Maimoona A. Zariwala

Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left–right asymmetry

Primary ciliary dyskinesia (PCD, MIM 242650) is characterized by recurrent infections of the respiratory tract due to reduced mucociliary clearance and by sperm immobility. Half of the affected offspring have situs inversus (reversed organs), which results from randomization of left-right (LR) asymmetry. We previously localized to chromosome 5p a PCD locus containing DNAH5, which encodes a protein highly similar to the Chlamydomonas γ-dynein heavy chain. Here we characterize the full-length 14-kb transcript of DNAH5. Sequence analysis in individuals with PCD with randomization of LR asymmetry identified mutations resulting in non-functional DNAH5 proteins.

Congenital Heart Disease and Other Heterotaxic Defects in a Large Cohort of Patients With Primary Ciliary Dyskinesia

Background— Primary ciliary dyskinesia (PCD) is a recessive genetic disorder that is characterized by sinopulmonary disease and reflects abnormal ciliary structure and function. Situs inversus totalis occurs in ≈50% of PCD patients (Kartagener’s syndrome in PCD), and there are a few reports of PCD with heterotaxy (situs ambiguus), such as cardiovascular anomalies. Advances in diagnosis of PCD, such as genetic testing, allow the systematic investigation of this association. Methods and Results— The prevalence of heterotaxic defects was determined in 337 PCD patients by retrospective review of radiographic and ultrasound data. Situs solitus (normal situs) and situs inversus totalis were identified in 46.0% and 47.7% of patients, respectively, and 6.3% (21 patients) had heterotaxy. As compared with patients with situs solitus, those with situs abnormalities had more ciliary outer dynein arm defects, fewer inner dynein arm and central apparatus defects (P<0.001), and more mutations in ciliary outer dynein arm genes (DNAI1 and DNAH5; P=0.022). Seven of 12 patients with heterotaxy who were genotyped had mutations in DNAI1 or DNAH5. Twelve patients with heterotaxy had cardiac and/or vascular abnormalities, and most (8 of 12 patients) had complex congenital heart disease. Conclusions— At least 6.3% of patients with PCD have heterotaxy, and most of those have cardiovascular abnormalities. The prevalence of congenital heart disease with heterotaxy is 200-fold higher in PCD than in the general population (1:50 versus 1:10 000); thus, patients with PCD should have cardiac evaluation. Conversely, mutations in genes that adversely affect both respiratory and embryological nodal cilia are a significant cause of heterotaxy and congenital heart disease, and screening for PCD is indicated in those patients.

Clinical and genetic aspects of primary ciliary dyskinesia/kartagener syndrome

Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (dynein axonemal heavy chain 5) or intermediate (dynein axonemal intermediate chain 1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for primary ciliary dyskinesia is available for the most common mutations. The respiratory manifestations of primary ciliary dyskinesia (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis, and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of patients with primary ciliary dyskinesia have laterality defects (including situs inversus totalis and, less commonly, heterotaxy, and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most patients with primary ciliary dyskinesia have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with primary ciliary dyskinesia.

A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort

Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening.Methods: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort.Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants.Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.Purpose: To determine short–term effects of intravitreal bevacizumab for subfoveal choroidal neovascularization (CNV) in pathologic myopia. Methods: In this prospective interventional case series, patients were treated with 2.5 mg of intravitreal bevacizumab and followed for 3 months. Best-corrected visual acuity (BCVA), optical coherence tomography (OCT), and fluorescein angiography (FA) were recorded. Indications for retreatment were active leaking CNV shown by FA and presence of subretinal fluid by OCT in combination with visual disturbances. Results: Fourteen patients were included, with a mean age of 53.86 ± 16.26 years (range 29–85). Mean spherical equivalent was −13.87 ± 3.68 diopters (−7.25 to −20.50). Minimum follow-up was 3 months. There were no adverse events. The mean initial visual acuity was 20/200 improving to 20/100 at 2 weeks, 20/80 at 4 weeks, and 20/60 at 8 and 12 weeks (P = 0.007; P = 0.001; P = 0.005; P = 0.001, respectively). Initial foveal thickness improved from 385.43 &mgr;m ± 125.83 &mgr;m to 257.64 ± 76.6 &mgr;m and 194.54 ± 54.35 &mgr;m after the first and third month, respectively (P = 0.001). Conclusions: Initial treatment results of patients with CNV due to pathologic myopia did not reveal any short-term safety concerns. Intravitreal bevacizumab resulted in a significant decrease in foveal thickness and improvement in visual acuity. These favorable initial results support further larger and long-term studies.

