Fatal Congenital Hypertrophic Cardiomyopathy and ... - SAGE Journals

Pediatric and Developmental Pathology 18, 237–244, 2015 DOI: 10.2350/14-07-1525-CR.1 ª 2015 Society for Pediatric Pathology

Fatal Congenital Hypertrophic Cardiomyopathy and a Pancreatic Nodule Morphologically Identical to Focal Lesion of Congenital Hyperinsulinism in an Infant with Costello Syndrome: Case Report and Review of the Literature BRANDON S. SHEFFIELD,1,2 STEPHEN YIP,1,3 EDUARDO D. RUCHELLI,4 CHRISTOPHER P. DUNHAM,1,2 ELIZABETH SHERWIN,1,5 PAUL A. BROOKS,1,5 AMITAVA SUR,1,6 AVASH SINGH,1,6 DEREK G. HUMAN,1,5 MILLAN S. PATEL,1,7 AND ANNA F. LEE1,2* 1

Faculty of Medicine, University of British Columbia, 4480 Oak Street, Room 2H47, Vancouver, BC, Canada Division of Anatomical Pathology, Children’s and Women’s Health Centre of British Columbia, and Department of Pathology and Laboratory Medicine, University of British Columbia, 4480 Oak Street, Room 2H47, Vancouver, BC V6H3V4, Canada 3 Centre for Translational and Applied Genomics, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, BC V6H3V4, Canada 4 Division of Anatomical Pathology, The Children’s Hospital of Philadelphia. 34th Street and Civic Center Boulevard, Philadelphia, PA 19104, USA 5 Division of Pediatric Cardiology, Children’s and Women’s Health Centre of British Columbia, 4480 Oak Street, Room 1C50, Vancouver, BC V6H3V4, Canada 6 Department of Neonatal Perinatal Medicine, Children’s and Women’s Health Centre of British Columbia, 4480 Oak Street, Vancouver, BC V6H3V4, Canada 7 Department of Medical Genetics and Child and Family Research Institute, Children’s and Women’s Health Centre of British Columbia, 4480 Oak Street, Room C234, Vancouver, BC V6H3V4, Canada 2

Received July 15, 2014; accepted February 9, 2015; published online February 10, 2015.

ABSTRACT Costello syndrome is characterized by constitutional mutations in the proto-oncogene HRAS, causing dysmorphic features, multiple cardiac problems, intellectual disability, and an increased risk of neoplasia. We report a male infant with dysmorphic features, born prematurely at 32 weeks, who, during his 3-month life span, had an unusually severe and ultimately fatal manifestation of hypertrophic cardiomyopathy and hyperinsulinemic hypoglycemia. Molecular studies in this patient demonstrated the uncommon Q22K mutation in the HRAS gene, diagnostic of Costello syndrome. The major autopsy findings revealed hypertrophic cardiomyopathy, congenital myopathy, and a 1.4-cm pancreatic nodule that was positive for insulin expression and morphologically identical to a focal lesion of congenital hyperinsulinism. Sequencing of KCNJ11 and ABCC8, the 2 most commonly mutated genes in focal lesion of congenital hyperinsulinThis work was completed at the Children’s and Women’s Health Centre of British Columbia, 4480 Oak Street, Vancouver, BC V6H3V4, Canada. *Corresponding author, e-mail: [email protected]

ism, revealed no mutations. While hyperinsulinism is a recognized feature of RASopathies, a focal proliferation of endocrine cells similar to a focal lesion of hyperinsulinism is a novel pathologic finding in Costello syndrome. Key words: congenital myopathy, Costello syndrome, focal lesion of congenital hyperinsulinism, HRAS, hypertrophic cardiomyopathy, RASopathy

INTRODUCTION The spectrum of disorders known as RASopathies encompasses a broad range of phenotypes, united by congenital dysregulation of the RAS and mitogen-activated protein kinase intracellular signaling pathway [1]. Noonan, Costello, and cardio-facio-cutaneous syndromes are prototypical RASopathies and feature overlapping phenotypes that include dysmorphic features, mental retardation, growth abnormalities, and neoplasia [2]. Other RASopathies have more restricted phenotypes, such as the neoplasms seen in neurofibromatosis type 1 [3] and the autoimmune diseases encountered in autoimmune lymphoproliferative syndrome [4].

