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A Novel PRKAR1A Gene Mutation Associated With Primary Pigmented Nodular Adrenocortical Disease
CASE-LETTER
A Novel PRKAR1A Gene Mutation Associated With Primary Pigmented Nodular Adrenocortical Disease
W
e studied a Chinese girl who has primary pigmented nodular adrenocortical disease (PPNAD), a rare form of adrenocorticotropic hormone (ACTH)-independent Cushing’s syndrome (CS).1 PPNAD may be isolated (iPPNAD) or associated with Carney complex (CNC),1 in which both cases inherited in an autosomal dominant manner. PRKAR1A gene, encoding the regulatory subunit type I-a (RIa) of the protein kinase A (PKA), was found responsible for PPNAD and CNC.2 Mutations in PDE11A, PDE8B and CTNNB1 gene are found in patients with PPNAD.3,4 We herein report a novel PRKAR1A gene mutation associated with PPNAD in a family. This observation is beneficial in clarifying the molecular action of mutant PRKAR1A in tumor formation and genetic counseling. A 10-year-old girl (memberIII-2; Figure 1A) was admitted because of progressive weight gain and growth deceleration. On physical examination, she was 121.5 cm tall and weighed 36 kg (body mass index, 24.4 kg/m2). She had an overly rounded face, mild hirsutism on her back, central adiposity and extra dorsocervical fat but no spotty skin pigmentation on her face. Laboratory tests showed elevated urinary free cortisol (977 nmol/24 h [normal range, 73–372 nmol/24 h]) and increased serum cortisol with low normal serum ACTH. Moreover, the cortisone level could not be refrained in high- and low-dose dexamethasone suppression test. Other tests revealed hyperinsulinmia with no impaired glucose tolerance. The results of imaging examinations showed multiple nodules in both adrenals in computed tomography, normal sellar in magnetic resonance imaging and normal ovarian and cardia in ultrasound. The bone age was 9 years. The patient was diagnosed with ACTH-independent CS, and on which she was performed a left adrenalectomy. Pathology examination of the left adrenal identified multiple dark-green nodules of various sizes with diameter less than 6 mm. The nodules were in the zona reticularis and composed of large eosinophilic cells with hyperchromatic nuclei and lipofuscin deposition. Immunohistochemistry of the nodules was positive for synaptophysin, a neuroendocrine marker of adrenal cortex. These findings reflected PPNAD syndromes. To further study genetic diagnosis, the available members of the family were evaluated for CS and CNC through detailed history taking, physical examination and laboratory image testing (dexamethasone suppression test was not included). The findings from the members were normal except for a 2-month-old girl who tested negative for physical and imaging examination. She was not evaluated by laboratory testing. The patient’s grandfather died from “lymphoma” in his 40s, hence, his findings were not available. DNA of 7 family members was extracted using BloodGen Mini Kit (ComWin Biotech Co., Beijing, China). All exons and the surrounding intron boundaries of the PRKAR1A, PDE11A, PDE8B and CTNNB1 gene were amplified. Polymerase chain reaction was performed by polymerase chain reaction amplification instrument (Bio Basic Inc, 20 Konrad Cres. Markham, Ontario L3R 8T4, Canada). Direct sequencing was carried out through an ABI 3730 automatic sequencer (Applied Biosystems Inc, Carlsbad, CA). We found a single base pair substitution in exon 2 (c.52T.G, C18G) of the PRKAR1A gene in the patient, her father (II-3) and her younger sister (III-3) The American Journal of the Medical Sciences
(Figures 1A and 1B). All 7 members in the family, including the subject, were negative for mutations in PDE11A, PDE8B and CTNNB1 gene. Five months after the operation, the subject’s cortisol level decreased, leading to regression of Cushing’s signs, while her height stayed unchanged. The insulin-like growth factor 1 value was low, and the growth hormone (GH) releasing test showed partial deficiency of GH: 0 minutes: 2.1 mg/L, 15 minutes: 3.9 mg/L, 30 minutes: 7.3 mg/L, 60 minutes: 5.9 mg/L, and 90 minutes: 4.1 mg/L. She then received recombinant human GH treatment for 9 months, resulting in a height increase of 7 cm (5.76%), and a cortisol level staying reflecting a 11 year old’s bone age. Postoperatively, she did not receive glucocorticoid replacement treatment. PPNAD occurs mostly in children and young adults and is characterized by multiple small-pigmented cortisolproducing adrenocortical nodules on adrenal glands.2 Histopathological manifestations give important reference to the diagnosis of PPNAD. In this case, the patient showed typical signs, which were reflected from corresponding laboratory results and was diagnosed with ACTH-independent CS. Computed tomography scan and the pathological examination confirmed PPNAD. Moreover, genetics of PPNAD is in line with the pathogenesis. The underlying mutation associated with PPNAD and CNC is the mutation in PRKAR1A, located on chromosome 17q22-24. PKA, which comprises a pair of 2 regulatory subunits and