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A Case of Parturient with Hereditary Thrombotic Thrombocytopenic Purpura: Case Report of a Novel VariantFunding None.
Hereditary thrombotic thrombocytopenic purpura (TTP) is a rare autosomal recessive inherited disease that represents a severe form of thrombotic microangiopathy (TMA). Hereditary TTP is due to the congenital absence or severe deficiency of the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which is required to enable cleavage of von Willebrand factor (VWF) multimers. Reduced levels of ADAMTS13 activity are responsible for an increase in the level and size of VWF multimers and thus an increased risk of microvascular thrombosis. The main manifestations of TTP include microvascular hemolytic anemia, thrombocytopenia, neuropsychiatric symptoms, fever, and kidney damage.
This correspondence reports a case of maternal hereditary TTP with multiple ecchymosis and thrombocytopenia as the first manifestation. Gene sequencing showed compound heterozygous mutation with ADAMTS13 c.1435_1436insG heterozygous frameshift mutation and c.1256G > T heterozygous missense mutation. We used the VarCards online platform (http://varcards.biols.ac.cn) to predict the effect of the missense mutations, which identified these as probably pathogenic. VarCards is an integrated clinical and genetic database of human genome mutations that integrates SIFT, polyphen-2, MutationTaster, PROVEAN, and other programs. The two types of mutations were located in exon 13 and exon 11 of ADAMTS13 gene respectively, which were both prespacer mutations. Prespacer mutations are usually associated with earlier onset of disease.
A 26-year-old female became pregnant 8 months prior to presentation, and did not suffer from vaginal bleeding or leakage of amniotic fluid during the intervening time. Due to the presence of ecchymosis all over her body and the presence of reddish urine 4 days prior to presentation, she came to our hospital on April 20, 2020. In addition to the above clinical symptoms, she claimed no dizziness, no abdominal pain, no vaginal bleeding, no cough, no fatigue, and no diarrhea. Outpatient examination showed that she had a single live fetus and that the fetal position was normal. The gestational age was 31 weeks and 6 days. Complete blood count (CBC) examination identified thrombocytopenia, with a platelet count of 6 × 109/L. Since this patient has no history of depression or impaired cognitive function, a brain magnetic resonance imaging was not done to look for silent cerebral infarction. At the same time, this patient denied headache, lethargy and abdominal pain, and other nonovert symptoms, and TTP had not been considered for the time being. Subsequently, she was admitted to the obstetrics department.
After admission, the patient underwent a further physical examination. She was clear-minded, with scattered ecchymoses all parts of the body. Temperature, heart rate, and blood pressure were all normal. No shortness of breath was found, breath sounds in both lungs were clear, and no dry or wet rales were heard. The superficial lymph nodes had not been significantly enlarged. Liver and spleen were out of reach, and contractions of the uterus could occasionally be felt. No obvious abnormality was observed in color ultrasound of heart, liver, and kidney. Chest computed tomography scan showed no obvious abnormality. She then underwent several additional blood tests, with the results shown in [Table 1].
Abbreviations: aPTT, activated partial thromboplastin time; INR, international normalized ratio; PT, prothrombin time; TT, thrombin time.
Note: Bold Values indicate values outside the expected range.
As the patient's hemoglobin dropped rapidly from 127 to 109 g/L within 24 hours, with percentage reticulocytes (RET%) being 5.63%, this suggested a serious hemolytic process was occurring. In addition to the very low platelet, the CBC showed a “warped-tail” in the platelet histogram. The horizontal axis (abscissa) of the platelet histogram represents the volume size, while the vertical axis (ordinate) represents the number of particles detected. Normally the platelet histogram is bell-shaped. When a large number of RBC are destroyed in patients with severe burns or with TTP, many fragmented RBCs of different sizes are generated in the peripheral blood. The fragmented RBCs, whose volumes are similar in size to platelets, would be “incorrectly” included in the platelet histogram, which would make the middle and tail of the curve rise. Thus, the “peak” of the curve is shifted to the right, and the curve resembles an upward trend rather than a bell shape. Since the tail of the platelet histogram showed this pattern, it indicated that there may be schistocytes present, which was also confirmed in the subsequent microscopic examination of blood smear. The number of schistocytes was more than 7%, and the platelet histogram and morphology of schistocyte are shown in [Fig. 1].
