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\begin{document}

\title{Hematopoietic stem cell transplantation in two sisters with bone marrow
failure associated with POLE gene variants}



\author[1]{Kentaro Fujimori}%
\author[1]{Norihito Ikenobe}%
\author[1]{Yoshihiro Gocho}%
\author[1]{Toru Uchiyama}%
\author[1]{Takao Deguchi}%
\author[1]{Hirotoshi Sakaguchi}%
\author[1]{Daisuke Tomizawa}%
\author[1]{Ichiro Takeuchi}%
\author[1]{Hirotaka Shimizu}%
\author[1]{Katsuhiro Arai}%
\author[1]{Akira Ishiguro}%
\author[1]{Kimikazu Matsumoto}%
\author[1]{Akihiro  Iguchi }%
\affil[1]{Kokuritsu Kenkyu Kaihatsu Hojin Kokuritsu Seiiku Iryo Kenkyu Center Byoin}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
POLE codes for DNA polymerase epsilon (Pol \selectlanguage{greek}ε\selectlanguage{english}) and variants of Pol \selectlanguage{greek}ε
\selectlanguage{english}catalytic subunit 1 can be pathogenic. We recently reported a novel POLE
gene variant (p.{[}D1131fs{]};{[}T1891del{]}) which leads to bone marrow
failure in two Japanese sisters. Here, we describe the successful course
of hematopoietic stem cell transplantation of these two sisters. Both
cases were born with congenital anemia and remained transfusion
dependent. Their bone marrow showed gradually proceeding trilineage
dysplasia. They both underwent HCT from an unrelated bone marrow donor
and successfully achieved engraftment. Ten and eight years have passed
respectively, and they are doing well without transfusion.%
\end{abstract}%



\sloppy


\textbf{Title: Hematopoietic stem cell transplantation in two sisters
with bone marrow failure associated with \emph{POLE} gene variants}

\textbf{Authors}

Kentaro Fujimori\textsuperscript{1}, Norihito
Ikenobe\textsuperscript{1}, Yoshihiro Gocho\textsuperscript{1}, Toru
Uchiyama\textsuperscript{1}, Takao Deguchi\textsuperscript{1}, Hirotoshi
Sakaguchi\textsuperscript{1}, Daisuke Tomizawa\textsuperscript{1},
Ichiro Takeuchi\textsuperscript{2}, Hirotaka Shimizu\textsuperscript{2},
Katsuhiro Arai\textsuperscript{2}, Akira Ishiguro\textsuperscript{1},
Kimikazu Matsumoto\textsuperscript{1}, Akihiro Iguchi\textsuperscript{1}

\textbf{Affiliated institutions}

Children's Cancer Center, National Center for Child Health and
Development

Division of Gastroenterology, National Center for Child Health and
Development

\textbf{Corresponding author:} Akihiro Iguchi

Children's Cancer Center, National Center for Child Health and
Development

2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan

TEL/FAX: +81-3-3416-0181/+81-3-3416-2222,
Email:\emph{iguchi-a@ncchd.go.jp}

\textbf{Word count} : Abstract 100 words, Main text 1185 words

\textbf{Running title} : Successful HCT for novel \emph{POLE} gene
variant associated bone marrow failure.

\textbf{Keywords} : POLE gene, Stem cell transplantation, Congenital
anemia, Bone marrow failure

Abbreviations key:\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
DNA & Deoxyribonucleic acid\tabularnewline
\midrule
\endhead
Pol \selectlanguage{greek}ε & Polymerase epsilon\tabularnewline
MDS & Myelodysplastic syndrome\tabularnewline
HCT & Hematopoietic stem cell transplantation\tabularnewline
RBCs & Red blood cells\tabularnewline
WBCs & White blood cells\tabularnewline
MCV & Mean corpuscular volume\tabularnewline
IBD & Inflammatory bowel disease\tabularnewline
HLA & Human leukocyte antigen\tabularnewline
MAC & Myeloablative conditioning\tabularnewline
GVHD & Graft-versus-host disease\tabularnewline
\bottomrule
\end{longtable}

\textbf{Abstract}

POLE codes for DNA polymerase epsilon (Pol \selectlanguage{greek}ε\selectlanguage{english}) and variants of Pol \selectlanguage{greek}ε
\selectlanguage{english}catalytic subunit 1 can be pathogenic. We recently reported a novel POLE
gene variant (p.{[}D1131fs{]};{[}T1891del{]}) which leads to bone marrow
failure in two Japanese sisters. Here, we describe the successful course
of hematopoietic stem cell transplantation of these two sisters. Both
cases were born with congenital anemia and remained transfusion
dependent. Their bone marrow showed gradually proceeding trilineage
dysplasia. They both underwent HCT from an unrelated bone marrow donor
and successfully achieved engraftment. Ten and eight years have passed
respectively, and they are doing well without transfusion.

