RESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.
Assuntos
Proteínas de Ciclo Celular/deficiência , Cílios/química , Transtornos da Motilidade Ciliar/genética , Proteínas dos Microfilamentos/genética , Axonema/química , Axonema/ultraestrutura , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/patologia , Códon sem Sentido , Estudos de Coortes , Análise Mutacional de DNA , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Heterogeneidade Genética , Homozigoto , Humanos , Mutação com Perda de Função , Masculino , Proteínas dos Microfilamentos/fisiologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Depuração Mucociliar/genética , Mutação , Mutação de Sentido Incorreto , Linhagem , Cultura Primária de Células , Situs Inversus/diagnóstico , Situs Inversus/genética , Situs Inversus/patologiaRESUMO
Nasal nitric oxide (NO) discriminates between patients with primary ciliary dyskinesia (PCD) and healthy individuals. We report feasibility of measurement and natural evolution of nasal NO and upon the impact of respiratory tract infection (RTI) on nasal NO in healthy infants (HI), followed from birth until age 2â years, with comparison to nasal NO in infant PCD.Tidal-breathing nasal NO measurements were performed at scheduled visits at 2â weeks old and at 4, 8, 12, 18 and 24â months old, with extra visits during RTIs. Historical nasal NO measurements for infant PCD were included for comparison.Altogether, 224 nasal NO measurements were performed in 44 enrolled infants. Median newborn nasal NO was 46 ppb (interquartile range (IQR) 29-69 ppb), increasing at a rate of 5.4% per month up to 283â ppb (IQR 203-389 ppb) at the age of 2â years. RTIs in 27 out of 44 infants temporarily suppressed nasal NO by 79%. Values for nasal NO in seven infants with PCD ranged from 6-80â ppb. The success rate to accept nasal NO sampling was 223 out of 224 measurements (99.6%).Tidal-breathing nasal NO measurement was indeed feasible in infancy and nasal NO in HI increased significantly up to 2â years of age, in opposition to nasal NO in PCD cases, which stayed low past 2â years of age. RTI episodes caused marked, temporary reductions in nasal NO in HI indistinguishable from that in infant PCD, suggesting that nasal NO should be measured in RTI-free intervals.
Assuntos
Síndrome de Kartagener/metabolismo , Óxido Nítrico/metabolismo , Infecções Respiratórias/metabolismo , Testes Respiratórios , Estudos de Casos e Controles , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Modelos Lineares , Estudos Longitudinais , Masculino , Estudos Prospectivos , Respiração , Sensibilidade e EspecificidadeRESUMO
Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.
