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\begin{document}

\title{Two novel gene mutations identified in a child with Pulmonary Alveolar
Microlithiasis complicated with bronchitis obliterans: a case report and
literature review}



\author[1]{meiyu zhang}%
\author[1]{Man Gao}%
\author[1]{Yuhuan Liu}%
\author[1]{Kun Wang}%
\author[1]{Siyan Zhou}%
\author[2]{Haoran Jing}%
\author[1]{Guo Yin}%
\author[1]{Fanzheng Meng}%
\affil[1]{The First Hospital of Jilin University}%
\affil[2]{Jilin University}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\textbf{Introduction}

Pulmonary alveolar microlithiasis (PAM) is an uncommon, autosomal
recessive lung disease with high penetrance (OMIM \#265 100) and is
considered to be a monogenic disorder. \textsuperscript{1} The only
known pathogenic gene is solute carrier family 34 member 2 (SLC34A2)
(Entrez Gene ID 10568).\textsuperscript{2-4} SLC34A2 mutations lead to
the accumulation of calcium phosphate in the alveoli, restrict alveolar
dilatation, and then progress to a restrictive lung function complicated
with reduced dynamic and static volumes.\textsuperscript{5, 6} Dyspnea
is the most frequent symptom, followed by dry cough, chest pain,
asthenia, pneumothorax, pulmonary fibrosis, and cor
pulmonale.\textsuperscript{7-10}While children are always in the onset
at the early stage of PAM and usually remain asymptomatic when
diagnosed; however, some can present with dry cough, exertional dyspnea,
and chronic hypoxic signs, including clubbing.\textsuperscript{3}
Recently, some complications with PAM have been reported, such as
asthma, pneumomediastinum, subcutaneous emphysema, and so
on.\textsuperscript{11-14}

PAM is difficult to be diagnosed because of nonspecific symptoms in
children. The diagnosis of PAM is often based on radiographic studies at
first, and an exact diagnosis requires at least one additional clinical
feature including genetic testing demonstrating a mutation in SLC34A2,
microlith analysis, or histopathology.\textsuperscript{15} Gradually, it
has been a tendency that gene analysis would play an increasingly
important role in the diagnostic procedure. Bendstrup et al. summarized
30 genetic variants of SLC34A2 in 2020.\textsuperscript{16}

In this case, we identified a PAM patient complicated with bronchitis
obliterans by computerized tomography (CT), bronchoscopy and
whole-exome-sequencing. Two novel compound heterozygous gene mutations,
gain (EXON:2-6 duplication) and c.1218C\textgreater{}A (p. Phe406Leu)
were identified to expand the spectrum of gene mutations.

\textbf{Case history}

A 2-year-old boy was admitted because of intermittent fever and cough in
the past 15 days. At that time, chest computed tomography (CCT) showed
bronchiectasis complicated with extensive pneumonia in both lungs
(\emph{Fig.1} A-D). The child remained paroxysmal irritating cough after
a 10-day course of antibiotic treatment. His father had a chronic cough
caused by smoking, his mother was in good health, and they were not
consanguineous. His grandmother was diagnosed with bronchiectasis and he
had no siblings. Genetic testing was recommended to his parents but was
rejected. Thereafter, he was lost from follow-up until he was
hospitalized at age 7 when he had been suffering a persistent cough and
expectoration for the past 2 months. He had a dry cough occasionally and
decreased exercise endurance over these past 5 years. He had not
received systematic cardiopulmonary function assessments or effective
treatments from age 2 to 7. He denied any previous history of allergies,
asthma, exposure to \emph{Mycobacterium tuberculosis}
or\emph{adenovirus} infections. Physical examinations were unremarkable.
Blood tests showed increased white blood cell count at 17.47*10\^{}9/L
(NE\%0.76, LY\%0.17, MO\%0.17). Pulmonary function tests (PFTs) showed a
restrictive syndrome with a forced vital capacity (FVC) of 1.21L (60.5\%
of predicted), a mild obstructive ventilation dysfunction with a forced
expiratory volume in 1 second/forced vital capacity ratio
(FEV\textsubscript{1}/FVC) of 69.8\% and a positive bronchodilation test
(BDT) with FEV\textsubscript{1} improvement ratio at 33.6\%. CCT at age
7 (Fig.1 E-H) showed bronchiectasis, interlobular septal thickening and
lung fibrosis. And some parts showed ``mosaic'' features. The
mediastinal window showed nodular calcifications in the upper and lower
lobes of both lungs (\emph{Fig.1} I-P). Bronchoscopy showed bronchitis
obliterans (\emph{Fig.2} ) and the Bronchoalveolar lavage fluid (BALF)
was still turbid after repeating lavage. BALF metagenomic
next-generation sequence (NGS) results suggested one positive pathogen
(\emph{Mycoplasma pneumoniae} ). The Tuberculin skin test (TST) was
medium positive (15mm). Two novel compound heterozygous mutations of the
SLC34A2 gene (\emph{Fig.3} ) were identified by whole-exome sequencing,
EXON:2-6 duplication (from the father) and c.1218 (EXON:11)
C\textgreater{}A (from the mother). According to the American College of
Medical Genetics (ACMG) guidelines in 2019, \textsuperscript{17} the
biological pathogenicity of the former was graded as ``LIKELY''
pathogenic, while the latter was graded as ``UNCERTAIN'' significance.
Based on the evidence above, the patient was finally diagnosed with
pulmonary alveolar microlithiasis (PAM). After intravenous injection of
amoxicillin-clavulanate (600mg, three times a day), and oral
azithromycin (250mg, every other day) for a week, his cough was partly
relieved and was discharged.