Costello syndrome is characterized by constitutional mutations in the HRAS gene (a member of the RAS family) [5]. Characteristic physical manifestations include polyhydramnios, short stature, mental retardation, poor feeding, facial dysmorphism, sparse curly hair, and hypotonia. Specific cardiac abnormalities include atrial tachycardia, hypertrophic cardiomyopathy, atrial and ventricular septal defects, and outflow tract obstruction [6]. Costello syndrome is also a tumor predisposition syndrome and is associated with malignancies, including rhabdomyosarcoma, neuroblastoma, and urothelial carcinoma, as well as benign perioral papillomata [7]. During infancy, affected individuals may encounter endocrine dysfunction such as growth hormone deficiency and hypoglycemia [8]. Literature reports have attributed hypoglycemia to aberrations in the secretion of insulin and other hormones, but no clear mechanism for this phenomenon has been established. Typically, diagnosis is based upon the above clinical features and can be difficult as a result of variable penetrance and expressivity. Furthermore, the phenotypes of the different RASopathy syndromes show a great degree of overlap. The recent characterization of the molecular underpinnings of the RASopathies opens additional diagnostic avenues that are being utilized with increased frequency [9]. Identification and documentation of an activating HRAS mutation can provide an earlier definitive diagnosis in neonates who have not yet manifested the phenotypic features of Costello syndrome [10]. In this report, we describe a male infant with Costello syndrome resulting from an HRAS Q22K mutation that has been rarely reported. He had severe hypertrophic cardiomyopathy, developing in utero, that proved fatal in infancy. The course was complicated by episodes of hyperinsulinemic hypoglycemia. Autopsy confirmed severe hypertrophic cardiomyopathy affecting the left and right ventricles and interventricular septum. There was also a 1.4-cm pancreatic nodule, morphologically identical to a focal lesion of congenital hyperinsulinism and immunohistochemically positive for insulin. To date, a focal endocrine proliferation nodule similar in appearance to a focal lesion of congenital hyperinsulinism has not been described in association with Costello syndrome.

CASE REPORT The patient was a male infant, born prematurely at 32 weeks of gestation to unrelated parents with unremarkable medical histories. Pregnancy history was significant for suspicion of nonimmune hydrops fetalis and polyhydramnios, the latter leading to an amnioreduction procedure at 29 weeks. The child was born by cesarean section secondary to premature, preterm rupture of membranes, and he had APGAR scores of 4 at 1 minute and 8 at 5 minutes. He had marked hypotonia and required resuscitation, intubation, and admission to the neonatal intensive care unit at an outside hospital. On the 1st day of life (DOL), a panel of laboratory tests

