HUMAN MUTATION 9:473�476 (1997)

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A Five-Basepair Deletion (7118 delTTTTA) Identified Within Neurofibromatosis (NF1) Exon 39; David I. Rodenhiser,1,2,3* JackH. Jung,1,2,3 Jane M.R. Gillett,2 Ken Hovland,1 Joseph Andrews,1 Peter J. Ainsworth,1,4 Marion Coulter-Mackie,5 and Shiva M.Singh1,2,3

1Molecular Medical Genetics Program, Child Health Research Institute, Children’s Hospital of Western Ontario, London, Ontario, CanadaN6C 2V5; Fax 519-685-8186; 2Department of Pediatrics, University of Western Ontario, London, Ontario, Canada; 3Department of Zoology, Univer-sity of Western Ontario, London, Ontario, Canada; 4Department of Medical Biochemistry and DNA Diagnostic Laboratory, Victoria Hospital, London,Ontario, Canada; 5Biochemical Diseases Laboratory, B.C. Children’s Hospital, Vancouver, British Columbia, Canada, V6H 3V4Communicated by Lap-Chee TsuiReceived 19 July 1995; accepted 12 September 1995.© 1997 Wiley-Liss, Inc.*Correspondence to D.I. Rodenhiser.

INTRODUCTIONWe have screened a panel of neurofibromatosis type 1 (NF1) patients representing 85 kindreds from Ontario, Canada. DNA was extracted

and polymerase chain reaction (PCR) used to amplify a 285-bp DNA fragment containing exon 39 (Abernathy et al., 1994). Heteroduplexanalysis (Ainsworth et al., 1994) of the PCR products revealed the presence of a heteroduplex variant from one patient (#70-630). PCRproduct sequenced directly and also cloned revealed a five-base deletion (7118 delTTTTA). Computer analysis of the resulting amino acidsequence showed that this deletion alters the reading frame of exons 39 and 40, where a novel stop codon is generated at position 7215 of thecDNA sequence.

We also used restriction enzyme analysis to verify the location of this mutation since the deletion eliminates a DraI site (TTT/AAA)partially contained within this sequence. DraI digestion of this PCR product gave 168- and 117-bp fragments from the normal allele but DraIdid not digest product amplified from the mutant allele. DNA from the patient’s affected mother also displayed the heteroduplex variant andloss of this DraI site.

DISCUSSIONThe patient is an 8-year-old boy who had numerous café-au-lait macules, axillary freckling, and some learning disabilities including attention

problems. To date, no Lisch nodules, orthopaedic abnormalities, or subcutaneous neurofibromas have been observed. He has a mild wide-basedgait and dysmetria on the left side. Magnetic resonance imaging (MRI) of his spine was normal while the MRI of his head revealed high signal inthe lenticular nuclei consistent with NF1 and mildly asymmetric optic nerves but no optic glioma.

This deletion is the second NF1 exon 39 mutation described. The other mutation is 6-bp deletion (7096del AACTTT) which completelyremoves one of four near-perfect hexameric repeat motifs [A(C)A(C)A(C)TTT] closely aligned at the 3´ end of exon 39 (Abernathy et al.,1994). Our patient’s deletion is partially contained within one of these repeat motifs, however since the entire repeat is not involved, we areunable to firmly conclude that this repeat motif is involved in the mechanism responsible for generating this particular mutation. This report doeshowever contribute to the growing literature of NF1 mutations which will permit the mechanism involved in NF1 mutagenesis to be determined.

ACKNOWLEDGMENTSWe acknowledge funding by the Child Health Research Institute, the support of the staff at the Regional Medical Genetics Centre and

Molecular Diagnostic Laboratory and the cooperation of our NF1 patients and their families.

REFERENCESAbernathy CR, Colman SD, Kouseff BG and Wallace MR (1994) Two NF1 mutations: Frameshift in the GAP related domain, and loss of two codons toward the

3´ end of the gene. Hum Mutat 3:347–352.Ainsworth PJ, Rodenhiser DI, Stuart A, and Jung JH (1994) Characterization of an intron 31 splice junction mutation in the Neurofibromatosis (NF1) gene.

Hum Mol Genet 3:1179–1181.

Three Novel Point Mutations in the Dystrophin Gene in DMD Patients; Adriana Lasa, Pia Gallano,* and Montserrat Baiget,Unitat de Genètica Molecular, Hospital de la Santa Creu i Sant Pau, Barcelona 08025, Spain; Fax: 34-3-2919192Communicted by Jean-Claude KaplanReceived 23 April 1996; accepted 19 June 1996.© 1997 Wiley-Liss, Inc.*Correspondence to Pia Gallano.

