Light transmittance in human atrial tissue and transthoracic illumination in rats support translatability of optogenetic cardioversion of atrial fibrillation.

BACKGROUND: Optogenetics could offer a solution to the current lack of an ambulatory method for the rapid automated cardioversion of atrial fibrillation (AF), but key translational aspects remain to be studied. OBJECTIVE: To investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall. METHODS: Atria of adult and aged rats were optogenetically modified to express light-gated ion channels (i.e., red-activatable channelrhodopsin), followed by AF induction and atrial illumination to determine the effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue. RESULTS: AF could be effectively terminated in the remodeled atria of aged rats (97%, n = 6). Subsequently, ex vivo experiments using human atrial auricles demonstrated that 565-nm light pulses at an intensity of 25 mW/mm2 achieved the complete penetration of the atrial wall. Applying such irradiation onto the chest of adult rats resulted in transthoracic atrial illumination as evidenced by the optogenetic cardioversion of AF (90%, n = 4). CONCLUSION: Transthoracic optogenetic cardioversion of AF is effective in the aged rat heart using irradiation levels compatible with human atrial transmural light penetration.

Design and Proof-of-Concept of a Matrix Transducer Array for Clamp-On Ultrasonic Flow Measurements.

Common clamp-on ultrasonic flow meters consist of two single-element transducers placed on the pipe wall. Flow speed is measured noninvasively, i.e., without interrupting the flow and without perforating the pipe wall, which also minimizes safety risks and avoids pressure drops inside the pipe. However, before metering, the transducers have to be carefully positioned along the pipe axis to correctly align the acoustic beams and obtain a well-calibrated flowmeter. This process is done manually, is dependent on the properties of the pipe and the liquid, does not account for pipe imperfections, and becomes troublesome on pipelines with an intricate shape. Matrix transducer arrays are suitable to dynamically steer acoustic beams and realize self-alignment upon reception, without user input. In this work, the design of a broadband 37×17 matrix array (center frequency of 1 MHz) to perform clamp-on ultrasonic flow measurements over a wide range of liquids ( c=1000-2000 m/s, α ≤ 1 dB/MHz · cm) and pipe sizes is presented. Three critical aspects were assessed: efficiency, electronic beam steering, and wave mode conversion in the pipe wall. A prototype of a proof-of-concept flowmeter consisting of two 36-element linear arrays (center frequency of 1.1 MHz) was fabricated and placed on a 1-mm-thick, 40-mm inner diameter stainless steel pipe in a custom-made flow loop filled with water. At resonance, simulated and measured efficiencies in water of the linear arrays compared well: 0.88 and 0.81 kPa/V, respectively. Mean flow measurements were achieved by electronic beam steering of the acoustic beams and using both compressional and shear waves generated in the pipe wall. Correlation coefficients of between measured and reference flow speeds were obtained, thus showing the operational concept of an array-based clamp-on ultrasonic flowmeter.

Optical ventricular cardioversion by local optogenetic targeting and LED implantation in a cardiomyopathic rat model.

AIMS: Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs (i) in the pathologically remodelled heart using an (ii) implantable multi-LED device for (iii) in vivo closed-chest, local illumination. METHODS AND RESULTS: In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place 3 weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin or Citrine for control experiments. At 8-10 weeks after TAC, VTs were induced ex vivo and in vivo, followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination. CONCLUSION: Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion.

The relation between the atrial blood supply and the complexity of acute atrial fibrillation.

BACKGROUND: Patients with a history of myocardial infarction and coronary artery disease (CAD) have a higher risk of developing AF. Conversely, patients with atrial fibrillation (AF) have a higher risk of developing myocardial infarction, suggesting a link in underlying pathophysiology. The aim of this study was to assess whether coronary angiographic parameters are associated with a substrate for AF in patients without a history of AF. METHODS: During cardiac surgery in 62 patients (coronary artery bypass grafting (CABG;n = 47), aortic valve replacement (AVR;n = 9) or CABG + AVR (n = 6)) without a history of clinical AF (age 65.4 ± 8.5 years, 26.2% female), AF was induced by burst pacing. The preoperative coronary angiogram (CAG) was assessed for the severity of CAD, and the adequacy of atrial coronary blood supply as quantified by a novel scoring system including the location and severity of right coronary artery disease in relation to the right atrial branches. Epicardial mapping of the right atrium (256 unipolar electrodes) was used to assess the complexity of induced AF. RESULTS: There was no association between the adequacy of right atrial coronary blood supply on preoperative CAG and AF complexity parameters. Multivariable analysis revealed that only increasing age (B0.232 (0.030;0.433),p = 0.03) and the presence of 3VD (B3.602 (0.187;7.018),p = 0.04) were independently associated with an increased maximal activation time difference. CONCLUSIONS: The adequacy of epicardial right atrial blood supply is not associated with increased complexity of induced atrial fibrillation in patients without a history of clinical AF, while age and the extent of ventricular coronary artery disease are.

