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Whole-exome sequencing for the genetic diagnosis of congenital red blood cell membrane disorders in Taiwan
Clinica Chimica Acta 487 (2018) 311–317
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Whole-exome sequencing for the genetic diagnosis of congenital red blood cell membrane disorders in Taiwan
T
Pei-Chin Lina,b,e, Shyh-Shin Chioua,b,e, Chien-Yu Linc,f, Shu-Chen Wangg, Hsi-Yuan Huangc, Ya-Sian Changc,f,h, Yu-Hsin Tsenge, Tzu-Min Kane, Yu-Mei Liaoa,e, Shih-Pien Tsaij, ⁎⁎ ⁎ Ching-Tien Pengd,i, , Jan-Gowth Changc,f,h,k,l, a
Division of Hematology and Oncology, Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Department of Pediatrics, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan c Department of Laboratory Medicine, China Medical University Hospital, China Medical University, Taichung, Taiwan d Department of Pediatrics, China Medical University Children's Hospital, Taichung, Taiwan e Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan f Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan g Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan h School of Medicine, China Medical University, Taichung, Taiwan i Department of Biotechnology, Asia University, Taichung, Taiwan j Department of Nursing, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan k Department of Bioinformatics and Medical Engineering, Asia University, Taichung, Taiwan l Center for Precision Medicine, China Medical University Hospital, Taichung, Taiwan b
ARTICLE INFO
ABSTRACT
Keywords: Next-generation sequencing Whole-exome sequencing Congenital red cell membrane disorder Hereditary spherocytosis Hereditary elliptocytosis
Purpose: Congenital hemolytic anemia caused by red blood cell (RBC) membrane defects is a heterogeneous group of disorders. The present study aimed to search the causative gene mutations in patients with RBC membrane disorders in Taiwan. Materials and Methods: Next-generation sequencing approach using whole-exome sequencing (WES) was performed. Sanger sequencing was performed for confirmation of variants detected in WES in patients and their family members. Results: Five causative variants, including two ANK1, two SPTA and one SPTB variants, were detected in four patients. All these variants, except one SPTA1 variant c.83G > A (p.R28H), are novel variants. Their pedigree analysis showed one de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY, one de novo ANK1 mutation c.1034C > A (p.A345E), one autosomal dominant combined SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P) mutations and one autosomal dominant ANK1 c.4462C > T (p.R1488X) mutation. Conclusions: Our data demonstrated that WES is an efficient tool for determining genetic etiologies of RBC membrane disorders and can facilitate accurate diagnosis and genetic counseling. Additional studies should be conducted on larger cohorts to investigate the distribution of gene mutations in patients with RBC membrane disorders in Taiwan.
1. Introduction Red blood cell (RBC) membrane disorders are categorized into two groups: disorders caused by red cell membrane dysfunction, including hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and
hereditary pyropoikilocytosis (HPP), and those caused by the passive flux of monovalent cations across the membrane, including hereditary stomatocytosis [1,2]. HS, characterized by the presence of spherocytes in peripheral blood smears, is the most common disorder in Northern Europe and North America, with an incidence of 1/2000 [3]. In Asia,
Abbreviations: RBC, red blood cell; WES, whole-exome sequencing; HS, hereditary spherocytosis; HE, hereditary elliptocytosis; HPP, hereditary pyropoikilocytosis; ANK1, ankyrin 1; SLCA1, band 3; EPB42, protein 4.2; SPTA1, α-spectrin; SPTB, β-spectrin; EPB41, protein 4.1R deficiency; NGS, next-generation sequencing; PCR, polymerase chain reaction; MAF, minor allele frequency; G-6PD, glucose-6-phosphate dehydrogenase ⁎ Correspondence to: J.G. Chang, Department of Laboratory Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichaung, Taiwan. ⁎⁎ Correspondence to: C.T. Peng, Department of Pediatrics, China Medical University Children's Hospital, No. 2, Yuh-Der Road, Taichaung, Taiwan. E-mail addresses: [email protected] (C.-T. Peng), [email protected] (J.-G. Chang). https://doi.org/10.1016/j.cca.2018.10.020 Received 17 May 2018; Received in revised form 21 September 2018; Accepted 10 October 2018 Available online 11 October 2018 0009-8981/ © 2018 Elsevier B.V. All rights reserved.
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HS has also been frequently described in Japan, and recently in Korea [4,5]. Although HS occurs in all racial and ethnic populations, it is less common in the African-American and Southeast Asian populations [3]. HE is another common red cell membrane disorder in Europeans and their descendants, with estimated incidence of 1 in 2000 to 4000 in the United States. Studies have demonstrated that the high frequencies of HE are observed in areas of endemic malaria, which are as high as 1.6% in Benin, Western Africa [6]. Recently, Wang et al. conducted a systematic review on HS epidemiology in Chinese patients and estimated the prevalence of HS in Mainland China. Their report included 2043 patients with HS during 1978–2013 and estimated a prevalence of
1.27–1.49 in 100,000 individuals [7]. In Taiwan, RBC membrane disorders have been reported occasionally; however, studies of genetic diagnosis are unavailable [8,9]. Many studies have demonstrated the role of mutations in ankyrin 1 (ANK1), band 3 (SLCA1), protein 4.2 (EPB42), α-spectrin (SPTA1), and β-spectrin (SPTB) genes in HS or mutations in spectrin self-association between the N-terminal of α-spectrin (SPTA1) and the C-terminal of βspectrin (SPTB) and protein 4.1R (EPB41) genes in HE [1–3,6,10]. In European and Americans populations, ANK1 mutations is the most common cause of HS, consisting of around 50% of cases, followed by SPTB mutations (~20%), SLC4A1 (~15%), EPB42 (~10%) and SPTA1
Fig. 1. Workflow of WES analysis. Numbers of variants were annotated in each step. 312
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(~5%). In Japan, the Yawata study group has reported a distribution of gene mutations in patients with HS which showed mostly in SLC4A1 (11 mutations) and/or in EPB42 genes (5 mutations) and fewer ANK1 mutations (2 mutations). Later on, the same group extended their study to total 49 patients and found 16/49 (33%) carry ANK1 mutations. In Korea, among 25 patients with HS, 13 ANK1 and 12 SPTB mutations were found and no mutations of SLC4A1, EPB42 or SPTA1 were found [5,6,11–13]. Because of the large size of these genes, the conventional Sanger-sequencing-based approach is not feasible to detect all possible disease-causing alterations. By contrast, next-generation sequencing (NGS) enables a high-throughput analysis and serves as a powerful tool for genetic disease diagnosis [14]. The present study used the wholeexome sequencing (WES) to elucidate the genetic mutations and the inheritance pattern in patients with RBC membrane disorders in Taiwan.
