CFAP300: Mutations in Slavic Patients with Primary Ciliary Dyskinesia and a Role in Ciliary Dynein Arms Trafficking.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous hereditary disease from a class of ciliopathies. In spite of the recent progress, the genetic basis of PCD in one-third of patients remains unknown. In search for new genes and/or mutations, whole-exome sequencing was performed in 120 unrelated Polish patients with PCD, in whom no genetic cause of PCD was earlier identified. Among a number of pathogenic variants in PCD genes, mutations in CFAP300 (alias C11orf70) were detected. Extended screening in the whole Polish PCD cohort revealed the relatively high frequency (3.6%) of otherwise rare c.[198_200 del_insCC] variant, indicating that it should be included in population-specific genetic tests for PCD in Slavic populations. Immunofluorescence analysis of the respiratory epithelial cells from patients with CFAP300 mutations revealed the absence or aberrant localization of outer and inner dynein arm markers, consistent with transmission electron microscope images indicating the lack of both dynein arms. Interestingly, the disparate localization of DNAH5 and DNALI1 proteins in patients with CFAP300 mutations suggested differential mechanisms for the trafficking of preassembled outer and inner dynein arms to the axoneme. The profile of CFAP300 expression during ciliogenesis in suspension culture was consistent with its role in cilia assembly. Gene silencing experiments, performed in a model organism, Schmidtea mediterranea (flatworm), pointed to the conserved role of CFAP300 in ciliary function.

CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

RPGR mutations might cause reduced orientation of respiratory cilia.

RPGR gene encodes retinitis pigmentosa guanosine triphosphatase regulator protein, mutations of which cause 70% of the X-linked retinitis pigmentosa (XLRP) cases. Rarely, RPGR mutations can also cause primary ciliary dyskinesia (PCD), a multisystem disorder characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis, and male subfertility. Two patients with PCD_RP and their relatives were analyzed using DNA sequencing, transmission electron microscopy (TEM), immunofluorescence (IF), photometry, and high-speed videomicroscopy. The Polish patient carried a previously known c.154G>A substitution (p.Gly52Arg) in exon 2 (known to affect splicing); the mutation was co-segregating with the XLRP symptoms in his family. The c.824 G>T mutation (p. Gly275Val) in the Australian patient was a de novo mutation. In both patients, TEM and IF did not reveal any changes in the respiratory cilia structure. However, following ciliogenesis in vitro, in contrast to the ciliary beat frequency, the ciliary beat coordination in the spheroids from the Polish proband and his relatives carrying the c.154G>A mutation was reduced. Analysis of the ciliary alignment indicated severely disturbed orientation of cilia. Therefore, we confirm that defects in the RPGR protein may contribute to syndromic PCD. Lack of ultrastructural defects in respiratory cilia of the probands, the reduced ciliary orientation and the decreased coordination of the ciliary bundles observed in the Polish patient suggested that the RPGR protein may play a role in the establishment of the proper respiratory cilia orientation.

Mutations in radial spoke head genes and ultrastructural cilia defects in East-European cohort of primary ciliary dyskinesia patients.

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2-5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2-3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2-5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes.

Population specificity of the DNAI1 gene mutation spectrum in primary ciliary dyskinesia (PCD).

BACKGROUND: Mutations in the DNAI1 gene, encoding a component of outer dynein arms of the ciliary apparatus, are the second most important genetic cause of primary ciliary dyskinesia (PCD), the genetically heterogeneous recessive disorder with the prevalence of ~1/20,000. The estimates of the DNAI1 involvement in PCD pathogenesis differ among the reported studies, ranging from 4% to 10%. METHODS: The coding sequence of DNAI1 was screened (SSCP analysis and direct sequencing) in a group of PCD patients (157 families, 185 affected individuals), the first ever studied large cohort of PCD patients of Slavic origin (mostly Polish); multiplex ligation-dependent probe amplification (MLPA) analysis was performed in a subset of ~80 families. RESULTS: Three previously reported mutations (IVS1+2-3insT, L513P and A538T) and two novel missense substitutions (C388Y and G515S) were identified in 12 families (i.e. ~8% of non-related Polish PCD patients). The structure of background SNP haplotypes indicated common origin of each of the two most frequent mutations, IVS1+2-3insT and A538T. MLPA analysis did not reveal any significant differences between patients and control samples. The Polish cohort was compared with all the previously studied PCD groups (a total of 487 families): IVS1+2-3insT remained the most prevalent pathogenetic change in DNAI1 (54% of the mutations identified worldwide), and the increased global prevalence of A538T (14%) was due to the contribution of the Polish cohort. CONCLUSIONS: The worldwide involvement of DNAI1 mutations in PCD pathogenesis in families not preselected for ODA defects ranges from 7 to 10%; this global estimate as well as the mutation profile differs in specific populations. Analysis of the background SNP haplotypes suggests that the increased frequency of chromosomes carrying A538T mutations in Polish patients may reflects local (Polish or Slavic) founder effect. Results of the MLPA analysis indicate that no large exonic deletions are involved in PCD pathogenesis.

Carrier status for 3 most frequent CFTR mutations in Polish PCD/KS patients: lack of association with the primary ciliary dyskinesia phenotype.

We screened a large group of primary ciliary dyskinesia/Kartagener syndrome (PCD/KS) patients and their siblings (148 patients from 126 unrelated families) for the presence of the CFTR mutations that are most frequently found in the Polish population: the severe F508del and 2,3del21kb, and the mild 3849+10kbC > T. No statistically significant increase in the frequency of these mutations was found in the studied group, as compared with the general population. This is consistent with an earlier observation in another population and indicates that the status of being a carrier of any of these CFTR mutations should not be considered as an important risk factor in PCD/KS pathogenesis.

[Cystic fibrosis diagnosed in 36-year old man]. / Mukowiscydoza rozpoznana u 36-letniego chorego.

UNLABELLED: Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasians. There is considerable variability in the clinical presentation and course of cystic fibrosis. Adult patients with mild symptoms are increasingly being diagnosed with CF. We present a case of a man diagnosed with CF at age 36 years. The diagnosis was suspected because of recurrent nasal polyposis, bronchiectases, male infertility, and a positive sweat tests. One CFTR mutation was identified. CONCLUSIONS: 1. Cystic fibrosis should be included into the differential diagnosis of chronic respiratory symptoms in adults. 2. CF patients diagnosed in adulthood usually have milder clinical course of the disease.