An automated hybrid bioelectronic system for autogenous restoration of sinus rhythm in atrial fibrillation.

Because of suboptimal therapeutic strategies, restoration of sinus rhythm in symptomatic atrial fibrillation (AF) often requires in-hospital delivery of high-voltage shocks, thereby precluding ambulatory AF termination. Continuous, rapid restoration of sinus rhythm is desired given the recurring and progressive nature of AF. Here, we present an automated hybrid bioelectronic system for shock-free termination of AF that enables the heart to act as an electric current generator for autogenous restoration of sinus rhythm. We show that local, right atrial delivery of adenoassociated virus vectors encoding a light-gated depolarizing ion channel results in efficient and spatially confined transgene expression. Activation of an implanted intrathoracic light-emitting diode device allows for termination of AF by illuminating part of the atria. Combining this newly obtained antiarrhythmic effector function of the heart with the arrhythmia detector function of a machine-based cardiac rhythm monitor in the closed chest of adult rats allowed automated and rapid arrhythmia detection and termination in a safe, effective, repetitive, yet shock-free manner. These findings hold translational potential for the development of shock-free antiarrhythmic device therapy for ambulatory treatment of AF.

Advice from the health insurer as a channelling strategy: a natural experiment at a Dutch health insurance company.

BACKGROUND: In a health care system based on managed competition it is important that health insurers are able to channel their enrolees to preferred care providers. However, enrolees are often very negative about financial incentives and any limitations in their choice of care provider. Therefore, a Dutch health insurance company conducted an experiment to study the effectiveness of a new method of channelling their enrolees. This method entails giving enrolees advise on which physiotherapists to choose when they call customer service. Offering this advice as an extra service is supposed to improve service quality ratings. Objective of this study is to evaluate this channelling method on effectiveness and the impact on service quality ratings. METHODS: In this experiment, one of the health insurer's customer service call teams (pilot team) began advising enrolees on their choice of physiotherapist. Three data sources were used. Firstly, all enrolees who called customer service received an online questionnaire in order to measure their evaluation of the quality of service. Enrolees who were offered advice received a slightly different questionnaire which, in addition, asked about whether they intended to follow the advice they were offered. Multilevel regression analysis was conducted to analyse the difference in service quality ratings between the pilot team and two comparable customer service teams before and after the implementation of the channelling method. Secondly, employees logged each call, registering, if they offered advice, whether the enrolee accepted it, and if so, which care provider was advised. Thirdly, data from the insurance claims were used to see if enrolees visited the recommended physiotherapist. RESULTS: The results of the questionnaire show that enrolees responded favorably to being offered advice on the choice of physiotherapist. Furthermore, 45% of enrolees who received advice and then went on to visit a care provider, followed the advice. The service quality ratings were higher compared to control groups. However, it could not be determined whether this effect was entirely due to the intervention. CONCLUSIONS: Channelling enrolees towards preferred care providers by offering advice on their choice of care provider when they call customer service is successful. The effect on service quality seems positive, although a causal relationship could not be determined.

Preoperative Planning of Transapical Beating Heart Mitral Valve Repair for Safe Adaptation in Clinical Practice.

OBJECTIVE: Transapical off-pump minimally invasive mitral valve repair (TOP-MINI) is a new technique for the surgical repair of degenerative mitral regurgitation based on mitral valve prolapse. The aim of this study is to demonstrate the preoperative planning tools available for starting this new procedure in a safe manner. METHODS: The first patients undergoing TOP-MINI by a single surgeon in 2016 were prospectively included. All patients underwent identical clinical pathways and underwent extensive preoperative planning for a safe start of the program. Patients were discussed in our dedicated mitral valve heart-team consisting of diagnostic and interventional mitral valve specialists. All patients underwent computed tomography, transthoracic and transesophageal echocardiography, and mitral valve replication using rapid prototyping. All procedures were performed by the same surgical team. RESULTS: Thirty-six patients were discussed for isolated mitral valve repair in our dedicated mitral valve heart team of which seven patients were deemed eligible for this novel approach. Three-dimensional (3D) reconstructions of computed tomography images allowed the surgical team to determine skin incision level and ideal level of device insertion near the apex of the heart. Echocardiography and rapid prototyping allowed us to assess surgical success probability by determining the amount of tissue overlap and was used intraoperatively for guidance. All patients were operated on successfully, without any major adverse events. CONCLUSIONS: We demonstrate a method to safely start the TOP-MINI program with precise patient selection and preoperative planning, allowing us to determine procedural strategy and assessment of surgical success probability.