ANNOVAR [19] to functionally annotate genetic variants and used the ratio of variants, minor allele frequency (MAF), clinical significance of ClinVar, report of dbSNP and three pathogenicity predictions software: SIFT, PolyPhen, and CADD_PHRED to select the important variants. 2.4. PCR and Sanger sequencing The variants of RBC membrane disorders candidate genes predicted as non-synonymous, deleterious, damaging, or pathogenic were selected for Sanger sequencing. Supplementary Table 2 provides a list of the designed primers. The detailed methods of PCR and Sanger sequencing were provided in the supplementary method. 3. Results 3.1. Causative variants of RBC membrane disorders detected from WES and pedigree analysis
2. Materials and methods 2.1. Patients and study design
Five causative variants of RBC membrane disorders in four patients were diagnosed (Table 1). The clinical characteristics and laboratory data were listed in Supplementary Table 3. A heterozygous SPTA1 variant c.83G > A (p.R28H) was detected in RMD05 and confirmed as a de novo variant because Sanger sequencing of RMD05's parents and brother showed a wild type. The pedigree of RMD05 was shown in Fig. 2. We further re-analyzed WES data of RMD05 for IVS45 as -12C > T, a splicing variant named low-expression α-spectrin allele (αLELY) with MAF higher than 1%. Allele αLELY may cause clinical significant disease when combined with a coding region mutation [6]. Allele αLELY was found with heterozygous status (ratio of variant: 53.85%) in WES data and confirmed using Sanger sequencing. Heterozygous status of allele αLELY was also found in the father, but not in the mother and brother using Sanger sequencing (Supplementary Fig. 1). The peripheral blood smear of RMD05 showed microspherocytosis and anisocytosis (Fig.2), and the EMA binding test revealed markedly decreased MCF units (Supplementary Table 3). No hemolytic anemia histories were noted in the parents or the brother. The RBC indices of the father showed within normal ranges. The EMA binding tests of the parents and the brother were within the reference range (Supplementary Table 4). Finally, a de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY was confirmed in RMD05. A heterozygous ANK1 variant (chr8:g.41577351 G > T: c.1034C > A: p.A345E) was detected only in RMD11, but not in her parents. The pedigree of RMD11 was shown in Fig. 3. RMD11's parents did not show any clinical or laboratory evidences of hemolytic anemia, and the EMA binding tests also showed within the reference range (Supplementary Table 4). RMD11 seems rather a congenital than an inherited RBC membrane disorder. The ANK1 variant c.1034C > A (p.A345E) is predicted as deleterious, probably damaging and with a high score by SIFT, PolyPhen, and CADD_PHRED and a high frequency of de novo mutations being reported in the ANK1 gene [20]. A de novo ANK1 mutation c.1034C > A (p.A345E) was confirmed in RMD11. Two heterozygous variants, SPTA1 (chr1:g.158612605 T > G:c.4604A > C:p.Q1535P) and SPTB (chr14:g.65234397A > G:c.6203 T > C:p.L2068P) were detected in SPH17. Sanger sequencing also showed these two variants in SPH17's father, but not in the mother. The pedigree of RMD17 was shown in Fig. 4. The hemograms showed a normocytic anemia with reticulocytosis in the father and showed normal RBC indices in the mother (Supplementary Table 4). The MCF units of EMA binding tests for RMD17 and the father were both 47.9 which are in the “grey area” (i.e. between the optimal cut-off value and the lower limit of the range for normal adults) [21], and it was within the reference range for the mother (Supplementary Table 4). Both of these two variants were predicted as deleterious, probably damaging and high score by SIFT, PolyPhen, and CADD_PHRED. An autosomal dominant combined SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P) mutations were confirmed in RMD17.
Seven patients with clinical evidence of congenital hemolytic anemia were enrolled for WES analysis. DNA samples of patients and their family members were extracted from peripheral blood mononuclear cells. The workflow is shown in Fig. 1. To search for the causative variants, panels of candidate genes of RBC membrane disorders, hyperbilirubinemia and other congenital hemolytic anemias were designed (Supplementary Table 1) [6,15,16]. Variants of candidate genes of RBC membrane disorders predicted as nonsynonymous, deleterious, damaging or pathogenic were further confirmed using Sanger sequencing. Additionally, Sanger sequencing of these variants was also performed in their family members for pedigree analysis, with all patients and their family members providing written consent. The study was approved by the Kaohsiung Institute Review Board (KMUHIRB-20140048). 2.2. WES A DNA library was constructed with a TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA, USA). In brief, 100 ng of genomic DNA was diluted in a tris-ethylenediaminetetraacetic acid buffer and sonicated to a fragment size of approximately 300–500 bp, followed by end repair, Atailing, adaptor ligation, and 12 cycles of polymerase chain reaction (PCR) amplification. A 300–400-bp band was gel-selected, and exome capture was performed using a TruSeq Exome Enrichment Kit (Illumina, USA). DNA capture probes were hybridized with the DNA library at the recommended concentration. The DNA library was denatured at 95 °C for 10 min. and subsequently hybridized with captured probes at 58 °C for 16 h. Streptavidin beads were used to bind biotin-labeled probes containing the targeted regions of interest. Three wash steps were performed to prevent nonspecific binding to the beads, and then the hybridization and washing process was repeated. A 10-cycle PCR enrichment process was performed after the second elution, and AMPure XP beads were utilized for purification. The enriched DNA library was quantified in the Qubit dsDNA HS assay (Invitrogen) and validated using the Experion Automated Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA). The library preparations were sequenced on an Illumina NextSeq 500 platform, and 150-bp paired-end reads were generated. 2.3. WES data analysis Base calling and quality scoring were performed by an updated implementation of Real-Time Analysis on the NextSeq 500 system. bcl2fastq Conversion Software was used to demultiplex data and convert BCL files to FASTQ file formats. Sequenced reads were trimmed for adaptor sequencing, and masked for low-complexity or low-quality sequencing, then mapped to the hg19 whole genome using BWA [17]. Finally, SNVs were detected using GATK [18] at their default settings. And then we utilized 313
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A heterozygous ANK1 variant (chr8:g.41542137 G > A: c.4462C > T: p.R1488X) and PIEZO1 variant (chr16:g.88786597 A > G:: c.6044 T > C: p.M2015 T) were identified in RMD21. Sanger sequencing confirmed both variants in RMD21. The ANK1 c.4462C > T (p.R1488X) was from the father and the PIEZO1 c.6044 T > C (p.M2015 T) was from the mother. The RBC indices and EMA binding test of RMD21 and the father both showed anemia, reticulocytosis and decreased MCF reading (Supplementary Table 3 and table 4). The RBC indices and EMA binding test of the mother were within reference ranges (Supplementary Table 4). Literature showed that PIEZO1 mutations are associated with hereditary xerocytosis, which is usually an autosomal dominant disease [6,22]. However, no hemolytic anemia was found in the mother. Whether the PIEZO1 c.6044 T > C (p.M2015 T) would exacerbate the hemolytic anemia while co-inheritance with ANK1 mutations is unknown. Finally, an autosomal dominant ANK1 c.4462C > T (p.R1488X) mutation was confirmed in RMD21. The pedigree of RMD21 was shown in Fig. 5.
