Two novel ANK1 loss‐of‐function mutations in Chinese families with hereditary spherocytosis

Abstract Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia disorder. ANK1 mutations account for most HS cases, but pathogenicity analysis and functional research have not been widely performed for these mutations. In this study, in order to confirm diagnosis, gene mutation was screened in two unrelated Chinese families with HS by a next‐generation sequencing (NGS) panel and then confirmed by Sanger sequencing. Two novel heterozygous mutations (c.C841T, p.R281X and c.T290G, p.L97R) of the ANK1 gene were identified in the two families respectively. Then, the pathogenicity of the two new mutations and two previously reported ANK1 mutations (c.C648G, p.Y216X and c.G424T, p.E142X) were studied by in vitro experiments. The four mutations increased the osmotic fragility of cells, reduced the stabilities of ANK1 proteins and prevented the protein from localizing to the plasma membrane and interacting with SPTB and SLC4A1. We classified these four mutations into disease‐causing mutations for HS. Thus, conducting the same mutation test and providing genetic counselling for the two families were meaningful and significant. Moreover, the identification of two novel mutations enriches the ANK1 mutation database, especially in China.

conditions range in severity from asymptomatic to severe transfusion-dependent states with several main clinical manifestations, including anaemia, jaundice, splenomegaly and cholelithiasis. 5 In the clinic, making an exact diagnosis of asymptomatic or atypical cases is relatively difficult based on clinical symptoms, family history and haematologic parameter tests alone. 6,7 Therefore, molecular detection is particularly important for the clinical diagnosis of HS.
Hereditary spherocytosis is caused by mutations of erythrocyte membrane-related genes, including ANK1 (ankyrin 1; OMIM 612641), SPTB (spectrin, beta, erythrocytic; OMIM 182870), SPTA1 (spectrin, alpha, erythrocytic 1; OMIM 182860), SLC4A1 (solute carrier family 4, member 1; OMIM 109270) and EPB42 (protein 4.2, erythrocytic; OMIM 177070), following an autosomal dominant (AD) and autosomal recessive inheritance mode. 8 Mutations in these genes usually produce decreased surface area per unit volume of erythrocytes or dysfunction of the erythrocyte membrane, leading to the detachment of the lipid bilayer from the spectrin-based cytoskeleton. Therefore, abnormal erythrocytes become spherocytes with increased osmotic fragility and are easily destroyed by the spleen, consequently resulting in haemolytic anaemia. 9 Mutations in the ANK1 gene can explain approximately half of all cases with HS.
Additionally, 80%-85% of ANK1 mutations are AD inherited. 10 The ANK1 gene is located at 8p11.21. 11 ANK1 contains 43 exons, and the cDNA is 8 300 bp in length, coding for erythroid ankyrin 1 protein of 1 880 amino acids with three main structural domains, an N-terminal membrane-binding domain, a central spectrin-binding domain and a C-terminal regulatory domain, which is the least conserved and subject to variation. 12 ANK1 protein serves as the key link between the SLC4A1 (band 3) complex on the lipid bilayer to the erythrocyte cytoskeleton and confers vertical stability and reversible deformability to the erythrocyte membrane. 13 A mouse model with truncated mutations lacking the spectrin-binding and C-terminal regulatory domains of Ank1 manifested severe HS. 8 To date, a total of 89 ANK1 mutations have been reported in the Human Gene Mutation Database (registration required, http://www. hgmd.cf.ac.uk, last accessed 24 May 2018), including 38 missense or nonsense mutations. Although mutations in ANK1 are common in HS, the pathogenic mechanism of ANK1 mutations is not entirely understood.
In this study, two novel mutations of ANK1 were found in two unrelated Chinese families with HS and predicted to be disease causing. Then, we carried out functional research in vitro to further confirm the pathogenicity of four ANK1 mutations, including the two novel mutations and two previously reported mutations (c.G424T, p.E142X and c.C648G, p.Y216X) in a Chinese population. 7

