Chai et al. BMC Medical Genetics(2020) 21:223
https://doi.org/10.1186/s12881-020-01161-4

CASE REPORTOpen Access

Novel nonsense mutation p. Gln264Ter inthe ANK1 confirms causative role forhereditary spherocytosis: a case report
Senmao Chai1, Rong Jiao2, Xiaodong Sun1, Pan Fu1, Qiang Zhao1 and Ming Sang1*

Abstract
Background: Hereditary spherocytosis (HS) is the most common haemolytic anaemia caused by congenital
membrane defects of red blood cells. The name derives from the presence of spherical red blood cells in the
peripheral blood. Clinical manifestations of HS are anaemia, haemolytic jaundice, and large spleen, and infection
can worsen the condition, often with cholelithiasis. HS is mainly caused by abnormal functions of the products ofsix genes. Splenectomy is the main treatment for HS.
Case presentation: Half a day after birth, the proband exhibited HS-related symptoms, with progressive
aggravation. Routine examination in the outpatient department showed an increase in white blood cells and adecrease in red blood cells. His mother had HS and a partial splenectomy. We suspected that the infant might also
have HS. Genomic DNA samples were extracted from the three members of the HS trio pedigree, and genomic
whole-exome sequencing (WES) was performed. The three DNA samples were amplified by polymerase chain
reaction (PCR), followed by Sanger sequencing to identify mutation sites. A novel nonsense heterozygous mutation,
c.790C > T (p. Gln264Ter), in the ANK1 gene, which causes premature termination of translation, was found in thisChinese family with autosomal dominant HS.
Conclusions: This de novo nonsense mutation can cause the onset of HS in early childhood, with severe
symptoms. Expanding the ANK1 genotype mutation spectrum will lay a foundation for the further application of
mutation screening in genetic counselling.
Keywords: Hereditary spherocytosis, Whole-exome sequencing, ANK1, Nonsense mutation* Correspondence: smxd2000@126.com
1
Hubei Institute of Parkinson’s Disease at Xiangyang No.1 People’s Hospital,
Hubei Key Laboratory of Wudang Local Chinese Medicine Research, Hubei
University of Medicine, Shiyan 442000, People’s Republic of China
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Chai et al. BMC Medical Genetics(2020) 21:223

Background
Hereditary spherocytosis (HS, OMIM:182900), also known
as congenital hemolytic anemia, refers to one type ofheterogeneous inherited anaemia characterized by the presence of spherocytes on the peripheral blood smear [1]. HS
occurs all over the world, affecting approximately 1/5000
people in Europe and North America [2, 3]. The main
result is defects in the erythrocyte membrane skeleton,
resulting in decreased surface area and spherical changes;
these changes lead to loss of erythrocyte membrane elasticity and mechanical stability and difficulty in passing
through the splenic capillaries, promoting erythrocyte
clearance by the spleen [4]. The increase in red blood cell
destruction leads to increases in bilirubin levels and causes
yellowing of the skin and sclera. The spleen also grows
larger due to the destruction of red blood cells and thestimulation of proliferation, followed by liver enlargement.
Therefore, anaemia, jaundice, large liver and large spleen
are the main symptoms of HS. Currently, the main clinicaltreatment is partial or total splenectomy and splenic artery
embolization. Theoretically, splenic circulation is not fully
developed until after birth, resulting in enhanced destruction of the globular cells surrounding the circulation andincreased haemolysis. Occasional visits to the hospital cause
a high rate of misdiagnosis and delayed treatment, which
threatens the life of patients with HS [4]. At present, 6abnormal genes which lead to HS have been identified.
They are ANK1(ankyrin; MIM: 182900), SPTB (beta-spectrin; MIM: 616649), SPTA1(alpha-spectrin; MIM: 270970),
SLC4A1(Band 3; MIM; 612,653) EPB41(protein 4.1; Phenotype MIM:611804) and EPB42(protein 4.2;MIM: 612690)[5]. There exist two genetic inheritance patterns for HS:
autosomal dominant (AD) (75%) and autosomal recessive(AR) (25%) [2]. HS-causative mutations in SPTB and
SLC4A1 show AD, whereas those in SPTA1, EPB41and
EPB42 show AR; alterations in ANK1 display AD or AR[2]. Although splenectomy greatly helps patients with HS, it
also produces many complications. Some patients even dieof infection of mesentery or portal vein occlusion after
splenectomy. The most common complication is infection,
especially among infants and young children.
In this study, we collected a trio pedigree of HS. The
proband showed HS-related symptoms half a day after
birth. Through gene sequencing, we identified a de novo
nonsense variant in ANK1 in this family, promoting further
application of mutation screening in genetic counselling.
Case presentation
Ethical compliance and human subjects

