Novel mutations in ADAMTS13 CUB domains cause abnormal pre‐mRNA splicing and defective secretion of ADAMTS13

Abstract Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss‐of‐function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice‐site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre‐mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra‐large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.

In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.

K E Y W O R D S
ADAMTS13, genetic mutations, thrombotic thrombocytopenic purpura, von Willebrand factor

| INTRODUC TI ON
Hereditary thrombotic thrombocytopenic purpura (TTP) is caused by a severe deficiency in the activity of the plasma ADAMTS13 (A disintegrin-like and metalloproteinase with a thrombospondin type 1 motif, member 13), resulting from loss-of-function mutations in ADAMTS13. 1 TTP is a rare yet life-threatening disorder, primarily characterized by severe thrombocytopenia, microvascular haemolytic anaemia and multiorgan damage. 2 ADAMTS13 specifically regulates the size of von Willebrand factor (VWF) multimers by cleaving the peptide bond between tyrosine and methionine in the exposed VWF A2 domain, preventing the development of microvascular thrombosis. 3 Hereditary TTP is inherited through an autosomal recessive pattern, comprises <5% of all TTP cases 4 and is associated with bi-allelic mutations in ADAMTS13 that result in the absence or severe deficiency of ADAMTS13 protein and activity. 1 ADAMTS13 is an approximately 190-kD multidomain protein, consisting of proximal metalloprotease, disintegrin-like, cysteine-rich and spacer domains, as well as thrombospondin domains, and two CUB (complement components C1r/C1s, Uegf and bone morphogenic protein 1) domains ( Figure 1A,B). 5 The CUB domains are unique to ADAMTS13, among the members of the ADAMTS family, and are composed of 10 β-strands, arranged into two β-sheets that are stabilized by two disulphide bonds. 6 To date, more than 100 mutations in ADAMTS13 have been reported. 7 However, mutations in the CUB domains are rare, and how these mutations affect the biosynthesis and function of ADAMSTS13 remains unclear. 8 We discovered two novel mutations in the ADAMSTS13 CUB domains in a sibling pair who displayed significantly reduced ADAMTS13 activity, although only the female developed acute TTP episodes. In vitro studies revealed that the mutations affected the mRNA splicing and protein secretion of ADAMTS13.

| Patient
The proband was a 31-year-old woman. Her blood and her family member's blood were collected to tubes coated with ethylenediaminetetraacetic acid (EDTA) or containing 3.2% of sodium citrate. This study was approved by the Ethic Committee of the First Affiliated Hospital of Soochow University. All participants provided informed consent.

| Genomic DNA analysis
Genomic DNA was isolated from peripheral blood leucocytes using a GenElute Blood Genomic DNA Kit (Millipore Sigma). All 29 exons and intron-exon boundaries of ADAMTS13 were sequenced by Next-generation sequencing (NGS) on NextSeq500 platform (Illumina). The regions containing abnormal sites identified by NGS were amplified by polymerase chain reaction (PCR) and then sequenced on an ABI 3130XL Genetic Analyzer (Applied Biosystems).
Sequencing results were compared against the reference sequence for ADAMTS13 (NCBI: NC_000009.11).

| Assay of plasma ADAMTS13 activity and inhibitor
To obtain plasma, whole-blood samples were centrifuged at 1000 g, for 15 minutes at 4°C. A modified FRETS-VWF73 (Peptides International) assay 9 and a chromogenic ADAMTS13 activity enzyme-linked immunosorbent assay (Kainos Laboratories Inc) 10 were performed to measure the levels of ADAMTS13 activity and inhibitor in the plasma.

| VWF multimer analysis
Plasma samples were separated by 1.3% SDS Seakem Gold agarose (LONZA) gel electrophoresis and Western blotted under non-reducing conditions. 11 The gel was first incubated with rabbit anti-human vWF antibody (DAKO) and horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (Thermo Fisher Scientific). The gel was then incubated with a chemiluminescence substrate (SuperSignal West Pico chemiluminescent substrate kit, Thermo Fisher Scientific) and exposed to X-ray films (Kodak).

| In vitro mini-gene expression
Exon 26 (147-bp) and the flanked introns (150-bp) of ADAMTS13 were amplified by PCR using genomic DNA from the patient and a healthy control. The fragments were then cloned into an Exontrap Cloning Vector pET01 (MoBiTec GmbH) for the mini-gene assay.
The ADAMTS13 vectors or empty control vector was then transfected into COS-7 cells. After 48 hours of incubation, total RNA was extracted using TRIzol (Millipore Sigma) and genomic DNA was digested by treating with DNase I (Millipore Sigma). Reverse transcription PCR (RT-PCR) was performed with M-MLV Reverse Transcriptase (Invitrogen). The PCR products were analysed on a 1.5% agarose gel and sequenced.

