Cardiac myxoma caused by fumarate hydratase gene deletion in patient with
cortisol-secreting adrenocortical adenoma
moderated kanůscript
Kentaro Suda ”, Hidenori Fukuoka ª, Yuto Yamazaki b, Katsumi Shigemura“, Miki Mukai ª,
Yukiko Odake ª, Ryusaku Matsumoto d, Hironori Bando ª, Michiko Takahashi a,e, Genzo Iguchi ª, Masato Fujisawa“, Masahiro Oka5, Katsuhiko Ono b, Kazuo Chihara8, Hironobu Sasano b, Wataru Ogawa ª, Yutaka Takahashi d
ªDivision of Diabetes and Endocrinology, Kobe University Hospital
Department of Anatomic Pathology, Tohoku University Graduate School of Medicine ‘Division of Urology, Department of Surgery Related, Kobe University Graduate School of Medicine ªDivision of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine
eDepartment of Nutrition, Kobe University Hospital Department of Dermatology, Tohoku Medical and Pharmaceutical University Hospital
§ Department of Diabetes and Endocrinology, Akashi Medical Center
Corresponding author: Hidenori Fukuoka Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Hospital, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan Tel .: +81-78-382-5861; Fax: +81-78-382-2080
@ Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. jc.2019-40885. See endocrine.org/publications for Accepted Manuscript disclaimer and additional information.
Grant or fellowships: Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology 15K09432 Conflict of interest disclosures: None to be declared
Accepted Manuscript
Abstract
Context: Germline mutations in fumarate hydratase (FH) gene are known to cause hereditary leiomyomatosis and renal cell carcinoma (HLRCC) and are occasionally accompanied with cutaneous and uterine leiomyoma or cortisol-producing adrenocortical hyperplasia. However, the association between FH mutations and cardiac or adrenocortical tumors has remained unknown. Here, we identified a novel deletion in FH, exhibiting cardiac myxoma and subclinical Cushing syndrome due to adrenocortical tumor.
Case Description: A 44-year-old man was referred to our hospital for cardiac and adrenal tumor evaluation. He had a history of multiple dermal painful papules and nodules diagnosed as cutaneous leiomyoma. The surgically resected cardiac tumor was diagnosed as myxoma. The adrenal tumor was clinically diagnosed as subclinical Cushing syndrome. Laparoscopically resected adrenal tumor was pathologically diagnosed as adrenocortical adenoma harboring unique histological findings similar to PPNAD (primary pigmented nodular adrenocortical disease). Deoxyribonucleic acid analysis revealed a germline deletion in FH c.737delT (p. Phe225Leufs*31) and loss of heterozygosity (LOH) in cardiac myxoma. As a functional analysis of FH in cardiac myxoma, low FH protein expression with elevated 2-succinocysteine (2SC), a marker of FH dysfunction, was immunohistochemically detected. However, in adrenocortical tumor, LOH of FH was not detected, and FH or 2SC expression was not altered.
Conclusion: This is the first case of HLRCC complicated with cardiac myxoma. LOH of FH deletion and its dysfunction were identified in cardiac myxoma. The association between FH deletion and adrenocortical lesion, however, needs to be further clarified.
Keywords: fumarate hydratase, cardiac myxoma, gene mutation, adrenocortical tumor
Précis
We identified LOH of FH in cardiac myxoma from HLRCC patient with subclinical Cushing syndrome. The dysfunction of FH was confirmed in cardiac myxoma.
Accepted Manuscript
Introduction
Cardiac myxoma is the most frequent cardiac tumor, and familial cardiac myxoma including the Carney complex constitutes approximately 7-10% of these cases (1). However, studies regarding the involvement of other genetic mutations in pathogenesis of myocardial myxoma have not been reported.
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare disease complicated by multiple cutaneous leiomyomas, symptomatic uterine leiomyoma, and papillary renal cell carcinoma. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. 15% of patients with HLRCC are known to harbor adrenal tumor and 14% of them to be associated with autonomous cortisol secretion (2). However, etiologic association between FH mutations and clinical phenotype has remained unknown.