The study was approved by the Ethics Committee of
Xiangyang No.1 People’s Hospital, Hubei. Informed
consent was obtained directly from the participants orparents/legal guardians of any participant under the ageof 18. Blood specimens were collected from the proband

Page 2 of 6

and his parents. The proband was a male infant born atfull term with an uneventful pregnancy. Apgar scores
were 9/1 min and 10/5 min. The proband showed HSrelated symptoms with gradual aggravation within half a
day after birth. Routine outpatient department examination showed an increased white blood cell count and adecreased red blood cell count. The proband’s motherhad a history of HS and underwent partial splenectomy.
We suspected the son might have HS. The probability oftriggering haemolysis and entire haemolysis is increasedin patients with erythrocyte brittleness (Table 1). Basedon differential diagnosis, physiological jaundice, neonatal
ABO haemolysis, neonatal sepsis, neonatal hepatitis syndrome and bilirubin encephalopathy were all excluded.
Amoxicillin and clavulanate potassium were given foranti-infection treatment. The patient was treated withhydration, phototherapy, sodium bicarbonate, and vitamin K1 supplementation. Neonatal diseases are treatedin the intensive care unit (ICU) of our hospital, withmother-infant separation.
Whole-exome sequencing (WES) and sanger sequencing
validation

Genomic DNA samples from each member of the HStrio pedigree (II:1, in Fig. 1a) were extracted from whole
blood and used to sequence the whole exome. The BGIExome V4 kit (BGI) was used for in-solution enrichmentof coding exons and flanking intronic sequences, as per
the manufacturer’s standard protocol. We used cutadapt(v1.15) to trim adaptor sequences at the tail of thesequencing reads and then aligned the sequencing reads
to the human reference genome (UCSC hg19) with
BWA (v0.7.15). Duplicated reads marked by Picard(v2.4.1). Qualimap [6] (v2.2.1) was used to calculate basequality metrics, the genome mapping rate, and thecoverage of targeted regions. Base quality score recalibration, indel realignment and variant (SNV & InDel)
calling were performed following the best practice protocol of Genome Analysis Toolkit (GATK, v3.8). Variant
filtering was performed by a finely tuned in-house script.
Pass-filter variants were annotated by the Pubvar variant
annotation engine (www.pubvar.com) and VEP [7].
Variant models for dominant inheritance and recessive
inheritance were separately identified in the genetic
analysis. Variants that met any of the following criteria
were excluded from the analysis: maximum population
frequency larger than 0.01, low genotype confidence, orpredicted as benign by all five algorithms, i.e., SIFT [8],PolyPhen 2 [9], MetaSVM [10], MCAP [11] and MutationTaster [12]. The pathogenic evidences of candidate
disease-causing variants were scored by InterVar [13](1.0.8) according to American College of Medical Genetics
and Genomics (ACMG) guidelines [14]. Subsequently, allvariants in previously reported HS genes (ANK1, SPTB,Chai et al. BMC Medical Genetics(2020) 21:223

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Table 1 Abnormal examination results on admission
Test item

