ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura

Congenital thrombotic thrombocytopenic purpura (TTP) (also known as Upshaw‐Schulman syndrome, USS) is a rare, life‐threatening disease characterized by thrombocytopenia and microangiopathic hemolytic anemia, associated with the deficiency of the von Willebrand factor‐cleaving protease (ADAMTS13) due to mutations in the corresponding gene. The spectrum of clinical phenotype in congenital TTP is wide, encompassing neonatal‐onset disease and adult‐onset disease, forms with a single disease episode and chronic‐relapsing forms. We review ADAMTS13 gene variants associated with inherited ADAMTS13 deficiency and congenital TTP. To date, 76 mutations of ADAMTS13 are reported in the literature. Missense mutations, which constitute nearly 60% of ADAMTS13 mutations, preferentially localize in the 5′‐half of the gene encoding the N‐terminal half of the protein, where the domains that are indispensable for ADAMTS13 catalytic function are situated. In vitro expression studies in cell cultures have shown that defects in protein secretion and catalytic activity are the main mechanisms responsible for the deficiency of ADAMTS13 in congenital TTP patients. Even if data from the literature suggest the existence of genotype–phenotype correlations, a clear relationship between the type and the effect of ADAMTS13 genetic defects with disease manifestations remains to be established. Hum Mutat 30:1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Thrombotic thrombocytopenic purpura: proposal of a new pathogenic mechanism involving Helicobacter pylori infection

Thrombotic thrombocytopenic purpura (TTP) is a severe, occlusive, thrombotic microangiopathy characterized by a systemic platelet aggregation, organ ischemia, profound thrombocytopenia and erythrocyte fragmentation. Recent observations have documented that a deficiency of a von Willebrand factor (VWF)-cleaving protease, termed ADAMTS13, that normally cleaves hyper-reactive unusually large VWF multimers into smaller and less adhesive VWF forms, may be responsible for many cases of TTP. Multiple mutations of the ADAMTS13 gene can result in ADAMTS13 deficiency and cause congenital TTP, while autoantibodies neutralizing ADAMTS13 protease activity have been associated with acquired TTP. However, in spite of the recent progresses in the pathophysiology of TTP, many aspects of this disease remain still controversial. In this study, basing on the laboratory results of a group of eight patients with an acquired form of TTP, an alternative pathogenic mechanism for TTP involving Helicobacter pylori infection is proposed. In fact, Helicobacter pylori, which has been recently implied in the pathogenesis of idiopathic thrombocytopenic purpura (ITP), could function as a triggering factor in TTP by inducing platelet aggregation through an interaction with VWF.     

一例P．Gly400Val和P．Arg532Ter导致的遗传性FXI缺陷症患者的临床表型CSCD

目的分析1例遗传性凝血因子XI（FXI）缺陷症患者的临床表型和基因突变特征。方法用凝固法检测先证者及家系成员活化部分凝血活酶时间（activated partial thromboplastin time, APTT）、凝血酶原时间（prothrombintime,PT）、凝血因子Ⅺ活性（FⅪactivity,FXI：C）,ELISA方法检测凝血因子Ⅺ抗原（FXI antigen,FXI：Ag）。对F11基因第1～15外显子及其侧翼序列进行PCR扩增、纯化和测序,寻找突变位点并用Pymol软件对突变进行分析。结果先证者APTT为70．3S,明显延长,FXI：C和FⅪ：Ag同时下降为6％和1．9％。先证者儿子FⅪ：C和FⅪ：Ag均下降为31％和39％。测序结果显示先证者携带F11基因第11外显子C．1296G〉T（P．Gly400Val）错义突变和第14外显子C．1691A〉T（P．Arg532Ter）无义突变;先证者儿子为C．1296G〉T（P．Gly400Val）杂合突变携带者。Pymol软件分析显示P．Gly400Val突变导致FⅪ蛋白氢键数量变化,使蛋白质二级结构改变。根据人类基因突变数据库（HGMD professional 2016．4）,F11 NM_13142C．1691A〉T（p．Arg532Ter）为未报道过的新突变,根据美国医学遗传学与基因组学学会（ACMG）2015年指南判断为功能缺失型突变。结论F11NM_13142 C．1296G〉T（p．Gly400Val）和F11 NM_13142C．1691A〉T（P．Arg532Ter）复合杂合突变是导致先证者遗传性FXI缺陷症的致病原因,引起FXI抗原和活性同时下降。     