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identified hyperinsulinemic hypoglycemia. Serum glucose was below 1.1 mmol/L (normal range 2.2–3.3 mmol/ L), insulin concentration was elevated at 1068 pmol/L (appropriate concentration during hypoglycemia: ,10 pmol/L), and beta-hydroxybutyrate was less than ,0.30 mmol/L (normal ,0.40 mmol/L). Repeat measurements on DOL 10 showed that during an episode of hypoglycemia (1.6 mmol/L), insulin was again elevated (70 pmol/L), and beta-hydroxybutyrate was low (,0.30 mmol/L). The inappropriately high insulin and low beta-hydroxybutyrate during hypoglycemic episodes were suggestive of hyperinsulinism. Additional workup to confirm this diagnosis (such as glucagon challenge) was not performed. He did not receive pharmacotherapy (such as diazoxide) for hyperinsulinemic hypoglycemia. Early hospital management for this baby included antibiotic therapy for possible sepsis and phototherapy for hyperbilirubinemia. He was weaned to continuous positive airway pressure. Investigations showed that the baby had severe left ventricular hypertrophy and left ventricular outflow tract obstruction with pulmonary hypertension. He was transferred to our neonatal intensive care unit on DOL 18 for evaluation and management of his cardiorespiratory issues. Echocardiography confirmed the presence of asymmetric septal thickening, consistent with hypertrophic cardiomyopathy. The infant was noted to have dysmorphic features including hypertelorism, short neck, and mild nuchal redundancy, but no webbing. The dysmorphic features and congenital cardiac abnormalities raised the possibility of a RASopathy. A complete metabolic workup, including testing for plasma amino acids, urine organic acids, transferrin isoelectric focusing, acylcarnitine, and alpha glucosidase, did not reveal any diagnostic abnormalities. Three more instances of hyperinsulinemic hypoglycemia were documented on DOL 18, with insulin levels of 125, 24, and 34 pmol/L. For the remainder of the baby’s 3-month hospitalization, serum glucose measurements fluctuated around the lownormal range, without any further episodes of frank hypoglycemia. The reason for this improvement was not known. Serum insulin was not further evaluated. While the hypoglycemia appeared to resolve without intervention, the baby’s cardiorespiratory status worsened, and he developed medically intractable pulmonary hypertension eventually requiring reintubation at 50 DOL. Ultimately, the primary care team and family elected to initiate comfort care measures, and the infant expired at the age of 13 weeks (5 weeks, corrected for prematurity). Permission for an autopsy examination restricted to the thorax and abdomen was granted by the next of kin. Autopsy confirmed hypertrophic cardiomyopathy, with markedly increased septal wall thickness, moderately increased left ventricle free wall and right ventricular thicknesses, and increased overall heart weight of 54 g (expected weight at 1 month postnatal: 23 6 7 g; at 3 months postnatal: 30 6 7 g) [11]. Multiple temporally separated myocardial infarctions were grossly identified

Figure 1. (A) Subacute myocardial infarction with fibrous scar (hematoxylin and eosin [H&E] stain, original magnification 310). (B) Acute myocardial infarction (H&E stain, original magnification 310). (C) Biventricular hypertrophy of the heart. This is an inferior view in the axial plane (left ventricle is on the right of the image, and anterior is at the top of the image). The septum measures 1.3 cm (120% increased from expected size for age). Note the dark patches of discoloration consistent with myocardial infarctions in the interventricular septum and papillary muscles. (D) Photomicrograph of section taken from diaphragm showing abnormal variation in myocyte size (H&E stain, magnification 320). (E) Immunohistochemical stain for myosin slow (type 1) fibers (original magnification 340). A color version of this figure is available online.

and confirmed on histologic sections (Fig. 1A–C). No cause for pulmonary hypertension was identified at autopsy. The pulmonary veins were explored and showed no evidence of stenosis. Multiple microscopic sections of the lungs showed no evidence of pre-acinar arteriolar, capillary, or pulmonary venous causes of pulmonary

hypertension. Based on these observations, elevated left atrial pressures during life remain the most likely explanation for the pulmonary hypertension. Consistent with the clinical observation of hypotonia, histologic sections from the diaphragm demonstrated congenital myopathy, with skeletal muscle showing

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Figure 2. (A) Gross photograph of pancreatic head (embedded in paraffin wax), showing generalized expansion of tissue with ill-defined borders. The duodenal mucosa is at the top of the photograph. (B) Photograph (taken without magnification) of a hematoxylin and eosin– stained, 4-mm section of pancreatic head, with the same orientation as for (A). Arrowheads indicate the ill-defined border of the pancreatic endocrine proliferation. A color version of this figure is available online.

marked variation and atrophy in type 1 fibers, confirmed by immunohistochemistry for slow myosin (Fig. 1D,E). No excess muscle spindles were observed in multiple sections. At autopsy, the head of the pancreas appeared mildly enlarged, and the papilla of Vater appeared prominent. A discrete nodule was not appreciated grossly on cut section, so sections of the pancreatic head were entirely

