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\begin{document}

\title{Novel mutation c.1224\_1225insGTCC (p.Cys409Valfs*41) of MEN1 gene in a
multiple endocrine neoplasia type 1 case with insulinoma and primary
hyperparathyroidism: first report of a Costa Rican case}



\author[1]{Paula Molina }%
\author[1]{Ernesto Ruiz}%
\author[2]{Ramses Badilla Porras}%
\author[1]{Giovanni Sedó}%
\affil[1]{CCSS}%
\affil[2]{National Children's Hospital}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant
disorder with tumor predisposition in the parathyroid gland, anterior
hypophysis and pancreatic islet cells. Here we describe the first Costa
Rican MEN1 case with a novel MEN1 mutation in a 37-year-old male with
history of nephrolithiasis and recurrent hypoglycemia%
\end{abstract}%



\sloppy


\textbf{Novel mutation c.1224\_1225insGTCC (p.Cys409Valfs*41)
of\emph{MEN1} gene in a multiple endocrine neoplasia type 1 case with
insulinoma and primary hyperparathyroidism: first report of a Costa
Rican case}

Paula Molina-C\selectlanguage{ngerman}éspedes\textsuperscript{1}, Ernesto José
Ruiz-Golcher\textsuperscript{2} , Ramsés
Badilla-Porras\textsuperscript{3}, Giovanni
Sedó-Mejía\textsuperscript{4}

\begin{enumerate}
\tightlist
\item
  Hospital San Juan de Dios, San José, Costa Rica, C.C.S.S
\item
  Hospital San Juan de Dios, San José, Costa Rica, C.C.S.S
\item
  Servicio de Genética y Metabolismo, Hospital Nacional de Niños, San
  José, Costa Rica, C.C.S.S
\item
  Unidad de Alergología, Hospital San Juan de Dios, San José, Costa
  Rica, C.C.S.S
\end{enumerate}

Corresponding Author: Dr. Ramsés Badilla-Porras, Servicio de Genética y
Metabolismo, Hospital Nacional de Niños, San José, Costa Rica, C.C.S.S.
rbadillap@tamizajecr.com\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Abstract} Multiple endocrine neoplasia type 1 (MEN1) is a rare
autosomal dominant disorder characterized by a predisposition for tumors
in the parathyroid gland, anterior hypophysis and pancreatic islet
cells. Here we describe the first Costa Rican MEN1 case with a novel
\emph{MEN1} mutation in a 37-year-old male with history of
nephrolithiasis and recurrent hypoglycemic episodes. Initial
laboratories revealed hypoglycemia and primary hyperparathyroidism. A
fasting test was positive at three hours demonstrating hyperinsulinemic
hypoglycemia. An abdominal CT SCAN showed one mass in the pancreatic
tail and bilateral nephrolithiasis. Direct sequence analysis of
\emph{MEN1} gene revealed heterozygosity for a novel pathogenic
insertion c.1224\_1225insGTCC (p.Cys409Valfs*41). Early genetic
diagnosis of MEN1 could identify individuals in a presymptomatic stage
of the disease which could introduce them in periodic surveillance
protocol for early identification and treatment of associated tumors.
\textbf{Key words} Multiple endocrine neoplasia-primary
hyperparathyroidism-insulinoma-hypoglycemia-nephrolithiasis-endocrine
and metabolic disorders-oncology-genetics. \textbf{Clinical Key Message}
Molecular confirmation of clinical suspicious cases, may lead to new
mutations in non-coding areas and will impact knowledge of the gene
biology, patient follow-up, family members screening and improves our
populational genetics understanding.\tabularnewline
\bottomrule
\end{longtable}

Introduction

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant
disorder with inter- and intra-familial variability, without a good
known genotype-phenotype correlation, characterized by a predisposition
for tumors in the parathyroid gland, anterior pituitary and pancreatic
islet cells (Falchetti, 2017; Falchetti et al., 2009; Jensen \& Norton,
2017; Lemos \& Thakker, 2008; Marini, Falchetti, Luzi, \& Maria Luisa,
2009; Thakker et al., 2012). Non-classic endocrine and non-endocrine
tumors may also develop such as adrenocortical tumors, thyroid tumors,
lipomas, collagenomas, angiofibromas, meningiomas and carcinoid tumors
(Falchetti, 2017; Lemos \& Thakker, 2008; Marini et al., 2009; Thakker,
2014; Thakker et al., 2012). MEN1 syndrome is highly penetrant with half
of patients developing signs and symptoms by 20 years of age (Marini et
al., 2009).