A novel homozygous missense ADAMTS13 mutation Y658C in a patient with recurrent thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a devastating systemic disorder that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurological dysfunction, and renal failure. In the hereditary form of TTP, severe deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor, is associated with the development of this disorder. A 34-year-old woman was diagnosed with TTP due to severely reduced ADAMTS13 activity; clinical manifestations resolved only by repeated total plasma exchanges or transfusion. Homozygous and heterozygous Y658C (c.1973A>G) alleles were detected in the patient and her child with severe and mild ADAMTS13 deficiencies, respectively. Herein, we report a novel missense mutation Y658C (c.1973A>G) on exon 17 of ADAMTS13 and discuss its clinical implications.     

Thrombotic thrombocytopenic purpura and other thrombotic microangiopathic hemolytic anemias: Diagnosis and classification

Thrombotic microangiopathies (TMAs) include several diseases, most prominently are thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS). TMAs are characterized by profound thrombocytopenia, microangiopathic hemolytic anemia and organ ischemia. In most cases TTP results from deficiency of ADAMTS13, the von Willebrand factor-cleaving protease leading to increase of ultra-large von Willebrand factor (ULVWF) multimers. Congenital TTP is due to mutations in the gene of ADAMTS13 whereas acquired TTP is due to production of autoantibodies against ADAMTS13. In both cases severe deficiency of ADAMTS13 exists. However, the presence of ADAMTS13 activity does not rule out TTP. Diagnostic criteria of TTP are based on clinical features of neurologic and renal disfunction along with anemia and thrombocytopenia, low ADAMTS13 activity, and the presence of ULVWF. The standard treatment of TTP includes plasma exchange, protein A immunoabsobtion, immunosuppressive drugs, CD20 antibodies against B cells, and splenectomy. HUS is commonly caused by infection with Shiga-toxin produced by Escherichia coli. HUS is characterized by thrombocytopenia, anemia, renal impairment and diarrhea. Rarely, atypical HUS appears as a consequence of mutations related to the alternative pathway for the compliment system. Plasmapheresis in HUS is not efficient. Alternatively, plasma therapy and in some cases dialysis are used. TMA diseases may be associated with other infections, bone marrow transplantation, pregnancy, systemic vasculitis, and certain drugs.     

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH‐cytochrome b5 reductase (NADH‐CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice‐site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three‐dimensional (3D) structure of human erythrocyte NADH‐CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH‐CYB5R deficiency.     

Thrombotic thromboytopenic purpura: another example of immunomediated thrombosis

Much progress has been made in recent years in understanding the mechanisms of TTP, through the demonstration of the role of ultra-large von Willebrand factor multimers and of ADAMTS13 deficiency in the pathogenesis of immune-mediated and genetic forms. There are, however, cases of TTP with normal or only slightly reduced levels of ADAMTS13, that may be due to the inhibition of the VWF cleaving activity ofADAMTS13 under the flow conditions of the microcirculation. The laboratory methods for assayingADAMTS13 are currently too complex to be used routinely on a large scale. In addition, they fail to reflect the conditions of blood flow that are necessary for the optimal interaction between VWF and its cleaving protease. The laboratory diagnosis of TTP is, therefore, still mainly clinical, being based on the presence of thrombocytopenia (due to increased intravascular consumption of blood platelets) and anemia (due to mechanical damage to red cells). There are still numerous questions to be answered. What are the pathogenic mechanisms of cases with normal or slightly reduced levels of ADAMTS13? What are the factors that periodically trigger the episodes of thrombocytopenia and anemia in the chronic recurrent forms, in the presence of constantly reduced levels of ADAMTS13? The behavior of anti-ADAMTS13autoantibodies following plasma exchange and therefore exposure to antigen (anamnestic response) is not well established. Likewise, the value of inducing immune tolerance by regular administrations of ADAMTS13, which will be possible when concentrates of the protease become available, has not been investigated.     