Duchenne muscular dystrophy (DMD) is an X-linked degenerative disorder of muscle, caused by gross rearrangements by the dystrophingene in two-thirds of cases. The remaining one-third of patients may carry more subtle mutations that are difficult to detect because of thelarge size and complexity of this gene (Roberts et al., 1994).

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We report three novel point mutations detected in 71 unrelated patients with no deletions or duplications of one or more exons in thedystrophin gene. The presence of these mutations was excluded in an additional sample of 90 DMD patients. Multiple single-strand confor-mation polymorphism (SSCP) (Lasa et al., 1995; Nigro et al., 1992) was performed for brain promotor (Pb), muscle promoter (Pm), and for 25different exons: 3, 4, 6, 8, 9, 12, 13, 16, 17, 19, 43–53, 60, 67, 70, and 74. The DNAs that presented abnormal mobility shifts in the SSCP weresingly amplified, purified using microconcentrators (Amicon) (Petroni et al., 1996) and sequenced by Sanger’s dideoxinucleotide method,using Sequenase 2.0 (US Biochemicals). A 11-bp deletion involving the acceptor splice site of exon 46 was identified in a sporadic DMD case.In accordance with the exon definition model (Robberson et al., 1990), this mutation will result in skipping of exon 46, giving an out-of-frametranscript (Arg2205 Ã frameshift). This patient shows a “typical” DMD phenotype with intellectual impairment; he was confined to a wheel-chair at the age of 12.

A G10431 + 1 Ã C transition was observed in the donor splice site of exon 70 of a sporadic DMD patient. Again, the result of the mutationis a frameshift. We have also detected a G-to-A transversion, at the same position of exon 70, which has been previously described by Lenk etal. (1993). Although the mutations described to date have not shown clustering within specific regions of the dystrophin gene, we point outthat there are at least three mutations affecting the same nucleotide in exon 70. This DMD boy became wheelchair bound at 13 years of age.A gradual deterioration in pulmonary function with reduced maximal inspiratory and expiratory pressures began 3 years later. He also showssome degree of mental handicap.

In exon 74, a deletion of two bases (CT) at cDNA position 10661 was characterized in another sporadic DMD patient. This deletioncauses a shift in the normal reading frame with the generation of a stop codon five triplets downstream. The deletion is predicted toresult in a truncated dystrophin. This patient, aged 6 years, showed classic onset and an expected progression of the disease. He is ofnormal intelligence.

The lack of available muscle specimens prevented us from carrying out any analysis in the expression products. As these three patientswere sporadic cases, SSCP analysis was performed for carrier diagnoses in their families.

ACKNOWLEDGMENTSThis work was supported by the Ministerio de Sanidad (FIS 94/1297). A.L. is recipient of a fellowship from Consejería de Sanidad del

Gobierno Vasco. We thank Vincenzo Nigro for his kind advice.

REFERENCESLasa A, Gallano P, Colomer J, Baiget M (1995) A novel insertional mutation of a single base in exon 12 of the dystrophin gene. Clin Genet 48:128–130.Lenk U, Hanke R, Thiele H, Speer A (1993) Point mutation at the carboxi terminus of the human dystrophin gene: Implications for an association with mental

retardation in DMD patients. Hum Mol Genet 2:1877–1881.Nigro V, Politano L, Nigro G, Romano SC, Molinari AM, Puca GA (1992) Detection of a nonsense mutation in the dystrophin gene by multiple SSCP. Hum Mol

Genet 1:517–520.Petroni D, Pastore C, Lasa A, Soler J, Baiget M, Nomdedéu J (1996) Rapid sequencing protocol using microconcentrators. Trends Genet 12, 5:167–168.Robberson BL, Cote GJ, Berget SM (1990) Exon definition may facilitate splice site selection RNAs with multiple exons. Mol Cell Biol 10:84–94.Roberts RG, Gardner RJ, Bobrow M (1994) Searching for 1 in 2,400,000: A review of dystrophin gene point mutations. Hum Mutat 4:1–11.

New Nonsense Mutation in the Breast Cancer-1 Gene in a French Site-Specific Breast Cancer Family; V. Laplace-Marieze, N.Presneau, C. Girodet, V. Vidal, C. Vaurs, P. Rio, and Y.-J. Bignon,* Laboratory of Molecular Oncology, INSERM CRI 9402, 63011Clermont-Ferrand Cedex 1, France; Fax: 33-73-27-80-42Communicated by R.G.H. CottonReceived 26 May 1996; accepted 23 July 1996.© 1997 Wiley-Liss, Inc.*Correspondence to Y.J. Bignon.