| Pedigrees and ethical statement
Two unrelated Chinese nuclear families with HS were recruited into the study ( Figure 1A). In pedigree 1, the male newborn (II: 2) had suspected HS with severe anaemia, jaundice and splenomegaly 3 months after birth and received several blood transfusions. His father (I: 1) underwent splenectomy because of HS at 1 year old.
His mother (I: 2) and elder brother (II: 2) were non-symptomatic. In F I G U R E 1 Two unrelated Chinese families with HS-associated ANK1 mutations. (A) Pedigrees: open symbols indicate unaffected individuals; filled black symbols indicate affected individuals; and black arrows indicate probands. The left is pedigree 1; member I: 1 and II: 2 carry the novel ANK1 mutation (c.T290G, p.L97R), and I: 2 and II: 1 do not have this mutation. The right is pedigree 2; the novel ANK1 mutation (c.C841T, p.R281X) was found in member II: 1, II: 2, I: 1 and was not identified in I: 2. (B) Peripheral blood smears of patients in two pedigrees. Spherocytes are observed in the blood films of the patients in two pedigrees (pedigree 1: member I: 1 and II: 2; pedigree 2: member II: 1) which are indicated by black arrows. (C) Partial DNA sequences containing the two novel heterozygous ANK1 mutations. The upper panels show the wild-type sequences, and in the lower panels, red arrows indicate the mutation sites pedigree 2, the proband (II: 1) was a 4-year-old girl with suspected HS who presented with severe haemolytic anaemia. Her mother (I: 2) underwent splenectomy due to HS when she was 10 years old. Her father (I: 1) showed abnormalities in the erythrocyte membrane, and her younger sister (II: 2) showed mild anaemia. In the two pedigrees, the probands underwent a series of detailed blood tests.
Glucose-6-phosphate dehydrogenase activity was normal. The results of an autoimmune antibody test were negative. Spherocytes were observed in a peripheral blood smear ( Figure 1B), and osmotic fragility was increased (

| Preparation of mutant constructs
A plasmid containing full-length ANK1 cDNA (BC030957) was purchased from abmgood (Applied Biological Materials, Inc, Richmond, Canada). Wild-type ANK1 was cloned into the pCDNA3.1(+)-3ΧFlag-C expression vector, and 5'-ATG  (TOYOBO, Osaka, Japan). The primers used were as follows: All expression constructs were validated by Sanger sequencing.  Gene expression was normalized to GAPDH and analysed according to the relative quantification method (2 −ΔΔCt ). Three independent experiments were performed. Substrate (Thermo Fisher, Rockford, IL) was used to visualize proteins on X-ray film.

| Immunofluorescence
Transfected cells grown on cover slips to 70%-80% confluence were fixed in 4% paraformaldehyde, then underwent permeation using PBS with 0.5% Triton and were blocked with 3% FBS in PBS-Triton.

| Cell surface biotinylation
The transfected HEK293T cells grown in 10-cm culture dishes were

| Statistical analysis
All statistical analyses were performed by paired two-tailed All experiments were repeated more than twice.

| Characterization of ANK1 mutations in two families with HS
In pedigree 1, a novel heterozygous mutation (c.T290G, p.L97R) in ANK1 was detected in two patients (II: 2 and I: 1), while two other unaffected members (I: 2 and II: 1) had wild-type ANK1 alleles ( Figure 1C). This missense mutation is located at exon 4 in the N-terminal membrane-binding domain and is highly conserved (Figure 2A and C). In pedigree 2, a novel heterozygous nonsense mutation in ANK1 (c.C841T, p.R281X) was identified in patients (II: 1, II: 2 and I: 2), but I: 1 did not carry it ( Figure 1C).
The mutation is located in exon 9 of the N-terminal membranebinding domain (Figure 2A and B). This mutation produced a truncated protein with a shortened membrane-binding domain and without a spectrin-binding domain as well as a regulatory domain ( Figure 2B). Both novel mutations were predicted to be disease causing (Table 2).

