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\begin{document}

\title{Heterozygous CTLA-4 splice site mutation c.458-1G\textgreater{}C
presenting with variable degree of immune dysregulation and autoimmunity
in three generation kindred of Caribbean descent}



\author[1]{UPENDRA MAHAT}%
\author[1]{Nila Ambani}%
\author[1]{seth rotz}%
\author[1]{Kadakkal  Radhakrishnan}%
\affil[1]{Cleveland Clinic}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is an immune
checkpoint, which downregulates T cell activation and T regulatory cell
function. CTLA-4 haploinsufficiency (CTLA4 HI) leads to T cell
hyperactivation, immune dysregulation, lymphoproliferation and cancer
predisposition. Less well understood is the penetrance and expressivity
of CTLA-4 mutations. We describe five members of a single family with
heterozygous CTLA-4 splice site mutation c.458-1G\textgreater{}C,
previously shown to result in CTLA-4 HI, who presented with variable
degree of immune dysfunction, lymphocytic infiltration and autoimmunity.
The host, environmental and the epigenetic factors affecting the
penetrance and expressivity of CTLA-4 mutations merits further
investigation.%
\end{abstract}%



\sloppy


\textbf{Introduction:}

CTLA-4 is an immunoglobulin superfamily receptor, primarily expressed by
activated T cells and FOXP3\textsuperscript{+} regulatory T
cells.\textsuperscript{1} CTLA-4 down-regulates the T cell mediated
immune response, and thus functions as an immune
checkpoint.\textsuperscript{2} Homozygous CTLA-4 deficiency in mice
causes severe fatal immune dysfunction.\textsuperscript{3,4} In humans,
CTLA-4 haploinsufficiency (HI) causes polyclonal T cell hyperactivation,
and present with immunodeficiency, antibody mediated autoimmunity and
lymphoproliferation in non-lymphoid organs such as gut, lungs, CNS, bone
marrow, etc.\textsuperscript{5-8} Furthermore, CTLA-4 HI predisposes
patients to development of various malignancies such as lymphoma and
gastric cancers.\textsuperscript{9,10}

\textbf{Method:}

We describe the incompletely penetrant and variably expressed clinical
phenotype of a patient of Caribbean descent with CTLA-4 HI, and his 4
related family members (Family pedigree, clinical presentation and
genetic defect as depicted in Figure 1A-C). The study was approved by
the institutional IRB. The informed consents were obtained as per the
IRB guidelines.

\textbf{Results:}

The index patient III.1 (previously published\textsuperscript{11}) is
24-year-old male, who was diagnosed with EBV positive classical Hodgkin
lymphoma (cHL) of nodular sclerosis histology at 11 year of age. He
achieved complete remission with multiagent chemotherapy and radiation.
Subsequently, he developed autoimmune hemolytic anemia (AIHA), and then
immune thrombocytopenia (ITP). At 17 years of age, he was diagnosed with
autoimmune lymphoproliferative syndrome (ALPS), and further genetic
testing revealed heterozygous CTLA-4 splice mutation
c.458-1G\textgreater{}C. Four years later, the patient developed
autoimmune lymphocytic enterocolitis (ALE). A year later, at 22 years of
age, he was diagnosed with a new EBV+ cHL of mixed cellularity subtype.
He was treated with further courses of chemotherapy, and complete
remission was achieved. Further, the patient underwent allogeneic
peripheral stem cell transplantation from matched unrelated donor. At
the present time, he remains in complete remission from both cHL, and
autoimmune conditions.

Patient III.3 is 14 years old male, and is the maternal cousin brother
of the proband. He developed chronic progressive diarrhea since he was 1
year old. At the age of 5 years\textbf{,} he developed multiple
recurrent anal warts. Esophagogastroduodenoscopy (EGD) and colonoscopy
revealed ALE.~He was started on oral tacrolimus, which improved his
symptoms. He was treated with multiple courses of corticosteroids. Few
months later, he developed right upper quadrant abdominal pain. Imaging
studies demonstrated multiple small gallbladder calculi and
choledocholithiasis, requiring ERCP and removal of CBD stones. A trial
of sirolimus worsened his symptoms and he had to be restarted back on
tacrolimus with improvement in his gastrointestinal symptoms. His
genetic testing revealed the same CTLA-4 spice site mutation as the
proband. Since the age of 12 years, he was started on abatacept, a
fusion protein composed of Fc region of the IgG1 fused to the
extracellular domain of CTLA-4, every 4 weeks, along with low dose
tacrolimus and sirolimus with good improvement of symptoms. He has
developed multiple nutritional deficiencies such as copper, vitamin A,
and vitamin D, which improved with supplementation. Of note, patient
also developed chronic EBV viremia since the age of 10 years. However,
no signs of lymphoma or lymphoproliferative disorders have been
identified.