According to the direct anti-human globulin test (DAHGT) negative, immunoglobulin A (IgA), IgG and IgM not increased, complement C3 and C4 basically normal, a PLASMIC score of 6 points, and the large number of schistocyte in her peripheral blood, she was suspected to have TTP rather than immune thrombocytopenia. Therefore, she was recommended for the ADAMTS13 activity test. ADAMTS13 activity was assessed using a commercial ACTIFLUOR ADAMTS13 Activity Kit (Sekisui Diagnostics, Stanford, CT) and the Mithras LB 940 Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany) using VWF86 substrate based on fluorescence resonance energy transfer (FRET). If the ADAMTS13 activity level is <30%, ADAMTS13 inhibitor (by Bethesda assay) is performed as a reflex test. The steps of the Bethesda method are as follows: first, the pretreated test plasma sample and pooled normal plasma (PNP) were mixed equally as “Test Mix,” and bovine serum albumin 2% in phosphate buffer saline (PBS BSA 2%) and PNP were mixed equally as “Control Mix.” Then Test Mix and Control Mix were incubated at 37°C for 2 hours and then cooled at 0°C for 10 minutes to terminate the reaction. The activity of ADAMTS13 was determined by FRET. Finally, the Bethesda Unit is calculated by the following formula: ADAMTS13 residual activity (RA%) = (Test Mix/Control Mix) * 100, Bethesda Unit (BU/mL) = [(2 − logRA%)/0.30103] * dilution factor. A Bethesda Unit is defined as the amount of antibody that will neutralize 50% of the ADAMTS13 activity.
Although the patient had no history of hypertension, proteinuria (3 + ) and elevated blood pressure (156/101 mmHg) occurred in late pregnancy. Pre-eclampsia was suspected, and then she was given oral nifedipine 10 mg twice a day for blood pressure control. The next morning after admission, the patient's fetal movement was significantly weakened, and fetal distress was suspected after examination. At the same time, ADAMTS13 activity was identified as 1.1% (normal range: 65–135%; less than 10% indicates that the activity is severely deficient) by the laboratory. Due to the diagnosis of TTP, a healthy baby girl was born immediately by caesarean section. As no inhibitor was detected, ADAMTS13 gene sequencing was subsequently performed. This identified an ADAMTS13 c.1435_1436insG heterozygous frameshift mutation, which could cause the 480th amino acid of the protein to be changed from aspartic acid to glycine, and cause the reading frame to shift, so that the protein translation is terminated prematurely (P.Asp480GlyfsTer54). This mutation had not been reported in the gnomAD, ExAC, 1000G, or HGMD databases. An ADAMTS13 c.1256G > T heterozygous missense mutation was also detected. This mutation could cause the amino acid at position 419th of the protein to be changed from glycine to valine (p.Gly419Val). The population frequency of this mutation in the gnomAD database was 3.24E-05, and there was no report in the ExAC, 1000G, or HGMD database. A family study found that the patient shared the same ADAMTS13 c.1256G > T heterozygous missense mutation with her mother and daughter, but presented the ADAMTS13 c.1435_1436insG heterozygous frameshift mutation alone. Her daughter's ADAMTS13 activity was 10.5% at the first 2 days of birth, and increased to 53.3% 2 months later. The earlier low result may be due to her preterm birth. The patient's husband exhibited a normal ADAMTS13 activity and no genetic mutation for ADAMTS13 gene was found. Unfortunately, the patient's father was not tested. For further details, see [Fig. 2].
To date, the most generally used method to prevent the symptoms of hereditary TTP caused by an ADAMTS13 mutation remains plasma infusion (or plasma exchange, especially in the case of ADAMTS13 antibodies). As TTP is a life-threatening disease with high morbidity, plasma exchange was used as a treatment for this patient, and caesarean section was performed immediately based on her condition. The patient was given 600 mL of fresh frozen plasma to treat her acute TTP, and 1,850 mL plasma exchange was performed twice in the following 2 days. Her ADAMTS13 activity increased to 129% and platelet count returned to normal level (168 × 109/L) after 3 days. At 14 days postsurgery, the patient's vital signs were stable and she was discharged. The recommendation for her future management for subsequent pregnancies is regular plasma infusion (10 mL/kg) every 2 weeks from 8 to 10 weeks of gestation in combination with low-dose aspirin. Plasma infusion usually increases from 20 weeks of gestation to once a week, and delivery is best performed at 36 to 38 weeks of gestation. If the platelet count drops below 150 × 109/L, treatment should be enhanced at any stage. In case of skin ecchymosis, dizziness and headache, significant thrombocytopenia, and other conditions, she was advised to return visit timely for further treatment. Two months after discharge, the platelet count was still normal (159 × 109/L), but the ADAMTS13 level had dropped to 12%. Unfortunately, the patient's financial status prevented more regular (preventative) prophylaxis.