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submitted for histology (Fig. 2). Microscopic examination revealed a 1.4-cm nonencapsulated nodule composed of cells with neuroendocrine features and mild nuclear size variation (Figs. 2,3). There was incorporation of exocrine (acinar) elements at the periphery of the lesion (Fig. 3B). Immunohistochemistry was positive for insulin in many, but not all, of the cells in the nodule (Fig. 3C). Cells positive for glucagon (Fig. 3D) and somatostatin (data not shown) by immunohistochemistry were also present. No other mass lesions were noted in the pancreas, and islets far away from the endocrine proliferation were normal in appearance (Fig. 3F). Loss of p57KIP2 protein expression was also observed in the nodule (Fig. 3E) and in apparently normal islets immediately outside the main confines of the nodule, suggesting that the border of the nodule trailed into the surrounding tissue. Expression of p57KIP2 was retained in islets away from the main endocrine nodule (Fig. 3F). Overall, the morphologic and immunohistochemistry features of this nodule were identical to those of a focal lesion of congenital hyperinsulinism [12]. Genetic testing was initiated during the patient’s hospitalization using a commercially available panel directed at genes implicated in hypertrophic cardiomyopathy. Results of the genetic screen for hypertrophic cardiomyopathy showed a heterozygous missense variant c. 64C.A (p. Q22K) in the HRAS gene, as well as a heterozygous missense variant c. 832C.T (p. R278C) in the TNNT2 gene (which codes for a subunit of the troponin complex). Commercial genetic testing on frozen spleen tissue collected at autopsy revealed no pathogenic mutations in ABCC8, KCNJ11, or other genes implicated in congenital hyperinsulinism (see ‘‘Materials and Methods’’ section for full list of the genes analyzed). Testing for BeckwithWiedemann syndrome was negative: the methylation status of H19DMR and KyDMR loci was normal, and additional sequencing of CDKN1C (p57KIP2) did not show any pathogenic variants. Germline testing by MLPA and STR analysis of 11p15 showed a normal gene dosage, methylation, and chromosome complement, indicating no alterations to the maternal or paternal alleles at that locus. The pancreatic nodule was also evaluated for mutations in ABCC8 and KCNJ11. Sanger sequencing of DNA extracted from the nodule was negative for pathogenic mutations in these 2 genes. The parents were both commercially tested for HRAS mutation, and neither parent carried Q22K or other variants, implying that the mutation arose de novo in the infant. The clinical, autopsy, and genetic findings were most consistent with Costello syndrome. The pancreatic nodule was considered to be secondary to Costello syndrome. The cause of death was attributed to complications of hypertrophic cardiomyopathy.

MATERIALS AND METHODS Immunohistochemistry Formalin-fixed, paraffin embedded (FFPE) autopsy tissues cut at 4-mm thickness and mounted onto charged

Figure 3. (A) Nodule (1.4 cm) at the head of the pancreas, consisting of cords of neuroendocrine cells. Note the darkerstaining compressed normal pancreas tissue in relation to the nodule (top of photomicrograph) (hematoxylin and eosin stain, original magnification 32). (B) Higher magnification (original magnification 320) of the edge of the endocrine nodule, showing ill-defined borders and incorporation of pancreatic acini (right of photograph). (C) Immunohistochemistry with anti-insulin primary antibody, showing cytoplasmic staining in many but not all cells in the pancreatic nodule. (D) Immunohistochemistry with anti-glucagon primary antibody, showing cytoplasmic staining of some (especially at the periphery of the photomicrograph) but not all cells in the pancreatic nodule. (E,F) Immunohistochemistry with anti-p57KIP2 primary antibody, showing lack of nuclear p57KIP2 expression in the cells of the nodule (E) but retention of nuclear p57KIP2 expression in islet cells of the pancreatic tail away from the pancreatic head nodule (F). Original magnification of E,F 340. A color version of this figure is available online.

glass slides were subjected to immunohistochemistry on the Ventana automated stainer. The antibodies used were as follows: p57KIP2 (p57p06 mouse monoclonal antibody, Neomarkers, Fremont, CA, USA), insulin (guinea pig

polyclonal antibody, Cell Marque, Rocklin, CA, USA), and myosin slow (NOQ7.5.4D mouse monoclonal antibody, Sigma-Aldrich, St Louis, MO, USA). Heatinduced antigen retrieval was performed with CC1 buffer


