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\begin{document}

\title{Clinical and genetic analysis of two patients with primary ciliary
dyskinesia caused by a novel mutation of DNAAF2}



\author[1]{Lili Dong}%
\author[1]{Lei Zhang}%
\author[1]{Xiao Li}%
\author[2]{Shiyue Mei}%
\author[3]{Yuelin Shen}%
\author[4]{Libing Fu}%
\author[3]{Shunying Zhao}%
\author[1]{xiaolei tang}%
\author[1]{Yu Tang}%
\affil[1]{Department of Respiratory Medicine Children’s Hospital Affiliated to Zhengzhou University/Henan Children’s Hospital/Zhengzhou Children’s Hospital Zhengzhou China}%
\affil[2]{Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases Children’s Hospital Affiliated to Zhengzhou University/Henan Children’s Hospital/Zhengzhou Children’s Hospital Zhengzhou China}%
\affil[3]{Department of Respiratory Medicine Beijing Children’s Hospital Capital Medical University National Center for Children’s Health Beijing China}%
\affil[4]{Department of Pathology Beijing Children’s Hospital Capital Medical University National Center for Children’s Health Beijing China}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Objective}: To investigate the clinical manifestations,
diagnosis and treatment processes of two siblings with PCD caused by the
same compound heterozygous mutations in DNAAF2. \textbf{Methods}: With
clinical diagnosis of PCD, the two siblings were recruited in the study.
We collected their clinical histories, laboratory tests, bronchoscopy,
otoscope images, and radiographic data. Whole blood of the siblings and
their parents were separately harvested for whole-exome sequencing and
Sanger sequencing. \textbf{Results}: The 7-year-old girl presented with
recurrent respiratory tract infection, sinusitis and otitis media.
Auxiliary examinations showed pneumonia, atelectasis, bronchiectasis,
low nitric oxide concentration (nNO), and conducting hearing loss. The
younger brother, 10-month boy, exhibited pneumonia, sinusitis, otitis
media, intestine malrotation and with lower nNO, atelectasis in chest
CT, obstructive ventilator dysfunction by pulmonary function and
conductive hearing loss. Two compound heterozygous mutations in DNAAF2
were detected in both of the siblings, nonsense mutation c.156C>A and
frameshift mutation c.177\_178insA, and the c.177\_178insA (p.E60Rfs*3)
mutation is a novel mutation. \textbf{Conclusion}: The study enriches
our knowledge of clinical manifestations and genetic information of PCD
caused by DNAAF2.%
\end{abstract}%



\sloppy


\textbf{Introduction}

With an estimated incidence of one in 10,000--20,000 live births
worldwide {[}1{]}, primary ciliary dyskniesia (PCD) is a rare inherited
autosomal recessive disease caused by impaired function of motile cilia,
resulting in recurrent respiratory inflammation, bronchiectasis,
sinusitis, otitis media, and neonatal respiratory distress.
Approximately 50\% of patients have situs inversus {[}2,8{]}. Besides
the clinical features, nasal NO concentration, high speed video
microscopy analysis (HSVMA) of cilia beat frequency and pattern, ciliary
ultrastructure in cross section through transmission electron microscopy
(TEM) are all helpful for the diagnosis of PCD{[}3{]}. Moreover, genetic
testing can help to confirm the diagnosis of PCD. The pathogenic
mechanism of PCD is cilia motility dysfunction, which depend on dynein
arms composing of the outer dynein arms (ODAs) and the inner dynein arms
(IDAs). The gene of~dynein axonemal assembly factor 2~(DNAAF2), also
known as KTU and PF13, has been reported to encode the cytoplasmic
proteins required for the assembly of both ODAs and IDAs, lead to loss
of cilia motility and cause PCD{[}6{]}. In this paper, we report the
clinical manifestations, diagnosis and treatment processes of two
siblings with PCD caused by the same compound heterozygous mutations in
DNAAF2, one of the mutations is novel.

\textbf{Methods}

After informed consent was obtained, we collected clinical histories,
laboratory tests, bronchoscopy, otoscope images, and radiographic data
of the two siblings. Identification of respiratory pathogens derived
from cultured bronchoalveolar lavage specimens was conducted. Whole
blood of the siblings and their parents were separately harvested for
whole-exome sequencing and Sanger sequencing. Then ciliary
ultrastructural defect was confirmed by Transmission Electron Microscopy
(TEM).

We also searched the literature of all the DNAAF2 mutations since first
reported. The clinical data of these patients were summarized.

