Received: 25 March 2024 | Accepted: 3 September 2024
DOI: 10.1002/ppul.27267
OR I G I NA L A R T I C L E
HYDIN variants cause primary ciliary dyskinesia
in the Finnish population
Thomas Burgoyne PhD1,2 | Mahmoud R. Fassad PhD3,4 | Rüdiger Schultz PhD5 |
Varpu Elenius PhD6 | Jacqueline S. Y. Lim PhD3 | Grace Freke PhD3 |
Ranjit Rai PhD2 | Mai A. Mohammed PhD3,7 | Hannah M. Mitchison PhD3 |
Anu I. Sironen PhD3,8
1Institute of Ophthalmology, University College London, London, UK
2PCD Diagnostic Team and Department of Paediatric Respiratory Medicine, Royal Brompton Hospital, London, UK
3Great Ormond Street Institute of Child Health, University College London, London, UK
4Human Genetics Department, Medical Research Institute, Alexandria University, Alexandria, Egypt
5Allergy Centre, Tampere University Hospital, Tampere, Finland
6Department of Pediatrics, Turku University Hospital, University of Turku, Turku, Finland
7Biochemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt
8Natural Resources Institute Finland (Luke), Jokioinen, Finland
Correspondence
Anu I. Sironen, PhD, Great Ormond St Institute
of Child Health, University College London,
30 Guilford St, London, WC1N 1EH, UK.
Email: a.sironen@ucl.ac.uk
Funding information
Wellcome Trust Collaborative Award in
Science; BEAT‐PCD network; H2020 Marie
Sklodowska‐Curie Actions; Biotechnology and
Biological Sciences Research Council; Ministry
of Higher Education in Egypt; NIHR Great
Ormond Street Hospital Biomedical Research
Centre
Abstract
Introduction: Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized
by chronic respiratory tract infections and in some cases laterality defects and
infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair‐like
organelles protruding out of the cell. Thus far, disease causing variants in over 50
genes have been identified and these variants explain around 70% of all known
cases. Population specific genetics underlying PCD has been reported highlighting
the importance of characterizing gene variants in different populations for devel-
opment of gene‐based diagnostics and management.
Methods: Whole exome sequencing was used to identify disease causing variants in
Finnish PCD cohort. The effect of the identified HYDIN variants on cilia structure
and function was confirmed by high‐speed video analysis, immunofluorescence and
electron tomography.
Results: In this study, we identified three Finnish PCD patients carrying homozygous
loss‐of‐function variants and one patient with compound heterozygous variants
within HYDIN. The functional studies showed defects in the axonemal central pair
complex. All patients had clinical PCD symptoms including chronic wet cough and
recurrent airway infections, associated with mostly static airway cilia.
Pediatric Pulmonology. 2024;59:3601–3609. wileyonlinelibrary.com/journal/ppul | 3601
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2024 The Author(s). Pediatric Pulmonology published by Wiley Periodicals LLC.
Hannah M. Mitchison and Anu I. Sironen are joint senior authors.
Conclusion: Our results are consistent with the previously identified important role
of HYDIN in the axonemal central pair complex and improve specific diagnostics of
PCD in different national populations.
K E YWORD S
axoneme, diagnostics, HYDIN, motile cilia, PCD, variant
1 | INTRODUCTION
Primary ciliary dyskinesia (PCD) is an inherited disease where impairment
of mucociliary clearance (MCC) leads to recurrent airway infections,
bronchiectasis and in some cases progressively severe lung damage. The
mucus accumulation in the airways is caused by malfunction of motile
cilia. Other syndromic features in PCD are situs inversus, otitis media,
hearing loss, complex congenital heart disease, and more rarely hydro-
cephalus and retinitis pigmentosa.1 Furthermore, PCD variants may affect
male and female fertility due to defective motile cilia in the oviduct and
male efferent duct, and defects in sperm tail development. Motile cilia
lining the airways remove mucus and pathogens by a forward beating
pattern, which is disrupted by variants in genes regulating aspects of
ciliogenesis or coding for dynein preassembly or structural proteins in
motile cilia. Thus far variants in over 50 genes have been identified to
cause PCD. The most commonly mutated genes code for components of
the dynein arms, which are required for creating the movement of motile
cilia.1,2 The axonemal structure of motile cilia consists of 9 + 2 micro-
tubules, inner and outer dynein arms, nexin links between the doublet
microtubules, radial spokes connecting the outer microtubule doublets to
the central pair and the central pair projection (Figure 1A).