Aortic elongation part I: the normal aortic ageing process.

OBJECTIVES: Differentiation between normal and abnormal features of vascular ageing is crucial, as the latter is associated with adverse outcomes. The normal aortic ageing process is accompanied by gradual luminal dilatation and reduction of vessel compliance. However, the influence of age on longitudinal aortic dimensions and geometry has not been well studied. This study aims to describe the normal evolution of aortic length and shape throughout life. METHODS: A total of 210 consecutive patients were prospectively enrolled in this cross-sectional single-centre study. All subjects underwent CT on a third-generation dual-source CT scanner. Morphometric measurements, including measurements of segmental length and tortuosity, were performed on three-dimensional models of the thoracic aorta. RESULTS: The length of the thoracic aorta was significantly related to age (r=0.54) and increased by 59 mm (males) or 66 mm (females) between the ages of 20 and 80 years. Elongation was most pronounced in the proximal descending aorta, which showed an almost 2.5-fold length increase during life. The lengthening of the thoracic aorta was accompanied by a marked change of its geometry: whereas the aortic apex was located between the branch vessels in younger patients, it shifted to a more distalward position in the elderly. CONCLUSIONS: The normal ageing process is accompanied by gradual aortic elongation and a notable change of aortic geometry. Part II of this two-part article investigates the hypothesis that excessive elongation could play a role in the occurrence of acute aortic dissection.

Aortic elongation part II: the risk of acute type A aortic dissection.

OBJECTIVES: Prophylactic surgery for prevention of acute type A aortic dissection (ATAAD) is reserved for patients with an ascending aortic aneurysm ≥55 mm. Identification of additional risk predictors is warranted since over 70% of patients presenting with ATAAD have a non-dilated aorta or an aneurysm that would not have met the diameter criterion for preventative surgery. Aim of the study was to evaluate ascending aortic elongation as a risk factor for ATAAD and to compare aortic lengths between ATAAD patients and healthy controls. METHODS: Aortic lengths and diameters of ATAAD patients were measured on three-dimensional modelled computed tomography and adjusted to predissection dimensions in this cross-sectional single-centre study. Logistic regression was used to evaluate the relation between ATAAD and aortic dimensions. Lengths of different aortic segments were compared with a healthy control group using propensity score matching. RESULTS: Two-hundred and fifty patients were included in the study (ATAAD, n=40; controls, n=210). Ascending aortic length and diameter proved to be independent predictors for ATAAD (OR=5.3, CI 2.5 to 11.4, p<0.001 and OR=8.6, CI 2.4 to 31.0, p=0.001). Eighty patients were matched based on propensity scores (ATAAD n=40, controls n=40). The ascending aorta was longer and more dilated in ATAAD patients compared with healthy controls (78.6±8.8 mm vs 68.9±7.2 mm, p<0.001, 34.4 mm ±3.2. vs 39.4 mm ±5.7, p<0.001, respectively). No differences were found in lengths of the aortic arch and descending aorta. CONCLUSIONS: Ascending aortic length could serve as an independent predictor for ATAAD. Future studies addressing indications for prophylactic surgery should also investigate aortic length.

Abnormal Myocardial Contractility After Pediatric Heart Transplantation by Cardiac MRI.