The protein encoded by FH is one of the components of Krebs cycle enzyme, which catalyzes fumarate to malate. FH dysfunction results in fumarate accumulation, causing mitochondrial respiratory chain activity dysfunction (3). Fumarate accumulation also inhibits hypoxia-inducible transcription factor la degradation, suppresses multiple a-ketoglutarate-dependent dioxygenases, and induces epithelial-to-mesenchymal transition (4), possibly contributing to tumorigenesis.
Here, we report a case carrying a novel deletion in FH, exhibiting cardiac myxoma and subclinical Cushing syndrome.
Case report
A 44-year-old Japanese man presented with obesity and painful skin nodules. At 40 years old, he experienced hypertension, dyslipidemia, and impaired glucose tolerance. He had no family history of papules and renal or adrenal tumors. His body mass index was 37.8 kg/m2; his blood pressure was 138/104 mmHg, and he was taking antihypertensive drugs (nifedipine
and doxazosin). He did not exhibit Cushingoid features. He had multiple painful skin nodules from the neck to the upper arm (Figure 1a). Abdominal computed tomography (CT) revealed a left adrenal tumor measuring 5 cm in diameter (18 Hounsfield units) and there was no swelling on the right adrenal gland (Figure 1b). Patient’s hormonal characteristics are summarized in Table 1. Serum cortisol levels were 3.0 µg/dL after overnight 1-mg dexamethasone suppression test. Adrenal 131I-60-iodomethylnorcholesterol scintigraphy exhibited ipsilateral adrenal uptake and contralateral adrenal suppression, demonstrating the diagnosis with subclinical Cushing syndrome (5). Simultaneously, echocardiography revealed a 7-mm tumor on the tricuspid valve (Figure 1b).
Skin biopsies were obtained from a nodule on the arm and a papule on the back. The pathological findings were consistent with leiomyoma (Figure 1c).
Surgical resection of the cardiac tumor was performed. The pathological findings were consistent with cardiac myxoma.
Laparoscopic adrenalectomy was performed for the left adrenal tumor (Figure 1c). Analysis of the expression kinetics of steroidogenic enzymes revealed distinctive findings, which were similar to the findings of primary pigmented nodular adrenocortical disease (PPNAD) (Figure 1d) and this adrenocortical lesion was histopathologically interpreted as adrenocortical adenoma harboring PPNAD like features.
Genomic deoxyribonucleic acid (DNA) was isolated from the lymphocyte and formalin-fixed paraffin-embedded samples of cardiac and adrenocortical tumor (QIAGEN). Coding sequences of FH and protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) gene were amplified by polymerase chain reaction and sequenced using BigDye Terminator Cycle Sequencing Kit and ABI PRISM 310 Genetic Analyzer. The primers are shown in Table 2. Gene mutations in all exons of PRKAR1A were not detected. On the contrary, a novel germline heterozygous deletion c.737delT in FH, resulting in a frameshift in
exon 5, and immature stop codon at c.737delT (p. Phe255Leufs*31) was identified. A loss of heterozygosity (LOH) of FH was identified in the cardiac myxoma, but not in the adrenocortical tumor (Figure 2a).
Fumarate measurement of the cardiac myxoma using mass spectrometry failed because of the small sample. Immunohistochemical analysis was performed via the streptavidin-biotin amplification method using Histofine Kit (Nichirei, Tokyo, Japan). Primary anti-FH antibodies (rabbit, polyclonal, 1:300, SIGMA, HPA025770), which recognize the domain of exons 9 and 10 of FH deleted in the mutation gene in this case, and primary anti-2-succinocysteine (2SC) antibody (rabbit, polyclonal, 1:250, provided by Dr. Norma Frizzell) (6) were employed. 2SC is well known to be produced by the intracellular accumulation of fumarate in response to cysteine, which reflects intracellular function of FH (6). FH protein expression was low in the cardiac myxoma, but not altered in the adrenocortical tumor (Figure 2b). In addition, 2SC immunoreactivity was marked in the cardiac myxoma, but not in the adrenocortical tumor (Figure 2b).
Totale
We then studied the FH mutation status in 26 cases of resected adrenocortical tumors from the patients with Cushing syndrome (n=9), subclinical Cushing syndrome (n=7), primary aldosteronism (n=7), and nonfunctioning adrenocortical adenoma (NFA) (n=3). Only one case (NFA) demonstrated a heterozygous germline mutation c.77C>T (p.P26L) in FH. However, LOH was not detected in the adrenocortical tumor, with no changes of both FH and 2SC levels (data not shown).