| The predicted effect of four mutations on protein structure
The results of structure predictions showed that mutation p.L97R led to the change of non-polar leucine to positively charged arginine at amino acid 97, thus altering the 3D structure of the N-terminal membrane-binding domain ( Figure 2D). The other three nonsense mutations resulted in truncated proteins, including the incomplete N-terminal membrane-binding domain ( Figure 2D). These changes might influence the combination of ANK1 and SLC4A1 and can lead to the non-union of ANK1 with SPTB.

| ANK1 mutations caused higher cell osmotic fragility
Through a preliminary test, the number of broken HEK293 cells was found to be significantly increased when the concentration of NaCl was less than 0.24%, and almost all cells were completely broken when the concentration was as low as 0.08%.
Therefore, we compared the change of cell rupture rate among wild-type and mutant cells in NaCl solution between 0.08% and isotonic concentration of 0.85% (0.08%, 0.12%, 0.16%, 0.2%, 0.24% and 0.85%). The detection and calculation results showed that the rate of cell rupture with ANK1 mutants was higher than that with wild-type protein at every concentration of NaCl ( Figure 3A). These results indicated that cell rupture occurred more easily in ANK1 mutant cells, thus leading to higher cell osmotic fragility.

| Protein stabilities were decreased in ANK1 mutants
To determine the pathogenicity of different ANK1 mutations, we successfully overexpressed wild-type and four mutant proteins in HEK293T cells. qPCR showed no differences in the transfection efficiency of different plasmids, and the mutations did not affect the mRNA expression level of ANK1 ( Figure 3B). However, western blot analyses revealed that the expression of all four mutants was weaker than that of wild-type. In particular, truncated mutant p.E142X led to the total degradation of ANK1 ( Figure 3C).

| ANK1 mutants had defect in being located on the plasma membrane
Mutations can affect the processing and structure of the protein, which may alter the cellular localization of protein. Thus, the subcellular localization of mutant ANK1 proteins was determined using Immunofluorescence (IF). Immunofluorescence indicated that wild-type ANK1 was localized to the plasma membrane of HEK293T, while three mutants (L97R, Y216X and R281X) were mainly localized to the endoplasmic reticulum (ER) ( Figure 4A).
Cell surface biotinylation assay was performed to determine the amount of wild-type and mutant ANK1-FLAG proteins bound by the plasma membrane of transfected HEK293T cells. The results indicated that the amount of wild-type ANK1-FLAG was relatively more than all the mutants combined with the plasma membrane. Especially, the binding signals of the two ANK1 mutants Y216X and R281X with plasma membrane were almost undetectable even when the exposure intensity was elevated ( Figure 4B).
The mutant ANK1 proteins were likely to be primarily retained in the ER rather than being targeted to the plasma membrane, thus they lost their function as anchoring protein. Note. The pathogenicity was tested using the bioinformatics software SIFT, PolyPhen2 and MutationTaster. Abbreviations: c, variation at cDNA level; ExAC, Exome Aggregation Consortium; Hete, heterozygote; p, variation at protein level; X, Stop codon.

| Mutations abolished ANK1 interaction with SPTB and SLC4A1
The four mutations occurred in the N-terminal membrane protein binding domain, and they failed to be transported to the plasma membrane, which indicated their combination with membrane protein SLC4A1 were impaired. Thus, the interaction between ANK1 mutants (L97R, Y216X and R281X) with SLC4A1 and another ANK1 major binding protein SPTB, was detected by co-immunoprecipitation. The biochemical interactions between two mutants (Y216X and R281X) and SLC4A1/SPTB were greatly abolished in HEK293T cells. The L97R mutant had a normal interaction with SPTB and an attenuated interaction with SLC4A1, while wild-type ANK1 could interact with the two proteins ( Figure 5). Therefore, ANK1 mutants could not anchor SLC4A1 to the SPTB due to their inability to be localized on the plasma membrane.

