KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

DNA combing versus DNA spreading and the separation of sister chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example, in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves sister chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading typically does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

DNA Combing versus DNA Spreading and the Separation of Sister Chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves single chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair.

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

Usefulness of Global Longitudinal Strain to Predict Heart Failure Progression in Patients With Nonobstructive Hypertrophic Cardiomyopathy.

While predicting prognosis to anticipate adverse disease course has long been an aspiration in hypertrophic cardiomyopathy (HC), reliable markers of progressive and unrelenting heart failure symptoms in the absence of obstruction are not well characterized. We sought to evaluate markers of systolic function, including the role of global longitudinal strain (GLS), to identify nonobstructive HC patients at risk for future heart failure. A cohort of 296 consecutive nonobstructive HC patients (42 ± 18years; 75% male) with NYHA class I/II symptoms and preserved systolic function at study entry (EF: 65 ± 6%), were followed for progressive heart failure symptoms (increase in ≥ 1 NYHA functional class) and/or development of systolic dysfunction (EF < 50%). Over median follow-up of 4 ± 3 years, 35 study patients (10%) experienced new heart failure events, including 31 with progressive symptoms and 4 who developed systolic dysfunction. Abnormal GLS < 16% was associated with a 5-fold increase in risk for heart failure compared to GLS > 18% (p < 0.001). GLS remained an independent predictor of heart failure even after adjustment for other relevant disease variables including EF (OR 1.23, p = 0.005). However, notably, when GLS and EF were combined, the prediction of heart failure for individual patients was enhanced (net reclassification improvement 0.55; p = 0.002). Together, GLS < 16% and EF 50% to 59% were associated with a 12.5-fold greater risk for heart failure versus patients with GLS > 18% and EF ≥ 60%, who were at the lowest risk. In conclusion, in nonobstructive HC with no or mild symptoms and preserved EF, abnormal GLS is a strong independent predictor for subsequent development of progressive heart failure symptoms and/or systolic dysfunction. Furthermore, the greatest power in predicting outcome in nonobstructive HC is achieved by combining GLS with EF to identify HC patients at the highest risk for heart failure progression and systolic dysfunction.

Outcomes in Patients With Hypertrophic Cardiomyopathy and Left Ventricular Systolic Dysfunction.

BACKGROUND: End-stage (ES) hypertrophic cardiomyopathy (HCM) has been considered a particularly grim and unfavorable disease complication, associated with substantial morbidity and mortality, frequently requiring heart transplant. Previous reports have included small numbers of patients with relatively short follow-up, predominantly in prior treatment eras. OBJECTIVES: The purpose of this study was to re-evaluate clinical profile and prognosis for end-stage heart failure in a large HCM cohort with contemporary management strategies. METHODS: Patients at Tufts HCM Institute, from 2004 to 2017, were identified with ES and systolic dysfunction (ejection fraction [EF] <50%), followed for 5.8 ± 4.7 years (up to 18 years). RESULTS: Of the 2,447 patients, 118 (4.8%) had ES-HCM (EF 39 ± 9%; range 12% to 49%) at age 48 ± 15 years. Notably, over follow-up, 57 patients (48%) achieved clinical stability in New York Heart Association functional classes I/II with medical treatment (or cardiac resynchronization therapy), including 6 patients ≥10 years from ES diagnosis (up to 14 years). In total, 61 other patients (52%) developed refractory heart failure to disabling New York Heart Association functional classes III/IV (5.2%/year); 67% have survived, including 31 with heart transplant. Of the 118 ES patients, 21 had appropriate implantable cardioverter-defibrillator (ICD) therapy terminating potentially lethal tachyarrhythmias, with no difference in frequency of events in patients with EF 35% to 49% versus EF <35% (17% vs. 19%; p = 0.80). With all available treatment modalities, ES-related mortality was 1.9%/year, with 10-year survival of 85% (95% confidence interval: 77% to 94%). Mortality was 4-fold lower than previously reported for ES (8.0%/year), but exceeded 10-fold HCM with preserved EF (0.2%/year; p < 0.001). CONCLUSIONS: Although ES remains an important complication of HCM, contemporary treatment strategies, including ICDs and heart transplant, are associated with significantly lower mortality than previously considered. Primary prevention ICDs should be considered when EF is <50% in HCM. Rapid heart failure progression is not an inevitable consequence of ES, and some patients experience extended periods of clinical stability.

Artificial Intelligence for the Measurement of the Aortic Valve Annulus.

OBJECTIVE: The authors aim to evaluate an automated echocardiography software as compared with computed tomography in measurement of the aortic valve annulus in patients with aortic stenosis. The authors hypothesize that aortic annular measurements by this software and computed tomography will show acceptable correlation. DESIGN: This study is an Institutional Review Board-approved, retrospective data collection of patients with aortic stenosis who underwent implantation of a transcatheter heart valve with intraprocedural transesophageal echocardiography, multidetector computed tomography, and use of the Siemens eSie Valves automated aortic valve software. SETTING: Intraprocedural in a single hospital institution. PARTICIPANTS: The participants are 47 patients who underwent implantation of an Edwards SAPIEN 3 transcatheter heart valve. INTERVENTIONS: The authors compared aortic valve annulus measurements by two-dimensional transesophageal echocardiography, computed tomography, and the automated software. MEASUREMENTS AND MAIN RESULTS: Aortic annulus measurements by the software correlated more closely to the computed tomography measurements than two-dimensional measurements. Bland-Altman analysis showed qualitative comparability of measurements performed by the automated software to computed tomography (95% limits of agreement between -4.62 mm and 1.26 mm for area-derived and -4.51 mm and 1.45 mm for perimeter-derived methods). Similarly, there was significant linear correlation with automated software use (r = 0.84, p < 0.0001 and r = 0.85, p < 0.0001). CONCLUSIONS: Periprocedural aortic valve measurement by automated echocardiographic software correlates with computed tomography in patients with severe aortic stenosis. This technology is helpful and accurate, but has limitations.

