The Challenging Management of Acute Thrombotic Microangiopathy in Pregnancy.

Thrombotic microangiopathy is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and end-organ injury. Pregnancy-associated thrombotic microangiopathy is a severe disorder with a high risk of maternal mortality and poor fetal outcomes. Preeclampsia/eclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome are the most common causes, and hemolytic uremic syndrome or thrombotic thrombocytopenic purpura are rare causes. Due to overlapping clinical findings, the differential diagnosis is challenging and should be managed by a multidisciplinary team. We present a case of a 38-year-old woman at 40 weeks of second gestation, admitted with thrombotic microangiopathy being the final diagnosis not immediately clear.

Identification of a new 2-amino acid insertion in the integrase coding region of HIV-1 subtype G isolates.

Amino acid insertions have been rarely found in the integrase (IN) coding region of Human immunodeficiency virus 1 (HIV-1), and have been considered as natural polymorphisms. It is still unclear the potential impact of these insertion mutations on the viral replication capacity and/or susceptibility to integrase strand transfer inhibitors (INSTIs). The objective of this study was to describe a previously unreported amino acid insertion in the IN coding region of HIV-1 isolates obtained from antiretroviral treatment-naïve infected individuals. Nucleotide sequences of HIV-1 isolates obtained from two infected individuals were analyzed for genotypic resistance to antiretroviral drugs. Phylogenetic inference was carried out for HIV-1 genetic variant identification. An unreported insertion of a threonine (T) and an asparagine (N) between codon 255 and 256 (S255N_TN) was identified in the IN C-terminal domain of HIV-1 subtype G isolates. No resistance-associated mutations to INSTIs were detected in the inserted sequences. Both individuals maintained undetectable HIV-1 RNA viral load, 24 months after undergoing antiretroviral treatment with an INSTI containing regimen. The results demonstrated the possibility of transmission of this insertion mutation and suggested that the codon 255 insert by itself may not affect susceptibility to INSTIs.

Clearance and persistence of SARS-CoV-2 RNA in patients with COVID-19.

Patients with coronavirus disease-2019 may be discharged based on clinical resolution of symptoms, and evidence for viral RNA clearance from the upper respiratory tract. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral clearance profile is crucial to establish a re-testing plan on discharge and ending isolation of patients. We aimed to evaluate the number of days that a patient needed to achieve undetectable levels of SARS-CoV-2 in upper respiratory tract specimens (nasopharyngeal swab and/or an oropharyngeal swab). The clearance and persistence of viral RNA was evaluated in two groups of positive patients: those who achieved two negative reverse transcription-polymerase chain reaction (RT-PCR) tests and those who kept testing positive. Patients were organized thereafter in two subgroups, mild illness patients discharged home and inpatients who had moderate to severe illness. Results from RT-PCR tests were then correlated with results from the evaluation of the immune response. The study evidenced that most patients tested positive for more than 2 weeks and that persistence of viral RNA is not necessarily associated with severe disease but may result from a weaker immune response instead.

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.

Venous thromboembolism risk associated with ABO, F11 and FGG loci.

: The current study aims to evaluate, for the first time in the Portuguese population, the association with venous thromboembolism (VTE) of five well known and replicated VTE-associated single nucleotide polymorphisms (SNPs) in genes ABO, F11 and FGG. A population sample of 96 cases of VTE, without strong or moderate inherited or noninherited predisposing factors, and 148 healthy controls were analyzed for variants in genes ABO (rs2519093; rs8176719), F11 (rs2036914; rs2289252) and FGG (rs2066865). SNPs were genotyped by real-time PCR with TaqMan probes or by PCR-restriction fragment length polymorphism. Logistic regression, adjusted for age and sex, revealed nominal significant association between the ABO rs8176719 C-allele and VTE in the additive model [odds ratio (OR) 1.62; P = 0.015] and significant association in the dominant model (OR 2.68; P = 0.001). A nominal significant association with VTE was found for the FGG rs2066865 minor T-allele in the dominant model (OR 1.82; P = 0.034). A genetic risk score created by using subjects who carry one or any combination of two to four risk alleles showed a cumulative effect on VTE: OR 2.31 (P = 0.025) and OR 3.23 (P = 0.0016), respectively, compared with individuals who have none of the risk alleles. Our data suggest that SNPs ABO rs8176719 and FGG rs2066865 may contribute individually to the VTE susceptibility in the Portuguese population. A genetic risk score combining the VTE-associated FGG and ABO alleles improved the risk prediction of VTE.

Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next-generation sequencing.

BACKGROUND: The 2 main forms of thrombotic microangiopathy (TMA) are thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). Deficiency of ADAMTS13 and dysregulation of the complement pathway result in TTP and aHUS, respectively; however, overlap of their clinical characteristics makes differential diagnosis challenging. OBJECTIVES AND METHODS: We aimed to develop a TMA diagnosis workflow based on ADAMTS13 activity and screening of ADAMTS13 and complement genes using a custom next-generation sequencing (NGS) gene panel. PATIENTS: For this, from a cohort of 154 Portuguese patients with acute TMA, the genotype-phenotype correlations were analyzed in 7 hereditary TTP (ADAMTS13 activity <10%, no inhibitor), 36 acquired TTP (ADAMTS13 activity <10%, presence of an inhibitor), and in 34 presumable aHUS. RESULTS: In total, 37 different rare variants, 8 of which novel (in ADAMTS13,CFH, and CD46), were identified across 7 genes. Thirteen TTP patients were homozygous (n=6), compound heterozygous (n=2), and heterozygous (n=5) for 11 ADAMTS13 variants (6 pathogenic mutations). Among the 34 aHUS patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause aHUS only when combined with other variants and/or CFH and CD46 risk haplotypes or CFHR1-3 deletion. CONCLUSIONS: Our study provides evidence of the usefulness of the NGS panel as an excellent technology that enables more rapid diagnosis of TMA, and is a valuable asset in clinical practice to discriminate between TTP and aHUS.

Genotype-phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGS.

The diagnosis of von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency and heterogeneous laboratory phenotype. The sequencing of the entire VWF coding region has not yet become a routine practice in diagnostic laboratories owing to its high costs. Nevertheless, next-generation sequencing (NGS) has emerged as an alternative to overcome this limitation. We aimed to determine the correlation of genotype and phenotype in 92 Portuguese individuals from 60 unrelated families with VWD; therefore, we directly sequenced VWF. We compared the classical Sanger sequencing approach and NGS to assess the value-added effect on the analysis of the mutation distribution in different types of VWD. Sixty-two different VWF mutations were identified, 27 of which had not been previously described. NGS detected 26 additional mutations, contributing to a broad overview of the mutant alleles present in each VWD type. Twenty-nine probands (48.3 %) had two or more mutations; in addition, mutations with pleiotropic effects were detected, and NGS allowed an appropriate classification for seven of them. Furthermore, the differential diagnosis between VWD 2B and platelet type VWD (n = 1), Bernard-Soulier syndrome and VWD 2B (n = 1), and mild haemophilia A and VWD 2N (n = 2) was possible. NGS provided an efficient laboratory workflow for analysing VWF. These findings in our cohort of Portuguese patients support the proposal that improving VWD diagnosis strategies will enhance clinical and laboratory approaches, allowing to establish the most appropriate treatment for each patient.

Familial thrombotic risk based on the genetic background of Protein C Deficiency in a Portuguese Study.

INTRODUCTION: Inherited protein C (PC) deficiency is a well-known risk factor for venous thrombosis (VT). Plasma PC levels are reliable in moderate to severe deficiencies; however, in mildly deficient individuals, the levels may overlap with those considered normal. Genetic studies of PROC, which encodes PC, could help identify carriers; genome-wide association studies (GWAS) have shown that approximately 50% of phenotypic variation in PC deficiency is caused by the cumulative effects of mutations in several other loci, namely in the PROCR. PATIENTS AND METHODS: With the main objective of determining the genotype/phenotype correlation in 59 Portuguese individuals from 26 unrelated families with history of thrombosis and repeatedly low/borderline PC plasma levels, we conducted a molecular study by direct sequencing of PROC; PROC promoter haplotypes and PROCR c.4600A>G polymorphism (rs867186), which are known to influence plasma PC concentrations, were also screened. RESULTS: Twelve different PROC mutations were identified, one of them not previously reported, p.Cys105Arg. The mutation types and locations as well as haplotype combinations correlated with the phenotypic severity. The most frequent mutation, p.Arg199X, correlated with the CGTC haplotype and was identified in nine families containing patients with higher numbers of VT episodes. This mutation in homozygous individuals for the CGTC haplotype is a significant risk factor for VT in Portuguese. CONCLUSION: These genetic family studies allowed the identification of the unknown carriers and individuals at a higher thrombotic risk within each family, thus permitting the evaluation of the need for prophylactic measures, particularly in at-risk situations.