\textbf{Discussion}

Pulmonary alveolar microlithiasis (PAM) is a rare genetic disease
characterized by the accumulation of microliths in the pulmonary
alveolar space.\textsuperscript{1, 3, 10, 18} These microliths induce a
chronic inflammation of the alveolar septa, responsible for a chronic
interstitial lung disease leading to respiratory failure and lung
fibrosis. Common symptoms are dyspnea, dry cough, chest pain,
hemoptysis, asthenia, and possible occurrence of pneumothorax. Children
are always in the onset at the early stage of PAM and are often
misdiagnosed because of nonspecific symptoms such as dry cough, acute
respiratory failure and asthenia.\textsuperscript{19} As early symptoms
are imperceptible, PAM has a low diagnostic rate in children aged [?] 5
years, accounting for only 2\%-3\% of all cases (28 cases to 1022 cases
in the most recent all-age cohort).\textsuperscript{19} In our study, we
reported a case of PAM in a 7-year-old boy diagnosed by genetic testing
and CT findings.

As reported in the literature, symptoms, signs, serological tests or
imaging features of PAM are not typical at the early stage, especially
in children. The diagnosis of PAM is often based on radiographic images
at first, and a definitive diagnosis requires at least one additional
clinical feature including genetic testing demonstrating a mutation in
SLC34A2, microlith analysis or
histopathology.\textsuperscript{15}Genetic testing demonstrates
pathogenic mutations in SLC34A2 are highly specific for
PAM,\textsuperscript{15} and because of less invasive than lung biopsy
or transbronchial biopsy, it is more frequently used to confirm PAM
diagnosis in children [?]5 years of age than in the all-age
cohort.\textsuperscript{1} Especially in families with unknown genetic
backgrounds, genetic investigations are highly recommended to identify
possible variants of SLC34A2. In the cases of suspected PAM with no
prior family history, it is also preferred to perform genetic analysis.
In our case, we tried to persuade the parents to perform a lung
biopsy/transbronchial biopsy or whole-exome sequencing on the patient at
age 2, but they rejected which caused delayed diagnosis, while finally
diagnosed by gene analysis.

Up to date, there have been approximately 40 pathogenic variants in
SLC34A2 reported. Based on the summary of SLC34A2 gene mutations by
Bendstrup et al,\textsuperscript{16} we searched PubMed and Web of
Science in these 3 years until Feb 1, 2023, and updated 7 novel
pathogenic variants including this case in\emph{Table 1} , namely c.286
C\textgreater{}T ,\textsuperscript{20} c.448 G\textgreater{}A
,\textsuperscript{21} c.524-1 G\textgreater{}C ,\textsuperscript{22}EXON
2-6 duplication, c.1218 C\textgreater{}A, c.1493 G\textgreater{}T
,\textsuperscript{23} c.1653\_1660del.\textsuperscript{24} Types of DNA
variants include 4 substitutions, 1 deletion, 1 splicing site and 1
duplication. According to the literature, there is no clear correlation
between genotype/phenotype. J\selectlanguage{ngerman}önsson et al. demonstrated that the disease
severity was associated with the pathogenicity of the
variants,\textsuperscript{6} but this needs to be investigated in a
larger patient population. In our case, we identified two heterozygous
mutations in SLC34A2, EXON:2-6 duplication and c.1218C\textgreater{}A in
EXON 11. The EXON:2-6 duplication was predicted to disrupt the reading
frame and leaded to the transcription factor degradation. Compared to
mutations of a single exon, 5 consecutive exons duplication in the
coding region tended to cause loss of function. In monogenic autosomal
recessive disease, duplications within one pathogenic gene could cause
dysfunctions or correspond different phenotypes.\textsuperscript{25} The
missense variant, c.1218C\textgreater{}A in EXON 11 was also predicted
to be pathogenic by forecasting tools, like PROVEAN, SIFT, Polyphen2,
Mutation Taster and Revel analyzing conservatism. And gene frequency in
normal general was below 0.0005. The compound heterozygous mutations
eventually leaded to dysfunction in SLC34A2.