\textbf{Results}

The probands, a 7-year-old girl and a 10-month-boy, were both born full
term. Their family histories were not remarkable. The elder sister
presented with wet cough and running nose for 2 months and had been
diagnosed with pneumonia and treated for 15 days in a local hospital
without improvement. Her computed tomography (CT) (Fig.1A, C) scanning
of lungs and sinus showed atelectasis and potential bronchiectasis in
the right middle lobe and sinusitis with hypertrophy of tonsils and
adenoids. Before transferred to the respiratory medicine department of
our hospital, she had suffered from recurrent respiratory tract
infection for almost 5 years. On physical examination, pharyngeal
congestion and swelling of tonsil were found, with normal growth and
development. Her nasal nitric oxide concentration (nNO) was much lower
(38.5ppb) than the PCD-specific nNO cutoff value (287ppb) {[}4{]}. While
the pulmonary function tests were normal. During bronchoscopy, massive
mucilage secretions were found in segments of lingula, left upper and
right middle lobes (Fig.1B). The culture of bronchoalveolar lavage (BAL)
showed positive for Haemophilus infuenzae (Hin). The pure-tone
audiometry proved conductive hearing loss. The air-bone gaps were 10dB
to 25dB for the right ear and 15dB to 35dB for the left one. The
otoscope and inner ear CT indicated otitis media in both of her ears,
which was responsible for the hearing loss (Fig.1D, E, F).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1 Clinical features of the elder sister. The chest
high-resolution computed tomography (HRCT) scan showed bronchiectasis
and atelectasis in the right middle lobe (A), sinusitis (C) and otitis
media (D). Bronchoscopy indicated large amount of mucilage secretions in
the right middle lobe. Otoscope findings of otitis media in left ear (E)
and the right one (F).

Azithromycin was given to treat Hin and play the anti-inflammatory
effect. Budesonide and acetylcysteine for inhalation were given for
their anti-inflammation effect and to dilute sputum. As for pulmonary
rehabilitation, mechanical vibration sputum expectoration, postural
drainage and effective breathing techniques were performed to accelerate
mucus clearance from the lungs. During follow-up, she had better
symptoms but persistent atelectasis in right middle lung and recurrent
sinusitis and otitis media.

The younger brother presented with cough and runny nose for one month.
He was diagnosed with pneumonia in both lungs, atelectasis in lingular
lobe, sinusitis, otitis media and hypertrophy of adenoids (Fig.2A-F)
Physical examination found phlegm sounds by stethoscope, and his growth
and development was normal. The pure-tone audiometry proved conductive
hearing loss. The air-bone gaps were 40dB to 60dB for the right ear and
30dB to 55dB for the left one. Bilateral tympanotomy with tube placement
and adenotomy were performed. His nNO was 32.8ppb, even lower than that
of the elder sister. Pulmonary function tests showed obstructive
ventilatory dysfunction. Transmission electron microscopy (TEM) of
bronchial cilia exhibit complete loss of ODAs and IDAs (Fig.3). Besides,
the echocardiography revealed residual left superior vena cava. With the
history of emesis, he was diagnosed with intestine malrotation by
abdominal ultrasound at 1.5 years old.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2 Clinical features of the younger brother. Otoscope findings of
otitis media of left and right ears (A \& B). The computed tomography
(CT) scan of nasopharynx showed sinusitis (C\&D). Chest HRCT scan
illustrated atelectasis in lingular lobe (E\&F).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3 TEM analysis of bronchial mucosal biopsy specimens displayed
complete loss of ODAs and IDAs.

After treated with azithromycin, budesonide inhalation solution, and
adjuvant mechanical vibration sputum expectoration, he was discharged
soon. He had better symptoms, better HRCT and well controlled sinusitis
and otitis media during following-up.

Furthermore, both of the siblings did not have the histories of fatty
diarrhea, malnutrition, meconium intestinal obstruction. Both of them
had normal results of cellular and humoral immune function, normal blood
amylase and lipase level, and negative results of tuberculosis infection
test (T-SPOT. TB).

With the informed consent of the child's parents, 2ml whole blood of the
siblings and their parents was separately collected for whole-exome
sequencing (WES) and Sanger sequencing. Two compound heterozygous
mutations in DNAAF2 were found in both siblings, one nonsense mutation
of NM\_018139: c.156C\textgreater{}A (p.Y52*) was inherited from the
mother and one frameshift mutation NM\_018139: c.177\_178insA
(p.E60Rfs*3) was inherited from the father (Fig.4 and 5). The mutation
of c.177\_178insA (p.E60Rfs*3) has not been reported before.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4 Pedigree of the family. Circles indicate females, squares
indicate males, solid symbols indicate patients.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5 Sanger DNA sequencing chromatogram of probands and their
parents. A novel mutation DNAAF2, c.177\_178insA (p.E60Rfs*3) was
identified in the probands, their father had a heterozygous mutation at
the