Acute cellular rejection (ACR) compromises graft function after heart transplantation (HTX). The purpose of this study was to describe systolic myocardial deformation in pediatric HTX and to determine whether it is impaired during ACR. Eighteen combined cardiac magnetic resonance imaging (CMR)/endomyocardial biopsy (EMBx) examinations were performed in 14 HTX patients (11 male, age 13.9 ± 4.7 years; 1.2 ± 1.3 years after HTX). Biventricular function and left ventricular (LV) circumferential strain, rotation, and torsion by myocardial tagging CMR were compared to 11 controls as well as between patients with and without clinically significant ACR. HTX patients showed mildly reduced biventricular systolic function when compared to controls [LV ejection fraction (EF): 55 ± 8% vs. 61 ± 3, p = 0.02; right ventricular (RV) EF: 48 ± 7% vs. 53 ± 6, p = 0.04]. Indexed LV mass was mildly increased in HTX patients (67 ± 14 g/m2 vs. 55 ± 13, p = 0.03). LV myocardial deformation indices were all significantly reduced, expressed by global circumferential strain (-13.5 ± 2.3% vs. -19.1 ± 1.1%, p < 0.01), basal strain (-13.7 ± 3.0% vs. -17.5 ± 2.4%, p < 0.01), mid-ventricular strain (-13.4 ± 2.7% vs. -19.3 ± 2.2%, p < 0.01), apical strain (-13.5 ± 2.8% vs. -19.9 ± 2.0%, p < 0.01), basal rotation (-2.0 ± 2.1° vs. -5.0 ± 2.0°, p < 0.01), and torsion (6.1 ± 1.7° vs. 7.8 ± 1.1°, p < 0.01). EMBx demonstrated ACR grade 0 R in 3 HTX cases, ACR grade 1 R in 11 HTX cases and ACR grade 2 R in 4 HTX cases. When comparing clinically non-significant ACR (grades 0-1 R vs. ACR 2 R), basal rotation, and apical rotation were worse in ACR 2 R patients (-1.4 ± 1.8° vs. -4.2 ± 1.4°, p = 0.01 and 10.2 ± 2.9° vs. 2.8 ± 1.9°, p < 0.01, respectively). Pediatric HTX recipients demonstrate reduced biventricular systolic function and decreased myocardial contractility. Myocardial deformation indices by CMR may serve as non-invasive markers of graft function and, perhaps, rejection in pediatric HTX patients.

Selective contracting and channelling patients to preferred providers: A scoping review.

Selective contracting by health insurers and channelling patients to contracted providers is crucial in a health care system based on managed competition, as this should lead to better value for money delivery of healthcare. However, an important consequence for enrolees is that health insurers interfere with their choice of care provider. This scoping review aims to find out what is known about selective contracting from the enrolee's perspective. Is it being done and how do enrolees feel about the role of their health insurer in their care provider choice? A literature search was conducted, and, in addition, experts were consulted for extra information and documents. Results show that selective contracting and channelling are practised in several countries. This is mostly through negative financial incentives, which are also found to be the most effective strategy. However, enrolees are very negative about restrictions on provider choice introduced by their insurer. This results in enrolees feeling less satisfaction with, and trust in, care providers and health insurers. Choice is crucial in this respect since enrolees are more satisfied with their health plans and care providers when they have chosen them themselves. Future research should focus on the role of trust and how people weigh different attributes of health plans if selective contracting and channelling is to be implemented in a manner acceptable to enrolees.

Health plan choice in the Netherlands: restrictive health plans preferred by young and healthy individuals.

In a health care system based on managed competition, health insurers negotiate on quality and price with care providers and are allowed to offer restrictive health plans. It is crucial that enrolees who need care choose restrictive health plans, as otherwise health insurers cannot channel patients to contracted providers and they will lose their bargaining power in negotiations with providers. We aim to explain enrolees' choice of a restrictive health plan in exchange for a lower premium. In 2014 an online survey with an experimental design was conducted on members of an access panel (response 78%; n=3,417). Results showed 37.4% of respondents willing to choose a restrictive health plan in exchange for a lower premium. This fell to 22% when the restrictive health plan also included a longer travelling time. Enrolees who choose a restrictive health plan are younger and healthier, or on lower incomes, than those preferring a non-restrictive one. This means that enrolees who use care will be unlikely to choose a restrictive health plan and, therefore, health insurers will not be able to channel them to contracted care providers. This undermines the goals of the health care system based on managed competition.

Optogenetic termination of ventricular arrhythmias in the whole heart: towards biological cardiac rhythm management.

AIMS: Current treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart. METHODS AND RESULTS: Systemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR-mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 97% of monomorphic and 57% of polymorphic VTs vs. 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light-induced arrhythmia termination indicating an important role for APD in this process. CONCLUSION: Brief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.

Evaluation of knowledge-based reconstruction for magnetic resonance volumetry of the right ventricle after arterial switch operation for dextro-transposition of the great arteries.

