Omar Khalifa

High Prevalence of Respiratory Ciliary Dysfunction in Congenital Heart Disease Patients With Heterotaxy

Background— Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. Methods and Results— We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. Conclusions— Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.

Airway ciliary dysfunction and sinopulmonary symptoms in patients with congenital heart disease

RATIONALE Patients with congenital heart disease with heterotaxy exhibit a high prevalence of abnormal airway ciliary motion and low nasal nitric oxide, characteristics associated with primary ciliary dyskinesia, a reflection of the role of motile cilia in airway clearance and left-right patterning. OBJECTIVES To assess the potential broader clinical significance of airway ciliary dysfunction in congenital heart disease, we assessed the prevalence of ciliary dysfunction versus respiratory symptoms in patients with congenital heart disease with or without heterotaxy. METHODS Patients with a broad spectrum of congenital heart disease were recruited (n = 218), 39 with heterotaxy. Nasal nitric oxide measurements and nasal biopsies for ciliary motion video microscopy were conducted. Sinopulmonary symptoms were reviewed by questionnaire. MEASUREMENTS AND MAIN RESULTS A high prevalence of ciliary motion defects (51.8%) and low or borderline low nasal nitric oxide levels (35.5%) were observed in patients with congenital heart disease with or without heterotaxy. Patients with ciliary motion defects or low nasal nitric oxide showed increased sinopulmonary symptoms, with most respiratory symptoms seen in those with both abnormal ciliary motion and low nitric oxide. Multivariate analysis showed that abnormal ciliary motion and low nasal nitric oxide were more important in determining risk of sinopulmonary symptoms than heterotaxy status. CONCLUSIONS Patients with congenital heart disease without heterotaxy exhibit a high prevalence of abnormal ciliary motion and low nasal nitric oxide. This was associated with more sinopulmonary symptoms. These findings suggest that patients with a broad spectrum of congenital heart disease and respiratory symptoms may benefit from screening for ciliary dysfunction and implementation of medical interventions to reduce sinopulmonary morbidities.

The complex genetics of hypoplastic left heart syndrome

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR–Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.

Respiratory motile cilia dysfunction in a patient with cranioectodermal dysplasia

Ciliopathies such as cranioectodermal dysplasia, Sensenbrenner syndrome, short‐rib polydactyly, and Jeune syndrome are associated with respiratory complications arising from rib cage dysplasia. While such ciliopathies have been demonstrated to involve primary cilia defects, we show motile cilia dysfunction in the airway of a patient diagnosed with cranioectodermal dysplasia. While this patient had mild thoracic dystrophy not requiring surgical treatment, there was nevertheless newborn respiratory distress, restrictive airway disease with possible obstructive airway involvement, repeated respiratory infections, and atelectasis. High‐resolution videomicroscopy of nasal epithelial biopsy showed immotile/dyskinetic cilia and nasal nitric oxide was reduced, both of which are characteristics of primary ciliary dyskinesia, a sinopulmonary disease associated with mucociliary clearance defects due to motile cilia dysfunction in the airway. Exome sequencing analysis of this patient identified compound heterozygous mutations in WDR35, but no mutations in any of the 30 known primary ciliary dyskinesia genes or other cilia‐related genes. Given that WDR35 is only known to be required for primary cilia function, we carried out WDR35 siRNA knockdown in human respiratory epithelia to assess the role of WDR35 in motile cilia function. This showed WDR35 deficiency disrupted ciliogenesis in the airway, indicating WDR35 is also required for formation of motile cilia. Together, these findings suggest patients with WDR35 mutations have an airway mucociliary clearance defect masked by their restrictive airway disease.

Brain Dysplasia Associated with Ciliary Dysfunction in Infants with Congenital Heart Disease

OBJECTIVE To test for associations between abnormal respiratory ciliary motion (CM) and brain abnormalities in infants with congenital heart disease (CHD) STUDY DESIGN: We recruited 35 infants with CHD preoperatively and performed nasal tissue biopsy to assess respiratory CM by videomicroscopy. Cranial ultrasound scan and brain magnetic resonance imaging were obtained pre- and/or postoperatively and systematically reviewed for brain abnormalities. Segmentation was used to quantitate cerebrospinal fluid and regional brain volumes. Perinatal and perioperative clinical variables were collected. RESULTS A total of 10 (28.5%) patients with CHD had abnormal CM. Abnormal CM was not associated with brain injury but was correlated with increased extraaxial cerebrospinal fluid volume (P < .001), delayed brain maturation (P < .05), and a spectrum of subtle dysplasia including the hippocampus (P < .0078) and olfactory bulb (P < .034). Abnormal CM was associated with higher composite dysplasia score (P < .001), and both were correlated with elevated preoperative serum lactate (P < .001). CONCLUSIONS Abnormal respiratory CM in infants with CHD is associated with a spectrum of brain dysplasia. These findings suggest that ciliary defects may play a role in brain dysplasia in patients with CHD and have the potential to prognosticate neurodevelopmental risks.