BRCA-1 is a tumor suppressor gene involved in ~45% of breast cancer families (Miki et al., 1994) and 92% of breast-ovarian cancerfamilies with no male breast cancer. We report a new nonsense mutation detected in a French breast-cancer family with three early-onsetbreast cancers but no ovarian cancers. By sequencing, a C to T substitution in codon 1604 (exon 16) was detected creating a stop codon(Gln1604ter).

The BRCA-1 mutation was detected in an affected woman and her unaffected mother, >80 years old. It strengthened the evidence ofincomplete penetrance of BRCA-1 mutations, which is estimated to be nearly 85%. Remarkably, the nonsense mutation is not found inanother sister with mammary dysplasia.

Until now, 90 mutations of BRCA-1 have been described; 64% are frameshift and 18% are nonsense mutations, which truncate theprotein product leaving no doubt about their deleterious effect in cancer hereditary predisposition. In exon 16, three other mutations havebeen detected: one missense mutation (Pro1637Leu) in an American ovarian cancer family (Futreal et al., 1994), one frameshift mutation atcodon 1656, generating a stop codon in an American breast/ovarian cancer family (Castilla et al., 1994), and one nonsense mutation (Tyr1563ter)in a French breast/ovarian cancer family (Serova et al., 1996).

The relationship between genotype and phenotype hints that mutations in the 3´ third of the gene are associated with lower incidence ofovarian cancer in affected families (Gayther et al., 1995). Interestingly, our mutation occurred in a site- specific breast cancer family corrobo-rating this trend.

ACKNOWLEDGMENTSWe thank the Ligue Nationale Contre le Cancer and its Comité du Puy de Dôme.

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REFERENCESCastilla LH, Couch FJ, Erdos MR, Hoskins KF, Calzone K, Garber JE, Lubin MB, Deshano ML, Brody LC, Collins FS, Weber BL (1994) Mutations in the

BRCA-1 gene in families with early-onset breast and ovarian cancer. Nature Genet 8:387–391.Futreal PA, Liu Q, Shattuck-Eidens D, Cochran C, Harshman K, Tavtgian S, Bennett LM, Haugen-Strano A, Swensen J, Miki Y, Eddington K, McClure M, Frye

C, Weaver-Feldhaus J, Ding W, Gholami Z, Söderkvist P, Terry L, Jhanwar S, Berchuck A, Inglehart JD, Marks J, Ballinger DG, Barrett JC, Skolnick MH,Kamb A, Wiseman R (1994) BRCA-1 mutations in primary breast and ovarian carcinomas. Science 266:120–122.

Gayther SA, Warren W, Mazoyer S, Russell PA, Harrington PA, Chiano M, Seal S, Hamoudi R, Van Rensburg EJ, Dunning AM, Love R, Evans G, Easton D,Clayton D, Stratton MR, Ponder BAJ (1995) Germline mutations of the BRCA-1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype correlation. Nature Genet 11:428–433.

Miki Y, Swensen J, Shattuck-Eidens D, Futreal A, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, Bell R, Rosenthal J, Hussey C, Tran T,McClure M, Frye C, Hattier T, Phelps R, Haugen-Strano A, Katcher H, Yakumo K, Gholami Z, Shaffer D, Stone S, Bayer S, Wray C, Bogden R, DayananthP, Ward J, Tonin P, Narod S, Bristow PK, Norris FH, Helvering L, Morrison P, Rosteck P, Lai M, Barrett JC, Lewis C, Neuhausen S, Cannon-Albright L,Goldgar D, Wiseman R, Kram B, Skolnick MH (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA-1. Science 266P66–71.

Serova O, Montagna M, Torchard D, Narod SA, Tonin P, Sylla B, Lynch HT, Feunteun J, Lenoir G (1996) A high incidence of BRCA-1 mutations in 20 breast-ovarian cancer families. Am J Hum Genet 58:42–51.