same location, whereas their mother did not. Another mutation DNAAF2,
c.156C\textgreater{}A (p.Y52*,786) was also identified in both of the
probands, their mother had a heterozygous mutation at the same location,
whereas their father did not.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 6 Mutations of DNAAF2 gene identified in the siblings with PCD.
The mutation in red was newly found in this study.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
cDNA change (Protein change)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Sex\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Age( years)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Ethnicity\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Clinical features\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Ultrastructural defect\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
c.1160A\textgreater{}G(p. Glu387Gly)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Turkish\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.472G\textgreater{}T(p.Glu158Term)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
VSD, Eisenmenger syndrome\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.23C\textgreater{}A(p.Ser8*)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Chronic otitis media and sinusitis, recurrent pneumonia, bronchiectasis,
removed middle lobe, complete situs inversus, impaired fertility\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ODA+IDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
c.1199\_1214dupACGATACCTGCGTGGC (p.Gly406Argfs*89)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Chronic otitis media and sinusitis, recurrent pneumonia, bronchiectasis,
complete situs inversus\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
ODA+IDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.564dupG(p.Lys189Glufs*5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ODA+IDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
c.2147C\textgreater{}T(p. Ala716Val)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
European\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Developmental disorder\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
c.156C\textgreater{}A(p.Tyr52Term)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
40\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Asian\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Recurrent bronchitis and pneumonia, chronic sinusitis, bronchiectasis,
situs inversus, infertility\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
ODA+IDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.26C\textgreater{}A(p.Ser9Term)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.727G\textgreater{}T(Glu243Term)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Swedish\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.2027\_2028delCT(p.S676Term)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.962C\textgreater{}A(p.Ser321Term)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Greek-Cypriot\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.998C\textgreater{}T(p.Ala333Val)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Dutch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ODA+partial IDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.1901T\textgreater{}C(p.Phe634Ser)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
c.796C\textgreater{}T(p.Gln266Term)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
30\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Asian\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Kartagener Syndrom, producting cough, hemoptysis, naso-sinusitis,
left-right laterality defects, azoospermia\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.1555delG(p.Glu519fs)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.1891G\textgreater{}A(p.Val631Ile)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Infetility\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.31delG(p.Glu11Argfs*5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Italian\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Neonatal RDS, bronchiectasis, sinusitis\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ODA+IDA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.491T\textgreater{}C (p.Leu164Pro)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
26\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Asian\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Scoliosis, bronchiectasis, sinusitis, situsinversus and
infertility\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ODA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
c.822del (p.Ala275Profs*10)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
53\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Asian\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Bronchiectasis, sinusitis and infertility\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
c.177\_178insA (p.E60Rfs*3) Novel\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Asian\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Recurrent respiratory tract infection, atelectasis, bronchiectasis,
sinusitis and otitis media\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10/12\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Asian\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pneumonia, atelectasis in lingular lobe, sinusitis, otitis media and
hypertrophy of adenoids\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ODA+IDA\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Table 1 Cases of DNAAF2 mutations. RDS, respiratory distress syndrome;
TEM, transmission electron microscopy; VSD, ventriculap septal defect;
IDA, inner dynein arm; ODA, outer dynein arm; - not available.

\textbf{Discussion}

In this study, two compound heterozygous mutations of DNAAF2 were
identified in two siblings with similar characteristic manifestations of
PCD including recurrent pneumonia with atelectasis mainly in right
middle and left lingular lobe, early bronchiectasis, sinusitis, otitis
media, significant reduction in nNO level and complete loss of ODAs and
IDAs of bronchial cilia found by the TEM. One of the mutations is a
nonsense mutation of NM\_018139: c.156C\textgreater{}A (p.Y52*) which
has already been reported in a Chinese family and proved to be
pathogenic{[}5{]}. Another mutation is a frameshift mutation
(NM\_018139: c.177\_178insA (p.E60Rfs*3) , which is a novel mutation.

DNAAF2 is located on chromosome 14q21.3 and consists of three exons
encoding cDNAs {[}6{]}. The mutation p.E60Rfs*3 locates in exon1 and
near another mutation p.Y52*. Exon1 is consists of 621 amino acids, and
the mutation of E60Rfs*3 leads to early transcription termination after
the 63 amino acids (Fig.6). The p.E60Rfs*3 mutation has been predicted
to be pathogenic with the evidences of PVS1+PM2+PM3+PP4 according to the
standards and guidelines of American College of Medical Genetics and
Genomics (ACMG). Multiple software, such as SIFT, Polyphen-2 and
Mutation Taster, also predict the p.E60Rfs*3 mutation to be pathogenic.
In addition, none of cystic fibrosis (CF) or primary immune deficiency
(PID) related genes which may also lead to recurrent pneumonia,
atelectasis and bronchiectasis were not detected. Based on the clinical
manifestations and hereditary mode of compound heterozygous mutations
from both parents, it's reasonable to highly suspected that the novel
p.E60Rfs*3 mutation of DNAAF2 is a disease causing mutation and is
responsible for the pathogenesis of PCD.