The manifestations of PAM are not classical at the early stage, since
the microliths have not caused obvious respiratory dysfunction. The
present patient became symptomatic at age 2 and was diagnosed at age 7.
His main symptoms were intermittent fever, cough, and expectoration
which were consistent with the characteristics of children suffering
from PAM. Intermittent fever and cough appear as first symptoms in
children cohort and CCT is performed for other reasons such as a viral
or bacterial lung infection. Combining with respiratory infections tends
to be the first reason for children's admissions, and it is also an
opportunity to find abnormal chest images. Further genetic testing or
biopsy confirm the diagnosis of PAM. We hypothesized that PAM combined
with recurrent respiratory infections, which could be rational to
explain why dry cough, fever, acute respiratory failure are frequent
symptoms in PAM. Furthermore, recurrent infections are one of the
factors resulting in bronchiectasis.

Notably, our patient showed unusual bronchiectasis at age 2. And CCT at
age 7 showed that central bronchiectasis had contracted. However, Deniz
and his partners found a different observation that peripheral
bronchiectasis was seen at a high incidence rate of 60\% (6/10) and none
of the group (mean age: 22±3.2) had central
bronchiectasis.\textsuperscript{26} Pathophysiological mechanisms of
bronchiectasis include persistent bacterial infections, dysregulated
immune responses, airway obstruction and impaired mucociliary
clearance.\textsuperscript{27} Most common pathogenic causes associated
with development of bronchiectasis in children are idiopathic factors,
post-infection, congenital immunodeficiency or associated with
dysplastic syndromes. \textsuperscript{28} For our patient, the
hereditary factor could be the primary reason when bronchiectasis was
noticed in early childhood since his grandmother and his father had
respiratory diseases. Following hereditary factor, airway epithelium was
destroyed due to respiratory infections. Especially complicated with
persistent infections, central bronchiectasis could be more severe at
young age.

In addition, our patient's PFTs showed a restrictive syndrome with FVC
of 1.21L (60.5\% of predicted), a mild obstructive ventilation
dysfunction with FEV\textsubscript{1}/FVC of 69.8\%, and a positive BDT
with FEV\textsubscript{1} improvement ratio at 33.6\%. PAM caused by
SLC34A2 mutations lead to the accumulation of calcium phosphate in the
alveoli, restrict alveolar dilatation, and then progress to a
restrictive lung function impairment. Our case also showed a mild
obstructive ventilation dysfunction possibly because of excessive
sputum. Furthermore, his positive BDT suggested airway spasm, possibly
associated with asthma or\emph{Mycoplasma pneumoniae} . The patient
denied history of allergies or asthma and lung auscultation had no
sonorous rhonchi or sibilant wheezes. There was no insufficient evidence
to diagnose asthma. \emph{Mycoplasma pneumoniae}is considered as a
factor resulting in a trigger in recurrent wheezing and exacerbations of
asthma in children.\textsuperscript{29} Besides, there may be bias in
this data of BDT, especially under the condition of a reduced vital
capacity, which needs multiple measurements.

As PFTs showed a restrictive ventilation dysfunction and CCT showed
mosaic sign, bronchitis obliterans was diagnosed by
bronchoscopy.\textsuperscript{30, 31}Histologically, bronchiolitis
obliterans is defined by obliteration of the lumen of bronchioles owning
to inflammation, granulation tissue or scarring.\textsuperscript{32}
Bronchial obliteration presents as a complete obliteration of the
bronchus by a smooth-surfaced membrane.\textsuperscript{33} Typically,
such changes are associated with chronic inflammation of the bronchial
walls and cartilage destructions, resulting in structural shifts such as
thickening, bronchiectasis, and fibrosis.\textsuperscript{34-36} Then
the bronchial or bronchiolar lumina may be contracted or dilated and
filled with mucopurulent debris\textsuperscript{34}, with partial or
complete luminal obliterans.\textsuperscript{35-37} In addition,
infections also play an important role in the development of bronchitis
obliterans and the most common post-infection pathogens occurrence of
bronchiolitis obliterans in children are \emph{Mycoplasma pneumoniae} ,
\emph{adenovirus} ,\emph{respiratory syncytial virus} , \emph{influenza}
, \emph{measles} and\emph{tuberculosis.} \textsuperscript{38}
\emph{Mycoplasma pneumonia}adheres to the ciliated columnar epithelium
of the respiratory tract and produces local
cytotoxic.\textsuperscript{39} And P1-adhesin, a transmembrane protein,
helps \emph{Mycoplasma pneumonia} in cell-to-cell transfer which
eventually increases the infective surface area and results in extensive
airway epithelium damage.\textsuperscript{40}\emph{Mycoplasma
pneumoniae} was detected in our case through BALF-NGS, therefore,
bronchitis obliterans in our case was considered to be the results of
bronchiectasis and \emph{Mycoplasma pneumoniae} infection. Currently,
there has been no evidence to contact PAM with infections, but it has
been predicted that environmental factors such as exposure to passive
smoking and infections may accelerate the process of PAM.