ResultCuereference range

Erythrocyte

3.26 × 1012/L↓(5.20–6.40) × 1012/L

Hemoglobin

112.0 g/L↓

180.0–190.0 g/L

Neutrophils percentage

79.2%

↑

31.0–40.0%

Lymphocyte percentage

13.6%

↓

40.0–60.0%

Red cell distribution width

19.0%

↑

11.5–14.5%

Red cell distribution width standard deviation

66.7 fL↑

35.0–56.0 fL

Began hemolysis in patients with erythrocyte brittleness5.40 g/L↑

3.8–4.6 g/L

Entirely hemolysis in patients with erythrocyte brittleness4.32 g/L↑

2.8–3.2 g/L

Total bilirubin

194.33 μmol/L↑

0.0–26.0 μmol/L

Direct bilirubin

12.47 μmol/L↑

0.0–8.0 μmol/L

Indirect bilirubin

181.86 μmol/L↑

0.0-18 μmol/L

γ-Glutamyl transferase

84.38 IU/L↑

10–60 IU/L

Alkaline phosphatase129.81 IU/L↑

45-125 IU/L

Creatine kinase

820 U/L↑

50-310 U/LCreatine kinase MB isoenzyme

44.48 U/L↑

0-24 U/L

Lactic dehydrogenase

385 U/L↑

120-250 U/L

α- Hydroxybutyrate Dehydrogenase

281.7 U/L↑

72-182 U/L

C- Reactive Protein

10.75 mg/L↑

0.0-8 mg/L

SPTA1, SLC4A1 EPB41 and EPB42) were investigated forsegregation. Finally, all variants following autosomal dominant and autosomal recessive inheritance were checked
for segregation.
A novel ANK1 (NM_000037.3: c.790C > T, p. Gln264Ter)
nonsense heterozygous mutation was identified in theinfant and his mother. According to the evidence classification of American College of Medical Genetics and Genomics (ACMG) pathogenicity, the mutation was judged to bepathogenic (PVS1 + PM2 + PP4). Frequency data in 1000
Genomes, gnomAD exomes and East Asian gnomAD
exomes were not found for this ANK1 mutation site.
Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/pri
mer-blast/) was used to design primers containing themutation site: forward CAGAAATGCCCTGACAACGC
and reverse ACTATTGCCTCCCACTGCAC. The annealing temperature was 60 °C, and the amplified fragment size
was 395 bp. To verify the ANK1 mutation, PCR followed bySanger sequencing were performed for the three individuals(I-1, I-2, II-1) of the pedigree. Our results confirmed that aheterozygous mutation was carried by the mother (I-2) andthe son (II-1, Table 2, Fig. 1b). This ANK1 nonsense
heterozygous mutation (NM_000037.3: c.790C > T, p.
Gln264Ter) is novel and not reported or listed in the
Human Gene Mutation Database (HGMD), Exome Variant
Server (EVS), the 1000 Genomes databases or any otherdatabase (gnomAD, ExAC, ClinVar, dbSNP). Taken together, the causative mutation was verified by examining
the trio pedigree via WES and Sanger sequencing.

Discussion and conclusion
The cytoskeleton plays a key role in maintaining themorphology and function of erythrocyte membranes.
Many proteins, such as ankyrin, spectrin α- and β-chains,proteins 4.1, or 4.1R and actin, cover the inner surface ofthe erythrocyte membrane to form two protein complexes,
the ankyrin and protein 4.1 complex (Fig. 2). These twocomplexes interact to maintain the circular pie-like structure of the central fovea of red blood cells. The former
complex consists of Band 3 tetramers, Rh, RhAG, CD47,
Glycophorin A and Protein 4.2; the latter consists of Band
3 dimers binding Adducins α and β, Glycophorin C,
GLUT1 and Stomatin [15, 16]. ANK1, the most commonof the 6 pathogenic genes of HS, is an important memberof the anchoring protein family, which is mainly expressedin the erythrocyte membrane. Anchoring proteins are connected to complete membrane proteins, further connectedto the spectrin and cytoskeleton. They play an importantrole in cell movement, activation and proliferation and participate in maintaining the specific morphology of cell
membranes. Mutation of the ANK1 gene can cause red
blood cells to change from a double concave disc shape toa spherical shape and reduce its deforming capacity. In capillary networks such as the liver and spleen, rupture andhaemolysis may occur due to mechanical damage [17].
There are more than 60 known mutations in the human
HS-related ANK1 gene, among which the most common
include missense mutations, nonsense mutations, frame
shifts and splice site mutations. The clinical severity of HS

Chai et al. BMC Medical Genetics(2020) 21:223

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Fig. 1 The novel pathogenic variant in the ANK1 gene. a The family tree. b The hemizygous variant confirmed by Sanger sequencing. The
heterozygous mutations has been carried by Mother and son. The red arrow indicates the mutation site

Table 2 ANK1 mutation of the family members
Family members

Exon

Reference sequence

Type

cDNA

ProteinFather8

NM_000037.3

Wild

c.790C

p. Gln264

Mather8

NM_000037.3

Mutantc.790C > T

p. Gln264Ter

propositus

8

NM_000037.3

Mutantc.790C > T

p. Gln264Ter

Chai et al. BMC Medical Genetics(2020) 21:223

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Fig. 2 A simplified cross section diagram of erythrocyte membrane. Phospholipid bilayer is the basic scaffold of red blood cell membrane. Protein
Ankyrin is marked in red to indicate damage due to gene mutation. At present, it has been found that 6 proteins Ankyrin; Spectrin alpha-chain;
Spectrin beta-chain; Band 3; Protein 4.1 and Protein 4.2 paly a pathogenic role in Hereditary spherocytosis. The relative positions of proteins invarious complexes are mostly unknown. The shapes of the major proteins are fictitious. GPA: glycophorin A; Rh: Rhesus polypeptide; B-4.1: proteinband 4.1; B-4.2: protein band 4.2; GPC: glycophorin C; RhAG: Rh-associated glycoprotein

