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\begin{document}

\title{Hemophagocytic Lymphohistiocytosis Caused by a Novel ZNFX1 Variant.}



\author[1]{suleimman Al-Sweedan}%
\author[1]{Mariam Almasri}%
\author[1]{Hassan Abushukair}%
\author[1]{Abdallah  Alhusan}%
\author[1]{Faris  Abu Za'nouneh }%
\author[1]{Dalia Alsweedan}%
\author[1]{hajer ayasreh}%
\affil[1]{Jordan University of Science and Technology}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Objective: We report this case of a 2.5 months old infant diagnosed with
HLH with an autosomal recessive ZNFX1 related immune-hematological
abnormalities in order to provide more information regarding the genetic
and clinical manifestations concerning this disorder. Method: Medical
file of the patient was reviewed including; patient profile, lab
results, and management. Results: we present a unique case of a 2.5
months HLH patient that presented with a unique genetic variant with a
mutated ZNFX1 gene. Conclusion: we report a homozygous ZNFX1 variant as
the base of HLH in this patient. HLH proposes a diagnostic challenge as
its signs and symptoms are concurrent with other differentials.%
\end{abstract}%



\sloppy


\textbf{Title} : Case Report:

Hemophagocytic Lymphohistiocytosis Caused by a Novel ZNFX1 Variant.

\textbf{Authors: Suleimman Al-Sweedan} , \textbf{Mariam Almasri, Hassan
Abushukair, Abdallah Alhusan, Faris Abu Za'nouneh, Dalia Alsweedan,
Hajar Ayasrah}

\emph{AFFILIATIONS:}

1-Department of Pediatrics/Faculty of Medicine, Jordan University of
Science\& Technology.

\emph{CORRESPONDING AUTHOR :}

Suleimman Al-Sweedan, MD, Ms

Department of Pediatric Hematology / Oncology,

Jordan University of Science \& Technology

P.O. Box 4614, Irbid, Jordan

Tel: (+962) 799051255

E-mail: sweedan@just.edu.jo

\textbf{Word count for:}

\textbf{Main Text: 1361}

\textbf{Number of Tables: 1}

\textbf{Number of Figures: 1}

\textbf{Short Running title:} \textbf{novel mutation in HLH}

\textbf{Keywords: HLH, Novel Mutation, Primary}

\textbf{CONFLICTS OF INTEREST AND SOURCE OF FUNDING:}

\textbf{The authors have no conflicts of interest relevant to this
article to declare. None of the authors have received any financial
support or funding from any pharmaceutical industry, sponsor or agency
for this work.}

\textbf{All the listed authors have contributed significantly to the
research process that led to the article and have participated in the
final manuscript.}

\textbf{Abstract:}

\textbf{Objective:} We report this case of a 2.5 months old infant
diagnosed with HLH with an autosomal recessive ZNFX1 related
immune-hematological abnormalities in order to provide more information
regarding the genetic and clinical manifestations concerning this
disorder.

\textbf{Method:} Medical file of the patient was reviewed including;
patient profile, lab results, and management.

\textbf{Results:} we present a unique case of a 2.5 months HLH patient
that presented with a unique genetic variant with a mutated ZNFX1 gene.

\textbf{Conclusion:} we report a homozygous ZNFX1 variant as the base of
HLH in this patient. HLH proposes a diagnostic challenge as its signs
and symptoms are concurrent with other differentials.

\textbf{Abreviations:}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{INF} & \textbf{Interferon}\tabularnewline
\midrule
\endhead
\textbf{dsRNA} & \textbf{Double-stranded RNA}\tabularnewline
\textbf{MIS-C} & \textbf{Multisystem Inflammatory Syndrome in
Children}\tabularnewline
\textbf{HLH} & \textbf{Hemophagocytic
lymphohistiocytosis}\tabularnewline
\textbf{NICU} & \textbf{Neonatal Intensive Care Unit}\tabularnewline
\textbf{MAP} & \textbf{Mean Arterial Pressure}\tabularnewline
\textbf{LDH} & \textbf{Lactate Dehydrogenase}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Introduction}

Innate immunity plays a major role in host defense when being exposed to
microorganisms, through pattern recognition receptors found on immune
cells like macrophages and natural killer cells leading to certain
cascades resulting in cytokine release such as interferon (IFN) and
interleukin-12 provoking an inflammatory response. IFN-mediated immunity
is essential in this defense throughout the binding of IFN molecules to
cell surface receptors leading to transcription of hundreds of
IFN-inducible genes evoking the needed antiviral state against RNA
viruses, DNA viruses, intracellular bacteria, and parasites {[}1,2{]}.