a pair of 2 catalytic subunits, is activated by cAMP connecting to the regulatory subunits. Then, the catalytic subunits dissociate from the regulatory subunits and regulate proliferation and differentiation. The increased PKA activity caused by mutation of PRKAR1A, considered a tumor suppressor gene, may introduce the formation of tumors.2 At least 129 mutations have been identified by the Human Gene Mutation Database. The majority of mutations were premature stop codons generated by nonsense, frameshift and splice mutations. Premature stop codons leads to nonsense mRNA mediated decay (NMD) and results in reduction in cellular RIa level. The small number of mutations, which contains missense and frameshift mutations, can escape NMD and give rise to the expression of a defective RIa. A defective RIa decreases the cAMP binding with the regulatory subunits and eliminates RIa binding with the catalytic subunit or other regular subunits, leading to elevation in PKA activity.2 Much like the small proportion of the PRKAR1A expressed mutations, the C18G mutation in our report is not subject to NMD but is caused by amino acid change and expression of altered protein in exon 2, whose expression affects the dimerization/docking domain (http://prkar1a.nichd.nih.gov/ hmdb/prkar1a.html). Based on x-ray crystal structural analysis, the intermolecular disulfide bonds between Cys18 and Cys39 are critical for dimerization of RIaD/D.5 The C18G mutation disrupts the formation of reciprocal disulfide bond between Cys18 and Cys39, resulting in the blockage of RIaD/D homodimer forming and adrenalcortical proliferation. The exact role of PRKAR1A and the actual consequences of the mutation may need further in vitro study. The mutation was inherited in an autosomal dominant manner because the father passes the mutation onto his daughter. Unlike the proband, the father manifested no signs of CS or CNC even after hormonal and radiological investigations despite the fact that he also carried the C18G mutation. This might be due to the low penetrance of the mutation. The
Volume 348, Number 2, August 2014
177
Case-Letter
FIGURE 1. (A), Pedigree of the Chinese family with PPNAD. Seven members volunteered for genetic analysis. Black and white symbols represent clinically affected and unaffected individuals. Individuals affected by the PRKAR1A mutation are indicated by the ‘‘+’’ symbol. Subject indicated by the arrow is the proposita. (B), Sequence analysis of gene. The arrow indicates the c.52T.G mutation in the exon 2 at codon 18 (TGT/GGT) encoding the substitution of cysteine by glycine. PPNAD, primary pigmented nodular adrenocortical disease.
sister will be re-evaluated for hormone investigation to confirm diagnosis. The genotype-phenotype correlation has not been completely established. Notably, a study of 380 patients with CNC showed that patients with CNC carrying the PRKAR1A mutations were at a younger age and had a higher frequency of developing myxomas, schwannomas, thyroid and gonadal tumors than patients without the mutations.2 In addition, the hot spot mutation c.709-7del6 and c.1A.G/p.M1V substitution, which affected exon 7 and the initiation codon of PRKAR1A, were associated mostly with iPPNAD.1,2 In this case, as a carrier of PRKAR1A mutation, the proband was at a higher risk of developing these tumors in CNC mentioned. Moreover, although the proband’s current manifestation accorded with iPPNAD, the phenotype may not be fully manifested because of her young age and short history, so active follow-up is necessary. Mutations in the PDE11A and PDE8B gene affected the ability of phosphodiesterase to degrade cAMP, leading to increased activity of cAMP signaling.3 Mutations in CTNNB1 led to abnormal activation of Wnt/b-catenin pathway, leading to tumorigenesis.4 In this study, screening for PDE11A, PDE8B and CTNNB1 was carried out in 1 family, and no mutation was found. The first-line treatment of CS due to PPNAD is bilateral adrenalectomy and hormone replacement therapy after surgery. Unilateral adrenalectomy was recommended in some young patients with mild symptoms.6 This proposita underwent left adrenalectomy. Postoperatively, the CS lowered significantly, and the cortisol level dropped. Early GH treatment is recommended for the children with CS who has deficiency of GH to achieve normal adult height.7 The proband was proved to be GH deficiency postoperatively, and catch-up growth occurred with the GH treatment in the proband. She will be followed up closely in case of relapse of CS. If such relapse should occur, adrenalectomy of the right side and hydrocortisone therapy should be applied. The family members, especially the currently
178
symptom-free carriers of C18G, will be followed up as well with periodic screening for CS and CNC, aiming for early diagnosis or treatment. In conclusion, the screening tests of a patient with PPNAD and her family members revealed the novel PRKAR1A C18G mutation. The finding provided insight to investigational study in PRKAR1A’s role in tumor formation and to genetic counseling of PPNAD or CNC.