Recurrent COL3A1 Mutation Results in EDS IV or Familial Aneurysms; David W. Anderson, Smita Thakker-Varia, and CatherineA. Stolle,* Department of Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854Communicated by Leena PeltonenReceived 29 May 1996; accepted 3 June 1996.© 1997 Wiley-Liss, Inc.*Correspondence to Catherine A. Stolle, now at the Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;Fax: 215-573-5892

EDS IV is a lethal genetic disease of collagen in which mutations in COL3A1 give rise to fragile skin, easy bruisability, and hollow organruptures (Kuivaniemi et al., 1991). Familial aneurysms is a vascular disease characterized by late -onset saccular aneurysms, probably multifac-torial in origin, but rare cases may be due to COL3A1 mutations. We investigated the cause of EDS IV in a 10-year-old white male with softskin, easy bruisability, and a family history of vascular ruptures. S1 nuclease analysis of RNA from patient fibroblasts indicated a defect aroundexon 20. Sequence analysis of cDNA clones identified 3 out of 5 clones that retained a portion of intron 20 and revealed a G+1 to A pointmutation in the splice donor site. The same mutation has been observed in a patient with familial aneurysms (Kontussari et al., 1990). In bothpatients, RT-PCR analysis revealed the presence of several RNA species: mRNA lacking exon 20, mRNA with 24 bases of intron 20 sequencein frame, mRNA containing all 132 bases of intron 20, and normally spliced mRNA. The four mRNA species can give rise to three translationproducts: protein lacking exon 20 sequence, protein with 8 noncollagenous amino acids in the middle of the triple helical domain, and normalprotein chains. The mRNA with all 132bp of intron 20 rersults in a truncated protein incapable of assembly into trimers. Consistent with thesefindings, cultured fibroblasts from the patient secreted less type III procollagen and retained abnormal timers in the cell layer. Pepsin digestionof cell layer proteins revealed multiple disulfide bonded trimers of type III procollagen, presumably the products of protein assembly from amixture of normal and multiple abnormal type III procollagen monomers.

REFERENCESKontusaari S, Tromp G, Kuivaniemi H, Ladda RL, Prockop DJ (1990) Inheritance of an RNA splicing mutation (G+1 IVS20) in the type III procollagen gene

(COL3A1) in a family having aortic aneurysms and easy bruisability: Phenotypic overlap between familial arterial aneurysms and Ehlers-Danlos syndrometype IV. Am J Human Genet 47:112–120.

Kuivaniemi H, Tromp G, Prockop DJ (1991) Mutations in collagen genes: Causes of rare and some common diseases in humans. FASEB J 5:2052–2060.

A New Missense Mutation in Exon 6 of the Proteolipid Protein Gene in a Patient With Pelizaeus-Merzbacher Disease; ChiakiKawanishi,1* Hitoshi Osaka,2 Kenji Owa, Ken Inoue,1 Tomohiro Miyakawa,1 Hideki Onishi,1 Yoshiteru Yamada,1 Kyoko Suzuki,1

Seiji Kimura,2 and Kenji Kosaka,2 1Department of Psychiatry, Yokohama City University, Yokohama 236, Japan; Fax: 81-45-783-2540; 2Depart-ment of Pediatrics, Urafune Hospital of Yokohama City University; Yokohama 232, Japan; 3Department of Pediatrics, Saiseikai Nakatsu Hospital,Osaka 530, JapanCommunicated by Kenshi HayashiReceived 30 April 1996; accepted 7 August 1996.© 1997 Wiley-Liss, Inc.Correspondence to C. Kawanishi

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder with dysmyelination restricted to the central nervous system. Proteo-lipid protein (PLP), the major myelin protein in the brain, is markedly decreased in the brains of PMD patients; molecular analysis has revealedvarious PLP gene defects in independent PMD families (Hodes et al., 1993). We report a new point mutation in exon 6 of the PLP gene in aJapanese patient with PMD.

The proband is a 2-year-old Japanese boy. Nystagmus was first detected during the neonatal period. He showed truncal hypotonia and couldnot hold his head upright until recently. T2-weighted magnetic resonance imaging (MRI) revealed a severe dysmyelinating pattern in the whitematter of the entire brain, and only the first waves were detected in the auditory brainstem response. Polymerase chain reaction (PCR) andheteroduplex analysis to screen single-base transitions in PCR products were performed as described previously (Osaka et al., 1995).

