Cell-Free DNA to Detect Heart Allograft Acute Rejection.

BACKGROUND: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients. METHODS: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. RESULTS: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P<0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. CONCLUSIONS: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Beta-blockers and Ambulatory Inotropic Therapy.

BACKGROUND: Continuous infusion of ambulatory inotropic therapy (AIT) is increasingly used in patients with end-stage heart failure (HF). There is a paucity of data concerning the concomitant use of beta-blockers (BB) in these patients. METHODS: We retrospectively reviewed all patients discharged from our institution on AIT. The cohort was stratified into 2 groups based on BB use. The 2 groups were compared for differences in hospitalizations due to HF, ventricular arrhythmias and ICD therapies (shock or antitachycardia pacing). RESULTS: Between 2010 and 2017, 349 patients were discharged on AIT (95% on milrinone); 74% were males with a mean age of 61 ± 14 years. BB were used in 195 (56%) patients, whereas 154 (44%) did not receive these medications. Patients in the BB group had longer duration of AIT support compared to those in the non-BB group (141 [1-2114] vs 68 [1-690] days). After adjusting for differences in baseline characteristics and indication for AIT, patients in the BB group had significantly lower rates of hospitalizations due to HF (hazard ratio [HR] 0.61 (0.43-0.86); P = 0.005), ventricular arrhythmias (HR 0.34 [0.15-0.74]; P = 0.007) and ICD therapies (HR 0.24 [0.07-0.79]; P = 0.02). CONCLUSION: In patients with end-stage HF on AIT, the use of BB with inotropes was associated with fewer hospitalizations due to HF and fewer ventricular arrhythmias.

Outcomes in patients with smaller body surface area after HeartMate 3 left ventricular assist device implantation.

BACKGROUND: Due to anatomic and physiologic concerns, prior generations of the left ventricular assist devices (LVAD) have frequently been denied to patients with small body size. However, outcomes in patients with small body surface area (BSA) following HeartMate 3 (HM3) LVAD implantation remain relatively unknown. METHODS: A cohort of 220 patients implanted at a single center was divided into two groups: BSA ≤1.8 m2 (small BSA, n = 37) and BSA >1.8 m2 (large BSA, n = 183). We investigated baseline characteristics and clinical outcomes including survival and incidence of adverse events. RESULTS: Small BSA patients were older (60 vs. 57 years), more likely female (60% vs. 20%), had a lower body mass index (24 vs. 32 kg/m2 ), lower incidence of diabetes (32% vs. 51%), history of stroke (5% vs. 19%), and left ventricular thrombus (0% vs. 11%). They had smaller left ventricular end diastolic diameter (64.8 vs. 69.3 mm). Pump speed and pump flows at discharge were lower in the small BSA group. Survival at 1 year and 2 years was 86% versus 87% and 86% versus 79% for small versus large BSA groups (p = 0.408), respectively. The rates of adverse events were similar between groups and there were no cases of confirmed pump thrombosis. The incidence of readmissions for low flow alarms was higher in the small BSA group (0.55 vs. 0.24 EPPY). CONCLUSIONS: These findings demonstrate comparable outcomes in patients with small body size and suggest that this parameter should not be an exclusion criterion on patients who are otherwise candidates for HM3 LVAD implantation.

Chronic Intravenous Inotropic Support as Palliative Therapy and Bridge Therapy for Patients With Advanced Heart Failure: A Single-Center Experience.

BACKGROUND: Many patients with American College of Cardiology/American Heart Association Stage D (advanced) heart failure are discharged home on chronic intravenous inotropic support (CIIS) as bridge to surgical therapy or as palliative therapy. This study analyzed the clinical trajectory of patients with advanced heart failure who were on home CIIS. METHODS: We conducted a single-institution, retrospective cohort study of patients on CIIS between 2010 and 2016 (n = 373), stratified by indication for initiation of inotropic support. Study outcomes were time from initiation of CIIS to cessation of therapy, time to death for patients who did not receive surgical therapy and rates of involvement with palliative care. RESULTS: Overall, patients received CIIS therapy for an average of 5.9 months (standard deviation [SD] 7.3). Patients on CIIS as palliative therapy died in an average of 6.2 months (SD 6.6) from the time of initiation of CIIS, and those on CIIS as bridge therapy who did not ultimately receive surgical therapy died after an average of 8.6 months (SD 9.3). Patients who received CIIS as bridge therapy were significantly less likely to receive palliative-care consultation than those on inotropes as palliative therapy, whether or not they underwent surgery. CONCLUSIONS: In this large cohort of patients with advanced HF, patients who on CIIS as palliative therapy survived for 6.2 months, on average, with wide variation among patients. Patients who were on CIIS as bridge therapy but did not ultimately receive surgical therapy received less palliative care despite the high mortality rate in this subgroup.

Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes.

The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.

Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B'. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

Impact of controlled hypothermic preservation on outcomes following heart transplantation.

BACKGROUND: Severe primary graft dysfunction (PGD) is a major cause of early mortality after heart transplant, but the impact of donor organ preservation conditions on severity of PGD and survival has not been well characterized. METHODS: Data from US adult heart-transplant recipients in the Global Utilization and Registry Database for Improved Heart Preservation-Heart Registry (NCT04141605) were analyzed to quantify PGD severity, mortality, and associated risk factors. The independent contributions of organ preservation method (traditional ice storage vs controlled hypothermic preservation) and ischemic time were analyzed using propensity matching and logistic regression. RESULTS: Among 1,061 US adult heart transplants performed between October 2015 and December 2022, controlled hypothermic preservation was associated with a significant reduction in the incidence of severe PGD compared to ice (6.6% [37/559] vs 10.4% [47/452], p = 0.039). Following propensity matching, severe PGD was reduced by 50% (6.0% [17/281] vs 12.1% [34/281], respectively; p = 0.018). The Kaplan-Meier terminal probability of 1-year mortality was 4.2% for recipients without PGD, 7.2% for mild or moderate PGD, and 32.1%, for severe PGD (p < 0.001). The probability of severe PGD increased for both cohorts with longer ischemic time, but donor hearts stored on ice were more likely to develop severe PGD at all ischemic times compared to controlled hypothermic preservation. CONCLUSIONS: Severe PGD is the deadliest complication of heart transplantation and is associated with a 7.8-fold increase in probability of 1-year mortality. Controlled hypothermic preservation significantly attenuates the risk of severe PGD and is a simple yet highly effective tool for mitigating post-transplant morbidity.

Racial Differences in Donor-Derived Cell-Free DNA and Mitochondrial DNA After Heart Transplantation, on Behalf of the GRAfT Investigators.

BACKGROUND: Black heart transplant patients are at higher risk of acute rejection (AR) and death than White patients. We hypothesized that this risk may be associated with higher levels of donor-derived cell-free DNA (dd-cfDNA) and cell-free mitochondrial DNA. METHODS: The Genomic Research Alliance for Transplantation is a multicenter, prospective, longitudinal cohort study. Sequencing was used to quantitate dd-cfDNA and polymerase chain reaction to quantitate cell-free mitochondrial DNA in plasma. AR was defined as ≥2R cellular rejection or ≥1 antibody-mediated rejection. The primary composite outcome was AR, graft dysfunction (left ventricular ejection fraction <50% and decrease by ≥10%), or death. RESULTS: We included 148 patients (65 Black patients and 83 White patients), median age was 56 years and 30% female sex. The incidence of AR was higher in Black patients compared with White patients (43% versus 19%; P=0.002). Antibody-mediated rejection occurred predominantly in Black patients with a prevalence of 20% versus 2% (P<0.001). After transplant, Black patients had higher levels of dd-cfDNA, 0.09% (interquartile range, 0.001-0.30) compared with White patients, 0.05% (interquartile range, 0.001-0.23; P=0.003). Beyond 6 months, Black patients showed a persistent rise in dd-cfDNA with higher levels compared with White patients. Cell-free mitochondrial DNA was higher in Black patients (185 788 copies/mL; interquartile range, 101 252-422 133) compared with White patients (133 841 copies/mL; interquartile range, 75 346-337 990; P<0.001). The primary composite outcome occurred in 43% and 55% of Black patients at 1 and 2 years, compared with 23% and 27% in White patients, P<0.001. In a multivariable model, Black patient race (hazard ratio, 2.61 [95% CI, 1.35-5.04]; P=0.004) and %dd-cfDNA (hazard ratio, 1.15 [95% CI, 1.03-1.28]; P=0.010) were associated with the primary composite outcome. CONCLUSIONS: Elevated dd-cfDNA and cell-free mitochondrial DNA after heart transplant may mechanistically be implicated in the higher incidence of AR and worse clinical outcomes in Black transplant recipients. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.

Higher levels of allograft injury in black patients early after heart transplantation.

