Germline RET Codon 918 Mutation in Apparently Isolated Intestinal Ganglioneuromatosis1

Charis Eng1, Debbie J. Marsh, Bruce G. Robinson, C. W. Chow, Michael A. Patton, Melissa C. Southey, Deon J. Venter, Bruce A. J. Ponder2, Peter J. Milla and Virpi V. Smith

Department of Adult Oncology, Charles A. Dana Human Cancer Genetics Unit, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School (C.E., D.J.M.), Boston, Massachusetts 02115; Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University (C.E.), Columbus, Ohio 43210; Molecular Genetics Laboratory, Kolling Institute for Medical Research, Department of Endocrinology, Royal North Shore Hospital, St. Leonards, New South Wales 2006; the Department of Medicine, University of Sydney (D.J.M., B.G.R.), Sydney NSW 2006, and the Department of Anatomical Pathology, Royal Children’s Hospital (C.W.C.), and the Departments of Pathology and Research, Peter MacCallum Cancer Institute (M.C.S., D.J.V.), Melbourne 3052, Australia; and the Department of Medical Genetics, St. George’s Hospital Medical School (M.A.P.), London SW7 ORE; Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge (C.E., B.A.J.P.), Cambridge CB2200; and the Gastroenterological Unit (P.J.M.) and Department of Histopathology (V.V.S.), Great Ormond Street Hospital for Sick Children and Institute of Child Health, London WCIN 3JH, United Kingdom

Address all correspondence and requests for reprints to: Dr. Charis Eng, Human Cancer Genetics, Ohio State University, 420 West 12th Avenue, 690MRF, Columbus, Ohio 43210.


    Introduction
 Top
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
THE RET proto-oncogene, which encodes a receptor tyrosine kinase, is the susceptibility gene for the autosomal dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes (reviewed in Refs. 1, 2). MEN 2 is divided into three clinical subtypes, depending on the tissues involved. MEN 2A is the most common and is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Familial MTC is characterized by the presence of MTC as the only phenotype. MEN 2B is similar to MEN 2A, except that the age of tumor onset is, on the average, 10 yr earlier, clinical hyperparathyroidism is absent, and classic stigmata such as ganglioneuromatosis of the intestines and mucous membranes, marfanoid habitus, and developmental delay are characteristic.

The International RET Mutation Consortium has reported germline RET mutations in over 92% of all MEN 2 cases (3). A mutation in one of codons 609, 611, 618, 620 (exon 10), 630, and 634 (exon 11) accounts for over 98% of MEN 2A cases, whereas a mutation in any of these codons or in codons 768 (exon 13) and 804 (exon 14) has been found in familial MTC cases. MEN 2B is unique, in that a single germline mutation at codon 918 (M918T) in exon 16 accounts for 95% of these cases, and a germline mutation at codon 883 (A883F) accounts for under 4% of these cases, whereas the remainder do not have any RET mutation (3, 4, 5). The Consortium study and other smaller studies, however, only address the issue of prevalence of germline RET mutation in classic MEN 2B cases. They do not answer such questions as what proportion of cases with partial MEN 2B phenotypic features have germline M918T. In this survey, we have ascertained three unrelated patients with apparently isolated intestinal ganglioneuromatosis at presentation and determined whether a germline RET mutation, specifically, either M918T or A883F, is present in these cases.


    Subjects and Methods
 Top
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Patients

Patients who initially presented with apparently isolated intestinal ganglioneuromatosis were considered for this study. Cases were excluded if they had any other feature characteristic of MEN 2B or if they had a family history consistent with MEN 2B. Medullated corneal nerve fibers, unless graded 4+, were not considered features of MEN 2B for purposes of this study because such a diagnosis, especially graded less than 4+, is highly subjective and unreliable.

RET mutation analysis

Germline genomic DNA was extracted from peripheral blood leukocytes using standard techniques (6, 7).

RET exon 16 was PCR amplified using 50–100 ng template DNA and the primer pairs CRT 5F and CRT 5G or FRET16 and RRET16 under previously described conditions (7, 8). Assessment of the presence of M918T was performed by direct sequence analysis or differential restriction enzyme digestion (7, 8, 9).


