TY - JOUR

T1 - SPEF2- and HYDIN-Mutant Cilia Lack the Central Pair-associated Protein SPEF2, Aiding Primary Ciliary Dyskinesia Diagnostics

AU - Cindrić, Sandra

AU - Dougherty, Gerard W

AU - Olbrich, Heike

AU - Hjeij, Rim

AU - Loges, Niki Tomas

AU - Amirav, Israel

AU - Philipsen, Maria C

AU - Marthin, June K

AU - Nielsen, Kim G

AU - Sutharsan, Sivagurunathan

AU - Raidt, Johanna

AU - Werner, Claudius

AU - Pennekamp, Petra

AU - Dworniczak, Bernd

AU - Omran, Heymut

PY - 2020/3

Y1 - 2020/3

N2 - 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.

AB - 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.

KW - Axoneme/chemistry

KW - Cell Cycle Proteins/deficiency

KW - Cilia/chemistry

KW - Ciliary Motility Disorders/diagnosis

KW - Codon, Nonsense

KW - Cohort Studies

KW - DNA Mutational Analysis

KW - Epithelial Cells/cytology

KW - Female

KW - Genetic Heterogeneity

KW - Homozygote

KW - Humans

KW - Loss of Function Mutation

KW - Male

KW - Microfilament Proteins/genetics

KW - Microscopy, Electron, Transmission

KW - Microscopy, Fluorescence

KW - Mucociliary Clearance/genetics

KW - Mutation

KW - Mutation, Missense

KW - Pedigree

KW - Primary Cell Culture

KW - Situs Inversus/diagnosis

U2 - 10.1165/rcmb.2019-0086OC

DO - 10.1165/rcmb.2019-0086OC

M3 - Journal article

C2 - 31545650

VL - 62

SP - 382

EP - 396

JO - American Journal of Respiratory Cell and Molecular Biology

JF - American Journal of Respiratory Cell and Molecular Biology

SN - 1044-1549

IS - 3

ER -