\textbf{Main Text}

\textbf{Introduction}

DNA polymerase epsilon (Pol \selectlanguage{greek}ε\selectlanguage{english}), which is composed of four subunits, is a
core component of DNA replication. Pathogenic variants in
\emph{POLE}encoding Pol \selectlanguage{greek}ε \selectlanguage{english}catalytic subunit 1 (the largest subunit) is
known to cause FILS and IMAGeI syndrome, two syndromes with similar
symptoms comprised of multiple malformations and organ dysfunctions,
including facial dysmorphism, congenital adrenal hypoplasia, and
immunodeficiency.\textsuperscript{1,2} Recently, we reported a novel
compound heterozygous variant in
\emph{POLE}(p.{[}D1131fs{]};{[}T1891del{]}) to be the cause of
congenital anemia in two Japanese sisters.\textsuperscript{3} Both
sisters developed multilineage dysplasia compatible with myelodysplastic
syndrome (MDS) and remained transfusion dependent. They both proceeded
to hematopoietic stem cell transplantation (HCT). Here, we report the
successful HCT outcomes of these two sisters.

\textbf{Cases}

Case 1 is a 10-year-old girl who was born to non-consanguineous healthy
Japanese parents. As we previously reported, she had severe macrocytic
anemia without reticulocytopenia (red blood cells {[}RBCs{]} 0.95 \selectlanguage{ngerman}×
10\textsuperscript{12}/L, hemoglobin level 43 g/L, mean corpuscular
volume {[}MCV{]} 123 fL, reticulocytes 158 × 10\textsuperscript{9}/L,
white blood cells {[}WBCs{]} 2.3 × 10\textsuperscript{9}/L, and
platelets 396 × 10\textsuperscript{9}/L) from birth. Anemia did not
improve with oral iron supplements or erythropoietin administration, and
she had to undergo recurring RBC transfusions. The first bone marrow
examination was performed when she was 6 months old, showing normal
cellularity and marked dysplasia only in the erythroid lineage.
G-banding karyotyping of the bone marrow aspirate showed a normal female
karyotype (46, XX). She also developed chronic bloody diarrhea at the
age of 2 months. Inflammatory bowel disease (IBD), unclassified type,
was diagnosed based on endoscopic and pathological findings at the age
of 8 months. She became corticosteroid dependent and required
intravenous hyperalimentation to treat malnutrition. A follow-up bone
marrow examination at the age of 17 months revealed hypercellularity and
dysplasia of trilineage cells without excess blasts. However, bone
marrow examination before HCT showed multilineage dysplasia and 7.5\%
blastic cells, reminiscent of progression to MDS with excess blast-1. As
she remained transfusion dependent, HCT was performed at the age of 1
year and 10 months from an unrelated human leukocyte antigen
(HLA)--matched donor (8/8 allele matched). She received myeloablative
conditioning (MAC) composed of busulfan (1.2 mg/kg) four times daily on
days \selectlanguage{english}-9 to \selectlanguage{english}-6 (total dose, 19.2 mg/kg) and cyclophosphamide (50 mg/kg)
once daily from days \selectlanguage{english}-5 to \selectlanguage{english}-2 (total dose, 200 mg/kg). The infused bone
marrow cells contained 3.0 x 10\textsuperscript{6}/kg of CD34-positive
cells. Graft-versus-host disease (GVHD) prophylaxis was performed with
tacrolimus and short-term methotrexate (15 mg/m\textsuperscript{2} on
day +1 and 10 mg/m\textsuperscript{2} on days +3, +6, and +11).
Engraftment was achieved on day +22 with full donor chimerism. She
developed grade II acute skin GVHD on day +94 which resolved immediately
after initiating corticosteroid treatment. However, she developed grade
III gastrointestinal GVHD after discontinuing corticosteroid on day
+140. The effect of reinitiated corticosteroid was insufficient, and
infliximab was added. Her bloody stool and diarrhea gradually resolved,
and corticosteroid was stopped approximately 1 year and 4 months after
developing gastrointestinal GVHD. Infliximab was discontinued 3 years
after discontinuing corticosteroid. Approximately ten years have passed
since HCT, and she is doing well without active complications or
transfusion demand and all her immunosuppressants have been
discontinued. However, due to prolonged tube feeding associated with
inflammation of the gastrointestinal tract, she is unable to ingest
orally. After HCT, a novel compound heterozygous variant in
\emph{POLE}(p.{[}D1131fs{]};{[}T1891del{]}) was found by genetic
analysis using her dermal fibroblasts.