The central pair projection is required for controlling the motility
pattern created by the dynein arms and variants in central pair
complex genes such as HYDIN have been shown to result in stiff and
static cilia.3 However, the central pair is not present in nodal cilia that
govern left‐right organ laterality and therefore variants in genes
coding for central pair proteins do not cause situs inversus.4 Recently,
it has been shown that lack of HYDIN causes depletion of SPEF2
from the ciliary central pair complex and thus immunostaining for
SPEF2 can be used as a diagnostic tool for PCD patients with HYDIN
variants.5 Variants in SPEF2 mainly cause male infertility due to
malformed and immotile sperm tails, and mild PCD symptoms have
also been reported that affect the airways.6–8
Two highly similar copies of HYDIN are present in the human
genome, HYDIN which is transcribed from chromosome 16 (hg38
16:70,802,084‐71,230,722) and a paralogue pseudogene HYDIN2,
which is located on chromosome 1 (hg38 1:146,547,367‐
146,898,974). HYDIN2 appears to be mainly expressed in the brain
and fetal tissues with low expression in the lung, due to a neuronal
promoter.9 HYDIN2 is a duplicate of HYDIN exons 6–84 with short
and long transcripts predicted to be transcribed, the most common
isoform containing exons 6–19.9 Due to their very high similarity, it is
difficult to distinguish HYDIN‐specific variants for diagnostics based
on short read DNA sequencing. We recently showed the ability for
long read sequencing to overcome this challenge,10 and here we
investigate the potential to apply RNA sequencing of nasal brushing
biopsies to confirm causality of HYDIN variants in PCD patients. In
this study, we have identified novel and rare HYDIN variants in the
Finnish population using exome and Sanger sequencing of DNA and
RNA, and confirmed the functional effect of compound heterozygous
variants using immunofluorescence and electron tomography.
2 | METHODS
2.1 | Subjects
Blood samples were collected for DNA extraction from 13 PCD pa-
tients, recruited at the University Hospitals of Turku, Kuopio and
Tampere after written informed consent was given. Patients were
selected for exome sequencing based on clinical symptoms under-
lining PCD; chronic airway and ear infections, low nasal nitric oxide
(nNO) and abnormal cilia beating pattern. Gene panel results were
negative for the selected patients. Nasal brushing samples were
collected for high‐speed video microscopy analysis (HSVA), electron
microscopy, immunohistochemistry and gene expression studies
when possible. The study was ethically approved by the University of
Turku Ethics Committee (ETMK 69–2017), London Bloomsbury
Research Ethics Committee approved by the Health Research
Authority (08/H0713/82), and the referring hospitals.
2.2 | Nasal nitric oxide analysis
For nNO analysis, a CLD 88sp analyser equipped with a Denox 88
module for flow control was used with standard techniques (Eco
Physics). If cooperativity was established, three consecutive trials
were taken, from which the highest value was recorded. Nasal nitric
oxide analysis was repeated on two different occasions.
2.3 | High‐speed video microscopy analysis (HSVA)
Nasal epithelial cells were suspended in DMEM medium and eval-
uated under a differential‐interference microscope (Zeiss) at x1000
magnification. Cilia beat was recorded with a digital high‐speed video
3602 | BURGOYNE ET AL.
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
(DHSV) camera (Hamamatsu Orca Flash 4.0) with a frame rate of
256Hz. The detailed protocol for HSVA can be found in.11 DHSV
video sequences were played back frame by frame to determine the
cilia beat frequency (CBF) by calculating the mean of all recorded cilia
beat cycles. The cilia beating pattern (CBP) was determined by two
independent expert operators. HSVA was repeated on two different
occasions and averaged.
2.4 | Whole exome sequencing
The Nonacus Cell3 Exome panel was used for whole exome sequencing
of patient DNA samples (https://nonacus.com/cell3tm-target).
Unmapped reads were aligned to the current Human reference genome
(GRCh38 build) by Burrows‐Wheeler Aligner tool (Bwa‐mem2 version) (Li
and Durbin, 2010), SAM files were produced and indexed and converted
to BAM format previous to marking and removing duplicates using Picard
(https://broadinstitute.github.io/picard/). Subsequent analysis was exe-
cuted following best practices guidelines for GATK from the Broad
Institute (https://gatk.broadinstitute.org/hc/en-us). Firstly, base quality
score recalibration (BQSR) was done to numerically correct individual
base calls. Variant discovery was carried out in a two‐step pipeline: var-
iant calling with HaplotypeCaller followed by joint genotyping with
GenotypeGVCFs. Once a multi‐sample VCF file was obtained containing
all definitive variant records, VariantRecalibrator was operated to fulfil
Variant Quality Score Recalibration (VQSR) and refinement of the
obtained variant callset.