Hui Ran, MD Xiaokun Ma, MD Qingzhu Wang, MD Ziyi Xie, MD *Guijun Qin, PhD Department of Endocrinology Department of Nuclear Medicine The First Affiliated Hospital of Zhengzhou University Zhengzhou, China *E-mail: [email protected] The authors have no financial or other conflicts of interest to disclose. ACKNOWLEDGMENTS The authors are grateful to the patient and her family who participated in our research and donated their time for this investigation. They also thank Xuejun Xu, PhD (Clinical Pharmacology Department, Basic Medical College, Zhengzhou University) for functional study. REFERENCES 1. Groussin L, Horvath A, Jullian E, et al. A PRKAR1A mutation associated with primary pigmented nodular adrenocortical disease in 12 kindreds. J Clin Endocrinol Metab 2006;91:1943–9. 2. Horvath A, Bertherat J, Groussin L, et al. Mutations and polymorphisms in the gene encoding regulatory subunit type 1‐alpha of protein kinase A (PRKAR1A): an update. Hum Mutat 2010;31:369–79. 3. Stratakis CA. New genes and/or molecular pathways associated with adrenal hyperplasias and related adrenocortical tumors. Mol Cell Endocrinol 2009;300:152–7. 4. Tadjine M, Lampron A, Ouadi L, et al. Detection of somatic b‐catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD). Clin Endocrinol (Oxf) 2008;69:367–73. 5. Sarma GN, Kinderman FS, Kim C, et al. Structure of D-AKAP2: PKA RI complex: insights into AKAP specificity and selectivity. Structure 2010;18:155–66. 6. Powell AC, Stratakis CA, Patronas NJ, et al. Operative management of Cushing syndrome secondary to micronodular adrenal hyperplasia. Surgery 2008;143:750–8. 7. Magiakou MA, Chrousos GP. Cushing’s syndrome in children and adolescents: current diagnostic and therapeutic strategies. J Endocrinol Invest 2002;25:181–94.
Volume 348, Number 2, August 2014
A Novel PRKAR1A Gene Mutation Associated With Primary Pigmented Nodular Adrenocortical Disease
W
e studied a Chinese girl who has primary pigmented nodular adrenocortical disease (PPNAD), a rare form of adrenocorticotropic hormone (ACTH)-independent Cushing’s syndrome (CS).1 PPNAD may be isolated (iPPNAD) or associated with Carney complex (CNC),1 in which both cases inherited in an autosomal dominant manner. PRKAR1A gene, encoding the regulatory subunit type I-a (RIa) of the protein kinase A (PKA), was found responsible for PPNAD and CNC.2 Mutations in PDE11A, PDE8B and CTNNB1 gene are found in patients with PPNAD.3,4 We herein report a novel PRKAR1A gene mutation associated with PPNAD in a family. This observation is beneficial in clarifying the molecular action of mutant PRKAR1A in tumor formation and genetic counseling. A 10-year-old girl (memberIII-2; Figure 1A) was admitted because of progressive weight gain and growth deceleration. On physical examination, she was 121.5 cm tall and weighed 36 kg (body mass index, 24.4 kg/m2). She had an overly rounded face, mild hirsutism on her back, central adiposity and extra dorsocervical fat but no spotty skin pigmentation on her face. Laboratory tests showed elevated urinary free cortisol (977 nmol/24 h [normal range, 73–372 nmol/24 h]) and increased serum cortisol with low normal serum ACTH. Moreover, the cortisone level could not be refrained in high- and low-dose dexamethasone suppression test. Other tests revealed hyperinsulinmia with no impaired glucose tolerance. The results of imaging examinations showed multiple nodules in both adrenals in computed tomography, normal sellar in magnetic resonance imaging and normal ovarian and cardia in ultrasound. The bone age was 9 years. The patient was diagnosed with ACTH-independent CS, and on which she was performed a left adrenalectomy. Pathology examination of the left adrenal identified multiple dark-green nodules of various sizes with diameter less than 6 mm. The nodules were in the zona reticularis and composed of large eosinophilic cells with hyperchromatic nuclei and lipofuscin deposition. Immunohistochemistry of the nodules was positive for synaptophysin, a neuroendocrine marker of adrenal cortex. These findings reflected PPNAD syndromes. To further study genetic diagnosis, the available members of the family were evaluated for CS and CNC through detailed history taking, physical examination and laboratory image testing (dexamethasone suppression test was not included). The findings from the members were normal except for a 2-month-old girl who tested negative for physical and imaging examination. She was not evaluated by laboratory testing. The patient’s grandfather died from “lymphoma” in his 40s, hence, his findings were not available. DNA of 7 family members was extracted using BloodGen Mini Kit (ComWin Biotech Co., Beijing, China). All exons and the surrounding intron boundaries of the PRKAR1A, PDE11A, PDE8B and CTNNB1 gene were amplified. Polymerase chain reaction was performed by polymerase chain reaction amplification instrument (Bio Basic Inc, 20 Konrad Cres. Markham, Ontario L3R 8T4, Canada). Direct sequencing was carried out through an ABI 3730 automatic sequencer (Applied Biosystems Inc, Carlsbad, CA). We found a single base pair substitution in exon 2 (c.52T.G, C18G) of the PRKAR1A gene in the patient, her father (II-3) and her younger sister (III-3) The American Journal of the Medical Sciences
