done by the Genome Analysis Toolkit (GATK ver 3.5). Variant calling was performed by GATK HaplotypeCaller. Variants were annotated by SnpEff ver 4.2. As a result, 1 heterozygous nonsense mutation, c.1956G>A; p.Trp652* (reference sequence: NM_001024858.2), was identified in the exon 13 of SPTB gene (Fig. 2). This novel mutation was not found in public population sequence databases such as 1000 Genomes Project Database, ESP6500, and ExAC, as well as in Korean population sequence databases (KRGDB, http://152.99.75.168/KRGDB/menuPages/intro.jsp). Any (likely) pathogenic variants were not identified in other 4 HS-associated genes according to the American College of Medicine and Genetics guidelines for the interpretation of sequence variation. Thus, this nonsense mutation of the SPTB was confirmed to cause HS in the proband and may affect symptomatic family members who could not be tested for the mutation.