Variants were individually selected for each sample. The called var-
iants, including both single nucleotide variants (SNVs) and indels, were
then annotated using ANNOVAR (http://www.openbioinformatics.org/
annovar/). This tool enables functional annotation, and thus the final VCF
files obtained contains detailed information for each variant site in the
sample, such as their impact within a gene, predicted pathogenicity
scores, minor allele frequency (MAF), zygosity status or reporting whether
they have been recorded in large‐scale databases like dbSNP.
2.5 | Variant prioritization
Variants were filtered for MAF<1%, their predicted functional impact on
the encoded protein (missense, splicing, frameshift or nonsense) and
frequency (<0.001) in the Genome Aggregation Database (gnomAD,
broadinstitute. org). A list of genes with high expression during motile cilia
development (n=652, reanalyzed gene list based on data in Marcet
et al.12) was used to further filter variants in genes with potential roles in
motile cilia. Finally, the pathogenicity of the identified variants was esti-
mated using the Combined Annotation Dependent Depletion tool
(CADD, https://cadd.gs.washington.edu/snv) with a CADD score >20
considered a significant pathogenicity score and associated softwares
(Polyphen‐2, SIFT, PROVEAN, Alphafold). All variants were reported ac-
cording to Human GenomeVariation Society recommendations.13 Variant
pathogenicity was scored according to American College of Medical
Genetics and Genomics and the Association for Molecular Pathology
(ACMG‐AMP) variant interpretation guidelines.14 Due to the known
inheritance pattern of PCD homozygous variants, these were prioritized,
but output files were also analyzed for the presence of compound het-
erozygous variants.
2.6 | RNA extraction and RT‐PCR
RNA was isolated from nasal brushing samples using a Qiagen
RNeasy kit (Qiagen) and RNA was reverse‐transcribed using a cDNA
Reverse Transcription Kit (Applied Biosystems). cDNA was amplified
using the TaqMan Fast Universal PCR Master Mix (Applied Biosys-
tems). Primers used for RT‐PCR are listed in Supplementary table 1.
2.7 | Sanger sequencing
The identified variants were confirmed in the probands and available
family members by Sanger sequencing. Primers flanking the variant
F IGURE 1 Stop gain variants in HYDIN cause PCD in Finnish patients S6‐S8. (A) HYDIN and SPEF2 form a complex in the axonemal central
pair projection. (B) Stop gain variant (c.1797 C > G) in HYDIN exon 14 was identified in three Finnish PCD patients and confirmed by Sanger
sequencing. (C) The c.1797 C > G variant results in a predicted truncated HYDIN protein product as visualized by the AlphaFold protein structure
prediction tool (https://alphafold.ebi.ac.uk/). PCD, primary ciliary dyskinesia.
BURGOYNE ET AL. | 3603
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
were designed using the NCBI Primer‐BLAST tool (Table S1). The
genomic or cDNA sequence was amplified using standard PCR con-
ditions and predicted primer annealing temperature. The specificity
of the PCR product was confirmed on agarose gel and purified using
ExoSAP‐IT (Thermo Fisher Sci) for Sanger sequencing.
2.8 | Transmission electron microscopy and
electron tomography
A nasal brush biopsy was collected from a patient using 0.6‐mm bronchial
cytology brush. The sample was fixed in 2.5% glutaraldehyde in caco-
dylate buffer before postfixing in 1% osmium tetroxide, and subsequently
centrifuging in 2% agar to generate a pellet. Using increasing concen-
trations of ethanol followed by propylene oxide, the pellet was dehy-
drated before embedding in Araldite resin. 100nm thick sections were
cut and stained with 2% methanolic uranyl acetate and Reynolds lead
citrate. Using a JEOL 1400+ transmission electron microscope (TEM)
fitted with an AMT 16X CCD camera, conventional TEM images were
acquired and 2D averaging was perform on TEM images using PCD‐
Detect.15 To generate tomograms, perpendicular tilt series (dual axis)
were collected using SerialEM (University of Colorado Boulder) with tilt
increments of 1° over a range of ±60°. The tilt series were processed
using IMOD16 to generate a dual axis tomogram. Subtomographic aver-
aging was performed in IMOD, averaging together the central pairs from
eight cilia of patient S10 and from six cilia of a healthy control.