All experiments were conducted in compliance with the protocol that was reviewed and approved by the Research Ethics Committee of Kobe University Hospital (Permit Number: #1351).
Discussion
We reported a case showing cutaneous nodules, cardiac myxoma, and adrenocortical tumor with subclinical Cushing syndrome. We also identified a novel germline deletion in FH instead of PRKAR1A. To the best of our knowledge, this is the first case exhibiting cardiac myxoma caused by a FH deletion.
In this case, LOH of FH was identified in cardiac myxoma, with low expression of FH protein. In a previous study, FH deletion was reported to result in low FH and high 2SC expression in renal cell carcinoma (7). In our present case, high expression of 2SC protein did indicate a causal association between cardiac myxoma and the FH deletion. These results indicate that some cardiac myxomas could be related to FH, and larger studies including these tumors are warranted.
In this case, however, LOH of FH was not detected in the tissues of adrenocortical tumor. In addition, the status of FH and 2SC protein was not altered. Somatic mutation of mediator complex subunit 12 (MED12), the most frequent driver mutation in uterine leiomyoma, has been reported to be associated with FH mutation without LOH in the uterine leiomyoma, suggesting possible tumorigenesis by different mechanisms including genetic instability caused by FH mutation (8). Further studies are required.
Regarding the pathological findings of adrenocortical adenoma of this patient, the expression kinetics of steroidogenic enzymes revealed that not 3-beta-hydroxysteroid dehydrogenase but both P-450 C17-hydroxylase and CYP11B1 were detected in eosinophilic-compact cells, similar to those of PPNAD (9). The typical adrenal morphological finding in HLRCC was macronodular adrenocortical hyperplasia (2), which is different from those in our present case. Pheochromocytoma has been reported as an adrenal complication of FH mutations (10), and the association between HLRCC and pheochromocytoma cannot be ruled out. No evident difference was reported in the type or
location of FH mutation in pheochromocytoma and HLRCC. However, this pathophysiological difference indicated that FH abnormalities could influence tumorigenesis as a driver gene.
This study has several limitations. We did not conduct functional analysis of FH. The association between FH and adrenocortical tumor could not be confirmed. In contrast, the presence of FH deletion and its LOH, insufficient FH protein expression, and 2SC protein overexpression in cardiac myxoma clearly indicated that this tumor was indeed caused by novel FH deletion.
In conclusion, we reported a case of cutaneous leiomyoma, subclinical Cushing syndrome, and cardiac myxoma with novel heterozygous FH deletion. LOH was identified in cardiac
Accepted M’a. tata wish wecan myxoma.
Acknowledgments
The authors thank C. Ogata and K. Imura for their excellent technical assistance, and special thanks for R.Nagai for the collaboration using liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS).
Accepted Manuscript
Figure legends
Figure 1. Pathological findings in the present case. (a) Macro findings of the skin nodules. Dermatological examination revealed multiple, brownish papules and nodules mainly on the extensor sides of both upper extremities. (b) Abdominal computed tomography and echocardiogram. (c) Hematoxylin and eosin staining of skin nodules, cardiac tumor, and adrenal tumor. Skin biopsies were obtained from a small flat nodule on the left arm and a papule on the back. The nodule and the papule were essentially similar. The epidermis showed hyperkeratosis and hyperpigmentation in the basal cell layer. In the dermis, upper and middle layer was replaced with interlacing bundles of smooth muscle fibers with which varying amounts of collagen bundles are intermingled. These collagen bundles were also scattered in the deep dermis. The cells of the smooth muscles had abundant eosinophilic cytoplasm and elongated nuclei. In the cardiac tumor, cells were sparsely distributed with low-dense intercellular substances inside the unstructured hyaline-like degeneration and mild fibrosis; endocardium covered the lesion. In the adrenal tumor, the basic construction of the adrenal cortex was not clear, and the nodules were consisted mainly with large and small clear cells that are proliferating. Among them, a relatively clear boundary nodule with eosinophilic cytoplasm and a nodule with enlarged nucleus with mixed clear cells and dense cells (arrow) were observed. (d) Immunostaining of steroidogenic enzymes in the adrenal tumor. P-450 C17-hydroxylase and CYP11B1 were remarkably expressed in dense cells (arrow). These cells had eosinophilic cytoplasm. These findings suggested that the entire adrenal cortex had an impaired formation or development, which was diagnosed as adrenal tumor.
