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\begin{document}

\title{Novel AP3B1 mutations in a Hermansky--Pudlak syndrome type2 with
neonatal pulmonary fibrosis}



\author[1]{Keigo Matsuyuki}%
\author[1]{Mizuki Ide}%
\author[1]{Keishirou Houjou}%
\author[1]{Saho Shima}%
\author[1]{Seiji Tanaka}%
\author[1]{Yoriko Watanabe}%
\author[2]{Tomoko Egashira}%
\author[2]{Hiroyuki Tomino}%
\author[2]{Toshimitsu Takayanagi}%
\author[3]{Katsuya Tashiro}%
\author[4]{Ken Okamura}%
\author[4]{Tamio Suzuki}%
\author[1]{Ryuta Nishikomori}%
\affil[1]{Kurume University School of Medicine}%
\affil[2]{National Hospital Organisation Saga National Hospital}%
\affil[3]{Karatsu Red Cross Hospital}%
\affil[4]{Yamagata University}%


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  \date{May 25, 2024}


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The authors declare no conflicts of interest.

Financial Support: Japan Society for the Promotion of Science 19K08299

Keywords: Patients with Hermansky--Pudlak syndrome, Pulmonary
fibrosis,\emph{AP3B1} mutation, Hemophagocytic lymphohistiocytosis,
Neonatal

To the Editor,

Hermansky--Pudlak syndrome (HPS) is an inherited disorder characterized
by albinism of the oculocutaneous region and hemorrhagic disease. Eleven
genes causative of HPS have been identified, and the clinical phenotypes
of the condition differ depending on which gene is affected. HPS type 2
(HPS2) is an autosomal recessive inherited disease caused by mutations
in \emph{AP3B1} , resulting in pulmonary fibrosis (PF) and
immunodeficiency\textsuperscript{1}. Patients with HPS2 also have
neutropenia and decreased NK cell cytotoxicity, which can lead to
hemophagocytic lymphohistiocytosis (HLH); however, the total number of
HPS2 cases reported was less than 40 as of March
2021\textsuperscript{1}, making it difficult to accurately assess the
risk of HLH associated with this disease. In addition, although PF
occurs earlier in HPS2 than in other types of HPS, HPS2 with PF in the
neonatal period has never been reported. Here, we report a case of HPS2
with neonatal PF harboring mutations in both \emph{AP3B1} alleles, one
of which is novel.

The patient is female, the second child of a non-consanguineous
marriage, and was born at 39 weeks 4 days (birth weight, 3798 g) by
spontaneous vaginal delivery. Apgar score was 9 at 1 minute; however,
respiratory failure gradually developed, resulting in endotracheal
intubation and mechanical ventilation. At 20 hours after birth, a right
tension pneumothorax developed, which required thoracentesis, thoracic
drainage, and nitric oxide inhalation therapy. A computerized tomography
scan at 6 days of age revealed PF (Fig. 1). Septic workup, including
blood and throat swabs, was negative. Respiratory distress syndrome was
less likely due to term delivery; therefore, we suspected hereditary
interstitial lung disease and tested the \emph{SFTPB} , \emph{SFTPC}
,\emph{ABCA3} , \emph{FOXF1} , \emph{NKX2.1} , \emph{GATA2} ,
\emph{CSF2RA} , and \emph{CSF2RB} genes, all of which were
negative\textsuperscript{2}. Since she had albinism, we performed
genetic analysis of 22 albinism-related genes, which revealed two
heterozygous variants in\emph{AP3B1} : c.1122\_1123insAG (p.Phe375fs*)
and c.2546T\textgreater{}A (p.Leu849Ter). The former variant is a novel
frameshift mutation, and the latter is a nonsense mutation that was
recently reported as the causative mutation in a patient with HPS2 in
Japan\textsuperscript{3}. Neither variant is recorded in the Exome
Aggregation Consortium (ExAC, http://exac.broadinstitute.org/) database,
and both are considered pathological. Each parent carried one of the
variants; therefore, we diagnosed the patient with HPS2 caused by
\emph{AP3B1} mutations. The patient also suffered from mitral
regurgitation, which did not require additional medication.