This case represents a successful case in which the laboratory staff assisted, initially observing CBC and cell morphology, to actively investigate the cause of the disease and to assist in her clinical diagnosis. HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome was considered instead of Evans syndrome at the first visit, because the patient had hemolysis and low platelets, and the aspartate aminotransferase (AST) was 78 U/L, and the DAHGT was negative ([Table 1]). Then, laboratory staff reported that a large number of schistocytes were found in blood cell morphology, and lactate dehydrogenase (LDH) >1,867 U/L and LDH/AST >22.12. It was thus suggested that TTP might be involved, and so clinicians reconsidered the diagnosis. The increase of LDH in patients with HELLP syndrome is mainly related to liver damage, while that of TTP is mainly related to extrahepatic factors such as hemolysis and myocardial injury.  The LDH/AST ratio theoretically reflects the proportion of extrahepatic factors, so TTP generally has a higher LDH/AST ratio. Although RBC fragments are seen in HELLP, the number of schistocytes is usually higher in TTP than in HELLP, as is the case here.   Therefore, it was speculated that the patient did not have HELLP, but TTP. TTP is a microvascular hemolytic anemia. The clinical manifestations are variable and can easily be confused with HELLP syndrome, disseminated intravascular coagulation, and atypical hemolytic uremic disease. The incidence of TTP, acquired and congenital combined, is very low, usually 2 to 6 per million, but congenital TTP is the rarer entity.  TTP can occur at any age, but is more commonly identified in women between 30 and 50 years old, especially pregnant women. Hereditary TTP accounts for only approximately 5% of all TTP, but accounts for approximately 25% of pregnancy-identified TTP. Hereditary TTP is caused by mutations in the ADAMTS13 gene. Usually, hereditary TTP does not develop into significant disease until there is a trigger, for example, pregnancy, injury, infection, and other triggers. The ADAMTS13 gene is located on chromatin 9q34, contains 29 exons, and encodes a 1427 amino acid multidomain protein. Mutations are distributed throughout the ADAMTS13 gene, including missense mutations (∼60%), deletions and insertions (∼20%), nonsense mutations, and splice site mutations. So far, more than 150 different ADAMTS13 mutations have been discovered, but only a few of them in Chinese patients.  A 5-year-old male Chinese boy with congenital TTP was reported by Hou and Du; the patient had a missense mutation 332G > A in exon 4 and a nonsense mutation 3121C > T in exon 24 and showed early onset of symptoms such as jaundice, thrombocytopenia, and dark urine at 18 hours after birth. Conboy et al reported a 9-year-old Chinese girl with two pathogenic variants in the ADAMTS13 gene, a missense mutation c.1787C > T and a duplication defined as c.1007_1025dup19. She had repeated thrombocytopenia and hemolysis, which were life-threatening. The level of ADAMST13 was less than 5% at the first test. Dai et al reported a 12-year-old boy with a history of intermittent thrombocytopenia and severe ADAMTS13 plasma deficiency in the prior 6 years, and a missense mutation c.577C > T and a nonsense mutation c.2397C > A were found. The three Chinese cases were all compound heterozygous for mutations, and all of them were earlier onset. Compared with previous reports, our case onset was first noted in pregnancy, instead of earlier in life, which was observed in the other cases. The difference of phenotype and severity of congenital TTP may be the result of multiple environmental and genetic factors acting in concert.
In summary, we report a case of a pregnant woman with hereditary TTP, which was identified as a compound heterozygous mutation of ADAMTS13 c.1435_1436insG heterozygous mutation and c.1256G > T heterozygous mutation, which were both the prespacer mutations. To our knowledge, this was the first such report in the world. The onset of symptoms appeared near the expected date of delivery, and the condition progressed rapidly. Although the patient had a clear state of mind and had no fever, there were many scattered ecchymoses on her skin, and the platelet count and hemoglobin level progressively decreased, and thus the fetus was at clear risk of dying. Meanwhile, the laboratory used the shape of platelet histogram to quickly determine that it was a suspected TMA, which was further confirmed by blood smear. Additional evidence suggested TTP, combined with a PLASMIC score of >5 points and an increased LDH/AST ratio, which saved valuable time to initiate further testing, and to thus facilitate timely diagnosis and treatment.
The study protocol was approved by the Tongji Hospital Ethics Committee for Research in Health. Informed consent was obtained from individuals included in this study.
13 August 2021 (online)
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