The gene that causes the disorder, \emph{MEN1} , was first identified in
1997 and since then more than 1800 mutations have been characterized
(Falchetti, 2017). However, up to 10\% of MEN1 patients will not have
mutations in the coding region or adjacent splice sites of
\emph{MEN1}(Falchetti, 2017; Falchetti et al., 2009). Other genes such
as\emph{AIP, CDKN1B, CDKN2B, CDKN2C,} and \emph{CDKN1A} have been
related to MEN1 (Arnold, 2019).

The diagnosis of MEN1 is made on the presence of two or more primary
MEN1 tumor types (parathyroid, pancreatic, and pituitary adenomas); the
occurrence of one of the MEN1-associated tumors in family members of a
patient with a diagnosis of MEN1; or if a positive germline mutation in
the \emph{MEN1} gene is present (Thakker, 2014; Thakker et al., 2012).
Here, we describe the first Costa Rican MEN1 case, molecularly
confirmed, with a novel \emph{MEN1} mutation, in a male patient with
primary hyperparathyroidism (PHPT) and insulinoma.

Case Report

In November 2017, a 37-year-old male with medical history of recurrent
nephrolithiasis was referred to the endocrinology department at San Juan
de Dios Hospital, C.C.S.S., in San Jos\selectlanguage{ngerman}é, Costa Rica. The patient had a
1-year history of recurrent episodes of diaphoresis and distal tremor in
fasting states that resolved with eating. Over time these episodes
became more frequent and symptomatic associating difficulty in waking
up, depressive symptoms, and paresthesia in extremities.

On physical examination we describe a male with a body mass index of
31.4 Kg/m\textsuperscript{2}. He had symmetrical neck without palpable
masses or adenopathies. His abdomen was soft and non-tender and there
were no palpable masses. In the dorsal region, the patient presented two
lipomas of approximately 2x2 cm and 1x1 cm. Neurologic examination was
normal.

Initial laboratories revealed a fasting serum glucose of 38 mg/dL, serum
calcium level of 11.3 mg/dL, and a serum intact parathyroid hormone
(PTH) level of 69.5 pg/mL (normal range, 11-67 pg/mL) consistent with
hypoglycemia and PHPT. All serum hypophysiary hormones levels were
within the normal range. Gastrin could not be measured as it was not
available at the hospital's laboratory. A fasting test was done. At
three hours we documented a serum glucose of 47 mg/dL, serum insulin of
243 pmol/L (normal range, 43.3-170.4 pmol/L) and serum C- peptide of
6.58 ng/mL (normal range, 0.90-7.10 ng/mL) compatible with the diagnosis
of insulinoma. Medical treatment with diazoxide and frequent feedings
was initiated.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

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\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

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An abdominal CT scan showed one slightly hyperdense solid focal lesion
of 28x27 mm localized in the pancreatic tail and bilateral
nephrolithiasis (see figure 1).

B)

\textbf{Figure 1.} CT scan of abdomen. A) Solid lesion located in
pancreatic tail (red arrowhead). B) Left nephrolithiasis.

Surgery was attempted and intraoperative direct pancreatic ultrasound
revealed a single homogeneous mass of 25x20 mm in the pancreatic tail. A
distal pancreatectomy plus splenectomy due to bleeding of the splenic
vessels was done. The microscopic study of the specimens showed multiple
nodules. Some of them revealed positivity for insulin and some for
glucagon. Immunohistochemical studies, revealed positivity for
cytokeratin 8-18, synaptophysin, chromogranin, E-cadherin, and a Ki-67
proliferation index \textless{}1\%. Beta-catenin was negative in the
nuclei and positive in membranes and cytokeratin 7 was negative. Those
findings were consistent with multifocal neuroendocrine tumors.

Approximately 36 hours after the surgery, the patient presented an
episode of hypoglycemia of 39 mg/dL. For this reason, medical treatment
with frequent feedings and diazoxide was continued. The patient
presented post-surgical complications with a pancreatic fistula,
ascites, bilateral pleural effusion and acute renal insufficiency. Once
he was stabilized the patient was discharged with medical treatment.
However, a subsequent hospitalization was required for persistent
hypoglycemic symptoms. During the two-month follow up CT scans and an
endoscopic US (EUS) were done. Since no residual neuroendocrine tumor
could be demonstrated it was decided, in conjunction with the patient,
to continue with medical management and not perform surgical
reintervention due to the high morbidity and mortality it represented.

