Unveiling the genetic etiology of primary ciliary dyskinesia: When standard genetic approach is not enough.

PURPOSE: Primary ciliary dyskinesia (PCD) is a ciliopathy caused by dysfunction of motile cilia. As there is still no standard PCD diagnostics, the final diagnosis requires a combination of several tests. The genetic screening is a hallmark for the final diagnosis and requires high-throughput techniques, such as whole-exome sequencing (WES). Nevertheless, WES has limitations that may prevent a definitive genetic diagnosis. Here we present a case that demonstrates how the PCD genetic diagnosis may not be trivial. MATERIALS/METHODS: A child with PCD and situs inversus totalis (designated as Kartagener syndrome (KS)) was subjected to clinical assessments, ultrastructural analysis of motile cilia, extensive genetic evaluation by WES and chromosomal array analysis, bioinformatic analysis, gene expression analysis and immunofluorescence to identify the genetic etiology. His parents and sister, as well as healthy controls were also evaluated. RESULTS: Here we show that a disease-causing variant in the USP11 gene and copy number variations in CRHR1 and KRT34 genes may be involved in the patient PCD phenotype. None of these genes were previously reported in PCD patients and here we firstly show its presence and immunolocalization in respiratory cells. CONCLUSIONS: This work highlights how the genetic diagnosis can turn to be rather complex and that combining several approaches may be needed. Overall, our results contribute to increase the understanding of the genetic factors involved in the pathophysiology of PCD/KS, which is of paramount importance to assist the current diagnosis and future development of newer therapies.

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex.

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.

New adenylate kinase 7 (AK7) mutation in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a congenital hereditary disease affecting 1/20,000-60,000 people that causes chronic sinusitis, bronchiectasis, sinus hypoplasia, secretory otitis media, and low fertility. The complexity and heterogeneity of the disease make diagnosis difficult. Although the genetic origin of PCD is clear, mutations in only five genes have been associated with the disease, and, to date, no disease-causing gene has been identified. Recently, low levels of AK7 gene expression have been linked to PCD. This study was designed to determine the mutational status of the AK7 gene in 31 PCD (17 PCD and 14 Kartagener syndrome diagnosed) patients compared with 40 healthy volunteers. We also determined the AK7 sequence in two families with members with PCD and investigated ciliary activity and ciliogenesis in one patient with a mutation in AK7. METHODS: We analyzed nasal mucociliary transport and cilial ultrastructure by electron microscopy and studied nasal ciliary beat frequency and beat pattern using high-resolution digital high speed video (DHSV) imaging. Mutation analyses were performed by direct resequencing of the 18 exons of the AK7 gene. Air-liquid interface differentiated cultures were studied using DHSV imaging and histochemistry. AK7 gene expression was studied by real-time reverse-transcription polymerase chain reaction. RESULTS: We identified two mutations in the AK7 gene, the described single nucleotide polymorphism (rs2369679), and a new mutation (c.1214insT) that, to the best of our knowledge, has not been described previously. Family and functional studies indicated that c.1214insT could be related to PCD. CONCLUSION: Our results indicate that AK7 may be involved in the development of PCD.

Plastic bronchitis developing 5 years after Fontan procedure in a girl with Kartagener's syndrome.

Although rare, plastic bronchitis (PB) is an important early complication after Fontan procedure. Kartagener's syndrome is characterized by mucociliary dysfunction of the respiratory tract and has a triad of features, including situs inversus totalis, chronic sinusitis, and bronchiectasia. We experienced PB in a patient with Kartagener's syndrome 5 years after Fontan procedure.

Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1.

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. 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. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the ß strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD.

Primary ciliary dyskinesia in mice lacking the novel ciliary protein Pcdp1.

