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\begin{document}

\title{A novel mutation in SLC39A7 identified in a patient with autosomal
recessive agammaglobulinemia}



\author[1]{Melinda Erdős}%
\author[2]{Kristina Mironska}%
\author[2]{Lidia Kareva}%
\author[2]{Katarina Stavrik}%
\author[2]{Arijeta Hasani}%
\author[3]{Arpad Lanyi}%
\author[3]{Judit Kallai}%
\author[4]{Laszlo Marodi}%
\affil[1]{University of Debrecen, Faculty of Medicine}%
\affil[2]{Ss Cyril and Methodius University in Skopje}%
\affil[3]{University of Debrecen}%
\affil[4]{Semmelweis Egyetem}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\textbf{A novel mutation in \emph{SLC39A7} identified in a patient with
autosomal recessive agammaglobulinemia}

\textbf{To the Editor,}

ZIP7 deficiency is the most recently described congenital
agammaglobulinemia with autosomal recessive inheritance (1). ZIP7,
encoded by \emph{SLC39A7,} is an endoplasmic reticulum-to-cytoplasm
Zn\textsuperscript{2+} transporter. Developing B cells are sensitive to
altered Zn\textsuperscript{2+}~distribution which cause developmental
blockade beyond the pre-B cell stage (2). Complete loss of ZIP7 in cell
lines causes a reduction in cytoplasmic Zn\textsuperscript{2+}~and an
increase in endoplasmic reticulum Zn\textsuperscript{2+}~concentration.
Since the original report in 2019, no additional cases of ZIP7
deficiency have been published. Here we describe a patient evaluated for
recurrent respiratory tract infections, meningitis, agammaglobulinemia
and B cell lymphopenia, ultimately found to carry a novel
\emph{SLC39A7}variant. We describe his clinical characteristics,
immunological findings, and genetic investigations.

The patient and his family members were interviewed, examined, treated
and monitored at the University Clinic for Children's Disease in Skopje.
Medical records were obtained from the electronic registry of the
University Clinic. The mother of the patient has given written informed
consent to conduct the study and for publication of data. All procedures
were performed in accordance with the ethical standards of the
Institutional Research Committee. Genomic DNA from the patient and his
family members was isolated with the Gen Elute Blood Genomic DNA kit
(Sigma-Aldrich) and subjected to whole-exome sequencing (WES) in the
patient and targeted gene sequencing in the patient and available family
members (3). WES was performed at the New York Genome Center and the
Rockefeller University using an Illumina HiSeq 2500 sequencing system
(Illumina). Exome capture was carried out with SureSelect human all
exome kit (Agilent) in accordance with the manufacturer's instructions.
Putative disease alleles found by WES were validated by dideoxy Sanger
sequencing in the patient as well as in family members.Exons and
flanking intronic regions of \emph{SLC39A7} were amplified by PCR.
Amplicons were sequenced with the Big Dye Terminator cycle sequencing
kit (Applied Biosystems) and targeted regions were analyzed by an ABI
3130 capillary sequencer (Applied Biosystems). Sequence variants were
determined by comparing to the appropriate GenBank reference sequence to
identify the position of mutations.

The patient, a 14-year-old male and the third child in a Macedonian
family with Albanian origin was born at term (Fig. 1). Birth weight and
length were in the normal ranges and neonatal adaptation was uneventful.
Consanguinity in the family was not reported. The patient received only
BCG vaccination at birth indicated by a small scar on the left shoulder.
He had suffered from recurrent upper and lower respiratory tract
infections since 6 months of age. He was first hospitalized for \emph{H.
influenzae} pneumonia at the age of 4 years. Treatment with ceftriaxone,
50 mg/kg/day for 10 days, resulted in clinical and radiological
recovery. He was suspected for immunodeficiency, but the family failed
to meet the medical appointments for immunological evaluation. By 6\selectlanguage{ngerman}½
years of age he developed bilateral chronic mastoiditis which was
treated with antibiotics and surgery with good therapeutic efficacy. At
the age of 7 he was hospitalized for purulent meningitis. Pathogenic
microorganisms did not yield from the cerebrospinal fluid which could be
due to the previously started cephalosporin antibiotic therapy. After
two weeks of treatment with 3\textsuperscript{rd} generation intravenous
cephalosporin, he was discharged from hospital but continued to develop
recurrent upper respiratory tract infections treated mostly by the
family pediatrician. At the age of 9 years, he was hospitalized again
because of severe bilateral pneumonia. Respiratory aspirate
yielded\emph{Pseudomonas aeruginosa} . He received intravenous
antibiotics, inhalative Colistin (polymyxin E), and antimycotic
treatment because\emph{C. albicans} was isolated from the tracheal
aspirate. Multiple rigid bronchoscopic lavages were performed as part of
the treatment. Chest computed tomography (CT) showed bronchiectasis and
perfusion scan of the lung showed hypoperfusion in the right lower lobe.
He was seen at the Immunology Department of the University Clinic for
Children's Diseases in Skopje, at age 9½. By physical examination
lymphoid tissue hypoplasia (lack of tonsillar tissues, small adenoids
and impalpable lymph nodes), hepatomegaly (liver edge was palpable 3 cm
below the costal margin), vesicular breathing with bilateral crepitation
above both lungs, and low body weight (18 kg,
\textless{}5\textsuperscript{th}percentile) were found. The family
history was negative for primary immunodeficiencies and hematological
disorders. His mother and three siblings (2 boys and 1 girl) are
healthy; his father died in a car accident at the age of 33 years.