Airway ciliary dysfunction and respiratory symptoms in patients with transposition of the great arteries

Background Our prior work on congenital heart disease (CHD) with heterotaxy, a birth defect involving randomized left-right patterning, has shown an association of a high prevalence of airway ciliary dysfunction (CD; 18/43 or 42%) with increased respiratory symptoms. Furthermore, heterotaxy patients with ciliary dysfunction were shown to have more postsurgical pulmonary morbidities. These findings are likely a reflection of the common role of motile cilia in both airway clearance and left-right patterning. As CHD comprising transposition of the great arteries (TGA) is commonly thought to involve disturbance of left-right patterning, especially L-TGA with left-right ventricular inversion, we hypothesize CHD patients with transposition of great arteries (TGA) may have high prevalence of airway CD with increased respiratory symptoms. Methods and results We recruited 75 CHD patients with isolated TGA, 28% L and 72% D-TGA. Patients were assessed using two tests typically used for evaluating airway ciliary dysfunction in patients with primary ciliary dyskinesia (PCD), a recessive sinopulmonary disease caused by respiratory ciliary dysfunction. This entailed the measurement of nasal nitric oxide (nNO), which is typically low with PCD. We also obtained nasal scrapes and conducted videomicroscopy to assess respiratory ciliary motion (CM). We observed low nNO in 29% of the patients, and abnormal CM in 57%, with 22% showing both low nNO and abnormal CM. No difference was observed for the prevalence of either low nNO or abnormal ciliary motion between patients with D vs. L-TGA. Respiratory symptoms were increased with abnormal CM, but not low nNO. Sequencing analysis showed no compound heterozygous or homozygous mutations in 39 genes known to cause PCD, nor in CFTR, gene causing cystic fibrosis. As both are recessive disorders, these results indicate TGA patients with ciliary dysfunction do not have PCD or cystic fibrosis (which can cause low nNO or abnormal ciliary motion). Conclusions TGA patients have high prevalence of abnormal CM and low nNO, but ciliary dysfunction was not correlated with TGA type. Differing from PCD, respiratory symptoms were increased with abnormal CM, but not low nNO. Together with the negative findings from exome sequencing analysis, this would suggest TGA patients with ciliary dysfunction do not have PCD but nevertheless may suffer from milder airway clearance deficiency. Further studies are needed to investigate whether such ciliary dysfunction is associated with increased postsurgical complications as previously observed in CHD patients with heterotaxy.

Low Nasal NO in Congenital Heart Disease With Systemic Right Ventricle and Postcardiac Transplantation

Background NO bioavailability has not been systematically examined in congenital heart disease (CHD). To assess NO in patients with CHD, we measured nasal NO (nNO) generated by the nasal epithelia, given blood NO is difficult to measure (half‐life, <2 ms). Given NOs role in hemodynamic regulation and the association of NO bioavailability with heart failure risk, we hypothesized NO levels may differ with varying severity of CHD physiologic characteristics. Methods and Results Six‐hundred eighteen subjects, 483 with CHD and 135 controls, had nNO measured noninvasively via the nares using American Thoracic Society/European Respiratory Society guidelines. Subjects were dichotomized as having low or normal nNO based on age‐specific cutoff values. Prevalence of low nNO was examined by various CHD physiologic feature types. Low nNO was more prevalent with CHD than controls (odds ratio, 2.28; P=0.001). A logistic regression model showed overall significance (P=0.035) for single ventricle, systemic right ventricle, ventricular dysfunction, oxygen desaturation, and heterotaxy predicting low nNO, with systemic right ventricle independently having twice the odds of low nNO (odds ratio, 2.04; P=0.014). Patients with low nNO had a higher risk of experiencing heart transplant or death (hazard ratio, 2.75; P=0.048), and heart transplant recipients (N=16) exhibited 5 times the odds of low nNO (69% versus 30%; odds ratio, 5.1; P=0.001). Conclusions Patients with CHD have increased prevalence of low nNO, with highest odds seen with systemic right ventricle and heart transplant. Further studies are needed to investigate heart failure risks in patients with CHD with left versus right systemic ventricle physiologic characteristics and utility of low nNO for predicting heart failure risk.

HIGH PREVALENCE OF RESPIRATORY CILIARY DYSFUNCTION ASSOCIATED WITH TRANSPOSITION OF THE GREAT ARTERIES

Heterotaxy patients have randomized left-right patterning and exhibit a high incidence of airway ciliary dysfunction similar to that of primary ciliary dyskinesia (PCD), a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. Transposition of the great