Patient III.4 is the paternal cousin sister of the proband, who is 20
years old. She developed ITP at 9 years of age. When she was 12 years
old, she was admitted with chronic diarrhea and excessive weight loss,
prompting EGD and colonoscopy, which revealed ALE. The genetic testing
confirmed CTLA4 c.458-1 G\textgreater{}C mutation. She was started on
oral tacrolimus since the age of 14 years, with near complete resolution
of GI symptoms associated with ALE. Of note, she was also found to have
EBV viremia since the age of 16 years. However, she has not developed
lymphoproliferative disorders or lymphoma.

Patient II.3 is the maternal uncle of the proband, and the father of
patient III.3 and III.4. He is 43 years old and was diagnosed with ALE
at 28 years of age. Later, he was found to have chronic
hypogammaglobinemia and was clinically diagnosed with common variable
immunodeficiency (CVID). He developed AIHA at the age of 29 years. When
he was 32 years old, he was diagnosed with atypical marginal zone
non-Hodgkin lymphoma with CNS metastasis. No genetic testing has been
performed.

Patient II.5 is the maternal uncle of the proband, who had history of
cataract at the age of 6 years. He also had history of multiple
surgeries for glaucoma in his childhood. He was diagnosed with juvenile
rheumatoid arthritis at the age of 9 months. Later, he developed ITP,
AIHA, ALE, hypothyroidism, hypogonadism and insulin dependent DM.

\textbf{Conclusion and discussion:}

The proband and the family members in our cohort harbors CTLA-4 splice
site mutation, c.458-1G\textgreater{}C, in the region of intron 2. As
described above, the patients presented with variable degree of immune
dysregulation and autoimmunity. The mutation has been previously shown
to disrupt the acceptor site of second CTLA-4 intron, and cause
defective transcription of third exon.\textsuperscript{12} The common
manifestations in the majority of the patients described by previous
reports have lymphoproliferation, lymphocytic infiltration of
non-lymphoid organs, autoimmune cytopenias and
hypogammaglobinemia.\textsuperscript{8,12} Mutations described in total
of 18 patients in those reports spanned across the CTLA-4 gene,
including non-sense mutations such as c.151C\textgreater{}t (p.R51X),
c.105C\textgreater{}A (p.C35*), c.208C\textgreater{}T (p.R70W),
c.371A\textgreater{}C (p.T124P), c.223C\textgreater{}T (p.R75W) and
c.2T\textgreater{}C (p.?), splice site mutations such as c110 +
1G\textgreater{}T in intron 1, c.458-1G\textgreater{}C in intron 2,
c.567+5G\textgreater{}C in intron 3, and frameshift deletion
c.75delG\textgreater{} p.L28Fs*44.\textsuperscript{8,12} Although the
nature and the location of these mutations were heterogeneous, all of
these mutations led to decreased CTLA-4 expression in Treg cells
compared to healthy controls. Furthermore, the reduction was more
pronounced on T cell activation for unclear reason. The Treg cells'
CTLA-4 dependent suppressive function, ligand binding and
transendocytosis of CD80 were uniformly impaired.