    Results
 Top
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Case 1

Patient 1 presented at 2 weeks of age with hypotonia and mild asymmetry of her lower extremities, with a smaller left leg and foot. Skeletal roentgenograms showed delayed bone age and ovoid vertebrae, but no definitive diagnosis could be made. Eight months later, she was noted to be short, with rolls of sc fat. At 11 months of age, she presented to a pediatric surgeon for severe constipation. Despite changing her diet and repeated bowel washouts, she had persistent constipation. Rectal biopsy was acetylcholine esterase negative and showed submucosal clusters of ganglion cells, thus excluding the diagnosis of Hirschsprung disease. A laparoscopic appendectomy was performed at the age of 33 months, and pathological examination demonstrated irregular, prominent clusters of neurons in the submucosa and muscle coat. Because the possibility of intestinal neuronal dysplasia was raised, a colostomy was performed at 36 months. Histopathology revealed marked proliferation of neural plexuses with numerous ganglion cells in the submucosa and muscle coat, consistent with the diagnosis of intestinal ganglioneuromatosis. Immunofluorescence studies showed increases in vasoactive intestinal polypeptide (VIP)- and substance P-immunoreactive nerve fibers in the mucosa and submucosa. Although the myenteric plexus was well labeled for substance P and VIP, and the circular muscle coat fibers were immunoreactive for VIP, immunoreactive fibers for substance P were absent. Persistent severe constipation resulted in total colectomy at the age of 49 months, with resolution of symptoms. Ophthalmological examination at 51 months revealed what was believed to be 1–2+ medullated corneal nerve fibers. No facial or oral mucosal neuromatosis was noted. The patient has a healthy older brother, and her parents are healthy. No signs of MTC or pheochromocytoma were found in the proband, her siblings, or her parents by physical examination and biochemical analyses (basal and pentagastrin-stimulated calcitonin levels and 24-h urine for catecholamine measurement).

Mutation analysis of RET exon 16 revealed a germline M918T in the patient, thus making the molecular diagnosis of MEN 2B (2, 3). Neither biological parent carries this germline mutation.

At 66 months, the patient was diagnosed with MTC after basal and peak pentagastrin-stimulated calcitonin levels were found to be elevated at 516 and more than 1000 ng/L (institutional normal, <30 ng/L), respectively. A total thyroidectomy was performed, and the diagnosis confirmed by pathological examination. When last seen at 93 months, the patient was well, with normal serum calcitonin and catecholamine levels.

Case 2

Patient 2, a 2-week-old girl, presented with intestinal obstruction. Rectal biopsy revealed extensive ganglioneuromatosis of the myenteric and submucosal plexuses. Intestinal decompression was achieved by a colostomy. Full-thickness colon from the colostomy site also showed extensive ganglioneuroma. Examination of this girl at the age of 3 yr revealed short stature, frontal bossing, and marked lumbar lordosis. However, no specific diagnosis of skeletal dysplasia could be made. At this time, because of recurrent episodes of obstruction, the colostomy was converted into an ileostomy. During this procedure, full-thickness samples taken from the both the large and small bowels showed extensive diffuse ganglioneuroma. Physical examination and biochemical testing (basal and pentagastrin-stimulated calcitonin levels; 24-h urine for catecholamine measurement) revealed no signs of MTC or pheochromocytoma. No family history of MEN 2 or skeletal dysplasia was elicited.

Germline mutation analysis revealed the presence of M918T in the patient, but not in her biological parents. The patient’s results are definitive for the molecular diagnosis of MEN 2B.

One year later, at the age of 4 yr, a routine pentagastrin-stimulated calcitonin level was elevated. The patient underwent a total thyroidectomy. Histopathological examination revealed two nodules of MTC. The patient has also undergone a total colectomy, which confirmed extensive ganglioneuromatosis throughout.

Case 3

Patient 3, a 13-day-old boy, developed vomiting and diarrhea. Necrotizing enterocolitis and large bowel obstruction were diagnosed. At another institution, Hirschsprung disease was excluded, and the diagnosis of intestinal neuronal dysplasia was suggested. A dysfunctioning colostomy was raised. At the age of 3 yr, a repeat rectal biopsy and biopsies taken distal and proximal to the stoma showed diffuse ganglioneuromatosis and an increase in acetylcholinesterase-positive fibers in the lamina propria. At 6 yr of age, ileo-rectal pull-through was carried out. The resected colon and terminal ileum both showed extensive ganglioneuromatosis in the submucosal and myenteric plexuses (Fig. 1Go). Abdominal and neck ultrasound scans revealed no obvious thyroid or adrenal masses. No other characteristic stigmata of MEN 2B were noted. No family history of MEN 2 was found.