Black patients suffer higher rates of antibody-mediated rejection and have worse long-term graft survival after heart transplantation. Donor-derived cell free DNA (ddcfDNA) is released into the blood following allograft injury. This study analyzed %ddcfDNA in 63 heart transplant recipients categorized by Black and non-Black race, during the first 200 days after transplant. Immediately after transplant, %ddcfDNA was higher for Black patients (mean [SE]: 8.3% [1.3%] vs 3.2% [1.2%], p = 0.001). In the first week post-transplant, the rate of decay in %ddcfDNA was similar (0.7% [0.68] vs 0.7% [0.11], p = 0.78), and values declined in both groups to a comparable plateau at 7 days post-transplant (0.46% [0.03] vs 0.45% [0.04], p = 0.78). The proportion of Black patients experiencing AMR was higher than non-Black patients (21% vs 9% [hazard ratio of 2.61 [95% confidence interval: 0.651-10.43], p = 0.18). Black patients were more likely to receive a race mismatched organ than non-Black patients (69% vs 35%, p = 0.01), which may explain the higher levels of early allograft injury.

Circulating microRNAs in cellular and antibody-mediated heart transplant rejection.

BACKGROUND: Noninvasive monitoring of heart allograft health is important to improve clinical outcomes. MicroRNAs (miRs) are promising biomarkers of cardiovascular disease and limited studies suggest they can be used to noninvasively diagnose acute heart transplant rejection. METHODS: The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter prospective cohort study that phenotyped heart transplant patients from 5 mid-Atlantic centers. Patients who had no history of rejection after transplant were compared to patients with acute cellular rejection (ACR) or antibody-mediated rejection (AMR). Small RNA sequencing was performed on plasma samples collected at the time of an endomyocardial biopsy. Differential miR expression was performed with adjustment for clinical covariates. Regression was used to develop miR panels with high diagnostic accuracy for ACR and AMR. These panels were then validated in independent samples from GRAfT and Stanford University. Receiver operating characteristic curves were generated and area under the curve (AUC) statistics calculated. Distinct ACR and AMR clinical scores were developed to translate miR expression data for clinical use. RESULTS: The GRAfT cohort had a median age of 52 years, with 35% females and 45% Black patients. Between GRAfT and Stanford, we included 157 heart transplant patients: 108 controls and 49 with rejection (50 ACR and 38 AMR episodes). After differential miR expression and regression analysis, we identified 12 miRs that accurately discriminate ACR and 17 miRs in AMR. Independent validation of the miR panels within GRAfT led to an ACR AUC 0.92 (95% confidence interval [CI]: 0.86-0.98) and AMR AUC 0.82 (95% CI: 0.74-0.90). The externally validated ACR AUC was 0.72 (95% CI: 0.59-0.82). We developed distinct ACR and AMR miR clinical scores (range 0-100), a score ≥ 65, identified ACR with 86% sensitivity, 76% specificity, and 98% negative predictive value, for AMR score performance was 82%, 84% and 97%, respectively. CONCLUSIONS: We identified novel miRs that had excellent performance to noninvasively diagnose acute rejection after heart transplantation. Once rigorously validated, the unique clinical ACR and AMR scores usher in an era whereby genomic biomarkers can be used to screen and diagnose the subtype of rejection. These novel biomarkers may potentially alleviate the need for an endomyocardial biopsy while facilitating the initiation of targeted therapy based on the noninvasive diagnosis of ACR or AMR.

Reliability of prehospital real-time cellular video phone in assessing the simplified National Institutes Of Health Stroke Scale in patients with acute stroke: a novel telemedicine technology.

BACKGROUND AND PURPOSE: The National Institutes of Health Stroke Scale (NIHSS) is the gold standard to assess patients with acute stroke. We aimed to examine the feasibility and reliability of prehospital real-time cellular video phone (VP) in performing the NIHSS. METHODS: Forty physicians prospectively performed a simplified NIHSS (sNIHSS) on a standardized patient remotely using VP with the assistance of a bedside emergency medical technician and later performed a bedside examination. We tested the hypothesis that there is high reliability between these 2 methods. Physicians were timed and sNIHSS scores were recorded. Finally, physicians were asked to rate the VP technology. RESULTS: A total of 480 pair comparisons of the sNIHSS scores between the VP and bedside examination were generated. After adjusting for the physician's specialty, level of training, and certification status, there was a strong positive linear correlation (r=0.97, P < 0.01) between the 2 methods with high average physician reliability (0.99; 95% CI, 0.992 to 0.995). The mean sNIHSS scores using VP and bedside examination were not different (6.82 ± 1.06 versus 6.63 ± 0.98; P=0.08). The mean time to perform the sNIHSS using VP was approximately 38 seconds longer than the bedside examination (3.38 ± 0.77 versus 2.93 ± 0.83 minutes; P=0.006). CONCLUSIONS: The VP is a feasible, reliable, and timely tool with the potential for remotely assessing the sNIHSS for patients presenting with acute stroke and may expedite the initial evaluation and treatment strategies.

Cellular video-phone assisted transmission and interpretation of prehospital 12-lead electrocardiogram in acute st-segment elevation myocardial infarction.