NFX1-type zinc finger-containing 1 (ZNFX1) is an IFN-stimulated
double-stranded RNA (dsRNA) sensor that limits viral replication in
infected host cells, thus a deficiency in ZNFX1 increases the liability
to serious viral infections, intracellular-bacterial infections, and
multisystem inflammatory syndrome in children (MIS-C) and adults, which
have been clinically compatible with Hemophagocytic lymphohistiocytosis
(HLH) {[}3{]}. HLH is described as both a familial and sporadic disorder
associated with specific genetic and/or environmental causes. Although
it's life-threatening, it's often misdiagnosed and confused with other
inflammatory disorders due to its rarity and the complexity of its
diagnostic criteria.

We report this case of a 2.5 months old infant diagnosed with HLH with
an autosomal recessive ZNFX1 related immune-hematological abnormalities
in order to provide more information regarding the genetic and clinical
manifestations concerning this disorder {[}4,5{]}.

\textbf{Case Presentation}

A 2.5-month-old Jordanian female patient born of second-degree
consanguineous parents and with no prior neonatal intensive care unit
(NICU) admission presented to the emergency room with respiratory
distress associated with vomiting, and diarrhea of 2 days duration. The
vomit was non-projectile, non-bilious, and non-bloody occurring after
each breastfeeding. The diarrhea was large in amount, foul-smelling,
with no blood or mucous, and occurring approximately 3-4 times per day.
A history of a spike of fever (38 c, axillary) was reported by the
mother. There's a positive ill contact and positive family history of 3
deaths with previous 3 siblings; a sister from HLH relapse, a sister
from cardiac disease, and a brother from liver cirrhosis at ages of 4.5
years-old, 1-year-old, and 5 months-old respectively. Her 4.5 years old
sister was previously diagnosed with HLH clinically based on the
modified HLH-2009 criteria. She was on infliximab and cyclosporine until
her condition suddenly deteriorated and was declared dead afterward.
Upon admission, our patient was not febrile (36.5\selectlanguage{ngerman}°C), with a pulse of
150 beats per minute, mean arterial pressure (MAP) of 75 mmHg,
respiratory rate of 45-50 breaths per minute, oxygen saturation on room
air was 75\%-85\% improved to 99\% on nasal cannula of 3L. Physical
examination revealed an ill looking, irritable patient with nasal
discharge, grunting, subcostal and suprasternal retractions. Upon
palpation, the abdomen was soft lax with no revealed tenderness.
Auscultation of the chest revealed decreased bilateral air-entry with no
added sounds. Examination for hepatosplenomegaly was inconclusive, no
rash, no bleeding, no lymphadenopathy, and normal neurological exam, the
rest of the systemic examination was unremarkable. The initial clinical
diagnosis was sepsis with gastroenteritis and bronchiolitis with
radiological features showing congested lung and fissures edema which
improved after administering furosemide.

The patient was admitted to the pediatric ICU, and a full septic workup
was done, she was given IV fluid and empirical antibiotics (Ceftriaxone,
IV Vancomycin) but her condition rapidly deteriorated and the culture
results turned out to be negative. COVID-19 by RT-PCR, Respiratory
syncytial virus, Clostridium difficile toxin, Rotavirus,
Cryptosporidium, and stool culture tests were all negative. The patient
was admitted several times after her first admission, upon her last
admission she developed severe metabolic acidosis along with respiratory
distress which ended with a cardiopulmonary arrest and death. Figure 1
illustrates a timeline from the patient's birth until death.

Lactate dehydrogenase (LDH) test was also done on 29/08 and it was 1311
U/L, then was repeated on 1/9/2021 along with serum ferritin and they
were further elevated to 613 ng/ml and 1335 U/L respectively, which
required the patient to be transferred into the pediatric
hematology/oncology department in the hospital for HLH full evaluation.
Detailed laboratory investigations are shown in Table 1 . A pre-therapy
HLA typing was performed to search for hematopoietic cell transplant
(HCT) donor, and the matching results were (3/6), (3/6), (2/6) for her
mother, father, and all her siblings respectively.

Lumbar puncture and echocardiogram did not show any abnormal findings.
Abdominal ultrasonography showed homogenous liver and spleen with no
focal lesions, a 6.2cm long axis for the liver, and 5.8cm for the
spleen. Both kidneys appeared normal in size, shape, and echotexture
with no hydronephrosis seen. A partially contracted gallbladder was
shown, with no intra- or extra-hepatic biliary dilatation.