Identification of novel mutations in the human ornithine transcarbamylase (OTC) gene of Korean patients with OTC deficiency and transient expression of the mutant proteins in vitro

The urea cycle plays key roles to prevent the accumulation of toxic nitrogenous compound and synthesize arginine de novo. Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea cycle, which is inherited in an X-linked manner. This study was undertaken to characterize molecular defects in Korean patients with OTC deficiency. With direct sequence analysis of OTC gene of 26 unrelated Korean patients with OTC deficiency, 23 different mutations were identified. Among these mutations, eleven were novel mutations. The novel mutations were p.Leu9X, p.Arg26Pro, p.Gly100Arg, p.Met205Thr, p.Lys221Asn, p.Asp249Gly, p.Phe281Ser, p.Val323Met, c.571delC, c.853delC, and c.796-805del. All the novel mutations in this study were tested in 100 normal alleles. In vitro expression study of some of novel missense mutations elucidated the correlation of genotype and phenotype of the OTC deficiency.     

一例多种羧化酶缺乏症的HLCS基因变异分析

目的对1例临床诊断为多种羧化酶缺乏症(multiple carboxylase deficiency,MCD)的患儿及其父母进行相关致病基因的变异分析,为临床诊断及遗传咨询提供依据。方法应用PCR技术和DNA测序技术对患儿的MCD致病基因BT和HLCS编码区进行变异检测,并对患儿父母进行相应基因变异分析。在80名正常人中对未报道过的基因变异进行PCR-限制性片段长度多态性分析。结果患儿的BT基因编码区未发现碱基改变,HLCS基因存在c.286delG(p.Val96Leufs*162)和c.1648G>A(p.Val550Met)复合杂合变异,其中c.286delG(p.Val96Leufs*162)经PCR-限制性片段长度多态性分析验证为新变异。结论HLCS基因c.286delG(p.Val96Leufs*162)和c.1648G>A(p.Val550Met)变异可能为患儿的致病原因,致病基因的检出为临床诊断及遗传咨询提供了依据,同时丰富了HLCS基因变异谱。     

Functional characterization of missense variants in the creatine transporter gene (SLC6A8): improved diagnostic application

Creatine transporter deficiency is an X-linked mental retardation disorder caused by mutations in the creatine transporter gene (SLC6A8). So far, 20 mutations in the SLC6A8 gene have been described. We have developed a diagnostic assay to test creatine uptake in fibroblasts. Additionally, we expanded the assay to characterize novel SLC6A8 missense variants. A total of 13 variants were introduced in the SLC6A8 cDNA by site-directed mutagenesis. All variants were transiently transfected in SLC6A8-deficient fibroblasts and tested for restoration of creatine uptake in deficient primary fibroblasts. Thus, we proved that nine variants (p.Gly87Arg, p.Phe107del, p.Tyr317X, p.Asn336del, p.Cys337Trp, p.Ile347del, p.Pro390Leu, p.Arg391Trp, and p.Pro554Leu) are pathogenic mutations and four variants (p.Lys4Arg, p.Gly26Arg, p.Met560Val, and p.Val629Ile) are nonpathogenic. The present study provides an improved diagnostic tool to classify sequence variants of unknown significance.     