BACKGROUND: Prehospital 12-lead electrocardiogram (ECG) reduces the time to reperfusion in acute ST-segment elevation myocardial infarction (STEMI). However, the reliability of using cellular video-phone (VP) assisted interpretation of ECG is unknown. METHODS: We studied the interphysician reliability in interpreting the ECG assisted with VP compared to print ECG interpretation. Twenty-seven physicians prospectively interpreted the ECG transmitted from the field in real-time using VP and later using the same printed ECG. The time to completion, accuracy of interpretation, and physician rating of the VP technology were recorded. RESULTS: Similar high interphysician reliability was observed with both VP assisted and printed ECG interpretation including presence of ST-segment elevation (intraclass correlation coefficient [ICC]= 0.98 [95% CI 0.96-1] vs. 0.99 [95% CI 0.99-1]) and pathologic Q wave (ICC = 0.99 [95% CI 0.98-1] vs. 1 [95% CI 1]), respectively. The mean time to transmit and interpret the ECG with VP versus printed ECG was 3.9 ± 1.9 versus 2.1 ± 0.9 minutes, respectively, P < 0.01. On a scale of 1 to 5 with 5 being the best, the average rating of VP ease of use was 4.4 ± 0.5 and utility to recommend treatment was rated a 5. CONCLUSION: Cellular VP-assisted transmission and interpretation in real-time of prehospital ECG has high interphysician reliability, similar to the printed ECG interpretation. Future studies testing whether VP decreases the ischemic time and expedites the reperfusion of STEMI patients are needed.

From Hip to Heart: A Comprehensive Evaluation of an Infiltrative Cardiomyopathy.

Infiltrative cardiomyopathies are an increasingly recognized cause of heart failure warranting systematic evaluation. Given overlap of clinical and imaging findings among etiologies of infiltrative cardiomyopathies, comprehensive evaluation, including a history and physical examination, advanced cardiac imaging, and sometimes endomyocardial biopsy, is required for diagnosis. We report a case of infiltrative cardiomyopathy in which endomyocardial biopsy confirmed diagnosis of cobalt-induced cardiomyopathy. The novel teaching points highlighted by this case report include identification of heavy-metal toxicity as a cause of infiltrative cardiomyopathy, and the outline of a diagnostic approach and management for cobalt-induced cardiomyopathy.

Le fait que les cardiomyopathies infiltrantes sont de plus en plus reconnues comme la cause de l'insuffisance cardiaque justifie une évaluation systématique. Puisque les résultats cliniques et d'imagerie se recoupent entre les étiologies des cardiomyopathies infiltrantes, l'évaluation exhaustive, y compris les antécédents et l'examen physique, les techniques avancées en imagerie cardiaque et parfois la biopsie endomyocardique, est nécessaire au diagnostic. Nous présentons un cas de cardiomyopathie infiltrante pour lequel la biopsie endomyocardique a permis de confirmer le diagnostic d'une cardiomyopathie induite par le cobalt. Parmi les points à enseigner illustrés dans cette observation, on cite la reconnaissance de la toxicité des métaux lourds comme une cause de cardiomyopathie infiltrante, et la vue d'ensemble de l'approche diagnostique et de la prise en charge de la cardiomyopathie induite par le cobalt.

Autoimmune Myocarditis Caused by Immune Checkpoint Inhibitors Treated With Antithymocyte Globulin.

The immune checkpoint inhibitors have brought about a paradigm shift in the treatment of many cancers and are being used as the first line therapy in increasing number of aggressive malignancies, including metastatic melanoma. Their adverse effects, mostly mediated by an uncontrolled overactivation of the immune system, may compromise the therapeutic benefit. Combination immune checkpoint therapies in particular, have higher therapeutic efficacy, but have also been associated with a higher incidence of severe immune-related adverse effects including autoimmune lymphocytic myocarditis. Recent clinical reports of this rare and life threatening condition indicated rapid progression of severe hemodynamic and electrical instability, with or without acute decompensated heart failure, reduced ejection fraction and shock, pointing to the need for early recognition, diagnosis and prompt management. Current guidelines for management of other immune-related adverse effects recommend high-dose glucocorticoids, with consideration of immunomodulators, such as infliximab in patients with severe colitis. However, knowledge about the treatment approaches in immune-related myocarditis remains extremely scarce. Here we report a case of severe, steroid refractory, lymphocytic myocarditis that occurred after the first cycle of combination immunotherapy with the programmed cell death protein-1 inhibitor, nivolumab, and the cytotoxic T-lymphocyte-associated protein 4 blocker, ipilimumab, for metastatic melanoma. We discuss treatment approaches including the role for transvenous pacemaker, advanced heart failure support, and interdisciplinary decision making.

Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.