Genomic DNA sequence analysis and deletion/duplication testing from
peripheral blood leukocytes was done in a clinical commercial laboratory
as part of a patient initiative. Direct sequence analysis of
\emph{MEN1}revealed heterozygosity for a novel pathogenic 4-bp
insertion: c.1224\_1225insGTCC(p.Cys409Valfs*41)

Molecular analysis of the variant showed that while this is not
anticipated to result in nonsense mediated mRNA decay, it is expected to
disrupt the last 202 amino acids of the MEN1 protein which result in
truncation of the protein and its Nuclear Localization Signal (NLS)
domains (see figure 2). This variant is not present in population
databases (ExAC no frequency) and has not been reported in the
literature in individuals with MEN1-related disease. ClinVar database
contains an entry for this variant (Variation ID: 428066). Literature
analysis showed that several different truncations located downstream of
this variant (p.Arg516Profs*15, p.Arg516Glyfs*43 and p.Gln554*) have
been determined to be pathogenic (Cardinal et al., 2005; Langer et al.,
2001; Lemmens et al., 1997; Lemos \& Thakker, 2008). This suggests that
deletion of this region of the MEN1 protein is causative of the disease.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 2. A)} Schematic representation of the \emph{MEN1} gene
mutation c.1224\_1225insGTCC. White and green boxes are translated
exons. Dark boxes are untranslated regions which include NLS1, NLSa,
NLS2 domains. \textbf{B)} Schematic representation of the mutant menin
with the p.Cys409Valfs*41 variation. White box represents the amino
acids of the menin protein that are transcribed. Dark boxes represent
amino acids that are disrupted. Diagonal lines represent the stop signal
at position 450 which results in truncation of the protein including the
NLS1, NLSa, and NLS2 domains. Ins: insertion. NLS: Nuclear localization
signal. Modified from: (Marini et al., 2009)

Discussion

The estimated prevalence of MEN 1 is between 1-10 per 100,000 (Jensen \&
Norton, 2017). MEN 1 syndrome was first described in 1903 by Erdheim in
a report of an autopsy of a patient with acromegaly and enlarged
parathyroid glands (Marini et al., 2009). In 1988, the \emph{MEN1} locus
was mapped to chromosome 11q13, and by 1997 \emph{MEN1} mutations were
confirmed to cause MEN1 (Chandrasekharappa et al., 1997; Jensen, Berna,
Bingham, \& Norton, 2008; Lemos \& Thakker, 2008; Marini et al., 2009).
To our knowledge this is the first confirmed case of a Costa Rican
patient with a novel pathogenic \emph{MEN1} mutation.

\emph{MEN1} is located on chromosome 11q13 and consists of 10 exons
which contain a coding region of 1.83 kb organized into nine exons that
encode a 610-amino acid protein called menin (Falchetti, 2017; Jensen et
al., 2008; Jensen \& Norton, 2017; Lemos \& Thakker, 2008; Marini et
al., 2009; Norton, Krampitz, \& Jensen, 2015; Thakker, 2014; Thakker et
al., 2012). Menin is an ubiquitous protein that has at least three
nuclear localization signals (NLS1, NLSa, and NLS2) at its C terminus,
at amino acids 479-497, 546-572, and 588-608, respectively (Falchetti,
2017; Falchetti et al., 2009; Jensen et al., 2008; Lemos \& Thakker,
2008; Marini et al., 2009; Thakker, 2014). These regions, rich in
positively charged residues, have been reported to directly bind to
double-stranded DNA which is thought to be necessary to target menin
into the nucleus (Falchetti et al., 2009; Lemos \& Thakker, 2008;
Thakker, 2014). Menin interacts with several proteins involved in DNA
transcriptional regulation, genome stability, cell division, cell
proliferation, and epigenetic regulation (Jensen et al., 2008; Lemos \&
Thakker, 2008; Norton et al., 2015; Thakker, 2014).

Inheritance of a germline \emph{MEN1} mutation (familial cases) or
a\emph{MEN1} mutation developed in an early embryonic stage (sporadic
cases) predisposes an individual to develop tumors (Marini et al.,
2009). \emph{MEN1} is a tumor suppressor gene that follows the Knudson\selectlanguage{ngerman}´s
two hit hypothesis (Falchetti et al., 2009; Marini et al., 2009; Thakker
et al., 2012).

\emph{MEN1} mutations can be identified in 70-95\% of MEN1 patients
(Lemos \& Thakker, 2008; Marini et al., 2009). Of these mutations, 41\%
are frameshift insertions or deletions, 23\% are nonsense mutations,
20\% are missense mutations, 6\% are in-frame insertions or deletions,
9\% are splice site mutations and 1\% large
deletions\textsuperscript{3}. Most frameshift and nonsense mutations are
predicted to result either in a truncated protein or in loss of the
translated protein because of nonsense-mediated mRNA decay (Lemos \&
Thakker, 2008). Our \emph{MEN1} mutation causes a premature
translational stop signal downstream to the 450 codon level and disrupts
the last 202 amino acids which results in truncation of the functionally
conserved NLS domains of menin 1 gene.