2.9 | Immunofluoresence
Respiratory epithelial ciliated cells from a nasal brushing of a PCD
patient and a control sample were stained after blocking (10% BSA, in
PBS) using DNAH5 (HPA037470, Cambridge Bioscience), RSPH4A
(HPA031196, Sigma‐Aldrich) and SPEF2 (HPA039606, Sigma‐
Aldrich) antibodies, colocalized with the cilia marker alpha‐tubulin
(322588, Invitrogen). After washes with PBST the slides were incu-
bated with secondary antibodies Alexa Fluor 488 anti‐mouse and
Alexa Fluor 594 anti‐rabbit (1:500). Slides were imaged using Zeiss
Axio Observer 7 and deconvoluted using Huygens Deconvolution
software (https://svi.nl/Huygens-Deconvolution).
3 | RESULTS
3.1 | HYDIN variants identified in the Finnish
population
Whole exome sequencing of Finnish PCD patients with chronic res-
piratory infections and otitis media (Table 1) identified one homo-
zygous variant (NM_001270974:exon14:c.1797 C > G:p. Tyr599Ter,
pathogenic) in three patients and two heterozygous variants
(NM_001270974:exon80:c.13801delG:p. Glu4601ArgfsTer17, path-
ogenic and NM_001270974:exon76:c.12899 G > C:p. Cys4300Ser,
likely pathogenic) in one patient. These variants had a low frequency
in the gnomAD database and were predicted pathogenic or likely
pathogenic, in keeping with ACMG/AMP guidelines,14 using Poly-
phen, Sift and Provean, also having high CADD scores (Table 2). The
homozygous variants were identified in two nonconsanguineous
families and were confirmed using HYDIN specific primers for Sanger
sequencing of patient DNA (Figure 1B). This variant results in a stop
gain in exon 14 and a predicted truncated protein product that would
severely disrupt HYDIN function. The protein 3D structure prediction
tool AlphaFold (https://alphafold.ebi.ac.uk/) was used to demon-
strate the effect of the stop gain variant on protein structure,
showing a clear reduction in size and a lack of several protein
domains (Figure 1C).
WES of patient S10 showed 4 variants within the HYDIN coding
region of which two were predicted to be likely pathogenic (Table 2).
A frameshift deletion (c.13801delG) was identified in exon 80 in cis
with amissense SNV (c.13800G > C). In trans with this allele, another
nonsynonymous SNV was found in exon 76 (c.12899 G >C), which
was predicted to be damaging by Polyphen, Sift and Provean with a
CADD score of 23.8 (Table 2). An additional benign nonsynonymous
SNV was detected in exon 22 (c.3291 A >G).
3.2 | HYDIN variants confirmed by sanger
sequencing and RNA expression
The stop gain variants were confirmed by Sanger sequencing of pa-
tients DNA using HYDIN specific primers (Table S1, Figure 1B). The
similarity of HYDIN and HYDIN2 across exons 72 to 85 hindered the
TABLE 1 Patient characteristics of four patients with mutations in the HYDIN gene.
Patient ID Ethnicity DOB Sex CBF Hz
Cilia beating
pattern (HSVA)
nNO
(nl/min) Clinical symptoms
S10 Finnish 1998 F ‐ Unsyncronized,
wavy
15 Recurrent sinusitis, otitis, respiratory infections,
bronchiectasis
S6 Finnish 2005 M 10.8 Stiff, static 29 Recurrent sinusitis, otitis, respiratory infections
S7 Finnish 1999 M 0 Stiff, static 23 Recurrent sinusitis, otitis, respiratory infections
S8 Finnish 1999 F 0 Static 5 Recurrent sinusitis, otitis, respiratory infections,
bronchiectasis
Abbreviation: CBF, cilia beat frequency
3604 | BURGOYNE ET AL.
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
confirmation of the identified variants in patient S10 since no HYDIN
specific DNA primers could be designed. Therefore, we extracted
RNA from the patient and her mother for transcript sequencing to
confirm the variants for which we failed to find HYDIN specific
primers for DNA sequencing. Sequencing of the variants at RNA level
confirmed the presence and heterozygosity of both potentially
causative HYDIN variants in the patient (Figure 2A) and the mother
was confirmed as a carrier of variant c.12899 G > C. Sanger
TABLE 2 HYDIN variants identified in Finnish PCD patients.