The total number of white blood cells, red blood cells and platelets
were normal but the patients had hypochromic anemia, neutropenia, and
CD19\textsuperscript{+} lymphopenia (Table 1). Immunochemistry tests
revealed agammaglobulinemia, and absence of pathogen-specific antibodies
to tetanus toxoid, \emph{H. influenza} type B, and~\emph{Str.
pneumoniae} (Table 1)\emph{.} Concentrations of complement components C3
and C4 were normal, and measurement of thyroid hormones showed normal
levels of fT3 and fT4. The Quantiferon-TB test was negative. Based on
these findings, monthly intravenous immunoglobulin (IVIG) therapy was
started.~Inborn errors of immunity (IEI) related genes were analyzed
based on the most recent updated classification of the International
Union of Immunological Societies Expert Committee (4).~Mutation
screening by WES revealed that the patient harbored a private homozygous
variant in \emph{SLC39A7} . The variant was predicted to be deleterious
(CADD score: 27.1). No other disease-causing variant in other IEI
related genes were identified by WES. The \emph{SLC39A7} sequence
variant was validated by targeted gene sequencing, and we found that the
patient was homozygous whereas his mother and three siblings were all
heterozygous for the c.1051A\textgreater{}G, p.Thr351Ala mutation
in\emph{SLC39A7} (Fig. 1).

We report here a novel private homozygous mutation in
\emph{SLC39A7}discovered in a boy with Albanian ancestry. This mutation
results in an amino acid substitution (alanine for threonine) located at
position 351 of the ZIP7 protein. The biallelic variant is associated
with a profound decrease of peripheral blood B cells and immunoglobulin
isotypes resulting in recurrent and severe infectious diseases. As such,
the present work supports earlier evidence in which homozygous or
compound heterozygous mutations in \emph{SLC39A7} were found in 6
patients with absent B cells and agammaglobulinemia and proposed as a
novel PID. These vary in that they occur at different residues within
the protein (1). Mutations including c.1051A\textgreater{}G described
here resulted in changes of amino acid residues mostly at the C terminal
end of the protein between residues 351 and 458 with no mutational
hotspot (Fig. 1).

Transmembrane transporters of Ca\textsuperscript{2+},
Mg\textsuperscript{2+} and Zn\textsuperscript{2+} control the movement
of these divalent cation mediators of lymphocyte signaling between the
cytosol and the cytosolic organelles or the extracellular space (5-7).
Monogenic germline mutations of genes may result in loss of function of
the encoded transporter proteins and impaired adaptive immunity mediated
by B and T cells (\emph{ORAI1} mutation; 5), T and NK cells
(\emph{MAGT1} mutation; 6) or B cells (\emph{SLC39A7} mutation; 7)
resulting in PIDs. Structural and functional integrity of thousands of
proteins are regulated by Zn\textsuperscript{2+} transporters (7).
Biallelic hypomorphic mutations of \emph{SLC39A7} which encodes the
Zn\textsuperscript{2+} transporter ZIP7 has recently been discovered.
Pertinent to this, in mouse B cell lines with homozygous mutation, a
diminished concentration of cytoplasmic free Zn\textsuperscript{2+} and
decreased phosphorylation of signaling molecules of pre-B cell and B
cell receptors were found. These findings highlight the critical role
for cytoplasmic Zn\textsuperscript{2+} in B cell receptor signaling
downstream of the pre-B cell and B cell receptors.