Skin and subcutaneous tumors may arise in 90\% of MEN1 patients (Jensen
\& Norton, 2017). Approximately 88\% of MEN1 patients present
angiofibromas, 72\% collagenomas, and 34\% lipomas (Norton et al.,
2015). The presence of cutaneous tumors may be helpful in the clinical
presymptomatic diagnosis of MEN1 patients, as often they appear before
any clinical manifestations of MEN1-associated hormone-secreting tumors
(Marini et al., 2009). Our patient on clinical examination had two
palpable lipomas.

PHPT affects 95\% of patients and presents as the first endocrine MEN1
manifestation in 90-100\% of cases(Marini et al., 2009; Norton et al.,
2015; Thakker, 2014). PHPT presents typically between 20 and 25 years of
age with 100\% of penetrance by the age of 50 (Falchetti et al., 2009;
Marini et al., 2009; Norton et al., 2015; Thakker, 2014). This is
consistent with our case given that he presented PHPT as his first
manifestation.

Insulinomas arise in about 10-30\% of MEN1 patients and are the second
most frequent functioning pancreatic islet tumor after gastrinomas
(Marini et al., 2009; Norton et al., 2015; Thakker et al., 2012).
Insulinomas usually occur in the third decade of life (Jensen et al.,
2008; Marini et al., 2009; Norton et al., 2015; Thakker et al., 2012).
The most reliable test for the diagnosis is a supervised 72h fast,
during which an increased concentration of plasma insulin in association
with hypoglycemia is demonstrated, along with an elevated C-peptide and
proinsulin concentrations (Marini et al., 2009; Norton et al., 2015;
Thakker et al., 2012). In our case, the diagnosis was made with a
fasting test positive at 3 hours when the patient was 37 years old.

Insulinomas usually present as multiple lesions which are generally
small (\textless{}2cm), benign, and distributed uniformly throughout the
whole pancreas (Marini et al., 2009; Norton et al., 2015). The
multiplicity makes it difficult to define which tumor is secreting the
excessive insulin, nonetheless the majority of MEN1 patients typically
have a dominant insulinoma predominantly found in the body or tail of
the pancreas (Norton et al., 2015). In our case, the patient's
recurrence of hypoglycemic episodes after surgery and the multiplicity
of the nodules in histological studies suggests the presence of residual
lesions in the remaining pancreas.

Insulinomas in MEN1 patients are almost invariably treated surgically
(Jensen et al., 2008; Marini et al., 2009; Norton et al., 2015; Thakker
et al., 2012). Diazoxide combined with frequent feedings is can also be
used to control the hypoglycemia (Jensen et al., 2008; Norton et al.,
2015). However, like in our case, approximately 40-50\% of the patients
do not respond adequately to this treatment

(Jensen et al., 2008). Long-acting somatostatin analogues such as
octreotide or lanreotide are an alternative treatment (Jensen et al.,
2008). Nevertheless, they are effective in only 40-50\% of
patients(Jensen et al., 2008).

Anterior pituitary tumors occur in 15-90\% of MEN1 patients (Marini et
al., 2009; Thakker et al., 2012). Approximately 60\% secrete prolactin,
fewer than 25\% secrete GH, 5\% secrete ACTH, and the remainder appear
to be non-functioning(Marini et al., 2009). Our patient presented serum
hypophysiary hormones within the normal range.

Individuals in which there is a high suspicion of clinical MEN1 or those
with familial MEN1, should be offered genetic counseling and
\emph{MEN1}mutation testing as early genetic diagnosis of \emph{MEN1} is
likely to reduce the morbidity and mortality related to the syndrome
(Falchetti, 2017; Falchetti et al., 2009; Marini et al., 2009; Thakker
et al., 2012). The identification of a germline \emph{MEN1} mutation
should prompt entry into a periodic clinical, biochemical, and
radiological screening program (Thakker et al., 2012).

To our knowledge this is the first report of a molecularly confirmed
case of MEN1 in our country and is the first report of the
c.1224\_1225insGTCC mutation related to a clinically affected patient.

Author contribution

All authors had equal contribution in conceptualization, writing,
reviewing, and editing the document.

Acknowledgements

The Authors would like to thank the patient and family for all the
support collecting information and their interest in getting the
patient's case published.

This paper is used on an preprint server:

\emph{https://www.authorea.com/users/323618/articles/452222-novel-mutation-c-1224\_1225insgtcc-p-cys409valfs-41-of-men1-gene-in-a-multiple-endocrine-neoplasia-type-1-case-with-insulinoma-and-primary-hyperparathyroidism-first-report-of-a-costa-rican-case?commit=bc88a3c18f7cca19219d1defc27e5fd535658736}

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