Patient ID Gene Function Variant rsID Zygozity Frequency GenomAD CADD score
S10 HYDIN Frameshift deletion HYDIN:NM_001270974:exon80:
c.13801delG:p. Glu4601ArgfsTer17
rs752405406 het 0.00002478 33
S10 Missense HYDIN:NM_001270974:exon80:
c.13800 G > C:p. Lys4600Asn
rs751812896 het 0.00005122 16
S10 HYDIN Missense HYDIN:NM_001270974:exon76:
c.12899 G > C:p. Cys4300Ser
rs200169224 het 0.001577 23.8
S10 HYDIN Missense HYDIN:NM_001270974:exon22:
c.3291 A >G:p. Ile1097Met
rs183427172 het 0.00367 5.758
S6 HYDIN Stop gain HYDIN:NM_001270974:exon14:
c.1797 C >G:p. Tyr599Ter
rs760517494 hom 0.0001524 35
S7 HYDIN Stop gain HYDIN:NM_001270974:exon14:
c.1797 C >G:p. Tyr599Ter
rs760517494 hom 0.0001524 35
S8 HYDIN Stop gain HYDIN:NM_001270974:exon14:
c.1797 C >G:p. Tyr599Ter
rs760517494 hom 0.0001524 35
Abbreviation: PCD, primary ciliary dyskinesia.
F IGURE 2 Compound heterozygous variants result in decreased HYDIN expression in a PCD patient S10. (A) Sanger sequencing of patient
RNA confirmed heterozygosity of the identified pathogenic HYDIN variants. (B) HYDIN transcript expression was decreased in the patient with
HYDIN variants c.12899 G >C and c.13801delG compared to control. GAPDH was used as a loading control. PCD, primary ciliary dyskinesia.
BURGOYNE ET AL. | 3605
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
sequencing also showed a variant c.12900 C > T predicted to result in
a nonsynonymous SNV (Figure 2A), which did not affect the protein
sequence. Therefore, we conclude that the PCD in patient S10 may
likely be caused by variants c.13801delG and c.12899 G > C. Fur-
thermore, HYDIN expression across exons 76–81 and at the end of
the gene across exons 85–86 was dramatically decreased compared
to the control (Figure 2B).
3.3 | The axonemal central pair structure is
affected in a patient with compound heterozygous
HYDIN variants
To further validate the causality of the heterozygous HYDIN variants
we investigated the structural defects in the ciliary axoneme of the
patient's nasal sample. TEM showed an apparently normal micro-
tubular organization in the ciliary axonemes and the presence of
dynein arms (Figure 3A), which was also confirmed by positive im-
munostaining with outer dynein arm component DNAH5 (Figure 4A).
Furthermore, the radial spokes appeared unaffected as demonstrated
by the presence of the radial spoke component RSPH4A (Figure 4A).
Electron tomography has been demonstrated as a useful tool for
identification of central pair structural defects17 and thus we utilized
this method to confirm the expected disruption of the central pair
complex arising from HYDIN variants. Tomograms showed a lack of
the proximal projection of the central pair complex in the patient with
compound heterozygous variants p. Glu4601Argfs*17 and p.
Cys4300Ser (Figure 3C), which was not obvious by the conventional
TEM (Figure 3A). PCD‐Detect15 was also able to identify the central
pair defect in the patient compared to control (Figure 3D) demon-
strating the usefulness of this tool in diagnostics. Since it has previ-
ously been shown that HYDIN is required for localization of SPEF2 in
the axonemal central pair projection5 we used SPEF2 immunostaining
to confirm the lack of the HYDIN/SPEF2 protein complex in the
axonemal central pair. SPEF2 was localized along the whole length of
cilia in airway epithelial cells from a healthy control individual, but
was completely depleted in the patient's cilia (Figure 3B).
Furthermore, we screened for the presence of the identified
causative variants and protein localizations in the nasal sample of the
mother of the patient. Localizations of SPEF2, DNAH5 and RSPH4A
were comparable to a control sample (Figure 4B). Sequencing of the
HYDIN variants showed inheritance of the c.12899 G > C variant from
the mother (Figure 4C). These results strongly support the conclusion
that the identified HYDIN variants in patient S10 are causative for
PCD and confirm the deleterious effect of the variants on HYDIN
localization to the ciliary axoneme.
F IGURE 3 The axonemal central pair projection is affected in the PCD patient S10 with compound heterozygous HYDIN variants. (A) TEM
showed apparently normal microtubular structure and the presence of dynein arms and radial spokes in the patient's axonemal cross sections. (B)
SPEF2 staining was depleted in cilia of the patient's nasal brushing samples. (C) Electron tomography showed a lack of the SPEF2/HYDIN
complex in the patient's central pair projection. (D) 2D image averaging using PCD Detect identified the lack of the SPEF2/HYDIN complex in
the patient's central pair. PCD, primary ciliary dyskinesia.
3606 | BURGOYNE ET AL.