Impact of Sex on Clinical Course and Survival in the Contemporary Treatment Era for Hypertrophic Cardiomyopathy.

Background The relation of sex to clinical presentation and course in hypertrophic cardiomyopathy (HCM) remains incompletely resolved. We assessed differences in clinical outcomes between men and women within our large HCM cohort. Methods and Results Of 2123 consecutive patients, a minority (38%) were women who were diagnosed with HCM at older ages or referred for subspecialty evaluation later than men (50±19 versus 44±16 and 55±18 versus 49±16; P<0.001). Women more commonly developed advanced New York Heart Association class III/IV symptoms (53% versus 35% in men; P<0.001), predominantly secondary to outflow obstruction. While end-stage heart failure with systolic dysfunction (ejection fraction <50%) was similar in men (5% versus 4% in women; P=0.33), women were 3-fold more likely to develop heart failure with preserved systolic function (7.5% versus 2.6%; P=0.002). Sudden death events terminated by defibrillator therapy were similar in women (0.9%/year) versus men (1.0%/year; hazard ratio, 0.92; 95% CI, 0.6-1.5; P=0.73). HCM mortality was uncommon, with identical rates in both sexes (0.3%/year; hazard ratio, 1.5; 95% CI, 0.7-3.4;, P=0.25). Age-adjusted all-cause mortality also did not differ between women and men (1.7% versus 1.3%/year; hazard ratio, 1.32; 95% CI, 0.92-1.91; P=0.13). Conclusions Survival was not less favorable in women with HCM. Contemporary treatments including surgical myectomy to reverse heart failure and defibrillators to prevent sudden death, were effective in both sexes contributing to low mortality. However, despite more frequent outflow obstruction, women with HCM are underrecognized and referred to centers later than men, often with more advanced heart failure. Greater awareness of HCM in women should lead to earlier diagnosis and treatment, with implications for improved quality of life.

Guideline-Based Referral for Septal Reduction Therapy in Obstructive Hypertrophic Cardiomyopathy Is Associated With Excellent Clinical Outcomes.

BACKGROUND: The outcome of medically refractory patients with obstructive hypertrophic cardiomyopathy treated according to the American College of Cardiology/American Heart Association consensus guideline recommendations is not known. The objectives of this study were to define the short- and long-term outcomes of medically refractory obstructive hypertrophic cardiomyopathy patients undergoing alcohol septal ablation (ASA) and surgical septal myectomy (SM) with patient management in accordance with these consensus guidelines, as well as to quantify procedural risk and burden of comorbid conditions at the time of treatment. METHODS AND RESULTS: Patients with obstructive hypertrophic cardiomyopathy referred for either ASA or SM from 2004 to 2015 were followed for the primary end point of short- and long-term mortality and compared with respective age- and sex-matched US populations. Of 477 consecutive severely symptomatic patients, 99 underwent ASA and 378 SM. Compared with SM, ASA patients were older ( P<0.001), had a higher burden of comorbid conditions ( P<0.01), and significantly higher predicted surgical mortality ( P<0.005). Procedure-related mortality was 0.3% and similarly low in both groups (0% in ASA and 0.8% in SM). Over 4.0±2.9 years of follow-up, 95% of patients had substantial improvement in heart failure symptoms to New York Heart Association class I/II (96% in SM and 90% in ASA). Long-term mortality was similar between the 2 groups with no difference compared with age- and sex-matched US populations. CONCLUSIONS: Guideline-based referral for ASA and SM leads to excellent outcomes with low procedural mortality, excellent long-term survival, and improvement in symptoms. These outcomes occur in ASA patients despite being an older cohort with significantly more comorbidities.

Clinical Profile of Nonresponders to Surgical Myectomy with Obstructive Hypertrophic Cardiomyopathy.

BACKGROUND: Surgical myectomy reverses heart failure symptoms in the vast majority of obstructive hypertrophic cardiomyopathy patients. However, a small subgroup fails to experience sustained postoperative improvement despite relief of obstruction. Clinical profile of such patients has not been well defined. METHODS: Consecutive obstructive hypertrophic cardiomyopathy patients undergoing myectomy at Tufts Medical Center for drug-refractory New York Heart Association III/IV heart failure symptoms, 2004 to 2017, were followed postoperatively for 2.5 ± 2.8 years and assessed for outcome. RESULTS: Of the 503 patients, there were 4 postoperative deaths (0.8%); 480 patients (96%) had sustained improvement to New York Heart Association classes I or II (responders), but 19 (3.8%) developed advanced symptoms (classes III or IV) in the absence of obstruction (nonresponders). Compared with responders, nonresponders were younger (40 ± 13 vs 53 ± 14 years; P < .001) and had greater septal thickness (25 ± 9 vs 20 ± 4 mm; P < .001). Massive hypertrophy (≥30 mm) was 5-fold more common in nonresponders (P < .01). Seven nonresponders developed systolic dysfunction (ejection fraction 20%-47%), 2 days to 6.1 years postoperatively. Four nonresponders underwent heart transplant 3.4 to 9.2 years after myectomy, and 2 others have been listed. CONCLUSIONS: Surgical myectomy is highly effective at reversing heart failure symptoms in the vast majority of patients with obstructive hypertrophic cardiomyopathy. However, a small minority experience persistent functional limitation despite surgical relief of outflow obstruction. Predictors of adverse postoperative course were substantial/massive septal thickness and youthful age. Patients who failed to respond symptomatically to myectomy were considered for advanced heart failure treatment, including heart transplantation.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.