disorders. We estimated the frequency of RBC membrane disorder by compared to β-thalassmia. The incidence of β-thalassemia carrier is around 1% in Taiwan. While β-thalassemia major results from two mutations, the inidence of β-thalassemia major is 1/10,000. Therefore, the incidence of RBC membrane disorder maybe around 1/100,000. Autosomal dominant diseases were confirmed in RMD17 and in RMD21, and de novo mutations were confirmed in RMD05 and in RMD11. The family histories were reported negative in patients with autosomal dominant inheritance because the mildness of the disease or a lack of accurate diagnosis of their parents. ANK1 encodes a 206-kD protein that links the spectrin–actin-based red cell membrane skeleton and transmembrane proteins [6,23,24]. Ankyrin is composed of three major domains: a band-3-binding site domain, a spectrin-binding site domain, and a regulatory domain [13,25]. The mutation in RMD11 (c.1034C > A, p.A345E) was located on exon 10 of ANK1, which involves the band-3-binding domain. The nonsense mutation in RMD21 (ANK1 c.4462C > T, p.R1488X) was located on exon 37 of ANK1, which involves the regulatory domain. Ankyrin defects lead to instabilities in spectrin heterodimers because spectrin heterodimers are only stable when bound to the membrane, and ankyrin provides a high-affinity binding site. Therefore, ankyrin defects are usually dominant defects, and de novo ANK1 mutations have been frequently detected [13,20,26]. Of all identified HS mutations, 35% have been ANK1 mutations [6]. Based on the causative mutations detected, RMD11 and RMD21 were diagnosed with HS. A de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY was identified in RMD05. The parents and brother had a wildtype genotype of SPTA1 p.R28H and the father has a αLELY polymorphism (Fig. 2). Carriers of αLELY are asymptomatic, even in the homozygous state. Combination of a αLELY opposite a severe HE allele results in clinically significant hemolysis [6]. The mutation involves CGT to CAT (arginine to histidine), CGT to AGT (arginine to serine), CGT to CTT (arginine to leucine), and CGT to TGT (arginine to cysteine) in codon 28 of α-spectrin have been reported to cause HE or the more severe form, HPP [27–29]. RMD05 had hemolytic anemia since early childhood with an initial diagnosis of glucose-6-phosphate dehydrogenase (G-6PD) deficiency. Microcytic spherocytosis, anisocytosis, increased fragility, and normal G-6PD activity were observed after he was transferred to our department. Intermittent transfusions were required during infection episodes, and marked splenomegaly and
3.2. Other variants detected in WES analysis Totally, five ClinVar pathogenic variants were detected in the WES analysis of four patients with RBC membrane disorders, including one RBC membrane disorders gene (SPTA1 chr1:g.158655079C > T c.83G > A: p.R28H) and other four mutations which have been reported in other diseases (Table 2). All variants detected in WES of the four patients with RBC membrane disorders predicted as non-synonymous, deleterious, probably damaging or pathogenic and the average read depths of target regions were listed in Supplementary Table 5. 4. Discussion In the present study, we successfully detected the causative gene mutations of RBC membrane disorders through WES and pedigree analysis in four patients from unrelated Taiwanese families. These four patients were selected from a pool of around 100 patients with congenital hemolytic anemia. Among them, 50 patients were β-thalassemia major, and 38 patients were hemoglobin H diseases. Seven patients with congenital hemolytic anemia other than thalassemias were enrolled in the inital WES analysis. Finally, four patients were diagnosed with RBC membrane
Fig. 2. The pedigree and red cell morphology of RMD05. The electropherograms indicate a SPTA1 mutation (chr1:g.158655079C > T:c.83G > A:p.R28H) in RMD05 and a wild-type genotype in the parents and younger brother, whose red cell indices and EMA binding tests yielded negative findings (a). Peripheral blood smear of RMD05 shows microspherocytosis and anisocytosis (b). The leftward hatching of the boxes is indicative of the αLELY mutation. The rightward hatching of the boxes is indicative of the SPTA1 mutation. The double-lined box schematically indicates detection of both defects in RMD05. The half-filled symbols indicated heterozygous. F, father; M, mother; C1, child 1; C2, child 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 314
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Fig. 3. Pedigree of a de novo ANK1 mutation in RMD11. The electropherograms indicate an ANK1 mutation (chr8:g.41577351 G > T: c.1034C > A: p.A345E) in RMD11 and a wild-type genotype in the parents. The leftward hatching of the boxes is indicative of the ANK1 mutation. The half-filled symbols indicated heterozygous. F, father; M, mother.
cholelithiasis were observed. Based on the mutations and the clinical course, RMD05 was subsequently diagnosed with HE/HPP. An autosomal dominant combination of two novel variants, SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P)
mutations, was confirmed in RMD17. RMD17 had been diagnosed with normocytic anemia at the age of 1 month and had recovered spontaneously without the need of transfusions. The SPTB c.6203 T > C (p.L2068P) was located in repeat 17 of the β-spectrin domain, which
Fig. 4. Pedigree of an autosomal dominant combined SPTA1 and SPTB mutations in RMD17. The electropherograms indicate a combined SPTA1(chr1:g.158612605 T > G:c.4604A > C:p.Q1535P) and SPTB (chr14:g.65234397 A > G: c.6203 T > C:p.L2068P) heterozygous missense mutation in both RMD17 and the father. The leftward hatching of the boxes is indicative of the SPTA1 mutation. The rightward hatching of the boxes is indicative of the SPTB mutation. The double-lined box schematically indicates the detection of both defects. The half-filled symbols indicated heterozygous. F, father; M, mother. 315
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Fig. 5. Pedigree of an autosomal dominant ANK1 mutation in RMD21. ANK1 (chr8:g.41542137 G > A: c.4462C > T:p.R1488X) was identified in RMD21 and his father. The Sanger sequencing of the mother showed wild type. The leftward hatching of the boxes is indicative of the ANK1 mutation. The half-filled symbols indicated heterozygous. F, father; M, mother.
