Artur Szkotak

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Regulation of Cl− secretion by α2-adrenergic receptors in mouse colonic epithelium

Previous studies have shown that α2 adrenoceptor (α2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of α2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that α2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that α2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated Cl− secretion but had no effect on CFTR activation by cAMP‐dependent phosphorylation. Apical administration of an ionophore, nystatin (90 μg ml−1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+–K+‐ATPase current, measured as ouabain‐sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 μm). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin‐activated Isc indicating that activation of α2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that α2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT‐PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by α2AR agonists required activation of pertussis toxin‐sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, α2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

The role of bestrophin in airway epithelial ion transport

The purpose of this study was to identify Cl− channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl− channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT‐PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36Cl− flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl− channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.

Proposed Approach to Thrombolysis in Dabigatran-Treated Patients Presenting with Ischemic Stroke

BACKGROUND Acute ischemic stroke thrombolysis in patients taking dabigatran is controversial because of a presumed increased risk of symptomatic hemorrhagic transformation. Using data from our local hematopathology laboratory, we developed a thrombolysis protocol for acute ischemic stroke patients taking dabigatran. METHODS A local thrombin time (TT)-dabigatran concentration relationship was calculated using dabigatran calibrators. The effect of dabigatran on activated partial thromboplastin time (aPTT) and prothrombin time (PT) (international normalized ratio [INR]) was also measured. A protocol was developed, in which a dabigatran concentration less than 10 ng/mL (corresponding to a TT<38 seconds or a normal aPTT) was selected as the upper limit for thrombolysis. Consecutive patients presenting with acute stroke were then enrolled in this prospective study. RESULTS In the 8 months after development of the protocol, 13 potential thrombolysis candidates taking dabigatran were assessed at a median (interquartile range) time of 192 (143) minutes. The median National Institutes of Health Stroke Scale score was 18 (20). The mean time from arrival to TT, aPTT, and PT (INR) results were 39±4.1 minutes, 21±6.5 minutes, and 21±6.5 minutes, respectively. Based on TT/aPTT, 4 patients were ineligible for thrombolysis. Six patients were not treated because of minor or resolving symptoms and another presented with intracerebral hemorrhage. Two patients were treated with intravenous tissue plasminogen activator (tPA), without symptomatic hemorrhagic transformation in either case. In 3 patients (42.8%), aPTT was normal, despite a prolonged TT. CONCLUSIONS Administration of intravenous tPA in dabigatran-treated patients is feasible. Although, the relationship between dabigatran concentrations and coagulation measures varies between laboratories, individual protocols, preferably based on TT, can be developed at acute stroke treatment centers.

Coupling of CFTR-mediated anion secretion to nucleoside transporters and adenosine homeostasis in Calu-3 cells.

The purpose of this study was to characterize the role of adenosine-dependent regulation of anion secretion in Calu-3 cells. RT-PCR studies showed that Calu-3 cells expressed mRNA for A2a and A2B but not A1 or A3 receptors, and for hENTl, hENT2 and hCNT3 but not hCNTl or hCNT2 nucleoside transporters. Short-circuit current measurements indicated that A2b receptors were present in both apical and basolateral membranes, whereas A2a receptors were detected only in basolateral membranes. Uptake studies demonstrated that the majority of adenosine transport was mediated by hENTl, which was localized to both apical and basolateral membranes, with a smaller hENT2-mediated component in basolateral membranes. Whole-cell current measurements showed that application of extracellular nitrobenzylmercaptopurine ribonucleoside (NBMPR), a selective inhibitor of hENTl-mediated transport, had similar effects on whole-cell currents as the application of exogenous adenosine. Inhibitors of adenosine kinase and 5′-nucleotidase increased and decreased, respectively, whole-cell currents, whereas inhibition of adenosine deaminase had no effect. Single-channel studies showed that NBMPR and adenosine kinase inhibitors activated CFTR Cl- channels. These results suggested that the equilibrative nucleoside transporters (hENTl, hENT2) together with adenosine kinase and 5′-nucleotidase play a crucial role in the regulation of CFTR through an adenosine-dependent pathway in human airway epithelia.

A comprehensive high-throughput sequencing test for the diagnosis of inherited bleeding, thrombotic and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Dabigatran assessment in patients with acute complications using routine coagulation assays.

Complications while on dabigatran therapy, particularly bleeding and thrombosis, are occurring, and require laboratory assessment. The utility of routine coagulation assays has been previously evaluated in stable patients, but not those with acute complications. The purpose of this study was to determine how to employ routine coagulation assays to assess dabigatran in patients with acute complications. Seventeen patients on dabigatran presenting with various complications were evaluated. In addition, plasma samples with various fibrinogen levels were spiked in vitro with dabigatran ranging from low trough levels to the highest supratherapeutic concentrations reported (5000 ng/ml). INR, partial thromboplastin time (PTT), thrombin time (TT, Diagnostica Stago reagent), and fibrinogen were assayed and results compared to that of the Hemoclot Thrombin Inhibitors assay. Interference in the Clauss fibrinogen assay was assessed using a variety of commercial reagents. The majority of patients on dabigatran with acute complications demonstrated a significant negative bias in PTT results compared to normal plasma. TT remained highly sensitive to the presence of dabigatran (at least 10 ng/ml) under all circumstances investigated. There was wide variation in the sensitivity of commercial fibrinogen assays to dabigatran, with some even showing interference in the therapeutic range but this could be mitigated. The PTT is unreliable as a method for assessment of dabigatran in patients with acute complications. The TT assay is a simple and reliable alternative, particularly when combined with a fibrinogen level.

Persistent cytogenetic abnormalities in patients undergoing intensive chemotherapy for acute myeloid leukemia

Abstract We evaluated the impact of bone marrow sample characteristics on the detection of persistent cytogenetic abnormalities (PCA) following induction chemotherapy for acute myeloid leukemia (AML). PCA’s were identified in 20.4% of patients and were more common with complete remission without count recovery (CRi) vs. those with count recovery (CR, 45.8 vs. 13.5%, p = .001), with  >2% blasts vs.  ≤2% blasts (42 vs. 12%, p =  .001) and with hypocellular trephine biopsies relative to those with normo/hypercellular biopsies (42.1 vs. 17.3%, p  = .03), although in a multivariate analysis only CRi and blast count >2% were independently associated with a PCA. PCA’s were not observed in patients with favorable risk karyotype. Amongst patients with intermediate and unfavorable risk karyotypes PCA were not associated with differences in overall or, amongst non-transplanted patients, relapse free survival. Thus, although PCAs are common post-induction it is unclear whether they provide any independent prognostic information beyond the diagnostic karyotype.

Crystals and Fatty Acid Abnormalities Are Not Present in Circulating Cells From Choroideremia Patients

Purpose To confirm whether choroideremia (CHM) is a systemic disease characterized by blood lipid abnormalities and crystals found in, or associated with, circulating peripheral blood cells of patients. Methods Peripheral blood samples obtained from three subjects with confirmed mutations in the CHM gene and three age-matched normal controls were processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Fatty acids from plasma of nine male CHM subjects were analyzed and compared to reference values for a sample from a Canadian population. Results Intracellular crystals were not observed in the cells from choroideremia-affected males. No crystals were found adherent to the external plasma membrane of red blood cells. Fatty acid profiles of patients were similar to reference values, with the exception of lower levels of nervonic acid. Conclusions This investigation failed to observe crystals previously reported in peripheral circulating blood cells derived from CHM subjects, and showed no significant fatty acid abnormalities, not supporting the view of CHM as a systemic disease.

Thrombosis and Hemostasis: A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.