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Figure 1. Ganglioneuromatosis of the gut. A, Section of bowel wall (case 3) showing extensive ganglioneuromatosis filling the submucosal and myenteric plexuses (magnification, x70). B, Extensive submucosal ganglioneuromatosis comprising thick nerve trunks with scattered mature neurons (magnification, x170).

 
Germline mutation analysis revealed the presence of M918T in this patient; thus the molecular diagnosis of MEN 2B was made. Germline M918T was not detected in his biological parents.

Six months later, he underwent prophylactic thyroidectomy, although no obvious thyroid or adrenal masses were seen on ultrasound scans, and serial pentagastrin-stimulated calcitonin levels and 24-h urine for catecholamine measurement had been consistently normal. Histopathological examination revealed multifocal micro-MTC.


    Discussion
 Top
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have found that all three unrelated patients who presented with apparently isolated intestinal ganglioneuromatosis have the MEN 2B-specific germline M918T mutation. Indeed, during follow-up, all three patients were found to have MTC; together with ganglioneuromatosis of the gut, the appearance of MTC would make the clinical diagnosis of MEN 2B. Although this is a small study, it is sufficient to note that the presence of germline M918T, even in the setting of isolated intestinal ganglioneuromatosis, is diagnostic of MEN 2B. No matter what the initial clinical presentation, the presence of M918T should prompt the clinician to view the patient as having MEN 2B and his/her family as at risk for MEN 2B, with full implications for prophylactic surgery, surveillance, and genetic testing. It is universally accepted that RET mutation analysis be performed as a clinical standard of care in known MEN 2 families and all presentations of apparently sporadic MTC because clinical management is altered subsequent to the genetic testing results (1, 10, 11, 12).

Our limited study does suggest that the histopathological recognition of ganglioneuromatosis is vital. Delays in diagnosis are evident in two of the three cases. All three cases have eventually developed MTC. Hence, in cases where the clinical differential diagnoses of neuronal intestinal dysplasia, intestinal ganglioneuromatosis, and Hirschsprung disease are entertained, it is imperative that the pathologist differentiate among these diagnoses. Ganglioneuromas comprise thickened nerve trunks within which there are scattered mature nerve cells.

The data from the latest analysis of the International RET Mutation Consortium has demonstrated that over 95% of classic MEN 2B cases carry germline M918T (3). Here, we demonstrate that at least a proportion of patients who present with apparently isolated intestinal ganglioneuromatosis do carry germline M918T and therefore represent a forme fruste of MEN 2B, at least at presentation. It is interesting to note that two of the three patients with M918T also seem to have some form of skeletal abnormality, which carries no formal diagnosis. These patients are in contrast to cases with pure mucosal neuroma syndrome (13). Four such patients with oral mucosal neuromas were examined for the presence of germline M918T, and none carried this mutation. These four patients are now all over 11 yr old (one is over 18 yr old) and without evidence of other features of MEN 2B, notably the tumors. Hence, although isolated mucosal neuroma syndrome might be unrelated to MEN 2B, apparently sporadic intestinal ganglioneuromatosis appears to be an entré to MEN 2B. Both of these series studying partial MEN 2B phenotypes are small, and so a concerted effort on the part of the International RET Mutation Consortium to study such "incomplete" phenotypes of various MEN 2 syndromes should be considered. Until then, however, patients presenting with apparently isolated intestinal ganglioneuromatosis should be offered RET mutation analysis at presentation. If germline M918T is found, then the risk of developing MTC is very high, and prophylactic thyroidectomy should be seriously considered.


    Acknowledgments
 
We thank Drs. Patricia L. M. Dahia and Oliver Gimm for critical review of the manuscript.


    Footnotes
 
1 Lawrence and Susan Marx Investigator, a Barr Investigator, and supported by the Cancer Research Campaign Dana-Farber Cancer Institute Fellowship. Back

2 Gibb Fellow of the Cancer Research Campaign. Back

Received August 4, 1998.

Revised September 2, 1998.

Accepted September 8, 1998.


    References
 Top
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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