forms a part of the spectrin self-association site [5]. Meanwhile, the SPTA1 c.4604A > C (p.Q1535P) was located beyond the self-association binding site, which tends to be less severe in clinical manifestations [6]. In humans, α-spectrin synthesis is four-fold of β-spectrin synthesis. Heterozygotes for α-spectrin defects usually produce adequate normal α-spectrin chains to pair with β-spectrin chains. Studies have indicated that α-spectrin production of less than 25% causes diseases [30]. Taken together, we suggested that the clinical manifestations of RMD17 mainly resulted from the SPTB c.6203 T > C (p.L2068P) mutation and was diagnosed with mild HE. However, further functional studies may be needed to elucidate the phenotypes of these two variants. Genetic diagnosis for red cell membrane disorders is laborious because of the wide genetic heterogeneity and the large size of candidate genes. Although the genetic diagnosis does not add extra information for the patient whose family is known to have the RBC membrane disorder, a recessive inheritance, de novo mutations or compound heterozygosity require further molecular studies [31]. NGS is a highthroughput genetic diagnosis tool, which can be used for the detection of rare diseases at the molecular level [32–34], and a cost-effective
approach for the molecular diagnosis of hereditary hemolytic anemia, particularly when the family history is non-informative or when routine laboratory tests cannot identify the causative hemolytic process [35]. Agarwal et al. reported the use of NGS of 28 genes' panel of cytoskeletal proteins and enzymes to determine the molecular basis of hemolytic anemia in cases of red cell membrane disorders despite the lack of family history. Ten of 12 of the mutations detected have not been previously described, including SPTB, SLC4A1 and ANK1 mutations. One of the patients has both ANK1 and SPTA1 mutations [35]. Barreto et al. used a 40 genes' panel for detecting new pathogenic mutations in patients with congenital hemolytic anemia [16]. Han et al. used WES to identify a novel nonsense mutation in ANK1 (p.Q1772X) in a Korean patient with HS and also confirmed a comorbid with UGT1A1 mutations [15]. Although WES can provide more comprehensive information than gene panels, it has limitations in sufficiently covering coding exons, especially GC-rich regions, and in copy number variation detection. Previous studies showed that WES failed to detect 0.42% of currently known pathogenic mutations and 0.81% if non-coding pathogenic variation were included as well [36].
Table 1 Causative mutations of RBC membrane disorders detected in WES and confirmed by Sanger sequencing. Patient
Gene
Coordinate
Ref/Var
Sequence depth, ratio of variant
Coding sequence
Amino acid
Genotype
ClinVar
SIFT
PolyPhen
CADD
Inheritance
RMD05
SPTA1
chr1:g.158655079
C/T
11/21 (52.38%),
c.83G > A
p.R28H
hetero
Pathogenic
deleterious
34
a
RMD11
ANK1
chr8:g.41577351
G/T
13/30 (43.33%),
c.1034C > A
p.A345E
hetero
deleterious
33
de novo
RMD17
SPTA1
chr1:g.158612605
T/G
c.4604A > C
p.Q1535P
hetero
deleterious
29.2
AD
SPTB
chr14:g.65234397
A/G
137/281 (48.58%) 90/195 (46.15%)
c.6203 T > C
p.L2068P
hetero
deleterious
ANK1
chr8:g.41542137
G/A
91/231 (39.39%)
c.4462C > T
p.R1488X
hetero
NA
Probably damaging Probably damaging Probably damaging Probably damaging NA
RMD21
31 36
hetero: heterozygous. AD: autosomal dominant. a A Combined SPTA1 p.R28H and αLELY polymorphism was detected in RMD05. The SPTA1 p.R28H was de novo and αLELY was from the father. 316
de novo
AD
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Table 2 ClinVar pathogenic mutations detected in WES analysis. Patient
Gene
Coordinate
Sequence depth, ratio of variant
ClinVar
RMD05 RMD11 RMD11 RMD17 RMD21
SPTA1 DCHS1 NDUFS3 HEXA LRRK2
chr1:g.158655079C > T:c.83G > A:p.R28H chr11:g.6645369C > T:c.7538G > A:p.R2513H chr11:g.47605974C > T:c.736C > T:p.R246C chr15:g.72638646G > C:c.1351C > G:p.L451 V chr12:g.40677699C > T:c.2264C > T:p.P755L
11/21 (52.38%) 9/29 (31.03%) 21/36 (58.33%) 86/178 (48.31%) 93/201 (46.27%)
Pathogenic Pathogenic Likely pathogenic Pathogenic Pathogenic
Our study is the first to use the WES approach for the genetic diagnosis of RBC membrane disorders in Taiwan and demonstrated the usefulness of the WES approach in the diagnosis of red cell membrane disorders. Additional studies involving larger cohorts must be conducted to investigate the distribution of causative gene mutations in patients with RBC membrane disorders in Taiwan. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cca.2018.10.020.
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Hartley, D.A. Dyment, C.L. Beaulieu, J. Schwartzentruber, A. Smith, H.M. Bedford, G. Bernard, F.P. Bernier, B. Brais, D.E. Bulman, J. Warman Chardon, D. Chitayat, J. Deladoey, B.A. Fernandez, P. Frosk, M.T. Geraghty, B. Gerull, W. Gibson, R.M. Gow, G.E. Graham, J.S. Green, E. Heon, G. Horvath, A.M. Innes, N. Jabado, R.H. Kim, R.K. Koenekoop, A. Khan, O.J. Lehmann, R. MendozaLondono, J.L. Michaud, S.M. Nikkel, L.S. Penney, C. Polychronakos, J. Richer, G.A. Rouleau, M.E. Samuels, V.M. Siu, O. Suchowersky, M.A. Tarnopolsky, G. Yoon, F.R. Zahir, F.C. Consortium, C. Care4Rare Canada, J. Majewski, K.M. Boycott, Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care, Clin. Genet. 89 (3) (2016) 275–284. [34] K. Danielsson, L.J. Mun, A. Lordemann, J. Mao, C.H. Lin, Next-generation sequencing applied to rare diseases genomics, Expert. Rev. Mol. Diagn. 14 (4) (2014) 469–487. [35] A.M. Agarwal, R.H. Nussenzveig, N.S. Reading, J.L. 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Acknowledgement We acknowledge Wallace Academic Editing for editing this manuscript. This study was supported by grants from the Ministry of Science and Technology, Taiwan, Republic of China (MOST 106-2314-B-037079-MY2) and Kaohsiung Medical University Hospital, Taiwan, Republic of China (KMUH106-6R48). References [1] J. Delaunay, The molecular basis of hereditary red cell membrane disorders, Blood Rev. 21 (1) (2007) 1–20. [2] M.J. King, A. Zanella, Hereditary red cell membrane disorders and laboratory diagnostic testing, Int. J. Lab. Hematol. 35 (3) (2013) 237–243. [3] S. Perrotta, P.G. Gallagher, N. Mohandas, Hereditary spherocytosis, Lancet 372 (9647) (2008) 1411–1426. [4] E. Han, A. Kim, J. Park, M. Kim, Y. Kim, K. Han, Y.J. Kim, Spectrin Tunis (Sp alpha (I/78)) in a Korean family with hereditary elliptocytosis, Ann Lab Med 33 (5) (2013) 386–389. [5] J. Park, D.C. Jeong, J. Yoo, W. Jang, H. Chae, J. Kim, A. Kwon, H. Choi, J.W. Lee, N.G. Chung, M. Kim, Y. Kim, Mutational characteristics of ANK1 and SPTB genes in hereditary spherocytosis, Clin. Genet. 