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
3.4 | HYDIN variants cause stiff cilia motility in
Finnish PCD patients
Patients S6, S7 and S8 who carry the homozygous stop gain HYDIN
variants all showed almost static, extremely stiff cilia in their airway
epithelial cell samples, which is in line with previous studies of pa-
tients with HYDIN variants (Table 1). Patient S10 with heterozygous
HYDIN variants had stiff cilia with some unsynchronized movement,
which may be due to residual HYDIN in some of the axonemal central
pair projections (Table 1). All patients were reported to have low NO
and recurrent sinusitis and otitis (Table 1). No situs inversus was
reported for any of the patients as is expected for individuals carrying
HYDIN variants based on the lack of central pair structure in nodal
cilia. Patients S8 and S10 had developed bronchiectasis. We conclude
that the symptoms reported for the patients can be explained by the
identified HYDIN variants and that this information can be used for
genetic counselling and management of PCD in these patients.
4 | DISCUSSION
Although genetic variants for PCD have been identified in over 50
genes across the world, the genetics of PCD is population specific in
many cases; the majority of identified variants causing PCD are found
in small number of patients. However, more widespread variants
have been identified within populations and across different ethni-
cities and geographical populations.2,18–20 Thus, it is important to
identify the population specific variants to improve diagnostics and
management of the disease. Identification of genetic causes of a
disease enables better prediction of the evolvement of symptoms in
F IGURE 4 Immunofluoresence confirmed the presence of the main axonemal structures and Sanger sequencing the inheritance pattern for
one HYDIN variant in patient S10. (A) Outer dynein arm protein DNAH5 and radial spoke protein RSPH4A protein localization in PCD patient
with c.12899 G > C and c.13801delG variants was comparable to the control. (B) SPEF2, DNAH5 and RSPH4A were present in the airway cilia of
the patient's carrier mother. (C) Sanger sequencing of patients mother showed heterozygosity of variant c.12899 G > C and control
homozygosity of variant c.13801delG confirming the inheritance of c.12899 G >C from the mother to the patient. PCD, primary ciliary
dyskinesia.
BURGOYNE ET AL. | 3607
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
each patient and development of treatments, including personalised
medicines to improve their quality of life.
There is tissue specificity and different severity of disease pro-
gression depending on the genes affected and types of variants in
PCD, hence the need for better understanding of its genetic causes at
the population level. The most severe PCD phenotypes arise from
genes affecting multiciliogenesis (CCNO, MCIDAS) due to either
complete lack or an inefficiently low number of motile cilia in the
airways. Although these genes do not affect sperm tail formation,
they may affect fertility via a lack of efferent duct cilia.21,22
Genotype‐phenotype relationships have also been identified in large‐
scale topological data analysis.1,23 PCD disease severity also appears
to be significantly worse in patients with variants in the molecular
ruler genes CCDC39 and CCDC40 compared to most other structural
gene variants in PCD, whilst other variants introducing more subtle
axonemal defects include those in DNAH11.
HYDIN variants are relatively common in PCD patients,3,10,24
although clinical diagnosis of these patients has been hindered due to
a lack of clear structural changes in the axoneme, lack of situs in-
versus and a pseudogene hindering the sequence‐based diagnostics.
HYDIN is a large gene containing 86 exons and spanning more than
423 kb and due to development of methods to improve diagnostics
of patients with HYDIN variants recent studies have identified HYDIN
as a common cause of PCD in many populations; 8.7% of cases in
Quebec cohort24 and 7% in UK cohort. For identification of HYDIN
variants in the UK cohort Fleming et al developed long‐read
sequencing to overcome the issues with the HYDIN2 pseudogene
sequence.10 Furthermore, electron tomography and immuno-
fluorescence microscopy have provided tools to confirm the struc-
tural defect in patients with HYDIN variants and demonstrating the
loss of HYDIN at the central pair complex through SPEF2 staining.
PCD Detect has been developed based on image averaging and en-
hancement as a diagnostic tool kit to assess cases which are difficult
to diagnose, but have a suggestive HSVA result, TEM result, or a
variant of unknown significance.15 In this study we utilized PCD
Detect and structural studies to confirm the causality of compound
heterozygous variants together with immunofluorescence and RNA
experiments.
In Finland genetic variants in PCD patients have previously been
identified only in the DNAH11 and CFAP300 genes‐3.11,25 The HYDIN
variants identified in this study contribute to the understanding of
the genetic background of PCD in Finland and enable development of
genetics diagnostics within the Finnish population.
Confirmation of the causality ofHYDIN variants is complicated by the
presence of the pseudogene HYDIN2. Here we have utilized RNA
sequencing and electron tomography to confirm the compound hetero-
zygous variants in HYDIN, which couldn't be verified with Sanger
sequencing of DNA or by conventional TEM. The homozygous stop gain
variant c.1797C>G was found in three PCD patients and is predicted to
truncate the protein product by 4522 aa, which likely causes degradation
of the protein by nonsense mediated decay (NMD). The variant is not
present in the GenomAD database and based on the results of this study
we concluded that this is the disease‐causing variant in these patients.