One patient reported by Kuehn et al harbored c.458-1G\textgreater{}C
mutation, as in our cohort, and presented with diffuse lymphadenopathy,
splenomegaly, AIHA, ITP, ALE and childhood onset EBV positive
HL.\textsuperscript{12} The severity and expressivity of immune
dysregulation in our cohort appears somewhat more penetrant in male
gender. Gastrointestinal manifestations especially ALE appear more
common and may pose management challenges. The role of abatacept in the
management of these patients may need to be studied further, alone or in
conjunction with tacrolimus and/or sirolimus. Two patients in our cohort
presented with lymphoma (two episodes of EBV driven cHL of different
histologies in one patient and one case of atypical marginal zone
non-Hodgkin lymphoma). An earlier published worldwide cohort of 184
patients with CTLA-4 mutations, has reported various malignancies in
these patients, such as lymphoma, gastric cancers, multiple myeloma and
metastatic melanoma.\textsuperscript{9}

In conclusion, heterozygous CTLA-4 mutation and the associated immune
dysregulation is a complex entity which is both phenotypically as well
as genotypically heterogeneous. Although the previous studies have
extensively investigated the essential role of CTLA-4 in T and B cell
homeostasis, our understanding of the molecular, genetic and epigenetic
regulation of its penetrance and expressivity remains limited, and
merits further investigation. Defining the genotypic and phenotypic
correlates may help refine the management options for this intriguing
disorder.

\textbf{Conflict of interest:}

Authors have no conflict of interest to disclose.

\textbf{Consent:}

Appropriate consents were obtained from the participants as per the IRB
guidelines.

\textbf{Acknowledgements:}

We would like to sincerely acknowledge the participants of this study.

\textbf{References:}

1. Buchbinder EI, Desai A. CTLA-4 and PD-1 Pathways: Similarities,
Differences, and Implications of Their Inhibition. \emph{Am J Clin
Oncol.} 2016;39(1):98-106.

2. Syn NL, Teng MWL, Mok TSK, Soo RA. De-novo and acquired resistance to
immune checkpoint targeting. \emph{Lancet Oncol.} 2017;18(12):e731-e741.

3. Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe
AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal
multiorgan tissue destruction, revealing a critical negative regulatory
role of CTLA-4. \emph{Immunity.} 1995;3(5):541-547.

4. Waterhouse P, Penninger JM, Timms E, et al. Lymphoproliferative
disorders with early lethality in mice deficient in
Ctla-4.\emph{Science.} 1995;270(5238):985-988.

5. Hudson LL, Rocca K, Song YW, Pandey JP. CTLA-4 gene polymorphisms in
systemic lupus erythematosus: a highly significant association with a
determinant in the promoter region. \emph{Hum
Genet.}2002;111(4-5):452-455.

6. Kouki T, Sawai Y, Gardine CA, Fisfalen ME, Alegre ML, DeGroot LJ.
CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory
function of CTLA-4 and contributes to the pathogenesis of Graves'
disease. \emph{J Immunol.} 2000;165(11):6606-6611.

7. Oaks MK, Hallett KM. Cutting edge: a soluble form of CTLA-4 in
patients with autoimmune thyroid disease. \emph{J
Immunol.}2000;164(10):5015-5018.

8. Schubert D, Bode C, Kenefeck R, et al. Autosomal dominant immune
dysregulation syndrome in humans with CTLA4 mutations. \emph{Nat
Med.}2014;20(12):1410-1416.

9. Egg D, Schwab C, Gabrysch A, et al. Increased Risk for Malignancies
in 131 Affected CTLA4 Mutation Carriers. \emph{Front
Immunol.}2018;9:2012.

10. Schwab C, Gabrysch A, Olbrich P, et al. Phenotype, penetrance, and
treatment of 133 cytotoxic T-lymphocyte antigen 4-insufficient
subjects.\emph{J Allergy Clin Immunol.} 2018;142(6):1932-1946.

11. Mahat U, Terzioglu MK, Buhtoiarov I. CTLA4 haploinsufficiency as a
predisposition to classical Hodgkin lymphoma. \emph{Pediatr Hematol
Oncol.} 2020;37(2):176-183.

12. Kuehn HS, Ouyang W, Lo B, et al. Immune dysregulation in human
subjects with heterozygous germline mutations in CTLA4.
\emph{Science.}2014;345(6204):1623-1627.

\textbf{Legends:}

\textbf{Figure 1:} Figure illustrating A. Family pedigree with members
from three generation family with heterozygous CTLA-4 mutation and
variable clinical phenotypic expression, B. Clinical presentation of the
members of the family depicting key immune manifestations and C. CTLA-4
mutation location and protein structure showing key domains.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

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