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(Ventana, Tucson, AZ, USA), and visualization was performed with the Ventana OptiView DAB system. Genetic testing Genetic testing for cardiomyopathy was performed at an outside laboratory using the Pan Cardiomyopathy DNA panel, which tests for 100 genes implicated in cardiomyopathy (Version 1.0, Updated May 2, 2013; Blueprint Genetics, Helsinki, Finland). Genetic testing for familial hyperinsulinism was performed at an outside laboratory using the comprehensive familial hyperinsulinism panel (University of Chicago Genetics Laboratory, Chicago, IL, USA). This panel identifies sequence changes in ABCC8, GCK, GLUD1, HADH, HNF1A, HNF4A, INSR, KCNJ11, SLC16A1, and UCP2 and copy number alterations in ABCC8, GLUD1, HADH, INSR, KCNJ11, SLC16A1, and UCP2. Deep cryptic intronic mutations in ABCC8 and HADH are also tested for in this panel. Testing for Beckwith-Wiedemann syndrome was also performed, including chromosome 11p15.5 assessment by both methylation-specific and dosage-specific multiplex ligation-dependent probe amplification (MLPA) and polymerase chain reaction (PCR)–based short tandem repeat assays (Hospital for Sick Children, Molecular Genetics Laboratory, Toronto, ON, Canada). Sanger sequencing of ABCC8 and KCNJ11 was performed at a research laboratory affiliated with our hospital on FFPE material from the pancreatic lesion and matched normal control (FFPE spleen). DNA was isolated using a modified QIAamp DNA FFPE kit method (Qiagen, Venlo, The Netherlands). A full mutational screening of all exons of the KCNJ11 and ABCC8 genes, corresponding to 7 and 39 individual PCR reactions, respectively, was performed on each sample. Amplified PCR products were then bidirectionally sequenced on the Applied Biosystems 31303l Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using the Big Dye v3.1 Terminator Cycle Sequencing Kit (Life Technologies). Analysis of DNA tracings was carried out using Mutation Surveyor version 3.2 (SoftGenetics, State College, PA, USA).

DISCUSSION HRAS mutations and Costello syndrome Costello syndrome is a rare genetic disorder, with an estimated incidence of 1 in 300 000 to 1 230 000 live births [7]. Activating mutations in HRAS causing Costello syndrome were first reported by Aoki and colleagues in 2005 [5]. Their report detailed a series of 12 individuals, each of whom harbored activating HRAS mutations in codons 12 or 13. This series did not feature any patients with a codon 22 mutation in HRAS (the mutation present in our patient). A 2007 report by van der Burgt and colleagues [13] identifies the HRAS Q22K mutation in a patient with a related syndrome, congenital myopathy with excess spindles (CMEMS). A review of Costello syndrome lists the HRAS Q22K variant as a rare cause of

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the syndrome but does not further describe the phenotype of patients affected by this variant [7]; it is unknown whether this review refers to the description of HRAS Q22K in the 2007 paper by van der Burgt [13] or to a previously unpublished patient. The identification of HRAS Q22K in our patient, who had phenotypic features of Costello syndrome and congenital myopathy (but no excess spindles), provides a genetic link between CMEMS and Costello syndrome, with the underlying problem being a disturbance in RAS pathway signaling. This report may be the 1st one to detail a patient with HRAS Q22K activating mutation, Costello syndrome, and congenital myopathy. Congenital hyperinsulinism and Costello syndrome The association between Costello syndrome and congenital hyperinsulinism has previously been reported [14]. A single case report described diffuse endocrine proliferation, a histologic pattern also described in BeckwithWiedemann syndrome [15], in the pancreas of a patient with Costello syndrome [16]. Another report [17] attributed the hyperinsulinism of Costello syndrome to abnormal expression of insulin-like growth factor genes. No definitive pathophysiologic mechanism has been established for the hyperinsulinemic hypoglycemia that is sometimes observed in Costello syndrome, but mechanisms such as growth hormone or cortisol deficiency have been proposed [16]. An association between hyperinsulinism and Noonan or cardio-facio-cutaneous syndromes does not appear to have been reported. The pancreatic histopathology of congenital hyperinsulinism can be separated into diffuse and focal lesions [15]. The focal lesion of congenital hyperinsulinism is usually associated with paternally inherited mutations in either the ABCC8 or KCNJ11 genes [18], followed by loss of the wild-type maternal allele [19]. This form of hyperinsulinism is important to recognize, as it may be amenable to surgical intervention in the face of medically intractable hypoglycemia [20]. The main differential diagnosis of the focal lesion is a pancreatic endocrine neoplasm. Focal lesions recapitulate normal islet cell organization, with alpha, beta, and delta cells being represented; may incorporate pancreatic exocrine (acinar) elements; and may have indistinct borders that make them difficult to appreciate grossly. In contrast, pancreatic endocrine neoplasms are generally well-circumscribed and, reflective of their clonal nature, have one secretory product [15]. A focal proliferation of endocrine cells in the pancreas, such as the one seen in our patient, is a previously unrecognized manifestation of Costello syndrome. Our patient’s pancreatic lesion is histologically identical to a focal lesion of congenital hyperinsulinism, but molecular analysis shows that the 2 entities are genetically distinct. Therefore, it would be reasonable to observe differences in biological behavior. Supportive of