The movements of cilia can help to clear airway mucous, cerebrospinal
fluid flow along the brain ventricular system, support the transport of
the oocyte to the uterus, and male germ cell move along the female
reproductive tract {[}6{]}. The normal structure of the cilia consists
of the conserved basic 9+2 microtubule-based axonemes and other several
functional modules{[}7{]}. Members of multi-subunit motor protein
complexes, ODAs and IDAs are responsible for the generation and
regulation of ciliary beating. Dynein arms are ATPase-based
protein-complexes, via ATP hydrolyzed by dynein heavy chain, the paired
microtubules slide relative to each other, leading to cilia curve over
the deformed microtubules{[}8-9{]}. During the process, IDA causes the
curvature of cilia, ODA accelerates active sliding of the outer
microtubules, leading to effective propulsion produced by the cilia
effectively paddling forward and reverting paddling backward.
Appropriate rheological properties and the functional structure of cilia
are the basis that maintain mucociliary clearance which is essential to
the respiratory tract clearance defense{[}5{]}.

Approximately 40 genes have been reported to be associated with PCD,
including DNAH5, DNAH11, DNAI1, DNAI2, DNAL1, TXNDC3, DNAAF1, and DNAAF2
et al. {[}1, 6, 10-14{]}. In 2008, two heterozygous mutations c.C23A
{[}pS8X{]} and c.1214-1215insACGATACCTGCGTGGC {[}p.G406Rfs89X mutations
of DNAAF2 were first identified in two consanguineous families with PCD
presented with recurrent respiratory tract infections, laterality
defects and impaired fertility{[}6{]}. DNAAF2 works as a facilitator of
dynein pre-assembly. The disfunction of DNAAF2 is associated with
specific defects in the interaction of intermediate and heavy chains in
the cytoplasm which leads to complete or partial loss of ODAs and IDAs
and loss of motility{[}6{]}. After that, only 15 PCD cases have been
reported caused by DNAFF2 with 19 mutations. The cases of DNAAF2
mutation are summarized in Tab.1{[}5-6, 15-27{]}. As for Chinese
children, DNAFF2 gene is rare. As Guan et al reported reported in 81
Chinese children, genes with the highest incidence of mutations that
caused PCD were DNAH11, followed by HYDIN, DNAH5, CCDC39, DNAH1 and
CCNO, no DNAFF2 gene mutation was detected{[}28{]}. In Chinese adult
patients, Sun et.at have identified two heterozygous mutations c.C156A
{[}p.Y52X{]} and c.C26A {[}p.S9X{]} in the DNAAF2 gene which lead to the
defect of outer dynein arms and inner dynein arms resulting in PCD with
the manifestation of male infertility{[}5{]}. Lu C et.at detected
another two heterozygous mutations c.491T\textgreater{}C {[}p.L164P{]}
and c.822del {[}p.A275Profs*10{]} in two females presents with the
bronchiectasis, sinusitis, situs inversus, and infertility, besides, the
one with c.491T\textgreater{}C mutation had scoliosis{[}27{]}.

Consequently, we made an early diagnosis of PCD in two siblings with a
novel mutation of the rare DNAFF2 gene. The early stagy of PCD may be
easily misdiagnosed as common pneumonia or sinusitis. When a child
presented with recurrent pneumonia or atelectasis accompanied with
recurrent sinusitis or otitis media, PCD should be considered. The
characteristic HRCT manifestations including atelectasis in the right
middle and left lingular lobe, thickened bronchial wall, and mild
bronchiectasis can provide a diagnostic clue for PCD. Besides, nNO and
TEM of bronchial cilia are useful for the further diagnosis , and
genetic tests can help to confirm the diagnosis.

In conclusion, our study suggests that the p.E60Rfs*3 mutation of DNAAF2
gene is a novel disease causing mutation of PCD. This study enriches our
knowledge of clinical manifestations and genetic information of PCD
caused by DNAAF2.

\textbf{Acknowledgements} We are grateful to the patients and their
family for

their collaboration.

\textbf{Funding} Medical Science and Technology Project of Henan
Province (2018020621); Demonstration application of remote collaborative
service network for complex genetic diseases (SJPT-03-01).

\textbf{Conflicts of interest} The authors declare that they have no
competeing conflicts.

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