The compound heterozygous variants in patient S10 were validated with
RNA expression and sequencing, immunohistochemistry and EM
tomography and the results showed convincing evidence that PCD is
caused by HYDIN defects in this patient as well. The c.13801delG
(rs752405406) variant is enriched in the Finnish population with an allele
frequency of 0.0005309, and is specific to the European population
(0.000001695). The c.12899G>C (rs200169224) variant is more com-
mon in the Finnish population (0.003242), but can also be found in
European, African and American populations (0.0002078–0.001935).
Thus, these variants can be expected to be a more common cause of PCD
in the Finnish population.
Our study has shown that HYDIN variants explain 31% of the
analysed cases (4/13, three families) in the Finnish patient cohort.
Other recurrent PCD‐causing variants were previously reported in
CFAP300 (three families) and DNAH11 (two families).11,25 There is
still work to be done to establish a more complete gene panel for
genetic diagnostics in Finland, but several Finnish population en-
riched variants have been identified thus far and this information can
be used for improving clinical diagnostics for PCD.
AUTHOR CONTRIBUTIONS
Thomas Burgoyne produced the electron tomography images and ana-
lysed the sample with PCD‐detect. Mahmoud R. Fassad performed the
variant calling. Rüdiger Schultz collected patient samples. Varpu Elenius
collected clinical data, patient samples and performed HSVA. Jacqueline
S. Y. Lim performed immunofluorescence staining. Grace Freke per-
formed Sanger sequencing. Ranjit Rai prepared electron microscopy
samples. Mai A. Mohammed extracted RNA. Hannah M. Mitchison
supervised the study. Anu I. Sironen wrote the manuscript, conducted
imaging, Sanger sequencing, variant filtering and bioinformatic analysis. All
authors contributed corrections and improvements to the manuscript.
ACKNOWLEDGMENTS
The authors would like to acknowledge the funding for this research
from BBSRC grant BB/V011251/1. Anu I Sironen was also funded by
Marie Sklodowska‐Curie individual fellowship No. 800556 from the
European Union's Horizon 2020 Research and Innovation Pro-
gramme. Hannah M. Mitchison was supported by NIHR GOSH BRC,
Ministry of Higher Education in Egypt and acknowledges support
from the BEAT‐PCD network (COST Action 1407 and European
Respiratory Society (ERS) Clinical Research Collaboration). Mahmoud
R. Fassad was supported by aWellcomeTrust Collaborative Award in
Science (210585/Z/18/Z).
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author, (Anu I Sironen), upon reasonable request.
ORCID
Anu I. Sironen http://orcid.org/0000-0003-2064-6960
3608 | BURGOYNE ET AL.
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
REFERENCES
1. Shoemark A, Rubbo B, Legendre M, et al. Topological data analysis
reveals genotype–phenotype relationships in primary ciliary dyski-
nesia. Eur Respir J. 2021;58(2):2002359.
2. Fassad MR, Patel MP, Shoemark A, et al. Clinical utility of NGS
diagnosis and disease stratification in a multiethnic primary ciliary
dyskinesia cohort. J Med Genet. 2020;57(5):322‐330.
3. Olbrich H, Schmidts M, Werner C, et al. Recessive HYDIN mutations
cause primary ciliary dyskinesia without randomization of left‐right
body asymmetry. Am J Hum Genet. 2012;91(4):672‐684.
4. Best S, Shoemark A, Rubbo B, et al. Risk factors for situs defects and
congenital heart disease in primary ciliary dyskinesia. Thorax.
2019;74(2):203‐205.
5. Cindrić S, Dougherty GW, Olbrich H, et al. SPEF2‐ and HYDIN‐
mutant cilia lack the central pair‐associated protein SPEF2, aiding
primary ciliary dyskinesia diagnostics. Am J Respir Cell Mol Biol.
2020;62(3):382‐396.
6. Liu C, Lv M, He X, et al. Homozygous mutations in SPEF2 induce
multiple morphological abnormalities of the sperm flagella and male
infertility. J Med Genet. 2020;57(1):31‐37.
7. Liu W, Sha Y, Li Y, et al. Loss‐of‐function mutations in SPEF2 cause
multiple morphological abnormalities of the sperm flagella (MMAF).
J Med Genet. 2019;56(10):678‐684.
