Primary Ciliary Dyskinesia Diagnostic Challenges: Understanding the Clinical Phenotype of the Puerto Rican RSPH4A Founder Mutation

Primary ciliary dyskinesia (PCD) is a rare, heterogeneous ciliopathy resulting in chronic oto-sino-pulmonary disease, bronchiectasis, newborn respiratory distress, and laterality defects. PCD diagnosis can be achieved by following diagnostic algorithms that include electron microscopy, genetics, and ancillary testing. Genetic mutations in more than 45 genes, including RSPH4A, can lead to PCD. RSPH4A mutations located on chromosome six, affect radial spokes and results in central complex apparatus abnormalities. The RSPH4A [c.921 + 3_6delAAGT] founder mutation was described as one cause of PCD without laterality defects in Puerto Rico. Additionally, there are further diagnostic challenges present in the Puerto Rican population to diagnose PCD. We describe the demographics, clinical features, and RSPH4A genetic variants in 13 patients with clinical PCD affecting 11 Puerto Ricans from unrelated families.


Introduction
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder characterized by motile cilia dysfunction with a prevalence of approximately 1:15,000 individuals in the United States [1]. The actual prevalence of PCD in Puerto Rico is unknown. Genetic mutations in more than 45 human genes can lead to PCD, affecting the function of around 200 ciliary structural proteins [2]. These abnormalities result phenotypically in neonatal respiratory distress in approximately 80% despite a full-term gestation, oto-sino-pulmonary disease since birth, infertility, and organ laterality defects in 40-50% of cases [3]. Diagnostic algorithms for suspected PCD patients recommend evaluation of individuals with at least 2 of the 4 key clinical features including unexplained neonatal respiratory distress in term infants, year-round daily cough and/or daily nasal congestion beginning before 6 months of age, and laterality defects [4,5].
PCD diagnosis has become a challenge for physicians since it is easily misdiagnosed with similar genetic syndromes such as cystic fibrosis and other chronic suppurative lung diseases [6]. Previous studies have shown that approximately 1 of 3 patients have a normal ciliary structure on electron microscopy, which increases the difficulty in confirming the PCD diagnosis [7]. As a key diagnostic test, electron microscopy evaluates the 9 + 2 ciliary normal configuration and assesses for ciliary ultrastructure defects. Updated guidelines incorporate genetic testing as part of the algorithm for PCD diagnosis [4]. Additional PCD mutations have been discovered as genetic testing has become more accessible to healthcare providers and patients.
Biallelic pathogenic variants in the RSPH4A gene, located on chromosome 6, affect ciliary radial spokes which alter the configuration of the central complex apparatus [8]. Mutations in RSPH4A affect the normal planar beating pattern of the cilia into a circular 2 of 9 ciliary motion [9]. The abnormal ciliary function impairs effective mucociliary clearance in PCD patients [10]. PCD mutations may interfere with the placement of the left-right organ, which is established during embryogenesis [11]. Normally, the embryonic node cilium lacks a central pair of microtubules following a 9 + 0 axonemal structure, which specifies the left-right axis [12]. Therefore, PCD mutations that affect radial spokes, like RSPH4A, will not present laterality defects like dextrocardia or complete situs inversus [13]. RSPH4A [c.921 + 3_6delAAGT] founder mutation was described as a PCD pathogenic variant without laterality defects. The splice site mutation [c.921 + 3_6delAAGT] in RSPH4A has been described as a common cause of PCD in the Hispanic population, specifically Puerto Rico [14]. We aim to describe our experience with clinical features, pulmonary function test, chest imaging, genetic profile, and electron microscopy findings diagnosing native Puerto Ricans with RSPH4A mutations. A better understanding of the clinical-genetic correlation of RSPH4A genetic variants will help to expose the diagnostic challenges for PCD patients without laterality defects in Puerto Rico.