Figure 2. Fumarate hydratase (FH) gene analysis in the present case. (a) Deoxyribonucleic acid sequencing data and schema of FH with a position of the deletion in the present case. (b)
Immunostaining of hematoxylin and eosin, FH, and 2-succinocysteine of the adrenocortical adenoma of control case (left), and cardiac tumor (middle), and adrenocortical tumor (right) of the present case.
Accepted Manuscript
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Reference
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6. Blatnik M, Frizzell N, Thorpe SR, Baynes JW. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes: formation of S-(2-succinyl)cysteine, a novel chemical modification of protein and possible biomarker of mitochondrial stress. Diabetes 2008; 57:41-49
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8. Kampjarvi K, Makinen N, Mehine M, Valipakka S, Uimari O, Pitkanen E, Heinonen HR, Heikkinen T, Tolvanen J, Ahtikoski A, Frizzell N, Sarvilinna N, Sjoberg J, Butzow R, Aaltonen LA, Vahteristo P. MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas. Br J Cancer 2016; 114:1405-1411
9. Sasano H, Miyazaki S, Sawai T, Sasano N, Nagura H, Funahashi H, Aiba M, Demura H. Primary pigmented nodular adrenocortical disease (PPNAD): immunohistochemical and in situ hybridization analysis of steroidogenic enzymes in eight cases. Mod Pathol 1992; 5:23-29
10. 0. Clark GR, Sciacovelli M, Gaude E, Walsh DM, Kirby G, Simpson MA, Trembath RC, Berg JN, Woodward ER, Kinning E, Morrison PJ, Frezza C, Maher ER. Germline FH mutations presenting with pheochromocytoma. J Clin Endocrinol Metab 2014; 99:E2046-2050
Accepted Marí die, welchedermed, ale auch
| Plasma ACTH (pg/mL) | 10.5 | ( 7.7 | - 63.1 | ) | |
| Serum Cortisol (mg/dL) | 8.4 | ( 5.9 | - 17.0 | ) | |
| Midnight serum cortisol (mg/dL) | 4.8 | ||||
| Serum cortisol after 1mg of dexamethasone (mg/dL) | 3.0 | ||||
| Urine free cortisol (mg/day) | 103 | ( 11.2 | - 80.3 | ) | |
| Serum DHEA-S (ng/mL) | 1067 | ( 700 | - 4950 | ) | |
| Plasma renin activity (ng/ml/hr) | 0.9 | ( 0.2 | - 2.3 | ) | |
| Plasma aldosterone concentration (pg/mL) | 140 | ( 30 | - 159 | ) | |
| Urine metanephrine concentration (mg/day) | 0.08 | ( | 0.05 | - 0.2 | ) |
| Urine normetanephrine concentration (mg/day) | 0.28 | ( | 0.1 | - 0.28 | ) |
Accepted Man
Table 2 Primers used for PCR and sequencing gene
| gene | name | primer sequence | gene | name | primer sequence |
|---|---|---|---|---|---|
| FH | exon1 F1 | ATTGGATAAGAGCGGAGGCC | PRKAR1A | exon1 F1 | AGGAGTCGCCCACCTGTCATCTGA |
| FH | exon1 F2 | CCCAGAAATTCTACCCAAGC | PRKAR1A | exon1 F2 | ACGTCAGTAGCCGAACGCTGATTG |