8. Mori M, Kido T, Sakamoto N, et al. Novel SPEF2 variant in a Japa-
nese patient with primary ciliary dyskinesia: a case report and lit-
erature review. J Clin Med. 2022;12(1):317.
9. Dougherty ML, Nuttle X, Penn O, et al. The birth of a human‐specific
neural gene by incomplete duplication and gene fusion. Genome Biol.
2017;18(1):49.
10. Fleming A, Galey M, Briggs L, et al. Combined approaches, including
long‐read sequencing, address the diagnostic challenge of HYDIN in
primary ciliary dyskinesia. Eur J Human Genet. 2024;32:1074‐1085.
11. Schultz R, Elenius V, Fassad MR, et al. CFAP300 mutation causing
primary ciliary dyskinesia in Finland. Front Genet. 2022;13:985227.
12. Marcet B, Chevalier B, Luxardi G, Coraux C, Zaragosi LE, Cibois M
et al. Control of vertebrate multiciliogenesis by miR-449 through
direct repression of the Delta/Notch pathway. Nat Cell Biol.
2011;13(6):693‐699. doi:10.1038/ncb2241
13. den Dunnen JT, Dalgleish R, Maglott DR, et al. HGVS recommen-
dations for the description of sequence variants: 2016 update. Hum
Mutat. 2016;37(6):564‐569.
14. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the inter-
pretation of sequence variants: a joint consensus recommendation of the
American college of medical genetics and genomics and the association
for molecular pathology. Genet Med. 2015;17(5):405‐424.
15. Shoemark A, Pinto AL, Patel MP, et al. PCD detect: enhancing ciliary
features through image averaging and classification. Am J Physiol
Lung Cell Mol Physiol. 2020;319(6):L1048‐L1060.
16. Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization
of three‐dimensional image data using IMOD. J Struct Biol.
1996;116(1):71‐76.
17. Shoemark A, Burgoyne T, Kwan R, et al. Primary ciliary dyskinesia
with normal ultrastructure: three‐dimensional tomography detects
absence of DNAH11. Eur Respir J. 2018;51(2):1701809. doi:10.
1183/13993003.13901809-13992017
18. Hannah WB, Seifert BA, Truty R, et al. The global prevalence and
ethnic heterogeneity of primary ciliary dyskinesia gene variants:
a genetic database analysis. Lancet Respir Med. 2022;10(5):
459‐468.
19. Rumman N, Fassad MR, Driessens C, et al. The Palestinian primary
ciliary dyskinesia population: first results of the diagnostic and
genetic spectrum. ERJ Open Res. 2023;9(2):00714‐2022.
20. Xu Y, Feng G, Yano T, et al. Characteristic genetic spectrum of pri-
mary ciliary dyskinesia in Japanese patients and global ethnic het-
erogeneity: population‐based genomic variation database analysis.
J Hum Genet. 2023;68(7):455‐461.
21. Hoque M, Kim EN, Chen D, Li FQ, Takemaru KI. Essential roles of
efferent duct multicilia in male fertility. Cells. 2022;11(3):341.
22. Terré B, Lewis M, Gil‐Gómez G, et al. Defects in efferent duct
multiciliogenesis underlie male infertility in GEMC1‐, MCIDAS‐ or
CCNO‐deficient mice. Development. 2019;146(8):dev162628.
doi:10.1242/dev.162628
23. Berger LM, Cancian M, Meyer DR. Maternal re‐partnering and new‐
partner fertility: associations with nonresident father investments in
children. Child Youth Serv Rev. 2012;34(2):426‐436.
24. Shapiro AJ, Sillon G, D'Agostino D, et al. HYDIN variants are a
common cause of primary ciliary dyskinesia in French Canadians.
Ann Am Thorac Soc. 2023;20(1):140‐144.
25. Schultz R, Elenius V, Lukkarinen H, Saarela T. Two novel mutations
in the DNAH11 gene in primary ciliary dyskinesia (CILD7) with
considerable variety in the clinical and beating cilia phenotype. BMC
Med Genet. 2020;21(1):237.
SUPPORTING INFORMATION
Additional supporting information can be found online in the Sup-
porting Information section at the end of this article.
How to cite this article: Burgoyne T, Fassad MR, Schultz R,
et al. HYDIN variants cause primary ciliary dyskinesia in the
Finnish population. Pediatr Pulmonol. 2024;59:3601‐3609.
doi:10.1002/ppul.27267
BURGOYNE ET AL. | 3609
 10990496, 2024, 12, D
ow
nloaded from
 https://onlinelibrary.w
iley.com
/doi/10.1002/ppul.27267 by Luonnonvarakeskus, W
iley O
nline Library on [13/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License