Right ventricular (RV) volume and function evaluation is essential in the follow-up of patients after arterial switch operation (ASO) for dextro-transposition of the great arteries (d-TGA). Cardiac magnetic resonance (CMR) imaging using the Simpson's method is the gold-standard for measuring these parameters. However, this method can be challenging and time-consuming, especially in congenital heart disease. Knowledge-based reconstruction (KBR) is an alternative method to derive volumes from CMR datasets. It is based on the identification of a finite number of anatomical RV landmarks in various planes, followed by computer-based reconstruction of the endocardial contours by matching these landmarks with a reference library of representative RV shapes. The purpose of this study was to evaluate the feasibility, accuracy, reproducibility and labor intensity of KBR for RV volumetry in patients after ASO for d-TGA. The CMR datasets of 17 children and adolescents (males 11, median age 15) were studied for RV volumetry using both KBR and Simpson's method. The intraobserver, interobserver and intermethod variabilities were assessed using Bland-Altman analyses. Good correlation between KBR and Simpson's method was noted. Intraobserver and interobserver variability for KBR showed excellent agreement. Volume and function assessment using KBR was faster when compared with the Simpson's method (5.1 ± 0.6 vs. 6.7 ± 0.9 min, p < 0.001). KBR is a feasible, accurate, reproducible and fast method for measuring RV volumes and function derived from CMR in patients after ASO for d-TGA.

Pain as a predictor of sleep problems in youth with autism spectrum disorders.

Evidence suggests that pain interferes with sleep in youth with developmental disabilities. This study examined the relationship between pain and sleep problems in a sample of youth with parent-reported autism spectrum disorder (N = 62). Mothers reported on standardized measures of pain and sleep problems. Youth demonstrated atypically high levels of both observed pain and sleep problems. Pain predicted overall sleep disturbance and three specific sleep problems: sleep duration, parasomnias, and sleep-disordered breathing. These specific sleep problems were predicted by specific modalities of nonverbal pain communication (e.g. sleep duration problems were predicted by social communication of pain). Effects were consistent across probing of relevant moderators. These findings suggest that comprehensive assessment of both pain and sleep problems may provide important information for medical and behavioral treatment planning for youth with autism spectrum disorder.

The mechanical fibrillation pattern of the atrial myocardium is associated with acute and long-term success of electrical cardioversion in patients with persistent atrial fibrillation.

BACKGROUND: Electrophysiological studies demonstrate that a short atrial fibrillation cycle length (AFCL) is related with poor outcome of electrical cardioversion (ECV) of atrial fibrillation (AF). We found previously that the mechanical AFCL (AFCL-tvi) and atrial fibrillatory velocity (AFV-tvi) may be determined noninvasively using color tissue velocity imaging (TVI) and closely relates to the electrophysiological AFCL. OBJECTIVE: To evaluate the relation between AFCL-tvi, AFV-tvi, and success of ECV in patients with AF. METHODS: We prospectively studied 133 patients with persistent AF by performing echocardiography before ECV and measured the AFCL-tvi and AFV-tvi in the right atrium and left atrium. Recurrent AF was monitored. RESULTS: Nineteen (14%) patients had failure of ECV, 42 (32%) remained in sinus rhythm after 1-year follow-up, and 72 (54%) had a recurrence of persistent AF. Patients with immediate ECV failure had a lower median AFV-tvi measured in the right atrium than did patients with a successful ECV: 0.7 cm/s (0.2-1.0 cm/s) vs. 1.7 cm/s (0.9-2.8 cm/s) (P = .008). Patients with maintenance of sinus rhythm after 1 year had a longer AFCL-tvi measured in the left atrium than did patients with recurrence of AF (150 ms vs 137 ms; P = .017) and had a higher AFV-tvi in both atria (1.4 vs. 0.9 cm/s in the left atrium; P = .013 and 2.2 vs 1.4 cm/s in the right atrium; P = .011). Multivariate analyses showed that all atrial TVI parameters were independently associated with the maintenance of sinus rhythm after 1 year. CONCLUSION: Higher atrial fibrillatory wall velocities and longer AFCLs determined by echocardiography are associated with acute and long-term success of ECV.

An FBN1 deep intronic mutation in a familial case of Marfan syndrome: an explanation for genetically unsolved cases?

Marfan syndrome (MFS) is caused by mutations in the FBN1 (fibrillin-1) gene, but approximately 10% of MFS cases remain genetically unsolved. Here, we report a new FBN1 mutation in an MFS family that had remained negative after extensive molecular genomic DNA FBN1 testing, including denaturing high-performance liquid chromatography, Sanger sequencing, and multiplex ligation-dependent probe amplification. Linkage analysis in the family and cDNA sequencing of the proband revealed a deep intronic point mutation in intron 56 generating a new splice donor site. This mutation results in the integration of a 90-bp pseudo-exon between exons 56 and 57 containing a stop codon, causing nonsense-mediated mRNA decay. Although more than 90% of FBN1 mutations can be identified with regular molecular testing at the genomic level, deep intronic mutations will be missed and require cDNA sequencing or whole-genome sequencing.