| FH | exon1 R | AGGGCTGAAGGTCACTGC | PRKAR1A | exon1 R1 | CTTATCCACAGCAGTTTCCTCACG |
| FH | exon2 F | TGATCCTGGGTTTCTTTTCAAC | PRKAR1A | exon1 R2 | ACGCCATCTTGGATCGGTCCAGCT |
| FH | exon2 R | ATGAATACAGCCTACTTCATCC | PRKAR1A | exon2 F | TCCCTGTGAATCAGTTGTCTAAT |
| FH | exon3 F | CCAAAATAATAAACTTCCATGC | PRKAR1A | exon2 R | ATGTAACAACTGTCACAATCACC |
| FH | exon3 R | ATGGGTCTGAGGTTATTAAG | PRKAR1A | exon3 F | GAATTGGTGTTTTCCTCTTAACTT |
| FH | exon4 F1 | CTGTATTCAAACTCTGTGGC | PRKAR1A | exon3 R | TATGATTCATTCATCAAAGGAGAC |
| FH | exon4 F2 | GGCAGACTGGATCAGGAACT | PRKAR1A | exon4 F | AATGTTTTTGGTTTATGGAATTGT |
| FH | exon4 R1 | CATTTCAATTGCTCTATTGCT | PRKAR1A | exon4 R | CACACCCTTACTTGAAAAATAGTG |
| FH | exon4 R2 | TTATAACCAAAAAACAGCAAAGC | PRKAR1A | exon5 F | GACAGTCTGGGGTCTTTAATTCTA |
| FH | exon5 F | GTTTTTGTTGCCTCTGATTTAAC | PRKAR1A | exon5 R | TCAAAGAGGAAAACAAACTTCAAT |
| FH | exon5 R | TGGCCATTTGTACCAAGCTC | PRKAR1A | exon6 F | TTTCTTTAATTTGGAATATGCTTC |
| FH | exon6 F | GAGTAACTTGTAAGCTATTAGG | PRKAR1A | exon6 R | ATCTGACATACAAGGGATGTAATG |
| FH | exon6 R | AATGTACAGACCACGTA | PRKAR1A 16 | exon7 F | TTTTTAAAACAAAGTTCAGGATTG |
| FH | exon7 F | TAACTTGTTCACCCATCTAGG | PRKAR1A | exon7 R | CTAAATCACACTCTCAAACACCAT |
|---|---|---|---|---|---|
| FH | exon7 R | CTAGTCAAGTTTTAGCTCCAAC | PRKAR1A | exon8 F | ATTATTCCATAGCATTATGTGGTG |
| FH | exon8 F | TTAGTCTTTACTCTGTCATTGG | PRKAR1A | exon8 R | AGTCACAGAGGAAATAACTGTGAA |
| FH | exon8 R | TAATAAGCCTTTGGTCAAAAAAC | PRKAR1A | exon9 F | GGCTATTTGGTTGAATCTCTTTAT |
| FH | exon9 F1 | ATTGTATATTTACTGTCAACCAG | PRKAR1A | exon9 F | TGAGTTCTTTACCTCTAAAATTCAA |
| FH | exon9 F2 | CTCTCACTCACTCAAAGATT | PRKAR1A | exon10 F | TTGTTTAGCTTTTTGGTGATTTTA |
| FH | exon9 R | AAACACTGATCCACTTGTCTCT | PRKAR1A | exon10 R | GGAGAAGACAAAATTATGGAAGAC |
| FH | exon10 F | CTGCTAACCCATATGTCGTC | PRKAR1A | exon11 F | TATTGTCTTCTTTCTCAGAAGTGC |
| FH | exon 10 R | CGTTTTTAAGAAATGGGAGTCTG | PRKAR1A | exon11 R | GTGCAATAAAAGCAACTTTCAATA |
| PRKACA | exon6 F | GTTTCTGACGGCTGGACTG | |||
| PRKACA | exon6 R | Accestated AGTTGGCAGGGAGGAAGG | |||
| PRKACA | exon7 F | CCTTCCTCCCTGCCAACT | |||
| PRKACA | exon7 R | AGTCCACGGCCTTGTTGTAG |
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a
b
RA
RV
C
Skin nodules
Cardiac tumor
Adrenal tumor
cript
HE
500um
500um
500um
HE
100um
100um
100um
d
3ßHSD
P450c17
P450c11B1
500um
500um
500um Accepted M
a
c.737 delT
1
2
3
4
5
6
7
8
9
10
Leu Phe
His Ala
Arg Gln
Stop
Lys
Glu
A A
A
A G
A
G G
T
T ₸
G T
C
A
C
G
A
A
A
G
A
T
G
C
A
C
A
G
Leukocyte
Cardiac tumor
ript
LOH(+)
Adrenal tumor
LOH(-)
b
FH wild type Adrenal tumor
c.737 delT Cardiac tumor
c.737 delT Adrenal tumor
HE
200um.
200um
FH
200um
200um
200um
2SC
200Lim
200um
200am
Accepted