As for PAM, we should distinguish between miliary tuberculosis (PAM
occurs frequently in countries where \emph{Mycobacterium}
\emph{tuberculosis}is common), hemosiderosis, silicosis, carcinomatosis,
or sarcoidosis.\textsuperscript{19} When there are extensive
calcifications in both lungs on mediastinal windows, we should
especially distinguish them from miliary tuberculosis
(TB).\textsuperscript{41} Miliary tuberculosis is a potentially fatal
form of disseminated disease due to the hematogenous spread of tubercle
bacilli to the lungs, and other organs. It results in the formation of
millet seed-sized (1 to 2 mm) tuberculous
foci.\textsuperscript{42}However, the size (3 to 6 mm) of calcifications
in our child's imaging was bigger than that of miliary TB and
calcifications were distributed in the middle and lower lobes. While in
miliary TB, innumerable micronodular (1 mm) infiltrates, diffusely
scattered in both lungs, especially the lung apices.\textsuperscript{43}
Besides, there was no obvious lymphadenopathy and no associated cavities
to spread satellite lesions. No evidence of tuberculosis was found in
BALF-NGS. Though the patient's TST was positive, probably because of
vaccination. To summarize, TB infection is not considered at this time
and calcifications in PAM are quite different from TB in location and
size.

\textbf{Conclusion}

PAM is rarely diagnosed in children, especially under 5 years old and
this may be related to the lack of obvious clinical symptoms and imaging
features. Our patient was diagnosed by identifying two novel gene
mutations which expanded the spectrum of genetic mutations in PAM,
however, no specific genotype-phenotype could be concluded and a larger
population review or further investigations are needed. This unique case
could help us to further explore the mechanism of PAM and emphasize the
role of gene analysis in diagnosing rare pediatric diseases.

\textbf{Author contributions}

\textbf{Meiyu Zhang:}Writing-original draft. \textbf{Man Gao:}
Writing-review and editing.\textbf{Yuhuan Liu} : Validation.
\textbf{Wang Kun:} Validation.\textbf{Siyan zhou:} Data curation.
\textbf{Haoran Jing:} Investigation.\textbf{Yin Guo:} Supervision.
\textbf{Fanzheng Meng:} Supervision.

\textbf{Acknowledgements}

We appreciate the patient and his parents for agreeing to use their data
for publication. We thank all the doctors in the Pediatric Department of
Respiration of the First hospital in Jilin University for supporting the
research.

\textbf{Funding statement}

This study was supported by the Natural Science Foundation of Jilin
Province (Grant No:20200201475JC) and the Finance Department Medical
Special Fund (Grant No: 2018SCZWSZX-051) of Jilin Province.

\textbf{Conflicts of interest}

The authors have no funding and conflicts of interest to disclose.

\textbf{Data availability statement}

All data generated or analyzed during this study are included in this
published article.

\textbf{Consent statement}

The patient's parent has provided informed consent for publication of
the case.

\textbf{ORCID}

Man Gao:\emph{https://orcid.org/0000-0002-2524-5034}

Fanzheng Meng:\emph{https://orcid.org/0009-0004-1632-4154}

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\par\null

\textbf{Figure legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1: The chest computed tomography of the patient. (A-D) The chest
computed tomography of the 2-year-old boy. Chest CT showed
bronchiectasis (red arrow) complicated with inflammation (black arrow)
in both lungs. (E-P) The imagings of profound diagnosed with PAM at age
7. Lung windows showed ``mosaic'' feature (yellow arrow), bronchiectasis
(red arrow), and interstitial changes (blue arrow). Mediastinal windows
showed extensive calcifications (white arrow) in both lungs.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2: Features of bronchitis obliterans under bronchoscopy. (A-B)
Bronchoscopy showed bronchitis obliterans was covered by a
smooth-surfaced membrane in involving many of the subsegmental airways
of both lungs.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\par\null

Figure 3: The results of whole-exome-sequencing. (A) The mutation of
gain (EXON:2-6 duplication) from his father. The variant was predicted
to disrupt the reading frame and leaded to transcription factor
degradation. (B) The mutation of c.1218 (exon11) C\textgreater{}A from
his mother. The missense variant was suspected as a pathogenic gene
mutation.

\textbf{Hosted file}

\verb`Table.docx`  available at \url{https://authorea.com/users/614642/articles/641399-two-novel-gene-mutations-identified-in-a-child-with-pulmonary-alveolar-microlithiasis-complicated-with-bronchitis-obliterans-a-case-report-and-literature-review}

\selectlanguage{english}
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