| Expression of recombinant wild-type (WT) and p.G1308D mutant ADAMTS13
The WT ADAMTS13 cDNA was cloned into the mammalian expression vector pSecTag2/Hygro (Invitrogen), and the p.G1308D mutant was then generated, by site-directed mutagenesis. The WT and mutant constructs were then transiently expressed in COS-7 cells. The serum-free conditioned medium was collected at 48 hours after transfection, and secreted polyhistidine-tagged ADAMTS13 was purified by Ni sepharose (GE Healthcare).

| A 31-year-old female was diagnosed as hereditary TTP with two novel mutations in CUB domains of ADAMTS13
The proband suffered from dizziness, vomiting and slurred speech during the 14th week of her second gestation, with a history of induced abortion during the 16th week of a previous gestation. Her 29-year-old brother and parents did not have any similar symptoms.
Upon admission, the level of plasma ADAMTS13 activity of the patient was <3%, without a detectable functional inhibitor in the plasma ( Figure 1C

| The sibling pair has compound heterozygous mutations of ADAMTS13 with reduced ADAMTS13 activity
We isolated the genomic DNA from the family members and sequenced all 29 exons and intron-exon boundaries in ADAMTS13.
Further Sanger sequencing analysis confirmed that the patient's brother had the same mutations as her ( Figure 1F,G). Their parents each carry one heterozygous mutation (Figure 1D, 1 and G).
ADAMTS13 activity assays were performed with fresh plasma. The siblings showed almost undetectable ADAMTS13 activity levels (normal range > 45%), without the presence of any ADAMTS13 functional inhibitors (normal range < 0.4 BU/mL) ( Figure 1C). Compared with normal controls and the parents of the sibling pair, VWF multimer analysis of the sibling pair showed increased levels of ULVWF multimers, indicating reductions in VWF proteolysis by ADAMTS13 ( Figure 1E).

| The c.3569−1 G>A mutation results in an abnormal pre-mRNA splicing with a premature termination codon (PTC)
To detect the effects of the G to A mutation at the splice acceptor site of intron 25, we performed an in vitro splicing assay by constructing a mini-gene expression system because patient mRNA was not available (Figure 2A after treated with cycloheximide as an inhibitor of non-sense-mediated mRNA decay (NMD). This result indicates that the abnormally spliced transcript from patient mini-gene is degraded through NMD ( Figure 2B). Therefore, we concluded that the splicing pattern of the patient ADAMTS13 mRNA is different from that of WT. Further sequence analysis identified a 73-bp intron inclusion that contains a PTC in exon 26 ( Figure 2C).

| The G1308D mutation causes an increased accumulation of ADAMTS13 in the ER
To determine the effects of the p.G1308D mutation on  Hereditary TTP is an autosomal recessive disorder; men and women would be equally affected by this disease. 4,7 However, in our study, only the patient had an acute episode of TTP after pregnancy. Studies have shown that in women who had TTP during their first pregnancy, the frequency of hereditary TTP was 25%-66%. 12 How pregnancy triggers TTP is unclear. Women with pregnancy are known to have elevated ULVWF multimers compared with the non-pregnant state. 12 In this family, although both siblings had ULVWF multimers in plasma and almost undetectable levels of ADAMTS13 activity, the brother has never had acute TTP. More interestingly, the female did not experience any TTP episodes during her first pregnancy, even though her first pregnancy failed during the second trimester. Our results suggested that unknown genetic or environmental factors, such as infections, cytokines, might also contribute to the development of acute TTP.  13 Recent studies have demonstrated that the distal T-CUB domains have allosteric properties, which may markedly inhibit substrate cleavage and be essential for relieving autoinhibition upon binding to VWF. 14,15 Several studies have shown that mutations in the CUB domains are likely to impair the secretion and/or extracellular degradation of ADAMTS13. 8,16 We found that the p.G1308D mutation causes an increased accumulation of ADAMTS13 in the ER, which leads to impaired secretion of ADAMTS13. Although how the CUB domain regulates the trafficking of ADAMTS13 in the ER remains to be studied, our results provide new molecular insights into the molecular pathogenesis of the hereditary TTP.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
YJ, ZW, CR and LX recruited the patient and designed the project; YJ, DH, MJ, ZM, LZ and XB collected and analysed the clinical data; YJ, YK and JS performed the experiments and analysed data; YJ, DH and LX organized data and wrote the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.