and young adults [7,21]. Aside from neuroblastoma, another endocrine neoplasm reported [22] in Costello syndrome patients is parathyroid adenoma. A report [23] of Sertoli cell tumor developing from a cryptorchid testis in a patient with Noonan syndrome has also been published. Activation of RAS family genes (HRAS, KRAS, NRAS) drives neoplasia through constitutive activation of downstream signaling pathways, leading to cell growth, proliferation, and evasion of apoptosis [24]. Presently, the role of Q22K as a driver of neoplastic transformation in HRAS has not been established in the literature; however, the Q22K mutation in KRAS, which is in a highly conserved region across RAS proteins [25], has been clearly established as a driver mutation [26,27]. It is tempting to speculate that in the absence of other genetic etiologies, an activating HRAS mutation may be a driving force behind the endocrine proliferation seen in this case.

Figure 4. (A) Spatial relationship of genes on chromosome 11p involved in Costello syndrome (HRAS), BeckwithWiedemann syndrome (H19 and IGF2), focal lesion of congenital hyperinsulinism (KCNJ11 and ABCC8), and p57KIP2, a marker for parent-specific imprinting in this region (CDKN1C). In addition to housing several genes involved in these related conditions, this region is heavily subject to imprinting, different methylation patterns with subsequent differential expression from the genes inherited maternally or paternally, giving rise to further complexity in the observed inheritance patterns. The region pictured constitutes approximately 0.5% of the entire genome. Drawing not to scale. (B) Proposed mechanism of neonatal death in a patient with hypertrophic cardiomyopathy, lesion morphologically identical to focal lesion of congenital hyperinsulinism, and underlying genetic changes [heterozygous missense variant c. 64C.A (p. Q22K) in the HRAS gene, and heterozygous missense variant c. 832C.T (p. R278C) in the TNNT2 gene].

this notion, the spontaneous resolution of our patient’s hypoglycemia is different than the expected course of patients with focal lesions and implies that our patient recovered a degree of responsiveness to insulin and glucose. We have not proven that the patient’s endocrine proliferation was the cause of his hyperinsulinemic hypoglycemia, and we cannot exclude the possibility that the nodule was nonfunctioning. This report expands the spectrum of observed phenotypic changes that can be seen in Costello syndrome [16] and provides a possible explanation for hyperinsulinism in a subset of these patients. Neoplasia and Costello syndrome Costello syndrome patients have increased susceptibility to developing rhabdomyosarcoma or neuroblastoma as young children and urothelial carcinoma as adolescents