Thrombotic thrombocytopenic purpura and pregnancy

Thrombotic thrombocytopenic purpura (TTP) is an acute life threatening disorder associated with a deficiency in the enzyme ADAMTS 13. It is diagnosed by thrombocytopenia, microangiopathic haemolytic anaemia (MAHA) and widespread microvascular thrombosis resulting in organ ischaemia. Approximately 70% of TTP cases occur in women and nearly half of these women are of childbearing age. Pregnancy is a recognised precipitating cause of TTP in between 10‐25% of all cases and includes patients with acquired antibody mediated TTP and congenital TTP, often presenting in adult hood. The availability of ADAMTS 13 assays allows differentiation between congenital and acquired TTP and appropriate treatment plans. There is also a subsequent risk of TTP relapse in women with previous non‐pregnancy related TTP. Presentation may occur at any stage in pregnancy or in the post partum period. This would suggest an hormonal influence as well as the reduction in ADAMTS 13 from the second trimester, related to pregnancy associated increase in Von Willebrand Factor. Differentiation from other pregnancy associated microangiopathies, such as pre‐eclampsia, HELLP (haemolysis, elevated liver enzymes and low platelets) or HUS (haemolytic Uraemic Syndrome) may be clinically difficult, but necessary, in part, because of differences in treatment. HELLP and pre‐eclampsia require delivery and HUS supportive care. TTP requires urgent treatment with plasma exchange (PEX) to attain remission, but also to prevent fetal abnormalities resulting from placental thrombosis. Presented is a review of the literature of pregnancy associated TTP and our experience of treatment of patients who present with TTP during pregnancy and monitoring of women who have had a history of TTP. Positive outcome in pregnancy has been associated with regular monitoring of routine laboratory parameters and ADAMTS 13 activity. All patients maintain low dose aspirin therapy and low molecular weight heparin is started in those women where an increased thrombotic risk is determined. The aim is to optimise implantation and preservation of placental function, especially in women with previous pregnancy loss, as abnormalities of the uteroplacental circulation resulting in insufficiency are established early in the first trimester. A significant reduction in ADAMTS 13 activity or reduction in platelet count below the normal range, PEX is undertaken to prevent any further deterioration. Frank relapse is treated with daily PEX. In women with a congenital TTP phenotype, regular treatment through pregnancy has been successfully undertaken. It is very difficult to devise evidence based guidelines for pregnancy in women with a history of TTP. In our cohort, patients with ADAMTS 13 activity < 5% at presentation in the current pregnancy had a history of TTP precipitated during pregnancy or recurrent TTP episodes, such that the chance of further exacerbation during pregnancy was considered to be high. Indeed, cases 1, 2 and 4 have demonstrated ADAMTS 13 activity < 5% before and after these pregnancies and only case 3 and 5 had further TTP episodes following pregnancy. Therefore the intensive monitoring and treatment was based on the high probability of relapse during pregnancy. Indeed, in Patients with normal ADAMTS 13 activity at the start of pregnancy were continually monitored, but did not have a TTP relapse. In Conclusion: a multidisciplinary approach to pregnancy care with regular monitoring of routine laboratory parameters and ADAMTS 13 activity during pregnancy allows pre‐emptive treatment of patients who are at risk from TTP relapse.     

Association of Single Nucleotide Polymorphisms in CYP1B1 and COMT Genes with Breast Cancer Susceptibility in Indian Women

Cytochrome P450 1B1 (CYP1B1) and catechol-$O$-methyltransferase (COMT) enzymes play critical roles in estrogen metabolism. Alterations in the catalytic activity of CYP1B1 and COMT enzymes have been found associated with altered breast cancer risk in postmenopausal women in many populations. The substitution of leucine (Leu) to valine (Val) at codon 432 increases the catalytic activity of CYP1B1, however, substitution of Val to methionine (Met) at codon 158 decreases the catalytic activity of COMT. The present study was performed to evaluate the associations of CYP1B1 Leu⁴³²Val and/or COMT Val¹⁵⁸Met polymorphisms with total, premenopausal and postmenopausal breast cancer risks in Indian women. COMT and CYP1B1 polymorphisms in controls and breast cancer patients were analyzed employing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by gel electrophoresis. Although CYP1B1 and COMT genotypes did not exhibit statistically significant association with breast cancer risks when analyzed individually, COMT wild type (Val¹⁵⁸Val) in combination with CYP1B1 heterozygous variant (Leu⁴³²Val) [OR: 0.21; 95% CI (0.05–0.82), p value; 0.021] and COMT heterozygous variant (Val¹⁵⁸Met) in combination with CYP1B1 wild type (Leu⁴³²Leu) [OR: 0.29; 95% CI (0.08–0.96), p value; 0.042] showed significant protective association with premenopausal breast cancer risk. The results demonstrate that CYP1B1 wild type in combination with COMT heterozygous or their inverse combination offer protection against breast cancer in premenopausal Indian women.     