HLH was suspected given the patient family history, critically ill
condition, cytopenia, and high serum ferritin. Whole-genome sequencing
was done showing a homozygous frameshift deletion variant of ZNFX1
(NM\_021035.3: c.5362\_5369del, NP\_066363.1:p.Val1788CysfsTer6) causing
the ZNFX1 related immuno-hematological abnormalities seen in our
patient.

\textbf{Discussion}

In this report, we present a unique case of a 2.5 months HLH patient
that presented with a unique genetic variant with a mutated ZNFX1 gene.
HLH is characterized by multisystem inflammation resulting from
prolonged activation of phagocytic cells and lymphocytes, which can
manifest idiopathically or secondary due to predisposing comorbidities.

Secondary or acquired HLH usually manifests in a strong immunological
activation background resulting from severe infection or malignancy,
whereas primary HLH occurs in a genetic background in which a mutation
is present at birth and manifestation appear in the first years of life
{[}6{]}. In our case, whole-exome sequencing revealed a frameshift
deletion mutation in the ZNFX1 gene, which is a member of the helicase
superfamily 1 stimulated by IFN {[}7{]}. ZNFX1 has a prominent antiviral
function as it recognizes viral RNA and interacts with mitochondrial
antiviral signaling protein to induce a type 1 IFN response {[}7{]}.

The literature on ZNFX1 involvement in immunodeficient states has been
growing lately, as it was first addressed in 13 patients with severe
viral infections {[}3{]}. Despite prior evidence which demonstrated that
ZNFX1 deficiency in mice or human cell lines does not predispose to DNA
virus infection, this study had six patients with DNA viruses, the
authors owed this to the insufficient resolution of the IFN response to
infection and the heavy viral load {[}3,7{]}. Another study reported on
four patients with intermittent monocytosis and mycobacterial infections
which were found to be homozygous for loss of function ZNFX1 variants.
Hematopoietic and nonhematopoietic cells from these patients' cell lines
responded normally to IFN-gamma indicating ZNFX1 is associated with
stress granule and is necessary for monocyte hemostasis and mycobacteria
immunity {[}8{]}.

In a recent study on ZNFX1-related familial immunodeficiency from the
middle east, nine patients were identified from seven families {[}9{]}.
Complete protein loss was predicted to occur in two ZNFX1 variants (p.
(Arg472Glyfs*16) and p. (Tyr362*)) through nonsense-mediated decay.
Patients with these variants have also manifested with more severe
clinical phenotypes including liver, spleen, and kidney involvement,
developmental delay, as well as death, compared to patients who were
able to produce truncated proteins missing only the C-terminal region
{[}9{]}. Interestingly, only the family from Jordan in this study had
the same variant as the one detected in our case (NM\_021035.3:
c.5362\_5369del, NP\_066363.1: p. Val1788CysfsTer6).

Other genetic conditions predisposing to HLH include familial causes
through the involvement of PRF1, UNC13D, STX11, STXBP2 genes {[}6{]} .
Pigmentary disorders were also found to be associated with HLH through
defective lymphocyte granule-mediated cytotoxicity with RAB27A, LYST,
AP3B1 genes involvement {[}6{]}. With the recent emerging evidence, the
ZNFX1 should be considered as well.

Treatment options for ZNFX1 deficient patients are still limited; in the
cohort reported by Vavassori et al. treatment with immunosuppressants
led to transient benefit while hematopoietic stem cells transplantation
led to complete arrest of HLH flares and neurologic development in one
patient {[}3{]}.

\textbf{Conclusion}

In conclusion, we report a homozygous ZNFX1 variant as the base of HLH
in this patient. HLH proposes a diagnostic challenge as its signs and
symptoms are concurrent with other differentials, so the need for high
clinical suspicion cannot be understated. Further studies on the novel
ZNFX1 variant (NM\_021035.3: c.5362\_5369del,
NP\_066363.1:p.Val1788CysfsTer6) can help in understanding the potential
role of ZNFX1 in its related immuno-hematological abnormalities and HLH
etiology, this insight will warrant enhanced targeted therapeutics in
this subset of HLH patients.