Case 2 is a 3-year-old girl, a younger sister of Case 1. She also had
severe macrocytic anemia without reticulocytopenia (RBCs 1.71 x
10\textsuperscript{12}/L, hemoglobin level 54 g/L, MCV 117 fL,
reticulocytes 30 x 10\textsuperscript{9}/L, WBCs 12.8 x
10\textsuperscript{9}/L, and platelets 306 x 10\textsuperscript{9}/L)
from birth and became transfusion-dependent. Genetic analysis of her
peripheral blood cells revealed the same variant of \emph{POLE} as in
Case 1. Bone marrow findings at 3 months of age were similar to those in
Case 1, with dysplasia only in the erythroid without hypoplasia.
Dysplasia of all three lineages was seen at 13 months of age (G-banding
karyotyping showed a normal female karyotype (46, XX), with 8\% blastic
cells. These findings indicated MDS with excess blast-1. However,
blastic cells decreased to 4.2\% at 16 months of age. Unlike Case 1 she
did not develop IBD. She remained transfusion dependent and there were
fluctuations in the proportion of blasts. HCT was performed at the age
of 1 year and 5 months from an unrelated HLA one allele mis-matched
donor. She received conditioning composed of fludarabine (25
mg/m\textsuperscript{2}) on days \selectlanguage{english}-8 to \selectlanguage{english}-4 (total dose, 125
mg/m\textsuperscript{2}), melphalan (90 mg/m\textsuperscript{2}) once
daily from days \selectlanguage{english}-4 to \selectlanguage{english}-3 (total dose, 180 mg/ m\textsuperscript{2}),
anti-thymocyte globulin (2.5 mg/kg) once daily from days \selectlanguage{english}-7 to \selectlanguage{english}-6 (total
dose, 5.0 mg/kg) and total body irradiation of 3 Gray on day 0. The
infused bone marrow cells contained 6.3 x 10\textsuperscript{6}/kg of
CD34-positive cells. GVHD prophylaxis was the same as in Case 1.
Engraftment was achieved on day +16 with full donor chimerism. She
developed grade III acute gastrointestinal GVHD on day +24 and was
treated with immunosuppressive agents including corticosteroid,
mycophenolate mofetil, and additional methotrexate. However, her
symptoms were refractory and required addition of mesenchymal stem cell
therapy. Meanwhile, pathological findings of her gastrointestinal biopsy
revealed intestinal thrombotic microangiopathy and recombinant human
soluble thrombomodulin was also administered. Her gastrointestinal
symptoms gradually resolved, and each treatment was able to be tapered
off without recurrence. Two years have passed since HCT, and she is
doing well without active complications or transfusion demand and all
her immunosuppressants have been discontinued.

\textbf{Discussion}

Recently, we described a novel human Pol \selectlanguage{greek}ε \selectlanguage{english}defect associated with
hematopoietic failure.\textsuperscript{3} The patients in our current
report are the same siblings reported there and thus may represent the
first HCT report of Pol \selectlanguage{greek}ε \selectlanguage{english}defect associated hematopoietic failure. Both
cases presented with trilineage dysplasia with fluctuations in the
proportion of blasts in bone marrow, which meets the diagnostic criteria
of MDS-EB-1.\textsuperscript{4} Along with persistent transfusion
dependency, HCT was performed according to protocol for MDS
patients.\textsuperscript{5,6} MAC was selected in Case 1 because her
bone marrow showed hypercellularity and, without precedent, MAC was
considered ideal to obtain definite engraftment. As expected, Case 1
achieved stable full donor chimerism. Based on this experience, we
decided to reduce the conditioning intensity of Case 2 to avoid cardiac
toxicity\textsuperscript{7} and late complications, including impaired
fertility.\textsuperscript{8} HCT certainly resolved their bone marrow
failure, and they became transfusion independent. In Case 1, IBD also
resolved after HCT. The association of IBD and \emph{POLE} gene variant
is uncertain; however, reconstitution of the immune system may be
beneficial, as in other monogenic forms of IBD.\textsuperscript{9} On
the other hand, as with variants in the exonuclease domain of
the\emph{POLE} ,\textsuperscript{10} there may be outcome factors other
than bone marrow failure in these cases, and continued careful follow-up
is essential.

\textbf{Author contributions}

KF, NI, IT, H. Shimizu, A. Ishiguro, KA, KM, and A. Iguchi were the
attending physicians. TU and TD supported laboratory examinations. KF
and A. Iguchi wrote the manuscript. YG, H. Sakaguchi, and DT supervised
the case and helped write the manuscript. All authors revised and
approved the manuscript.

\textbf{Conflict of interests}

The authors declare no conflicts of interest.

\textbf{Acknowledgements}

Consent for publishing the case was obtained by the parents of the
patients.

Financial disclosure: Supported by Child Health and Development grants:
2022A-5.



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2 Logan CV, Murray JE, Parry DA, Robertson A, Bellelli R, Tarnauskait\selectlanguage{polish}ė
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8 Majhail NS, Brazauskas R, Rizzo JD, Sobecks RM, Wang Z, Horowitz
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9 Nambu R, Warner N, Mulder DJ, Kotlarz D, McGovern DPB, Cho J \emph{et
al.} A systematic review of monogenic inflammatory bowel
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10 Elsayed FA, Kets CM, Ruano D, van den Akker B, Mensenkamp AR,
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\textbf{Hosted file}

\verb`04.POLE case report_Table 1.docx`  available at \url{https://authorea.com/users/756458/articles/727542-hematopoietic-stem-cell-transplantation-in-two-sisters-with-bone-marrow-failure-associated-with-pole-gene-variants}

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