Inhibitory influence of amino acids on secretagogues induced exocrine secretion in isolated perfused rat pancreas

Influence of amino acids upon pancreatic exocrine secretion has been investigated in the isolated perfused pancreas of rats. Arg produced significant and dose-related inhibition of pancreatic juice flow, protein output and amylase output evoked by CCK-PZ (1.25 pM). The secretory response evoked by CCK-PZ was inhibited by other amino acids (Ala, Asp, Asn, Gly, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Val, in each 20 mM). A similar inhibitory pattern was observed using 10 mixed amino acids of 2 mM each (Pro, Phe, Thr, Met, Lys, Asp, Leu, Trp, Val, Gly). Gly at a concentration of 20 mM produced significant inhibition of exocrine secretion evoked by ACh (50 nM) or GRP (36 pM). The inhibitory response induced by amino acids could not be repeated by using exogenous insulin (1 microM) and glucagon (280 nM). The inhibitory response was also not changed by increased extracellular Ca (5 or 10 mM). However, Gly (20 mM) produced inhibition of exocrine secretion evoked by Ca reintroduction into a pancreas which was pretreated with A 23187. It was suggested that the inhibitory effects of some amino acids on exocrine secretion are mainly caused by suppression of Ca influx in a stimulus-secretion coupling process.     

Development and application of a new ADAMTS13 assay for TTP diagnosis

A plasma glycoprotein, von Willebrand factor (VWF), is essential for normal platelet aggregation. In healthy individuals, the homo-multimeric forms (VWF multimers) are partially cleaved by a plasma metalloprotease, ADAMTS13. Congenital or acquired deficiency of ADAMTS13 activity leads to the accumulation of hyperactive large VWF multimers, inducing a life-threatening disease, thrombotic thrombocytopenic purpura (TTP). As measuring ADAMTS13 activity is important in TTP diagnosis, a number of assay methods have been developed in the past few years. However, the time and skill required for these methods prohibited the progress of clinical usage. Recently, we have developed a fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity. A synthetic 73-amino-acid peptide, FRETS-VWF73, which is now commercially available, is used as a substrate. Cleavage of this peptide between two modified residues relieves the fluorescence quenching in the intact form. Incubation of FRETS-VWF73 with normal plasma quantitatively increased fluorescence over time, while TTP-patient plasma had little or no effect. The measurement can be achieved within a one-hour period using a 96-well format in commercial plate readers with common filters. The FRET assay will be useful not only for TTP diagnosis but also characterization of thrombotic microangiopathies.     

Clinical features and laboratory findings of thrombotic thrombocytopenic purpura associated with ticlopidine

Ticlopidine is an antiplatelet agent that interferes with platelet membrane function by inhibiting adenosine diphosphate-induced platelet activation. It is used in an increasing number of cases of cerebrovascular disease, unstable angina, coronary artery stenting, and peripheral vascular diseases. It has rare but serious adverse reactions, including thrombotic thrombocytopenic purpura (TTP). TTP is a life-threatening disease, characterized by Moschcowitz's pentad: thrombocytopenia, microangiopathic hemolytic anemia, fluctuating neurological signs, renal failure, and fever. Recent advances in elucidating the proteolytic processing of plasma von Willebrand factor (VWF) multimers have established assays for VWF-cleaving protease (VWF-CP) activity and its inhibitor(autoantibodies). These assays apparently demonstrated that TTP patients have defective enzymatic activity with or without presence of the inhibitor. VWF-CP is now identified as a metalloproteinase belonging to the ADAMTS (A Disintegrin And Metalloproteinase domain, with ThromboSpondin type 1 motif) family, termed ADAMTS13. Cases of ticlopidine-associated TTP were first reported in 1991. This complication occurs in 1 in 1600 to 1 in 5000 patients who receive ticlopidine. It is known that they develop TTP within 2 to 8 wk of starting ticlopidine treatment and show severely deficient of ADAMTS13 activity with the presence of the inhibitor. These results suggest that ticlopidine or its metabolites induce the production of autoantibodies against ADAMTS13. As treatment, discontinuation of ticlopidine therapy and rapid initiation of plasma exchange is effective: the majority of patients completely recover and relapse is uncommon. It is thus recommended that physicians should perform complete blood count every 2 weeks for 12 weeks for rapid diagnosis. Physicians and patients should be aware of this fatal but curable complication of ticlopidine therapy.     

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK‐deficient patients from 35 unrelated families. Twenty‐nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in‐frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N‐terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three‐dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme‐linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype‐to‐phenotype correlation in PK deficiency as well as the enzyme's structure and function. Hum Mutat 0, 1–8, 2008. © 2008 Wiley‐Liss, Inc.     