Similarly to defects of B cell development to rearrange heavy and then
light chain immunoglobulin genes that lead to AR and X-linked
agammaglobulinemias, loss of function alleles of \emph{SLC39A7} may lead
to impaired B cell signaling. The patient described in this report had
agammaglobulinemia, lack of B cells, and hypoplastic lymph nodes and
tonsils despite recurrent upper respiratory tract infections. We thought
of XLA and searched for \emph{BTK} mutation first but found wild type
sequences in the patient (not shown). Next, WES and targeted gene
sequencing were used to search for known AR and AD agammaglobulinemia
genes and we found in the patient's DNA a previously unknown
c.1051A\textgreater{}G, p.Thr351Ala \emph{SLC39A7} mutation. By using
blood derived DNA samples from all available family members, we
confirmed homozygosity of this \emph{SLC39A7} mutation in the patient
and heterozygosity in family members who were clinically healthy (Fig.
1). The gene encoding the zinc transporter protein ZIP7 has been
implicated to cause autosomal recessive agammaglobulinemia in white
European, South Asian, and Hispanic ancestries (7). To our knowledge the
patient reported here is the first diagnosed with inherited ZIP7
deficiency in Central and Eastern Europe.

Hematologic stem cell transplantation (HSCT) was found beneficial in two
of the 6 patients in the previous study (7); these two patients had the
most severe disease phenotype including failure to thrive, blistering
dermatitis and thrombocytopenia in addition to early onset infections,
agammaglobulinemia and B cell depletion which were present in all
patients. The other 4 patients including those two with failure to
thrive, liver dysfunction and seborrheic dermatitis, responded well to
intravenous immunoglobulin replacement alone. HSCT in our patient has
not been performed until now, because we did not have an accurate
molecular diagnosis, so we treated him with regular IVIG substitution
dose of 400 mg/3-4 weeks, since his 9½ years of age. The adherence to
the treatment has often been poor and he has signs of chronic pulmonary
damage. He does not attend school, does not cooperate and spirometry
cannot be performed which make clinical management challenging.

In summary, the report of this patient adds to the current clinical and
genetic knowledge on ZIP7 deficiency in humans. Early recognition and
diagnosis of this condition is pivotal for improved outcomes and
prevention of complications like lung tissue damage developed over years
in this patient. Unfortunately, most patients with PID in Eastern and
Central Europe still remain without molecular diagnosis (8-10).
Therefore, collaborative programs with more advanced centers are
critically important especially in countries with lower socioeconomic
condition and limited resources to molecular diagnostics. Such a program
has been established by the J Project physician education and clinical
research collaboration network (www.thejpnetwork.com). The J Project
program provides help for early diagnosis, treatment, and family
counseling of known and even very recently described PIDs like ZIP7
deficiency (10). In conclusion, diagnosis of ZIP7 deficiency should be
contemplated in patients with autosomal recessive agammaglobulinemia and
recurrent, early onset infectious diseases.

KEYWORDS

zinc transporter proteins, SLC39A7 mutation, B cell deficiency,
agammaglobulinemia

\textbf{ACKNOWLEDGEMENTS}

We thank the mother of the patient for helpful collaboration. This
research was supported by the J Project and the Foundation for Children
with Immunodeficiencies, and from the European Society for
Immunodeficiencies to ME.

\textbf{CONFLICT OF INTEREST}

The authors declare that they have no competing financial interests.

\textbf{AUTHOR CONTRIBUTIONS}

\selectlanguage{polish}\textbf{Melinda Erdős:}\selectlanguage{english} performing bioinformatics analysis and writing
the initial draft. \textbf{Kristina Mironska:} conducting clinical
research and patient care, editing the initial draft. \textbf{Lidia
Kareva:} conducting clinical research and patient care, data
collection.\textbf{Katarina Stavric:} conducting clinical research and
patient care, data collection. \textbf{Arijeta Hasani:} conducting
clinical research and patient care, data collection. \selectlanguage{ngerman}\textbf{Árpád
Lányi:}conducting targeted gene sequencing. \textbf{Judit Kállai:}
conducting targeted gene sequencing. \textbf{László Maródi:} formulation
of research goals, writing the final draft\textbf{.} All authors
reviewed the manuscript.

\textbf{AVAILABILITY OF DATA AND MATERIALS}

The data that support the findings and the materials used in this study
are available on request. The data are not publicly available due to
ethical restrictions.

\textbf{Figure legend}

\textbf{Figure 1}

\textbf{Homozygous \emph{SLC39A7} mutation in a patient with autosomal
recessive agammaglobulinemia.}

Family Pedigree with \emph{SLC39A7} allele segregation. The black-filled
symbol indicates the proband (P) having the novel homozygous
\emph{SLC39A7} mutation. Symbols consisting of black and white colours
indicate heterozygous disease carriers as determined by targeted
sequencing. Diagonal bar indicates diseased individual. Generations are
designated by a Roman numeral (I and II), and each individual by an
Arabic numeral (from left to right). DNA was obtained for genetic
analysis from all individuals to whom a number is assigned. Automated
sequencing profiles show homozygous c.1051A\textgreater{}G, p.T351A
mutation in the proband (II.3; P) and heterozygous mutations in four
family members (I.2, II.1, II.2, II.4).~C, Control.