90 (1) (2016) 69–78. [6] S.E. Lux, Disorders of the Red Cell Membrane, in: D.E.F. Stuart H. Orkin, David Ginsburg, A. Thomas Look, Samuel E. Lux, David G. Nathan (Eds.), Nathan and Oski's Hematology and Oncology of Infancy and Childhood, Elsevier-Saunders, Philadelphia, 2015, pp. 515–579. [7] C. Wang, Y. Cui, Y. Li, X. Liu, J. Han, A systematic review of hereditary spherocytosis reported in Chinese biomedical journals from 1978 to 2013 and estimation of the prevalence of the disease using a disease model, Intractable Rare Dis Res 4 (2) (2015) 76–81. [8] Y.H. Chang, C.F. Shaw, S.H. Jian, K.H. Hsieh, Y.H. Chiou, P.J. Lu, Compound mutations in human anion exchanger 1 are associated with complete distal renal tubular acidosis and hereditary spherocytosis, Kidney Int. 76 (7) (2009) 774–783. [9] S.W. Cheng, Y.W. Chiu, Y.H. Weng, Etiological analyses of marked neonatal hyperbilirubinemia in a single institution in Taiwan, Chang Gung Med. J. 35 (2) (2012) 148–154. [10] E. Miraglia Del Giudice, B. Nobili, M. Francese, L. D'Urso, A. Iolascon, S. Eber, S. Perrotta, Clinical and molecular evaluation of non-dominant hereditary spherocytosis, Br. J. Haematol. 112 (1) (2001) 42–47. [11] X. An, N. Mohandas, Disorders of red cell membrane, Br. J. Haematol. 141 (3) (2008) 367–375. [12] Y. Yawata, A. Kanzaki, A. Yawata, W. Doerfler, R. Ozcan, S.W. Eber, Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population, Int. J. Hematol. 71 (2) (2000) 118–135. [13] H. Nakanishi, A. Kanzaki, A. Yawata, O. Yamada, Y. Yawata, Ankyrin gene mutations in japanese patients with hereditary spherocytosis, Int. J. Hematol. 73 (1) (2001) 54–63. [14] J. Zhang, P. Barbaro, Y. Guo, A. Alodaib, J. Li, W. Gold, L. Ades, B.J. Keating, X. Xu, J. Teo, H. Hakonarson, J. Christodoulou, Utility of next-generation sequencing technologies for the efficient genetic resolution of haematological disorders, Clin. Genet. 89 (2) (2016) 163–172. [15] J.H. Han, S. Kim, H. Jang, S.W. Kim, M.G. Lee, H. Koh, J.H. Lee, Identification of a novel p.Q1772X ANK1 mutation in a Korean family with hereditary spherocytosis, PLoS One 10 (6) (2015) e0131251. [16] R. Del Orbe Barreto, B. Arrizabalaga, A.B. De la Hoz, A. Garcia-Orad, M.I. Tejada, J.C. Garcia-Ruiz, T. Fidalgo, C. Bento, L. Manco, M.L. Ribeiro, Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using nextgeneration sequencing, Int. J. Lab. Hematol. 38 (6) (2016) 629–638.
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Whole-exome sequencing for the genetic diagnosis of congenital red blood cell membrane disorders in Taiwan
T
Pei-Chin Lina,b,e, Shyh-Shin Chioua,b,e, Chien-Yu Linc,f, Shu-Chen Wangg, Hsi-Yuan Huangc, Ya-Sian Changc,f,h, Yu-Hsin Tsenge, Tzu-Min Kane, Yu-Mei Liaoa,e, Shih-Pien Tsaij, ⁎⁎ ⁎ Ching-Tien Pengd,i, , Jan-Gowth Changc,f,h,k,l, a
Division of Hematology and Oncology, Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Department of Pediatrics, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan c Department of Laboratory Medicine, China Medical University Hospital, China Medical University, Taichung, Taiwan d Department of Pediatrics, China Medical University Children's Hospital, Taichung, Taiwan e Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan f Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan g Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan h School of Medicine, China Medical University, Taichung, Taiwan i Department of Biotechnology, Asia University, Taichung, Taiwan j Department of Nursing, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan k Department of Bioinformatics and Medical Engineering, Asia University, Taichung, Taiwan l Center for Precision Medicine, China Medical University Hospital, Taichung, Taiwan b
ARTICLE INFO
ABSTRACT
Keywords: Next-generation sequencing Whole-exome sequencing Congenital red cell membrane disorder Hereditary spherocytosis Hereditary elliptocytosis
Purpose: Congenital hemolytic anemia caused by red blood cell (RBC) membrane defects is a heterogeneous group of disorders. The present study aimed to search the causative gene mutations in patients with RBC membrane disorders in Taiwan. Materials and Methods: Next-generation sequencing approach using whole-exome sequencing (WES) was performed. Sanger sequencing was performed for confirmation of variants detected in WES in patients and their family members. Results: Five causative variants, including two ANK1, two SPTA and one SPTB variants, were detected in four patients. All these variants, except one SPTA1 variant c.83G > A (p.R28H), are novel variants. Their pedigree analysis showed one de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY, one de novo ANK1 mutation c.1034C > A (p.A345E), one autosomal dominant combined SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P) mutations and one autosomal dominant ANK1 c.4462C > T (p.R1488X) mutation. Conclusions: Our data demonstrated that WES is an efficient tool for determining genetic etiologies of RBC membrane disorders and can facilitate accurate diagnosis and genetic counseling. Additional studies should be conducted on larger cohorts to investigate the distribution of gene mutations in patients with RBC membrane disorders in Taiwan.
1. Introduction Red blood cell (RBC) membrane disorders are categorized into two groups: disorders caused by red cell membrane dysfunction, including hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and
hereditary pyropoikilocytosis (HPP), and those caused by the passive flux of monovalent cations across the membrane, including hereditary stomatocytosis [1,2]. HS, characterized by the presence of spherocytes in peripheral blood smears, is the most common disorder in Northern Europe and North America, with an incidence of 1/2000 [3]. In Asia,
Abbreviations: RBC, red blood cell; WES, whole-exome sequencing; HS, hereditary spherocytosis; HE, hereditary elliptocytosis; HPP, hereditary pyropoikilocytosis; ANK1, ankyrin 1; SLCA1, band 3; EPB42, protein 4.2; SPTA1, α-spectrin; SPTB, β-spectrin; EPB41, protein 4.1R deficiency; NGS, next-generation sequencing; PCR, polymerase chain reaction; MAF, minor allele frequency; G-6PD, glucose-6-phosphate dehydrogenase ⁎ Correspondence to: J.G. Chang, Department of Laboratory Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichaung, Taiwan. ⁎⁎ Correspondence to: C.T. Peng, Department of Pediatrics, China Medical University Children's Hospital, No. 2, Yuh-Der Road, Taichaung, Taiwan. E-mail addresses: [email protected] (C.-T. Peng), [email protected] (J.-G. Chang). https://doi.org/10.1016/j.cca.2018.10.020 Received 17 May 2018; Received in revised form 21 September 2018; Accepted 10 October 2018 Available online 11 October 2018 0009-8981/ © 2018 Elsevier B.V. All rights reserved.