Materials and Methods
A descriptive retrospective chart review of 31 Puerto Rican subjects was completed. Patients were followed up at a single private outpatient clinic in Puerto Rico with expertise in PCD from September 2018 to September 2020. Detailed histories, physical examinations, pulmonary function tests, imaging studies, and nasal ciliary biopsies were completed. Criteria for PCD evaluation were achieved by documenting the presence of 2 of the 4 key clinical features described on the suggested diagnostic algorithm as previously published [5]. Measurement of nasal nitric oxide (nNO) was not available in our institution. The sweat test was completed to rule out cystic fibrosis. Genetic testing was evaluated using commercially available sequence analysis (Invitae Corporation, San Francisco, CA, USA), using buccal swabs or saliva samples. Gene panel testing evaluated for deletion/duplication on 35 PCD-related genes. The cystic fibrosis transmembrane conductance regulator (CFTR) gene was also evaluated as part of the initial patient PCD evaluation. Genetic testing included a sequence of exons and introns for the RSPH4A gene (Transcript: NM_001010892.2). Nasal biopsies were submitted for pathology review at Focus Pathology Medical Laboratories in Chester, New York. A total of thirteen (n = 13) PCD patients with the presence of RSPH4A mutations were included in the analysis. High-Resolution chest CT (HRCT) scans were reviewed, if available. Pulmonary function tests were completed in accordance with the American Thoracic Society guidelines [15]. Airflow limitation was determined using Zscore values of −1.64 as the lower limit of normal. The Global Lung Function Initiative-2012 (GLI) was used as a reference equation. Descriptive statistics presented in percentages and means. The Institutional Review Boards approved data collection and analysis for the Protection of Human Subjects from University of Puerto Rico, Medical Sciences Campus in San Juan, Puerto Rico.

Electron Microscopy Findings
Ciliary biopsies were abnormal in 100%, (11/11). Abnormal findings on electron microscopy were related to central apparatus defects and abnormal microtubule configuration, including loss of central and outer doublets, presence of central solitary microtubule, and extra gain of central singlets, triplets, and quadruplets ( Figure 1). two females homozygous for RSPH4A [c.921 + 3_6delAAGT] were sexually active and self-reported to be diagnosed with infertility. Laterality defects were present in 0%, (0/13) of the subjects with RSPH4A mutations (Table 1 and Table A1).

Electron Microscopy Findings
Ciliary biopsies were abnormal in 100%, (11/11). Abnormal findings on electron microscopy were related to central apparatus defects and abnormal microtubule configuration, including loss of central and outer doublets, presence of central solitary microtubule, and extra gain of central singlets, triplets, and quadruplets ( Figure 1).

RSPH4A Genetic Sequencing
RSPH4A genetic variants were identified in all 13 subjects from 11 unrelated families. Biallelic pathogenic variants in RSPH4A gene were noted in 62%, (8/13) subjects of the cohort (Table A1)

Imaging
HRCT were available for review in a total of 9 of 10 patients. Homozygous RSPH4A [c.921 + 3_6delAAGT] patients displayed bronchiectasis on 100% (8/8) of those above age seven. Case 9 and 10 with compound heterozygous genetic variants for RSPH4A displayed bronchiectasis on HRCT. The most common findings on HRCT were: cylindrical and varicose bilateral bronchiectasis, bibasilar parenchymal scarring tissue, centrilobular nodules, and tree-in-bud opacities. Representative views of the HRCT of both pediatric and adult PCD patients who are homozygous, compound heterozygous, or VUS for RSPH4A are presented in Figure 2.