\textbf{References}

1. Kaur BP, Secord E. Innate Immunity. Pediatr. Clin. 2019; 66:905--911

2. Schneider WM, Chevillotte MD, Rice CM. Interferon-stimulated genes: a
complex web of host defenses. Annu. Rev. Immunol. 2014; 32:513--545

3. Vavassori S, Chou J, Faletti LE, et al. Multisystem inflammation and
susceptibility to viral infections in human ZNFX1 deficiency. J. Allergy
Clin. Immunol. 2021; 148:381--393

4. Griffin G, Shenoi S, Hughes GC. Hemophagocytic lymphohistiocytosis:
An update on pathogenesis, diagnosis, and therapy. Best Pract. Res.
Clin. Rheumatol. 2020; 34:101515

5. Jordan MB, Allen CE, Greenberg J, et al. Challenges in the diagnosis
of hemophagocytic lymphohistiocytosis: Recommendations from the North
American Consortium for Histiocytosis (NACHO). Pediatr. Blood Cancer
2019; 66:e27929

6. Canna SW, Marsh RA. Pediatric hemophagocytic lymphohistiocytosis.
Blood 2020; 135:1332--1343

7. Wang Y, Yuan S, Jia X, et al. Mitochondria-localised ZNFX1 functions
as a dsRNA sensor to initiate antiviral responses through MAVS. Nat.
Cell Biol. 2019; 21:1346--1356

8. Le Voyer T, Neehus A-L, Yang R, et al. Inherited deficiency of stress
granule ZNFX1 in patients with monocytosis and mycobacterial disease.
Proc. Natl. Acad. Sci. U. S. A. 2021; 118:

9. Alawbathani S, Westenberger A, Ordonez-Herrera N, et al. Biallelic
ZNFX1 variants are associated with a spectrum of immuno-hematological
abnormalities. Clin. Genet. 2021; n/a:\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figures and Tables}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& 27/08/2021 & 08/09/2021 & 22/09/2021 & 06/10/2021 &
07/10/2021\tabularnewline
\midrule
\endhead
\textbf{Hb} & 9.2 g/dl & 11.2 g/dl & 11.3 g/dl & 11.6 g/dl &
10.1g/dl\tabularnewline
\textbf{HCT} & 28.8\% & 35.1\% & 34.3\% & 35.3\% & 32.6\%\tabularnewline
\textbf{Reticulocytes} & 5.48\% & & & &\tabularnewline
\textbf{WBCs} & 37.3 10*3/mm*3 & 28.05 10*3/mm*3 & 17.3 10*3/mm*3 &
22.63 10*3/mm*3 & 52.5 10*3/mm*3\tabularnewline
\textbf{Platelets} & 261 10*3/mm*3 & 265 10*3/mm*3 & 105 10*3/mm*3 & 262
10*3/mm*3 & 351 10*3/mm*3\tabularnewline
\textbf{PMN} & 29\% & 13.6\% & 20.7\% & 32.9\% & 35\%\tabularnewline
\textbf{Lymphocytes} & 64\% & 79\% & 71.6\% & 58.6\% &
59\%\tabularnewline
\textbf{Monocytes} & 4\% & 6.4\% & 6.1\% & 6.8\% & 4\%\tabularnewline
\textbf{Serum ferritin} & & & 502.55 ng/ml & &\tabularnewline
\textbf{Fibrinogen} & 187 mg/dl & & 259 mg/dl & &\tabularnewline
\textbf{AST} & 49 U/L & & & & 37.6 UL\tabularnewline
\textbf{ALT} & 15 U/L & & & & 11.6 U/L\tabularnewline
\textbf{ALP} & 281 U/L & & & & 274 U/L\tabularnewline
\textbf{Albumin} & 34 g/L & & & & 29.8 g/L\tabularnewline
\textbf{GGT} & 32 U/L & & & & 17 U/L\tabularnewline
\textbf{LDH} & 1168 U/L & 984 U/L & & & 933 U/L\tabularnewline
\textbf{Total Bilirubin} & 7 umol/L & & & & 3.4 umol/L\tabularnewline
\textbf{Direct bilirubin} & 2 umol/L & & & & 0.1 umol/L\tabularnewline
\textbf{PT-INR} & 1.14 & & & &\tabularnewline
\textbf{TG} & & & & & 1.27 mmol/L\tabularnewline
\textbf{Blood NH3} & & & & & 89.5 umol/L\tabularnewline
\textbf{Creatinine} & 21 umol/L & & & 21 umol/L & 31
umol/L\tabularnewline
\textbf{Na} & 141 mmol/L & & & 139 mmol/L & 134 mmol/L\tabularnewline
K & 4.9 mmol/L & & & 5.41 mmol/L & 6 mmol/L\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
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