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HS has also been frequently described in Japan, and recently in Korea [4,5]. Although HS occurs in all racial and ethnic populations, it is less common in the African-American and Southeast Asian populations [3]. HE is another common red cell membrane disorder in Europeans and their descendants, with estimated incidence of 1 in 2000 to 4000 in the United States. Studies have demonstrated that the high frequencies of HE are observed in areas of endemic malaria, which are as high as 1.6% in Benin, Western Africa [6]. Recently, Wang et al. conducted a systematic review on HS epidemiology in Chinese patients and estimated the prevalence of HS in Mainland China. Their report included 2043 patients with HS during 1978–2013 and estimated a prevalence of
1.27–1.49 in 100,000 individuals [7]. In Taiwan, RBC membrane disorders have been reported occasionally; however, studies of genetic diagnosis are unavailable [8,9]. Many studies have demonstrated the role of mutations in ankyrin 1 (ANK1), band 3 (SLCA1), protein 4.2 (EPB42), α-spectrin (SPTA1), and β-spectrin (SPTB) genes in HS or mutations in spectrin self-association between the N-terminal of α-spectrin (SPTA1) and the C-terminal of βspectrin (SPTB) and protein 4.1R (EPB41) genes in HE [1–3,6,10]. In European and Americans populations, ANK1 mutations is the most common cause of HS, consisting of around 50% of cases, followed by SPTB mutations (~20%), SLC4A1 (~15%), EPB42 (~10%) and SPTA1
Fig. 1. Workflow of WES analysis. Numbers of variants were annotated in each step. 312
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(~5%). In Japan, the Yawata study group has reported a distribution of gene mutations in patients with HS which showed mostly in SLC4A1 (11 mutations) and/or in EPB42 genes (5 mutations) and fewer ANK1 mutations (2 mutations). Later on, the same group extended their study to total 49 patients and found 16/49 (33%) carry ANK1 mutations. In Korea, among 25 patients with HS, 13 ANK1 and 12 SPTB mutations were found and no mutations of SLC4A1, EPB42 or SPTA1 were found [5,6,11–13]. Because of the large size of these genes, the conventional Sanger-sequencing-based approach is not feasible to detect all possible disease-causing alterations. By contrast, next-generation sequencing (NGS) enables a high-throughput analysis and serves as a powerful tool for genetic disease diagnosis [14]. The present study used the wholeexome sequencing (WES) to elucidate the genetic mutations and the inheritance pattern in patients with RBC membrane disorders in Taiwan.
ANNOVAR [19] to functionally annotate genetic variants and used the ratio of variants, minor allele frequency (MAF), clinical significance of ClinVar, report of dbSNP and three pathogenicity predictions software: SIFT, PolyPhen, and CADD_PHRED to select the important variants. 2.4. PCR and Sanger sequencing The variants of RBC membrane disorders candidate genes predicted as non-synonymous, deleterious, damaging, or pathogenic were selected for Sanger sequencing. Supplementary Table 2 provides a list of the designed primers. The detailed methods of PCR and Sanger sequencing were provided in the supplementary method. 3. Results 3.1. Causative variants of RBC membrane disorders detected from WES and pedigree analysis
2. Materials and methods 2.1. Patients and study design
Five causative variants of RBC membrane disorders in four patients were diagnosed (Table 1). The clinical characteristics and laboratory data were listed in Supplementary Table 3. A heterozygous SPTA1 variant c.83G > A (p.R28H) was detected in RMD05 and confirmed as a de novo variant because Sanger sequencing of RMD05's parents and brother showed a wild type. The pedigree of RMD05 was shown in Fig. 2. We further re-analyzed WES data of RMD05 for IVS45 as -12C > T, a splicing variant named low-expression α-spectrin allele (αLELY) with MAF higher than 1%. Allele αLELY may cause clinical significant disease when combined with a coding region mutation [6]. Allele αLELY was found with heterozygous status (ratio of variant: 53.85%) in WES data and confirmed using Sanger sequencing. Heterozygous status of allele αLELY was also found in the father, but not in the mother and brother using Sanger sequencing (Supplementary Fig. 1). The peripheral blood smear of RMD05 showed microspherocytosis and anisocytosis (Fig.2), and the EMA binding test revealed markedly decreased MCF units (Supplementary Table 3). No hemolytic anemia histories were noted in the parents or the brother. The RBC indices of the father showed within normal ranges. The EMA binding tests of the parents and the brother were within the reference range (Supplementary Table 4). Finally, a de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY was confirmed in RMD05. A heterozygous ANK1 variant (chr8:g.41577351 G > T: c.1034C > A: p.A345E) was detected only in RMD11, but not in her parents. The pedigree of RMD11 was shown in Fig. 3. RMD11's parents did not show any clinical or laboratory evidences of hemolytic anemia, and the EMA binding tests also showed within the reference range (Supplementary Table 4). RMD11 seems rather a congenital than an inherited RBC membrane disorder. The ANK1 variant c.1034C > A (p.A345E) is predicted as deleterious, probably damaging and with a high score by SIFT, PolyPhen, and CADD_PHRED and a high frequency of de novo mutations being reported in the ANK1 gene [20]. A de novo ANK1 mutation c.1034C > A (p.A345E) was confirmed in RMD11. Two heterozygous variants, SPTA1 (chr1:g.158612605 T > G:c.4604A > C:p.Q1535P) and SPTB (chr14:g.65234397A > G:c.6203 T > C:p.L2068P) were detected in SPH17. Sanger sequencing also showed these two variants in SPH17's father, but not in the mother. The pedigree of RMD17 was shown in Fig. 4. The hemograms showed a normocytic anemia with reticulocytosis in the father and showed normal RBC indices in the mother (Supplementary Table 4). The MCF units of EMA binding tests for RMD17 and the father were both 47.9 which are in the “grey area” (i.e. between the optimal cut-off value and the lower limit of the range for normal adults) [21], and it was within the reference range for the mother (Supplementary Table 4). Both of these two variants were predicted as deleterious, probably damaging and high score by SIFT, PolyPhen, and CADD_PHRED. An autosomal dominant combined SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P) mutations were confirmed in RMD17.