\textbf{B)} Schematic representation of domain structure of ZIP7 and
localization of mutations. Numbers above and below the scheme indicate
the amino acid residue numbers. Numbers below the scheme show the
borders of histidine-rich domains. Positions of the identified SLC39A7
variants including six missense (P190A, L217P, T351A, E363K, T395I,
G458A) and two nonsense (Q372X, E451X) mutations. The novel T351A
mutation is marked in red. Mutations in bold were observed in homozygous
form. TM, transmembrane; His, histidine.

\selectlanguage{polish}\textbf{Melinda Erdős\textsuperscript{1,2}}\selectlanguage{english}

\textbf{Kristina Mironska\textsuperscript{3}}

\textbf{Lidia Kareva\textsuperscript{3}}

\textbf{Katarina Stavric\textsuperscript{3}}

\textbf{Arijeta Hasani\textsuperscript{3}~}

\selectlanguage{ngerman}\textbf{Árpád Lányi\textsuperscript{4}}

\textbf{Judit Kállai\textsuperscript{4}}

\textbf{László Maródi\textsuperscript{1,2}}

\textsuperscript{1}Primary Immunodeficiency Clinical Unit and
Laboratory, Department of Dermatology, Venereology and Dermatooncology,
Semmelweis University, Budapest, Hungary

\textsuperscript{2}St. Giles Laboratory of Human Genetics of Infectious
Diseases, Rockefeller Branch,

The Rockefeller University, New York, NY

\textsuperscript{3}Department of Immunology, University Clinic for
Children's Disease, Skopje, Republic of North Macedonia

\textsuperscript{4}Department of Immunology, Faculty of Medicine,
University of Debrecen, Debrecen, Hungary

\textbf{Correspondence}

Melinda Erd\selectlanguage{polish}ő\selectlanguage{english}s or L\selectlanguage{ngerman}ászló Maródi, Primary Immunodeficiency Clinical Unit
and Laboratory, Department of Dermatology, Venereology and
Dermatooncology, Semmelweis University, Budapest, Hungary

Email: Erdos.Melinda@med.semmelweis-univ.hu

or

Marodi.Laszlo@ med.semmelweis-univ.hu

\textbf{ORCID}

Erd\selectlanguage{polish}ő\selectlanguage{english}s Melinda: https://orcid.org/0000-0001-6770-8490

Mar\selectlanguage{ngerman}ódi László: https://orcid.org/0000-0002-9692-3402

\textbf{REFERENCES}

\begin{enumerate}
\tightlist
\item
  Anzilotti C, Swan DJ, Boisson B, et al. An essential role for the
  Zn~\textsuperscript{2+}~transporter ZIP7 in B cell
  development.\emph{Nat Immunol} . 2019;20:350-361.
\item
  Kambe T, Hashimoto A, Fujimoto S. Current understanding of ZIP and ZnT
  zinc transporters in human health and diseases.~\emph{Cell Mol Life
  Sci} .~2014;71:3281-3295.
\item
  Erd\selectlanguage{polish}ő\selectlanguage{english}s M, L\selectlanguage{ngerman}ányi Á, Balázs G, Casanova JL, Boisson B,~Maródi L.
  Inherited TOP2B mutation: Possible confirmation of mutational hotspots
  in the TOPRIM domain. J Clin \emph{Immunol} . 2021;41:817-819.
\item
  Tangye SG, Al-Herz W, Bousfiha A, et al. Human inborn errors of
  immunity: 2019 update on the classification from the international
  union of immunological societies expert committee. \emph{J Clin
  Immunol} . 2020;40:24-64.
\item
  Feske S, Gwack Y, Prakriya M,~et al. A mutation in Orai1 causes immune
  deficiency by abrogating CRAC channel function. \emph{Nature} .
  2006;441:179-185.
\item
  Li FY, Chaigne-Delalande B, Kanellopoulou C, et al. Second messenger
  role for Mg2+ revealed by human T-cell immunodeficiency.\emph{Nature}
  . 2011;475:471-476.
\item
  Woodruff, G. et al. The zinc transporter SLC39A7 (ZIP7) is essential
  for regulation of cytosolic zinc levels.~\emph{Mol Pharmacol}
  .~2018;94:1092-1100.
\item
  Maródi L and the J Project Study Group. Fifteen years of the J
  Project. \emph{J Clin Immunol} . 2019; 39:636-639.
\item
  Tuzankina I, Bolkov M, Nabieva U, Lázár I, Maródi L. The J Daughter
  Siberia Project. \emph{J Clin Immunol} . 2021;41:262-265.
\item
  Maródi L and the J Project Study Group. The
  10\textsuperscript{th}anniversary of World Primary Immunodeficiency
  Week: A J Project celebration. \emph{Eur J Immunol} . 2021; 51:
  2364--2366.
\end{enumerate}\selectlanguage{english}
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