depends on the degree of membrane loss, and most ANK1
mutations show AD inheritance [17, 18]. Ankyrin is themain component of erythrocyte membranes and composedof three structural domains: a 24 homologous repeat Nterminal membrane-binding domain (MBD) involved in
the binding of Band 3 protein; a Spectrin-binding central
domain (SBD); and a least conserved carboxy-terminal
regulatory domain subject to variation [19].
This clinical report shows that the proband and his
mother carry a de novo nonsense heterozygous mutationin the ANK1 (NM_000037.3: c.790C > T, p. Gln264Ter)
gene. In contrast to most affected newborns, thisproband presented severe anaemia in a very early lifeand remained transfusion dependent. Half a day after
birth, his skin appeared yellow dye, and suffered fromprogressive aggravation, his white blood cell counts andc-reactive protein both were increased significantly. Ourfindings show the de novo nonsense mutation can cause
the onset of the disease in early childhood, even withmore severer symptoms. Expanding the genotypic mutation spectrum of ANK1, can work for further applicationof mutation screening in the genetic counseling.
Abbreviations
HS: Hereditary spherocytosis; AD: Autosomal dominant; AR: Autosomal
recessive; ANK1: Ankyrin 1; SPTB: Beta-spectrin; SPTA1: Alpha-spectrin;
SLC4A1: Band 3; EPB41: Protein 4.1; EPB42: Protein 4.2; WES: Whole exome
sequencing; ACMG: American College of Medical Genetics and Genomics

Acknowledgements
We thank the patient’s family for participating in this study, Kd ofReproductive Medicine Center, Renmin Hospital, Hubei University of
Medicine. We thank all the staff of Hubei Institute of Parkinson’s Disease at
Xiangyang No.1 People’s Hospital. They were very helpful.

Authors’ contributions
MS, Sm C and RJ Conceived and designed the experiments. Sm C and, Xd S
Performed the sample recruitment and Whole exome and sanger
sequencing. QZ and PF Conducted the data analysis. MS Provided thereagents/ materials/ analysis tools to carry out the experiments. Sm C Wrotethe paper. All authors read and approved the final manuscript.
Funding
This study was funded by the Natural Science Foundation of Hubei Province.
Award Number: 2019AHB068, 2017CFB674,2018CFB701. Open Project of
Hubei Key Laboratory of Wudang Local Chinese Medicine Research. Award
Number: WDCM2018009. The Innovative Team Project from the Institute ofMedicine and Nursing at Hubei University of Medicine. Award Number:
2017YHKT02. The recipient is Ming Sang for All of the funding.MS is also thecorresponding author.
The funding bodies played no role in the design of the study and collection,
analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
All relevant data are within the paper, including raw data from WES, Further
information is available from the authors on request. All raw data related toour study has been submitted on NCBI Sequence Read Archive (SRA) (SRA
RunSelector: https://www.ncbi.nlm.nih.gov/sra/PRJNA671560). Accession
number is PRJNA671560. Reference datasets were listed in the “Wholeexome sequencing (WES) and Sanger sequencing validation” section, such as
the human reference genome (UCSC hg19) and transcription number(NM_000037.3).
Ethics approval and consent to participate
The Ethical approval for the study was taken from Ethics Committee of
Xiangyang No.1 People’s Hospital. Written informed consents were obtained
from the parents or guardians of the propositus and 74 healthy control
cases.
Consent for publication
Written informed consent for publication of individual/genetic data was
obtained from the participants or parents/legal guardians of any participant
under the age of 18.
Competing interests
The authors declare no competing interest.

Chai et al. BMC Medical Genetics(2020) 21:223

Author details
1
Hubei Institute of Parkinson’s Disease at Xiangyang No.1 People’s Hospital,
Hubei Key Laboratory of Wudang Local Chinese Medicine Research, Hubei
University of Medicine, Shiyan 442000, People’s Republic of China.
2
Department of New pediatric, Xiangyang No.1 People’s Hospital, Hubei
University of Medicine, Xiangyang, Hubei, China.
Received: 4 July 2020 Accepted: 29 October 2020

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