The 11p15 locus and Costello syndrome It is noteworthy that HRAS resides at 11p15.5, close to ABCC8 and KCNJ11 at 11p15.1. Even closer to the HRAS locus, and also located on 11p15.5, are the IGF2 and H19 loci, which are subject to epigenetic alterations in Beckwith-Wiedemann syndrome [28]. The spatial relationship of these genes is presented in Figure 4A. Based on the loss of p57KIP2 expression in the patient’s endocrine proliferation, we can infer that this lesion does not express allele(s) from the maternal copy of 11p15. Molecular analysis of 11p15 in the patient’s germline DNA, as part of the workup for Beckwith-Wiedemann syndrome, showed a normal gene dosage, methylation, and chromosome complement, suggesting that maternal and paternal copies of 11p15 were not structurally or epigenetically altered. Having excluded the possibility of structural or epigenetic alterations at the 11p15 locus, our genetic studies do not clearly explain why p57KIP2 expression was lost in the pancreatic lesion only. An unknown mechanism contributing to the suppression of p57KIP2 expression from the maternal allele may have worked in concert with the known genetic alterations in this patient to drive the endocrine proliferation seen in the pancreas. Our genetic data also do not completely explain how our patient’s endocrine lesion developed. We propose that the activating HRAS mutation plays a role in its development, but the precise genetic basis for this patient’s endocrine proliferation remains to be discovered. Final remarks Neonatal death due to severe cardiomyopathy is rare in Costello syndrome [21]. Since insulin excess can independently cause hypertrophic cardiomyopathy [29], the unusual degree of severity in our patient could be explained, at least in part, by the effects of excess insulin secretion on a heart with a separate predisposition to develop hypertrophic cardiomyopathy. A complicating factor in understanding this patient’s course is the


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mutation in TNNT2 that was detected on the commercial sequencing panel (Fig. 4B). Although TNNT2 mutations can cause familial and sporadic hypertrophic or dilated cardiomyopathy, reported cases describe onset in adulthood [30]. The significance of the R278C variant in our patient is unknown, but it is possible that it further worsened the severity of our patient’s symptoms, as multiple gene mutations with additive or synergistic effects have previously been described in a subset of hypertrophic cardiomyopathy patients [31]. Our results illustrate how individual components of monogenic diseases can be multifactorial in origin. Costello syndrome should be considered in the differential diagnosis of any patient who presents with early-onset hypertrophic cardiomyopathy and congenital hyperinsulinism. The spectrum of activating mutations extends beyond the common mutations found in codons 12 and 13, and this fact should be considered when choosing an appropriate genetic test. Consideration of a pancreatic insulin-secreting endocrine lesion should be made when managing neonates with severe hypertrophic cardiomyopathy or hyperinsulinemic hypoglycemia. ACKNOWLEDGMENTS We would like to thank Dr M. Seidman and Dr C. Stanley for helpful discussions of this case; Ms A. Lum for assistance with Sanger sequencing; Ms M. Dittrick for critical review of the manuscript; and the University of British Columbia anatomical pathology residency program for continued support. REFERENCES 1. Tidyman WE, Rauen KA. The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 2009;19:230–236. 2. Cizmarova M, Kostalova L, Pribilincova Z, et al. Rasopathies— dysmorphic syndromes with short stature and risk of malignancy. Endocr Regul 2013;47:217–222. 3. Williams VC, Lucas J, Babcock MA, et al. Neurofibromatosis type 1 revisited. Pediatrics 2009;123:124–133. 4. Oliveira JB, Bidere N, Niemela JE, et al. NRAS mutation causes a human autoimmune lymphoproliferative syndrome. Proc Natl Acad Sci USA 2007;104:8953–8958. 5. Aoki Y, Niihori T, Kawame H, et al. Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 2005;37:1038– 1040. 6. Zenker M. Clinical manifestations of mutations in RAS and related intracellular signal transduction factors. Curr Opin Pediatr 2011;23: 443–451. 7. Gripp KW, Lin AE. Costello syndrome. In: Pagon RA, Adam MP, Ardinger HH, et al., eds. GeneReviews. Seattle, WA: University of Washington, Seattle; 1993–2004. Available at http://www.ncbi.nlm. nih.gov/books/NBK1507/. Accessed January 2, 2015. 8. Stein RI, Legault L, Daneman D, et al. Growth hormone deficiency in Costello syndrome. Am J Med Genet A 2004;129A:166–170. 9. RASopathy diagnosis advances: quick, accurate diagnoses are now commonplace. Am J Med Genet A 2012;158A:xi.

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