(Figures 1A and 1B). All 7 members in the family, including the subject, were negative for mutations in PDE11A, PDE8B and CTNNB1 gene. Five months after the operation, the subject’s cortisol level decreased, leading to regression of Cushing’s signs, while her height stayed unchanged. The insulin-like growth factor 1 value was low, and the growth hormone (GH) releasing test showed partial deficiency of GH: 0 minutes: 2.1 mg/L, 15 minutes: 3.9 mg/L, 30 minutes: 7.3 mg/L, 60 minutes: 5.9 mg/L, and 90 minutes: 4.1 mg/L. She then received recombinant human GH treatment for 9 months, resulting in a height increase of 7 cm (5.76%), and a cortisol level staying reflecting a 11 year old’s bone age. Postoperatively, she did not receive glucocorticoid replacement treatment. PPNAD occurs mostly in children and young adults and is characterized by multiple small-pigmented cortisolproducing adrenocortical nodules on adrenal glands.2 Histopathological manifestations give important reference to the diagnosis of PPNAD. In this case, the patient showed typical signs, which were reflected from corresponding laboratory results and was diagnosed with ACTH-independent CS. Computed tomography scan and the pathological examination confirmed PPNAD. Moreover, genetics of PPNAD is in line with the pathogenesis. The underlying mutation associated with PPNAD and CNC is the mutation in PRKAR1A, located on chromosome 17q22-24. PKA, which comprises a pair of 2 regulatory subunits and a pair of 2 catalytic subunits, is activated by cAMP connecting to the regulatory subunits. Then, the catalytic subunits dissociate from the regulatory subunits and regulate proliferation and differentiation. The increased PKA activity caused by mutation of PRKAR1A, considered a tumor suppressor gene, may introduce the formation of tumors.2 At least 129 mutations have been identified by the Human Gene Mutation Database. The majority of mutations were premature stop codons generated by nonsense, frameshift and splice mutations. Premature stop codons leads to nonsense mRNA mediated decay (NMD) and results in reduction in cellular RIa level. The small number of mutations, which contains missense and frameshift mutations, can escape NMD and give rise to the expression of a defective RIa. A defective RIa decreases the cAMP binding with the regulatory subunits and eliminates RIa binding with the catalytic subunit or other regular subunits, leading to elevation in PKA activity.2 Much like the small proportion of the PRKAR1A expressed mutations, the C18G mutation in our report is not subject to NMD but is caused by amino acid change and expression of altered protein in exon 2, whose expression affects the dimerization/docking domain (http://prkar1a.nichd.nih.gov/ hmdb/prkar1a.html). Based on x-ray crystal structural analysis, the intermolecular disulfide bonds between Cys18 and Cys39 are critical for dimerization of RIaD/D.5 The C18G mutation disrupts the formation of reciprocal disulfide bond between Cys18 and Cys39, resulting in the blockage of RIaD/D homodimer forming and adrenalcortical proliferation. The exact role of PRKAR1A and the actual consequences of the mutation may need further in vitro study. The mutation was inherited in an autosomal dominant manner because the father passes the mutation onto his daughter. Unlike the proband, the father manifested no signs of CS or CNC even after hormonal and radiological investigations despite the fact that he also carried the C18G mutation. This might be due to the low penetrance of the mutation. The
Volume 348, Number 2, August 2014
177
Case-Letter
FIGURE 1. (A), Pedigree of the Chinese family with PPNAD. Seven members volunteered for genetic analysis. Black and white symbols represent clinically affected and unaffected individuals. Individuals affected by the PRKAR1A mutation are indicated by the ‘‘+’’ symbol. Subject indicated by the arrow is the proposita. (B), Sequence analysis of gene. The arrow indicates the c.52T.G mutation in the exon 2 at codon 18 (TGT/GGT) encoding the substitution of cysteine by glycine. PPNAD, primary pigmented nodular adrenocortical disease.