The patient's respiratory condition improved, and she was discharged
from hospital at 190 days of age, with the introduction of home oxygen
therapy. At 9 months old, she was hospitalized due to HLH. Physical
examination revealed no abnormal findings, other than pharyngeal
injection and slight fever. Laboratory tests showed thrombocytopenia,
with platelets 3.0 \selectlanguage{ngerman}× 10\textsuperscript{4}/\selectlanguage{greek}μ\selectlanguage{english}L and white blood cell count
1100/\selectlanguage{greek}μ\selectlanguage{english}L (2.8\% neutrophils). Ferritin was not raised; however, soluble
IL2 receptor levels were elevated at 7469 U/mL. Bone marrow examination
showed evidence of hemophagocytosis. CD107a expression was decreased in
CD3\textsuperscript{--}CD56\textsuperscript{+} NK cells compared with
control NK cells, detected as previously described\textsuperscript{4},
suggesting a decrease in cytotoxic degranulation (Fig. 2). Blood samples
were negative for Epstein--Barr virus, cytomegalovirus, and human
parvovirus B12. The patient was diagnosed with HLH associated with viral
infection. A primary immunodeficiency gene panel, comprising
approximately 400 genes, was evaluated; no mutations in \emph{PFR1}
,\emph{UNC13D} , \emph{STX11} , \emph{STXBP2} , \emph{RAB27A} ,
or\emph{LYST} were detected. She was treated with dexamethasone (10
mg/m\textsuperscript{2}/day) and immunoglobulin (2.5 g/dose), with good
clinical response. We started G-CSF for continuous neutropenia after
recovery from HLH. She had another episode of fever with
thrombocytopenia due to viral infections at the age of 1 year 9 months.

Among the primary immunodeficiency diseases affecting cytotoxic
granules, patients with hereditary granulopathies, such as
Chediak--Higashi and Griscelli syndrome type 2, are at high risk of
severe HLH, and early hemopoietic stem cell transplantation (HSCT) is a
treatment option\textsuperscript{5}. By contrast, complication of HLH
occurs less frequently in HPS2, although there is a risk of developing
HLH in some patients with the condition. Jessen et al. reported that the
risk of developing HLH was relatively low in a mouse HPS2 model
affecting \emph{Ap3b1} (the \emph{pearl} mouse)\textsuperscript{6},
which is compatible with reports regarding patients with HPS2; however,
Dell'Acqua et al. cautioned of the risk of HLH in HPS2, in their report
of a case with lethal HLH\textsuperscript{7}.

Further delineation of genotype--phenotype correlations
between\emph{AP3B1} mutations and complication of HLH indicated that
truncating mutations, rather than non-truncating changes such as
missense mutations, may be associated with a higher risk of developing
HLH, which is consistent with our case having nonsense and frameshift
mutations in\emph{AP3B1.} Regarding genotype--phenotype correlation of
PF in HPS2, 17 of 37 cases are reported to have developed PF, all of
which had homozygous or compound heterozygous truncating mutations. The
lack of missense mutations among HPS2 cases with HLH and/or PF suggests
that missense variants may be less pathogenic, in terms of HLH and PF.
It will be necessary to accumulate additional cases of HPS2 and follow
their clinical courses in detail to understand these genotype--phenotype
correlations more precisely.

To the best of our knowledge, this is the first case of HPS2 with PF
observed in the neonatal period. Among types of HPS, PF occurs mainly in
HPS1, HPS2, and HPS4. In HPS1 (the most frequent type), 100\% of
patients develop PF in their thirties or forties\textsuperscript{8}.
Hengst et al. reported six cases of HPS2 with PF, with a mean age of 8.8
years, respiratory symptoms 3.3 years before diagnosis, and a minimum
age of 0.8 years\textsuperscript{9}. It has not previously been reported
that patients with HPS2 suffer from mitral regurgitation, which may
contribute to early onset of PF. In any case, the onset of PF in our
case is the earliest recorded to date, and suggests that neonatologists
should consider HPS2 as a diagnosis for neonates presenting with PF and
albinism.

In conclusion, we report a case of HPS2 with neonatal-onset PF and an
episode of HLH, caused by truncating compound heterozygous mutations of
\emph{AP3B1} . Clinicians should be aware of the importance of accurate
genetic analysis of \emph{AP3B1} to evaluate the severity of clinical
phenotypes, particularly the onset of PF and morbidity from HLH.

