From April 2015 to June 2021, 20 Japanese families with suspected DHSt were enrolled in this study. This study was performed in accordance with the principles of the Declaration of Helsinki and approved by the ethics committee of the institution. After obtaining written informed consent, blood samples were collected from all patients. In addition, we collected detailed clinical information from the attending doctors, including family histories, clinical courses, and physical findings.
In most patients, when possible, we first performed additional red cell membrane functional examinations, including the acidified glycerol hemolysis time (AGLT) test, flow-cytometric osmotic fragility (FCM-OF) test, and eosin-5’-maleimide (EMA) binding test with a negative direct antiglobulin test as per previously reported methods10 (link). DHSt was suspected when clinical findings such as hemolytic anemia with stomatocytosis and hemochromatosis not due to transfusion, positive family history, and past history of perinatal edema were observed, and laboratory tests revealed elevated MCV, increased % residual red cells (%RRC) in the FCM-OF test, and normal or increased EMA binding.
Genomic DNA was extracted from the patient’s peripheral blood using a QIAamp DNA extraction kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). The Haloplex HS target enrichment system (Agilent Technologies, Santa Clara, CA, USA) was used for TCS. Using SureDesign (https://earray.chem.agilent.com/suredesign/home.htm ), the target panel was designed to include all coding exons and intron‒exon boundaries of the 74 possible candidate genes10 (link). Massive parallel sequencing was performed using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Raw data were aligned to the human genome sequence GRCh37/hg19. The generated FASTQ files were imported into SureCall v3.5 (Agilent Technologies) for variant calling. Analysis following the filtering of the obtained variants was described previously10 (link). The obtained variants were filtered according to the following strategy: (1) variant frequencies were below 1% in 1000G_EAS and ALL (1000 Genomes), HGDV, and dbSNP; (2) synonymous variants were excluded (nonsynonymous variants, variants associated with frameshift, insertion/deletion variants, and variants in splicing donor/acceptor sites were included); (3) variants with allele frequencies less than 30% of the total read depth were excluded; and (4) the CADD_phred was higher than 20 if obtained. Variant information obtained using wANNOVAR (http://wannovar.wglab.org/ ) was used for curation. Integrative Genomics Viewer (https://software.broadinstitute.org/software/igv/ ) was used for visual evaluation. All variants were evaluated using the guidelines proposed by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP)11 (link).
The existence of the identified variants in the probands of the enrolled patients was confirmed using conventional PCR-Sanger sequencing. Genotyping for αLELY (low expression allele of SPTA1), UGT1A1, and Memphis I and II (SLC4A1) was also performed using conventional PCR-Sanger sequencing for all patients12 (link)–15 (link).
In most patients, when possible, we first performed additional red cell membrane functional examinations, including the acidified glycerol hemolysis time (AGLT) test, flow-cytometric osmotic fragility (FCM-OF) test, and eosin-5’-maleimide (EMA) binding test with a negative direct antiglobulin test as per previously reported methods10 (link). DHSt was suspected when clinical findings such as hemolytic anemia with stomatocytosis and hemochromatosis not due to transfusion, positive family history, and past history of perinatal edema were observed, and laboratory tests revealed elevated MCV, increased % residual red cells (%RRC) in the FCM-OF test, and normal or increased EMA binding.
Genomic DNA was extracted from the patient’s peripheral blood using a QIAamp DNA extraction kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). The Haloplex HS target enrichment system (Agilent Technologies, Santa Clara, CA, USA) was used for TCS. Using SureDesign (
The existence of the identified variants in the probands of the enrolled patients was confirmed using conventional PCR-Sanger sequencing. Genotyping for αLELY (low expression allele of SPTA1), UGT1A1, and Memphis I and II (SLC4A1) was also performed using conventional PCR-Sanger sequencing for all patients12 (link)–15 (link).
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