sister will be re-evaluated for hormone investigation to confirm diagnosis. The genotype-phenotype correlation has not been completely established. Notably, a study of 380 patients with CNC showed that patients with CNC carrying the PRKAR1A mutations were at a younger age and had a higher frequency of developing myxomas, schwannomas, thyroid and gonadal tumors than patients without the mutations.2 In addition, the hot spot mutation c.709-7del6 and c.1A.G/p.M1V substitution, which affected exon 7 and the initiation codon of PRKAR1A, were associated mostly with iPPNAD.1,2 In this case, as a carrier of PRKAR1A mutation, the proband was at a higher risk of developing these tumors in CNC mentioned. Moreover, although the proband’s current manifestation accorded with iPPNAD, the phenotype may not be fully manifested because of her young age and short history, so active follow-up is necessary. Mutations in the PDE11A and PDE8B gene affected the ability of phosphodiesterase to degrade cAMP, leading to increased activity of cAMP signaling.3 Mutations in CTNNB1 led to abnormal activation of Wnt/b-catenin pathway, leading to tumorigenesis.4 In this study, screening for PDE11A, PDE8B and CTNNB1 was carried out in 1 family, and no mutation was found. The first-line treatment of CS due to PPNAD is bilateral adrenalectomy and hormone replacement therapy after surgery. Unilateral adrenalectomy was recommended in some young patients with mild symptoms.6 This proposita underwent left adrenalectomy. Postoperatively, the CS lowered significantly, and the cortisol level dropped. Early GH treatment is recommended for the children with CS who has deficiency of GH to achieve normal adult height.7 The proband was proved to be GH deficiency postoperatively, and catch-up growth occurred with the GH treatment in the proband. She will be followed up closely in case of relapse of CS. If such relapse should occur, adrenalectomy of the right side and hydrocortisone therapy should be applied. The family members, especially the currently
178
symptom-free carriers of C18G, will be followed up as well with periodic screening for CS and CNC, aiming for early diagnosis or treatment. In conclusion, the screening tests of a patient with PPNAD and her family members revealed the novel PRKAR1A C18G mutation. The finding provided insight to investigational study in PRKAR1A’s role in tumor formation and to genetic counseling of PPNAD or CNC.
Hui Ran, MD Xiaokun Ma, MD Qingzhu Wang, MD Ziyi Xie, MD *Guijun Qin, PhD Department of Endocrinology Department of Nuclear Medicine The First Affiliated Hospital of Zhengzhou University Zhengzhou, China *E-mail: [email protected] The authors have no financial or other conflicts of interest to disclose. ACKNOWLEDGMENTS The authors are grateful to the patient and her family who participated in our research and donated their time for this investigation. They also thank Xuejun Xu, PhD (Clinical Pharmacology Department, Basic Medical College, Zhengzhou University) for functional study. REFERENCES 1. Groussin L, Horvath A, Jullian E, et al. A PRKAR1A mutation associated with primary pigmented nodular adrenocortical disease in 12 kindreds. J Clin Endocrinol Metab 2006;91:1943–9. 2. Horvath A, Bertherat J, Groussin L, et al. Mutations and polymorphisms in the gene encoding regulatory subunit type 1‐alpha of protein kinase A (PRKAR1A): an update. Hum Mutat 2010;31:369–79. 3. Stratakis CA. New genes and/or molecular pathways associated with adrenal hyperplasias and related adrenocortical tumors. Mol Cell Endocrinol 2009;300:152–7. 4. Tadjine M, Lampron A, Ouadi L, et al. Detection of somatic b‐catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD). Clin Endocrinol (Oxf) 2008;69:367–73. 5. Sarma GN, Kinderman FS, Kim C, et al. Structure of D-AKAP2: PKA RI complex: insights into AKAP specificity and selectivity. Structure 2010;18:155–66. 6. Powell AC, Stratakis CA, Patronas NJ, et al. Operative management of Cushing syndrome secondary to micronodular adrenal hyperplasia. Surgery 2008;143:750–8. 7. Magiakou MA, Chrousos GP. Cushing’s syndrome in children and adolescents: current diagnostic and therapeutic strategies. J Endocrinol Invest 2002;25:181–94.
Volume 348, Number 2, August 2014