Seven patients with clinical evidence of congenital hemolytic anemia were enrolled for WES analysis. DNA samples of patients and their family members were extracted from peripheral blood mononuclear cells. The workflow is shown in Fig. 1. To search for the causative variants, panels of candidate genes of RBC membrane disorders, hyperbilirubinemia and other congenital hemolytic anemias were designed (Supplementary Table 1) [6,15,16]. Variants of candidate genes of RBC membrane disorders predicted as nonsynonymous, deleterious, damaging or pathogenic were further confirmed using Sanger sequencing. Additionally, Sanger sequencing of these variants was also performed in their family members for pedigree analysis, with all patients and their family members providing written consent. The study was approved by the Kaohsiung Institute Review Board (KMUHIRB-20140048). 2.2. WES A DNA library was constructed with a TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA, USA). In brief, 100 ng of genomic DNA was diluted in a tris-ethylenediaminetetraacetic acid buffer and sonicated to a fragment size of approximately 300–500 bp, followed by end repair, Atailing, adaptor ligation, and 12 cycles of polymerase chain reaction (PCR) amplification. A 300–400-bp band was gel-selected, and exome capture was performed using a TruSeq Exome Enrichment Kit (Illumina, USA). DNA capture probes were hybridized with the DNA library at the recommended concentration. The DNA library was denatured at 95 °C for 10 min. and subsequently hybridized with captured probes at 58 °C for 16 h. Streptavidin beads were used to bind biotin-labeled probes containing the targeted regions of interest. Three wash steps were performed to prevent nonspecific binding to the beads, and then the hybridization and washing process was repeated. A 10-cycle PCR enrichment process was performed after the second elution, and AMPure XP beads were utilized for purification. The enriched DNA library was quantified in the Qubit dsDNA HS assay (Invitrogen) and validated using the Experion Automated Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA). The library preparations were sequenced on an Illumina NextSeq 500 platform, and 150-bp paired-end reads were generated. 2.3. WES data analysis Base calling and quality scoring were performed by an updated implementation of Real-Time Analysis on the NextSeq 500 system. bcl2fastq Conversion Software was used to demultiplex data and convert BCL files to FASTQ file formats. Sequenced reads were trimmed for adaptor sequencing, and masked for low-complexity or low-quality sequencing, then mapped to the hg19 whole genome using BWA [17]. Finally, SNVs were detected using GATK [18] at their default settings. And then we utilized 313
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A heterozygous ANK1 variant (chr8:g.41542137 G > A: c.4462C > T: p.R1488X) and PIEZO1 variant (chr16:g.88786597 A > G:: c.6044 T > C: p.M2015 T) were identified in RMD21. Sanger sequencing confirmed both variants in RMD21. The ANK1 c.4462C > T (p.R1488X) was from the father and the PIEZO1 c.6044 T > C (p.M2015 T) was from the mother. The RBC indices and EMA binding test of RMD21 and the father both showed anemia, reticulocytosis and decreased MCF reading (Supplementary Table 3 and table 4). The RBC indices and EMA binding test of the mother were within reference ranges (Supplementary Table 4). Literature showed that PIEZO1 mutations are associated with hereditary xerocytosis, which is usually an autosomal dominant disease [6,22]. However, no hemolytic anemia was found in the mother. Whether the PIEZO1 c.6044 T > C (p.M2015 T) would exacerbate the hemolytic anemia while co-inheritance with ANK1 mutations is unknown. Finally, an autosomal dominant ANK1 c.4462C > T (p.R1488X) mutation was confirmed in RMD21. The pedigree of RMD21 was shown in Fig. 5.
disorders. We estimated the frequency of RBC membrane disorder by compared to β-thalassmia. The incidence of β-thalassemia carrier is around 1% in Taiwan. While β-thalassemia major results from two mutations, the inidence of β-thalassemia major is 1/10,000. Therefore, the incidence of RBC membrane disorder maybe around 1/100,000. Autosomal dominant diseases were confirmed in RMD17 and in RMD21, and de novo mutations were confirmed in RMD05 and in RMD11. The family histories were reported negative in patients with autosomal dominant inheritance because the mildness of the disease or a lack of accurate diagnosis of their parents. ANK1 encodes a 206-kD protein that links the spectrin–actin-based red cell membrane skeleton and transmembrane proteins [6,23,24]. Ankyrin is composed of three major domains: a band-3-binding site domain, a spectrin-binding site domain, and a regulatory domain [13,25]. The mutation in RMD11 (c.1034C > A, p.A345E) was located on exon 10 of ANK1, which involves the band-3-binding domain. The nonsense mutation in RMD21 (ANK1 c.4462C > T, p.R1488X) was located on exon 37 of ANK1, which involves the regulatory domain. Ankyrin defects lead to instabilities in spectrin heterodimers because spectrin heterodimers are only stable when bound to the membrane, and ankyrin provides a high-affinity binding site. Therefore, ankyrin defects are usually dominant defects, and de novo ANK1 mutations have been frequently detected [13,20,26]. Of all identified HS mutations, 35% have been ANK1 mutations [6]. Based on the causative mutations detected, RMD11 and RMD21 were diagnosed with HS. A de novo SPTA1 mutation c.83G > A (p.R28H) combined with αLELY was identified in RMD05. The parents and brother had a wildtype genotype of SPTA1 p.R28H and the father has a αLELY polymorphism (Fig. 2). Carriers of αLELY are asymptomatic, even in the homozygous state. Combination of a αLELY opposite a severe HE allele results in clinically significant hemolysis [6]. The mutation involves CGT to CAT (arginine to histidine), CGT to AGT (arginine to serine), CGT to CTT (arginine to leucine), and CGT to TGT (arginine to cysteine) in codon 28 of α-spectrin have been reported to cause HE or the more severe form, HPP [27–29]. RMD05 had hemolytic anemia since early childhood with an initial diagnosis of glucose-6-phosphate dehydrogenase (G-6PD) deficiency. Microcytic spherocytosis, anisocytosis, increased fragility, and normal G-6PD activity were observed after he was transferred to our department. Intermittent transfusions were required during infection episodes, and marked splenomegaly and
3.2. Other variants detected in WES analysis Totally, five ClinVar pathogenic variants were detected in the WES analysis of four patients with RBC membrane disorders, including one RBC membrane disorders gene (SPTA1 chr1:g.158655079C > T c.83G > A: p.R28H) and other four mutations which have been reported in other diseases (Table 2). All variants detected in WES of the four patients with RBC membrane disorders predicted as non-synonymous, deleterious, probably damaging or pathogenic and the average read depths of target regions were listed in Supplementary Table 5. 4. Discussion In the present study, we successfully detected the causative gene mutations of RBC membrane disorders through WES and pedigree analysis in four patients from unrelated Taiwanese families. These four patients were selected from a pool of around 100 patients with congenital hemolytic anemia. Among them, 50 patients were β-thalassemia major, and 38 patients were hemoglobin H diseases. Seven patients with congenital hemolytic anemia other than thalassemias were enrolled in the inital WES analysis. Finally, four patients were diagnosed with RBC membrane
Fig. 2. The pedigree and red cell morphology of RMD05. The electropherograms indicate a SPTA1 mutation (chr1:g.158655079C > T:c.83G > A:p.R28H) in RMD05 and a wild-type genotype in the parents and younger brother, whose red cell indices and EMA binding tests yielded negative findings (a). Peripheral blood smear of RMD05 shows microspherocytosis and anisocytosis (b). The leftward hatching of the boxes is indicative of the αLELY mutation. The rightward hatching of the boxes is indicative of the SPTA1 mutation. The double-lined box schematically indicates detection of both defects in RMD05. The half-filled symbols indicated heterozygous. F, father; M, mother; C1, child 1; C2, child 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 314
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Fig. 3. Pedigree of a de novo ANK1 mutation in RMD11. The electropherograms indicate an ANK1 mutation (chr8:g.41577351 G > T: c.1034C > A: p.A345E) in RMD11 and a wild-type genotype in the parents. The leftward hatching of the boxes is indicative of the ANK1 mutation. The half-filled symbols indicated heterozygous. F, father; M, mother.