Discussion
Diagnostic challenges in rare lung disorders like PCD could be numerous depending on the prevalence, clinical phenotype, and genetic variability of the population to be studied. In Puerto Rico, there is a lack of information about rare lung diseases, including PCD. The actual prevalence of PCD on the island is unknown. Considering a prevalence of PCD of 1:15,000 and the actual population of Puerto Rico to be around 2.8 million individuals, we can estimate a population of 187 patients living with PCD on the island. This estimation doesn't count the presence of a founder genetic mutation on the RSPH4A gene previously described [14], which may contribute to an increased amount of patients living in Puerto Rico with PCD. The fact that in 24 months our clinic was able to diagnose a total of 13 patients with RSPH4A, shows that PCP in Puerto Rico has been underdiagnosed. We understand that genetic testing and electron microscopy are limitations for the Puerto Rican population to follow the established diagnostic algorithms as recommended by the PCD foundation in the United States. The lack of nNO test as a screening and diagnostic PCD tool for Puerto Rico and the Caribbean are limitations in some challenging cases. The genetic variability of the Puerto Rican population introduces additional challenges as commercial genetic testing panels with 35 genes may not be able to unmask rare PCD genetic variants as pathogenic to confirm the diagnosis when more than 45 [14,16], we saw fewer chronic secretory otitis media cases. Central complex defects on electron microscopy have been associated with severe otologic features in children with PCD [17]. Our results showed that central complex defects were present in 100% of the cohort. However, the severity of otologic features was not as prevalent as expected. Laterality defects were absent in our cohort as previously documented on mutations that affect the central complex apparatus. We know that RSPH4A mutations are not necessarily a requirement for abnormal left-right axis orientation [12].
The genetic diversity of Puerto Rican ancestry may explain the range and combination of VUS genetic variants found. Three patients (Cases 4, 7 and 8) were "purely" homozygous for the RSPH4A [c.921 + 3_6delAAGT] mutation. All other patients had a combination of pathogenic RSPH4A with other PCD related genetic heterozygous VUS. The interactions of VUS with pathogenic PCD variants are unknown. Additional studies are needed to explore the role of digenic inheritance in PCD as seen in other ciliopathies [18]. On the other hand, we show in Table A1,  Another atypical patient is presented in Case 10, which exhibited two of the main features: daily wet cough and year-round daily nasal congestion but lack laterality defects or recurrent secretory otitis media. Electron microscopy findings showed central apparatus defects and HRCT revealed extensive bilateral bronchiectasis, reticular-nodular infiltrates with tree-in-bud changes at RML and RLL (Figure 2c). Pulmonary function test demonstrated severe airflow obstruction with an FEV1: 24%. Genetic testing resulted in three VUS genetic variants including RSPH4A, DNAAF3, and DNAH11. Although none of these genetic VUS had been associated with PCD pathogenic phenotype; the medical history, clinical findings, electron microscopy, and the severity of her pulmonary disease evidenced by pulmonary function test and imaging, highly suggest PCD. Ancillary testing like nNO, if available, will help to confirm challenges in PCD cases like this. Two rare RSPH4A variants were detected in our cohort. First, the RSPH4A [c.1103T>G], which replace a valine with glycine at the codon 368 of the RSPH4A protein. This missense mutation found in Case 9 promotes physicochemical differences between both amino acids. Although the effect of this missense mutation is unclear, this variant is currently classified as pathogenic and seen in individuals with PCD phenotype. The second variant found on Case 10 is RSPH4A [c.902A > C], which replace glutamine with proline at the codon 301. This missense mutation was not previously reported on individuals with RSPH4A-related diseases and is currently classified as VUS. Additional testing and evaluation including nNO and electron microscopy will be needed to confirm the diagnosis of PCD.

Conclusions
In Puerto Rico, PCD diagnosis remains under-recognized by the general population and healthcare providers. To understand the presence of a PCD founder mutation on the island, it is important to address the medical history of recurrent pulmonary infections, bronchiectasis, and sinus disease during the initial encounter. Although asthma may be a concomitant disease in PCD, a detailed history of PCD symptoms is vital in those with Diagnostics 2021, 11, 281 7 of 9 severe persistent asthma not responding to standard care. Referral to knowledgeable medical doctors with PCD expertise is imperative for early patient diagnosis that will prevent or delay additional PCD comorbidities later in life. Awareness about PCD clinical features on the island must be a priority for early recognition of potential PCD cases. Collaboration among neonatologists, otorhinolaryngologists, pediatricians, infertility clinics, pulmonologists, and radiologists, among other providers, is essential to reveal the actual prevalence of PCD on the island. The presence of a founder mutation in RSPH4A warrants the development of a multidisciplinary center for PCD for the evaluation of suspected cases in Puerto Rico. The development of a PCD center is urgently needed to explore new founder mutations in the genetic pool of individuals living in the Caribbean and Latin-America using state-of-the-art diagnostic tools following ATS and PCD foundation diagnosis guidelines. Collaboration with PCD Centers in the United States is important considering that, to date, we do not have the capacity for lung transplantation in Puerto Rico and lung transplantation in Latin-America is limited.  Informed Consent Statement: Patient consent was waived by IRB due to the methodology used in this article: a retrospective chart review.

Data Availability Statement:
All data analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.