| D ISCUSS I ON
Hereditary spherocytosis is a common inherited erythrocyte membrane disorder, and the clinical symptoms are variable even within one family. 7,13,20 Cases with atypical clinical phenotypes or without family history defy diagnosis. The molecular genetic analysis of the proband and as many other family members as possible would be helpful to confirm the diagnosis, especially for situations in which the clinical phenotype of the proband is more severe than that of other affected members in the family. 9 Mutations in ANK1 are a common genetic cause of HS. More than half of HS patients bear ANK1 mutations in many countries, such as the USA, Europe and South Korea, but the prevalence of ANK1 mutations in China is unclear.
For a long time, the conventional mutation detection method for ANK1 genes has been Sanger sequencing. However, the cost of direct sequencing is high because of the large size of the ANK1 gene, its high-allelic heterogeneity and the lack of hot-spot mutations.
Currently, the development of NGS technology provides a valid way to find the exact genetic cause of diseases. NGS could be rapider, more thorough and less expensive than Sanger sequencing, especially for the detection of large genes. 21 As NGS is used more widely glycolytic enzymes and haemoglobin. 22 The 55 kDa regulatory domain of ANK1 is involved in regulating its association with SPTB and SLC4A1. 23,24 As the key to maintaining the shape of erythrocytes, all three domains of ANK1 are essential for appropriate function and assembly of the erythrocyte membranous-cytoskeletal network. According to previous reports, ANK1 mutations, particularly nonsense and frameshift mutations carried by HS patients, could decrease the expression of ANK1. 25,26 Based on our in vivo experiments, the E142X mutant was completely degraded and non-functional, and the expression of the other three mutants was also reduced. What's more, the ER retention of the misfolded ANK1 mutants (L97R, Y216X and R281X) was observed, which may lead to the degradation of the protein. 27 The SPTB-ANK1-SLC4A1 complex was identified with the help of IP. 28 SLC4A1 could be tightly linked to the spectrin-based cytoskeleton through binding to specific domains of ANK1. 28,29 Three mutants in addition to the E142X mutant lost their function as anchoring proteins linked to SPTB and SLC4A1 were determined by Co-IP. These proteins were retained in ER instead of being localized to the plasma membrane. Therefore, all three mutants were dysfunctional.
Mutations in ANK1 could contribute to spherocytes with high osmotic fragility, which were observed in the blood of most HS patients. 30 Here, the HEK293T cell line was used to verify whether the four ANK1 mutants could increase the osmotic fragility of cells. HEK293T cells are adherent and do not contain Here, we reported two novel mutations in the ANK1 gene in two unrelated HS Chinese families. We also provided powerful evidence for the pathogenicity of two novel and two previously reported ANK1 mutations in China. Thus, conducting the same mutation test and providing genetic counselling for the relatives of mutation carriers are meaningful and significant. If other healthy family members at risk have this demand, we can provide presymptomatic gene diagnoses for them. This method will help us to identify asymptomatic mutation carriers and provide diagnosis of HS. For example, in the present study, the two daughters in pedigree 2 carried the same mutation with different clinical phenotypes; therefore, molecular genetic analysis helped to provide diagnosis for the asymptomatic daughters in this family. More generally, the identification of the two new mutations enriches the genetic profile of HS, especially in the Chinese population, which will contribute to the clinical understanding of HS caused by mutations in the ANK1 gene. Our study also provided an example to test the pathogenicity of ANK1 mutations found in HS patients by in vivo experiments. Accumulating more mutation data will aid in molecular diagnosis for patients with HS.

ACK N OWLED G EM ENTS
This work was supported in part by National Key Research and Development Program of China (2016YFC1000503)

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. F I G U R E 5 Co-immunoprecipitation (Co-IP) of wild-type and mutant ANK1 with SPTB/SLC4A1 in human embryonic kidney 293T (HEK293T) cells. Co-IP analysis of physiologically relevant ANK1-SLC4A1/SPTB interactions in HEK293T cells using FLAG antibodies. None or little SCLC4A1/SPTB was captured when ANK1 was mutated in the three sites. The asterisks indicate non-specific or protein degradation product. Semi-quantitative analysis of the captured amount of SPTB/SLC4A1 relative to the amount of immunoprecipitated ANK1 was performed by greyscale scanning of strips. Data are the mean values of duplicate samples and revised the manuscript. All authors read and approved the manuscript.