cholelithiasis were observed. Based on the mutations and the clinical course, RMD05 was subsequently diagnosed with HE/HPP. An autosomal dominant combination of two novel variants, SPTA1 c.4604A > C (p.Q1535P) and SPTB c.6203 T > C (p.L2068P)
mutations, was confirmed in RMD17. RMD17 had been diagnosed with normocytic anemia at the age of 1 month and had recovered spontaneously without the need of transfusions. The SPTB c.6203 T > C (p.L2068P) was located in repeat 17 of the β-spectrin domain, which
Fig. 4. Pedigree of an autosomal dominant combined SPTA1 and SPTB mutations in RMD17. The electropherograms indicate a combined SPTA1(chr1:g.158612605 T > G:c.4604A > C:p.Q1535P) and SPTB (chr14:g.65234397 A > G: c.6203 T > C:p.L2068P) heterozygous missense mutation in both RMD17 and the father. The leftward hatching of the boxes is indicative of the SPTA1 mutation. The rightward hatching of the boxes is indicative of the SPTB mutation. The double-lined box schematically indicates the detection of both defects. The half-filled symbols indicated heterozygous. F, father; M, mother. 315
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Fig. 5. Pedigree of an autosomal dominant ANK1 mutation in RMD21. ANK1 (chr8:g.41542137 G > A: c.4462C > T:p.R1488X) was identified in RMD21 and his father. The Sanger sequencing of the mother showed wild type. The leftward hatching of the boxes is indicative of the ANK1 mutation. The half-filled symbols indicated heterozygous. F, father; M, mother.
forms a part of the spectrin self-association site [5]. Meanwhile, the SPTA1 c.4604A > C (p.Q1535P) was located beyond the self-association binding site, which tends to be less severe in clinical manifestations [6]. In humans, α-spectrin synthesis is four-fold of β-spectrin synthesis. Heterozygotes for α-spectrin defects usually produce adequate normal α-spectrin chains to pair with β-spectrin chains. Studies have indicated that α-spectrin production of less than 25% causes diseases [30]. Taken together, we suggested that the clinical manifestations of RMD17 mainly resulted from the SPTB c.6203 T > C (p.L2068P) mutation and was diagnosed with mild HE. However, further functional studies may be needed to elucidate the phenotypes of these two variants. Genetic diagnosis for red cell membrane disorders is laborious because of the wide genetic heterogeneity and the large size of candidate genes. Although the genetic diagnosis does not add extra information for the patient whose family is known to have the RBC membrane disorder, a recessive inheritance, de novo mutations or compound heterozygosity require further molecular studies [31]. NGS is a highthroughput genetic diagnosis tool, which can be used for the detection of rare diseases at the molecular level [32–34], and a cost-effective
approach for the molecular diagnosis of hereditary hemolytic anemia, particularly when the family history is non-informative or when routine laboratory tests cannot identify the causative hemolytic process [35]. Agarwal et al. reported the use of NGS of 28 genes' panel of cytoskeletal proteins and enzymes to determine the molecular basis of hemolytic anemia in cases of red cell membrane disorders despite the lack of family history. Ten of 12 of the mutations detected have not been previously described, including SPTB, SLC4A1 and ANK1 mutations. One of the patients has both ANK1 and SPTA1 mutations [35]. Barreto et al. used a 40 genes' panel for detecting new pathogenic mutations in patients with congenital hemolytic anemia [16]. Han et al. used WES to identify a novel nonsense mutation in ANK1 (p.Q1772X) in a Korean patient with HS and also confirmed a comorbid with UGT1A1 mutations [15]. Although WES can provide more comprehensive information than gene panels, it has limitations in sufficiently covering coding exons, especially GC-rich regions, and in copy number variation detection. Previous studies showed that WES failed to detect 0.42% of currently known pathogenic mutations and 0.81% if non-coding pathogenic variation were included as well [36].
Table 1 Causative mutations of RBC membrane disorders detected in WES and confirmed by Sanger sequencing. Patient
Gene
Coordinate
Ref/Var
Sequence depth, ratio of variant
Coding sequence
Amino acid
Genotype
ClinVar
SIFT
PolyPhen
CADD
Inheritance
RMD05
SPTA1
chr1:g.158655079
C/T
11/21 (52.38%),
c.83G > A
p.R28H
hetero
Pathogenic
deleterious
34
a
RMD11
ANK1
chr8:g.41577351
G/T
13/30 (43.33%),
c.1034C > A
p.A345E
hetero
deleterious
33
de novo
RMD17
SPTA1
chr1:g.158612605
T/G
c.4604A > C
p.Q1535P
hetero
deleterious
29.2
AD
SPTB
chr14:g.65234397
A/G
137/281 (48.58%) 90/195 (46.15%)
c.6203 T > C
p.L2068P
hetero
deleterious
ANK1
chr8:g.41542137
G/A
91/231 (39.39%)
c.4462C > T
p.R1488X
hetero
NA
Probably damaging Probably damaging Probably damaging Probably damaging NA
RMD21
31 36
hetero: heterozygous. AD: autosomal dominant. a A Combined SPTA1 p.R28H and αLELY polymorphism was detected in RMD05. The SPTA1 p.R28H was de novo and αLELY was from the father. 316
de novo
AD
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Table 2 ClinVar pathogenic mutations detected in WES analysis. Patient
Gene
Coordinate
Sequence depth, ratio of variant
ClinVar
RMD05 RMD11 RMD11 RMD17 RMD21
SPTA1 DCHS1 NDUFS3 HEXA LRRK2
chr1:g.158655079C > T:c.83G > A:p.R28H chr11:g.6645369C > T:c.7538G > A:p.R2513H chr11:g.47605974C > T:c.736C > T:p.R246C chr15:g.72638646G > C:c.1351C > G:p.L451 V chr12:g.40677699C > T:c.2264C > T:p.P755L
11/21 (52.38%) 9/29 (31.03%) 21/36 (58.33%) 86/178 (48.31%) 93/201 (46.27%)
Pathogenic Pathogenic Likely pathogenic Pathogenic Pathogenic
Our study is the first to use the WES approach for the genetic diagnosis of RBC membrane disorders in Taiwan and demonstrated the usefulness of the WES approach in the diagnosis of red cell membrane disorders. Additional studies involving larger cohorts must be conducted to investigate the distribution of causative gene mutations in patients with RBC membrane disorders in Taiwan. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cca.2018.10.020.
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