Investigation of citrullinemia type I variants by in vitro expression studies

Mild citrullinemia is an allelic variant of classical citrullinemia type I also caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). Affected patients comprise a biochemical but no clinical phenotype. However, there is no reliable parameter allowing conclusions regarding the course of the disorder or its type of manifestation. The aim of this study was to test the importance of varying levels of ASS residual activities for the severity at diagnosis. Bacterial in vitro expression studies allowed the enzymatic analysis of purified wild-type and the mutant ASS proteins p.Ala118Thr (c.352G>A), p.Trp179Arg (c.535T>C), p.Val263Met (c.787G>A), p.Arg265Cys (c.793C>T), p.Met302Val (c.904A>G), p.Gly324Ser (c.970G>A), p.Gly362Val (c.1085G>T), and p.Gly390Arg (c.1168G>A). In the chosen system, classical mutations do not show any significant enzymatic activity, whereas mutations associated with a mild course yield significant ASS activity levels. The mutation p.Ala118Thr (c.352G>A) impresses by a high residual activity (62%) but a severe reduction of affinity toward the substrates citrulline and aspartate. This mutation was identified in a hitherto healthy female adult with no history of known citrullinemia who had died during the postpartum period from hyperammonemic coma. The results of this study suggest that even a high level of residual ASS activity is not a reliable prognostic marker for an uneventful clinical course. Determination of ASS residual activities, therefore, cannot help in anticipating the risk of metabolic derangement. This study should guide clinicians as well as patients with mild citrullinemia toward a lifelong awareness of the disorder.     

A new large CFTR rearrangement illustrates the importance of searching for complex alleles

The p.Val754Met variant, described in 1996 in a CF patient, has been considered a CF mutation. However, biochemical aspects, results of functional studies and, finally, the identification of a complex deletion removing exons 3 to 10 and 14b to 16 in cis of p.Val754Met in a CF patient, argue against a strong deleterious effect. An inventory through the French CF network of patients carrying p.Val754Met led to the registration of seven patients (CF: n=4; idiopathic chronic pancreatitis: n=3) and six healthy individuals, all heterozygous for the variation. Extensive CFTR gene analysis was carried out, including the search for large rearrangements and other possible mutations. The complex deletion, whose breakpoints are described here, was found only in the four CF patients, in association with the same haplotype. This data, added to the fact that the p.[Phe508del]+[Val754Met] genotype was found in a healthy individual, bring further arguments against the association of p.Val754Met with CF. We thus suggest looking for a possible complex allele whenever p.Val754Met is detected and considering it neutral regarding genetic counseling when found in isolation.     

Thrombotic thrombocytopenic purpura: a case report

We report a case of thrombotic thrombocytopenic purpura (TTP) in a 60 years-old woman with Sjogren's syndrome. Symptomatology on admission leads to evoke the diagnosis of TTP. Biological results allow to set the diagnosis. Actually, association of haemolytic anaemia, schizocytes and thrombocytopenia are in favour of TTP. Undetectable ADAMTS 13 activity (below 5%) confirms the diagnosis. In congenital TTP, plasma ADAMTS 13 is absent or severely reduced as a consequence of mutations in the two ADAMTS 13 gene. In acquired TTP, circulating antibodies inhibit plasma ADAMTS 13 activity. In those cases, further biological studies are needed to find a cause of TTP. Follow-up implies standard laboratory tests. Plasma exchanges are progressively tapered after normalization of platelets count.     

Von Willebrand factor,ADAMTS13, and thrombotic thrombocytopenic purpura

Von Willebrand factor (vWF), a glycoprotein critical for supporting platelet adhesion and aggregation at sites of vessel injury, exists in the plasma as a series of multimers. Recent studies have shown that a metalloprotease cleaves endothelial vWF to a series of multimers. A deficiency of the protease activity due to autoimmune IgG inhibitors or genetic mutations is associated with thrombotic thrombocytopenic purpura (TTP). Positional cloning based on kindreds with a genetic deficiency of the protease and amino acid sequencing of the purified protein have identified the protease as a novel member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 repeat) zinc metalloprotease family located on the long arm of chromosome 9. Mutations of the gene are detected in patients with the congenital form of TTP. These findings support the view that vWF proteolysis is critical in regulating vWF-platelet interaction and set the stage for improving the diagnosis and treatment of thrombotic thrombocytopenic purpura.