Deletions and point mutations of LRRC50 cause primary ciliary dyskinesia due to dynein arm defects.

Genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility in primary ciliary dyskinesia (PCD). The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Here, we demonstrate that large genomic deletions, as well as point mutations involving LRRC50, are responsible for a distinct PCD variant that is characterized by a combined defect involving assembly of the ODAs and IDAs. Functional analyses showed that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5- and DNAI2-containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia. On the basis of these findings, we assume that LRRC50 plays a role in assembly of distinct dynein-arm complexes.

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

High-Resolution CT of Patients with Primary Ciliary Dyskinesia

OBJECTIVE High-resolution CT is an important tool in the detection and management of bronchiectasis, but there is little information about high-resolution CT findings in primary ciliary dyskinesia (PCD). We analyzed all high-resolution CT studies of the chest available for a cohort of PCD patients to identify an associated pattern of high-resolution CT changes. MATERIALS AND METHODS High-resolution CT studies were available for 45 PCD patients from 42 families with ranges of age and disease severity. The images were assessed for severity and distribution of bronchiectasis, peribronchial thickening, mucous plugging, and other findings. A bronchiectasis severity score was calculated. CT findings were correlated with phenotypic findings, including situs type, ciliary ultrastructural defect, nasal level of nitric oxide, forced expiratory volume in 1 second, and microbiologic findings in the airways. RESULTS Twenty-nine adults (mean age, 42 +/- 15 years; age range, 21-73 years) and 16 children (mean age, 8 +/- 4 years; age range, 1-14 years) were included; 26 (58%) of the patients were women or girls. Situs inversus totalis (38%) or heterotaxy (18%) was identified in 56% of the patients. A high (9%) prevalence of pectus excavatum was identified. High-resolution CT of all of the adult and 56% of the pediatric patients showed bronchiectasis in a predominantly middle and lower lobe distribution. The right middle lobe was most commonly involved. Bronchiectasis severity score correlated with older age and worse pulmonary function. CONCLUSION High-resolution CT shows that pulmonary disease related to PCD predominantly involves the middle and lower lobes of the lungs. In adults, high-resolution CT findings negative for bronchiectasis may have a role in excluding the diagnosis of PCD. Correlation of severity of disease on high-resolution CT with patient phenotype gives further insight into the diversity and natural history of PCD.

Standardizing Nasal Nitric Oxide Measurement as a Test for Primary Ciliary Dyskinesia

RATIONALE Several studies suggest that nasal nitric oxide (nNO) measurement could be a test for primary ciliary dyskinesia (PCD), but the procedure and interpretation have not been standardized. OBJECTIVES To use a standard protocol for measuring nNO to establish a disease-specific cutoff value at one site, and then validate at six other sites. METHODS At the lead site, nNO was prospectively measured in individuals later confirmed to have PCD by ciliary ultrastructural defects (n = 143) or DNAH11 mutations (n = 6); and in 78 healthy and 146 disease control subjects, including individuals with asthma (n = 37), cystic fibrosis (n = 77), and chronic obstructive pulmonary disease (n = 32). A disease-specific cutoff value was determined, using generalized estimating equations (GEEs). Six other sites prospectively measured nNO in 155 consecutive individuals enrolled for evaluation for possible PCD. MEASUREMENTS AND MAIN RESULTS At the lead site, nNO values in PCD (mean ± standard deviation, 20.7 ± 24.1 nl/min; range, 1.5-207.3 nl/min) only rarely overlapped with the nNO values of healthy control subjects (304.6 ± 118.8; 125.5-867.0 nl/min), asthma (267.8 ± 103.2; 125.0-589.7 nl/min), or chronic obstructive pulmonary disease (223.7 ± 87.1; 109.7-449.1 nl/min); however, there was overlap with cystic fibrosis (134.0 ± 73.5; 15.6-386.1 nl/min). The disease-specific nNO cutoff value was defined at 77 nl/minute (sensitivity, 0.98; specificity, >0.999). At six other sites, this cutoff identified 70 of the 71 (98.6%) participants with confirmed PCD. CONCLUSIONS Using a standardized protocol in multicenter studies, nNO measurement accurately identifies individuals with PCD, and supports its usefulness as a test to support the clinical diagnosis of PCD.

Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure

Rationale Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11). Objectives To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology. Methods 82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease. Results Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations. Conclusions Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.

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.