Keigo Matusyuki\textsuperscript{1}

Mizuki Ide\textsuperscript{1}

Keishirou Houjou\textsuperscript{1}

Saho Shima\textsuperscript{1}

Seiji Tanaka\textsuperscript{1}

Yoriko Watanabe\textsuperscript{1}

Tomoko Egashira\textsuperscript{2}

Hiroyuki Tomino\textsuperscript{2}

Toshimitsu Takayanagi\textsuperscript{2}

Tashiro Katsuya\textsuperscript{3}

Ken Okamura\textsuperscript{4}

Tamio Suzuki\textsuperscript{4}

Ryuta Nishikomori\textsuperscript{1}

\textsuperscript{1}Department of Pediatrics and Child Health, Kurume
University School of Medicine

Kurume, Fukuoka, Japan

\textsuperscript{2}National Hospital Organization Saga National Hospital

Saga, Saga, Japan

\textsuperscript{3}Karatsu Red Cross Hospital

Karatu, Saga, Japan

\textsuperscript{4}Department of Dermatology, Faculty of Medicine,
Yamagata University

Yamagata, Yamagata, Japan

Acknowledgments

We thank Ms. Ohhinata for technical assistance.

Key Message: Hermansky--Pudlak Syndrome type2 can develop pulmonary
fibrosis in the neonatal period

References

1. Huizing M, Malicdan M, Gochuico B, Gahl W. Hermansky-Pudlak
Syndrome.\emph{GeneReviews} 2000;
https://www.ncbi.nlm.nih.gov/books/NBK1287/, may 2021.

2. Okamura K, Hayashi M, Abe Y, et al. NGS-based targeted resequencing
identified rare subtypes of albinism: Providing accurate molecular
diagnosis for Japanese patients with albinism. \emph{Pigment Cell
Melanoma Res.} 2019;32(6):848-853.

3. Nishikawa T, Okamura K, Moriyama M, et al. Novel AP3B1 compound
heterozygous mutations in a Japanese patient with Hermansky-Pudlak
syndrome type 2. \emph{J Dermatol.} 2020;47(2):185-189.

4. Shibata H, Yasumi T, Shimodera S, et al. Human CTL-based functional
analysis shows the reliability of a munc13-4 protein expression assay
for FHL3 diagnosis. \emph{Blood.} 2018;131(18):2016-2025.

5. Sharma P, Nicoli ER, Serra-Vinardell J, et al. Chediak-Higashi
syndrome: a review of the past, present, and future. \emph{Drug Discov
Today Dis Models.} 2020;31:31-36.

6. Jessen B, Bode SF, Ammann S, et al. The risk of hemophagocytic
lymphohistiocytosis in Hermansky-Pudlak syndrome type 2.
\emph{Blood.}2013;121(15):2943-2951.

7. Dell'Acqua F, Saettini F, Castelli I, Badolato R, Notarangelo LD,
Rizzari C. Hermansky-Pudlak syndrome type II and lethal hemophagocytic
lymphohistiocytosis: Case description and review of the
literature.\emph{J Allergy Clin Immunol Pract.} 2019;7(7):2476-2478
e2475.

8. Vicary GW, Vergne Y, Santiago-Cornier A, Young LR, Roman J. Pulmonary
Fibrosis in Hermansky-Pudlak Syndrome. \emph{Ann Am Thorac
Soc.}2016;13(10):1839-1846.

9. Hengst M, Naehrlich L, Mahavadi P, et al. Hermansky-Pudlak syndrome
type 2 manifests with fibrosing lung disease early in
childhood.\emph{Orphanet J Rare Dis.} 2018;13(1):42.

Figure legends

Fig 1. CT image of interstitial pneumonia at 6 days of age.

Bilateral lung fields showing diffuse ground-glass opacity and
bronchioles dilated to obliteration. Some dorsal lung fields are
hyperintense, possibly reflecting the effect of gravity.

Fig 2. Patient genotype and NK cell activity.

(a) Chromatogram demonstrating compound heterozygous mutations,
c.1122\_1123insAG (p.Phe375fs)* and c.2546T\textgreater{}A
(p.Leu849Ter), in \emph{AP3B1} .

(b) Analysis of NK cell degranulation by flow cytometric analysis.
Profiles show CD56 versus CD107a staining on
CD3-CD56\textsuperscript{+}NK cells after stimulation. CD107a expression
was significantly decreased in patient cells (above) relative to control
cells (below). The assay was performed as previously
described\textsuperscript{4}.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/HPS-Figure-2/HPS-Figure-2}

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