Primary ciliary dyskinesia (PCD) results from ciliary dysfunction and is commonly characterized by sinusitis, male infertility, hydrocephalus, and situs inversus. Mice homozygous for the nm1054 mutation develop phenotypes associated with PCD. On certain genetic backgrounds, homozygous mutants die perinatally from severe hydrocephalus, while mice on other backgrounds have an accumulation of mucus in the sinus cavity and male infertility. Mutant sperm lack mature flagella, while respiratory epithelial cilia are present but beat at a slower frequency than wild-type cilia. Transgenic rescue demonstrates that the PCD in nm1054 mutants results from the loss of a single gene encoding the novel primary ciliary dyskinesia protein 1 (Pcdp1). The Pcdp1 gene is expressed in spermatogenic cells and motile ciliated epithelial cells. Immunohistochemistry shows that Pcdp1 protein localizes to sperm flagella and the cilia of respiratory epithelial cells and brain ependymal cells in both mice and humans. This study demonstrates that Pcdp1 plays an important role in ciliary and flagellar biogenesis and motility, making the nm1054 mutant a useful model for studying the molecular genetics and pathogenesis of PCD.

Establishing a connection between cilia and Bardet-Biedl Syndrome.

Bardet-Biedl Syndrome (BBS) is a gentic disorder with primary features of retinal dystrophy, obesity, polydactyly, structural and functional renal abnormalities, and learning disabilities. In addition to displaying remarkable pleiotropy, BBS is a heterogeneous disorder with linkage to at least eight loci. The identification of the first five BBS genes provided little insight into BBS protein function. Ansley at al. have now identified a sixth BBS gene (BBS8) and provide evidence that the BBS8 protein and other BBS proteins localize to the basal body of ciliated cells, suggesting that BBS is a ciliary dysfunction disorder.

Absence of nexin links as a possible cause of primary ciliary dyskinesia.

Transmission electron microscopy of nasal cilia was performed in three patients, two of them siblings, with repeated respiratory infections. Number of microtubuli and dynein arms were within normal limits and they had an ordered arrangement except for a disarray of the microtubuli in some areas of the biopsies from two of the patients. In the normal areas radial spokes and sheaths were easily found but nexin links could not be discerned in any of the patients. The orientation of the cilia was partly random. As all patients repeatedly and constantly had very low nasal NO (range 9-15 ppb; normal findings for persons <10 years old are > 50 ppb), the diagnoses were very likely primary ciliary dyskinesia (PCD). Absence of nexin links may be an ultrastructural variant of PCD. Deficiency of these structures might be the cause of the microtubular disarray observed in some areas of the biopsies.

Nitric oxide in chronic airway inflammation in children: diagnostic use and pathophysiological significance.

BACKGROUND: The levels of exhaled and nasal nitric oxide (eNO and nNO) in groups of patients with inflammatory lung diseases are well documented but the diagnostic use of these measurements in an individual is unknown. METHODS: The levels of nNO and eNO were compared in 31 children with primary ciliary dyskinesia (PCD), 21 with non-CF bronchiectasis (Bx), 17 with cystic fibrosis (CF), 35 with asthma (A), and 53 healthy controls (C) using a chemiluminescence NO analyser. A diagnostic receiver-operator characteristic (ROC) curve for PCD using NO was constructed. RESULTS: The median (range) levels of nNO in parts per billion (ppb) in PCD, Bx, CF, and C were 60.3 (3.3-920), 533.6 (80-2053), 491.3 (31-1140), and 716 (398-1437), respectively; nNO levels were significantly lower in PCD than in all other groups (p<0.05). The median (range) levels of eNO in ppb in PCD, Bx, CF, A, and C were 2.0 (0.2-5.2), 5.4 (1.0-22.1), 2.6 (0.8-12.9), 10.7 (1.6-46.7), and 4.85 (2.5-18.3), respectively. The difference in eNO levels in PCD reached significance (p<0.05) when compared with those in Bx, A and C but not when compared with CF. Using the ROC curve, nNO of 250 ppb showed a sensitivity of 97% and a specificity of 90% for the diagnosis of PCD. CONCLUSIONS: eNO and nNO cannot be used diagnostically to distinguish between most respiratory diseases. However, nNO in particular is a quick and useful diagnostic marker which may be used to screen patients with a clinical suspicion of PCD.

Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus.

Primary ciliary dyskinesia (PCD), also known as Kartagener's syndrome, is a human syndrome that results from ciliary dysfunction. This syndrome is characterized by recurrent respiratory infections, situs inversus and infertility. In some cases, hydrocephalus is also observed. We have characterized an insertional mutation in a mouse axonemal dynein heavy chain gene (Mdnah5) that reproduces most of the classical features of PCD, including recurrent respiratory infections, situs inversus and ciliary immotility. These mice also suffer from hydrocephalus and die perinatally. Electron microscopic studies demonstrate the loss of axonemal outer arms. These results show that mutations in Mdnah5 are a primary cause of PCD and provide direct evidence that mutations in an axonemal dynein can cause hydrocephalus. Mutations in the human DNAH5 have recently been identified in PCD patients. Comparison of the mouse model and the human data suggests that the degree of ciliary dysfunction is causally related to the severity of human PCD, particularly the presence of hydrocephalus.

Isolation and expression of the human hPF20 gene orthologous to Chlamydomonas PF20: evaluation as a candidate for axonemal defects of respiratory cilia and sperm flagella.

Primary ciliary dyskinesia (PCD) is a heterogeneous congenital disorder characterized by bronchiectasis and chronic sinusitis, sometimes associated with situs inversus (i.e., Kartagener's syndrome) and male infertility. At the cell level, the disease phenotype includes various axonemal abnormalities of respiratory cilia and sperm flagella. We have previously isolated DNAI1, the first gene involved in these diseases in patients lacking outer dynein arms. In this study, designed to find additional genes for other axonemal defects, we report the isolation of a novel human gene, hPF20, which is orthologous to Chlamydomonas pf20. The hPF20 gene is expressed as two major transcripts: one is expressed in testis only, whereas the second is weakly expressed in many other tissues. As flagella of Chlamydomonas strains carrying pf20 mutations lack the axonemal central complexes, we tested the involvement of the hPF20 gene in the disease phenotype of five patients in whom cilia or flagella display abnormal central complexes. Five intragenic polymorphisms were identified and used to exclude hPF20 in two consanguineous patients, while no mutation was found in the remaining patients. However, given the genetic heterogeneity of PCD, we consider that this gene remains a good candidate to be investigated in patients with abnormal central complexes.

[Sinobronchial syndrome].

A condition characterized by chronic paranasal sinusitis and simultaneous chronic pulmonary infection was recognized and reported as long ago as the early 1900's. This condition was termed "sinobronchial syndrome" (SBS) in 1966. Many types of SBS are reported including Kartagener's syndrome/primary ciliary dyskinesia, Young's syndrome, bare-lymphocyte syndrome, cystic fibrosis and various types of immunoglobulin deficiency. SBS is also found in Japan. Among them, the most important is diffuse panbronchiolitis. The pathogenesis of SBS might involve inherited predisposition, probably accompanied by some deficiencies in the host defense of the respiratory system.

[Three cases of Kartagener's syndrome in children]. / Trzy przypadki zespolu Kartagenera u dzieci.

On the basis of the most recent works, etiology and the symptoms of Kartagener's syndrome have been described, as well as three cases of this disease in children.

Sindrome de Kartagener / Kartagener's syndrome

Kartagener descreveu uma associação entre dextrocardia, sinusite e bronquiectasia, que leva o seu nome. Sabe-se que esta síndrome completa é a forma extrema em gravidade da doença dos cílios imóveis, causada por defeitos ciliares em diferentes partes do organismo. Os autores relatam um caso desta síndrome e discutem os aspectos clínicos, diagnósticos e terapêuticos, bem como a necessidade de uma ampla avaliação de pacientes que se apresentem com qualquer sintoma isolado, para possibilitar um diagnóstico precoce