Heteroduplex analysis revealed heteroduplex patterns with the mixed PCR product containing exon 6 of the proband. A G-to-C substitution

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was observed at nucleotide position 721 by sequencing DNA, resulting in an A241P substitution. The mutation was confirmed by digesting PCRproducts with the restriction enzyme, BsoFI. The normal product has three BsoFI sites that cut the 277-bp fragment into 140-, 119-, 15-, and 3-bp fragments. The mutation in the proband results in the loss of one site, so that 155- and 119-bp fragments were separated on a 3% NuSieveelectrophoresis gel (FMC Bio Products) after digestion with BsoFI. The mother showed the heterozygous pattern. Then, 120 X chromosomes fromunrelated Japanese subjects were examined to check the absence of the transition; it was not present in any of the alleles analyzed. A241 isconceivably located in a putative transmembrane domain of PLP (Pham-Dinh et al., 1991). Mutant PLP in our proband may be dysfunctionalbecause of aberrant peptide structure induced by the replacement of Ala with Pro, a residue known to disrupt ~-helices. Gow et al. (1994)examined the subcellular localization of wild-type and five mutant forms of PLP, including the A242V mutant, which was observed in the jimpymsd

mutant mouse (Gencic and Hudson, 1990), using Cos-7 cells transiently transfected with wild-type and mutant PLP cDNA. Unlike the wild-type, all mutant PLP molecules were retained in the endoplasmic reticulum and were not expressed on the cell surface. A241P substitution mayalso affect the transport and processing of the protein.

REFERENCESDiehl H-J, Schaich M, Budzinski R-M, Stoffel W (1986) Individual exons encode the integral membrane domains of human myelin proteolipid protein. Proc Natl

Acad Sci USA 83:9807–9811.Gencic S, Hudson LD (1990) Conserved amino acid substitution in the myelin proteolipid protein of jimpymsd mice. J Neurosci 10:117–124.Gow A, Friedrich VL Jr, Lazzarini RA (1994) Many naturally occurring mutations of myelin proteolipid protein impair its intracellular transport. J Neurosci Res

37:574–583.Hodes ME, Pratt VM, Dlouhy SR (1993) Genetics of Pelizaeus-Merzbacher disease. Dev Neurosci 15:383–394.Osaka H, Kawanishi C, Inoue K, Uesugi H, Kurisaki H, Nishiyama K, Yamada Y, Suzuki K, Kimura S, Kosaka K (1995) Novel nonsense protolipid protein gene

mutation as a cause of X-linked spastic paraplesia in twin males. Biochem Biophys Res Commun 215:835–841.Pham-Dinh D, Popot J-L, Boespflug-Tanguy O, Landrieu P, Delenze J-F, Boué J, Jollès P, Dautigny A (1991) Pelizaeus-Merzbacher disease: A valine to phenylalanine

point mutation in a putative extracellular loop of myelin proteolipid. Proc Natl Acad Sci USA 88:7562–7566.

Identification of a Novel Somatic Mutation in the RET Proto-Oncogene in a Patient With Sporadic Medullary Thyroid Carcinoma;Xavier Matias-Guiu,* Elena Lagarda, Monica Calaf, Arturo Azpiroz, Alberto De Leiva, Jaime Prat, and Montserrat Baiget, Depart-ments of Pathology, Endocrinology and Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Departmentof Pathology Clínica Naval, Madrid, Spain; Fax: 34-3-2919344Communicted by Michel GoossensReceived 6 May 1996; accepted 16 September 1996.© Wiley-Liss, Inc.*Correspondence to Xavier Matias-Guiu.

Germline mutations in exons 10, 11, and 13 of the RET proto-oncogene occur in the vast majority of patients suffering from multipleendocrine neoplasia (MEN) type IIA and familial medullary thyroid carcinoma (Donis-Keller et al., 1993; Mulligan et al., 1993), whereas patientswith MEN IIB usually carry germline mutations in codon 918 of exon 16 (Eng et al., 1994). In contrast, somatic mutations in the RET proto-oncogene are detected in a subset of sporadic medullary thyroid carcinomas (SMTC). These somatic mutations are located in exon 16 and, morerarely, in exons 13 and 15. The detection of a point mutation in exons 10 and 11 in either the DNA of the tumour or of peripheral blood issuggestive of a hereditary disease. In one case of SMTC, Dou and Donis-Keller detected a six base-pair somatic deletion that removed a cysteineresidue at codon 630 of the exon 11 (Donis-Keller, 1993). Recently, Romei et al. (1996) identified three somatic mutations in codon 634 of exon11. In the present report, we describe a novel somatic point mutation in codon 630 of exon 11 of the RET proto-oncogene in a SMTC, demon-strating that somatic mutations may occur in such location.

A 62-yr-old woman with no previous family history of thyroid cancer was found to have a thyroid nodule. After thyroidectomy, a 1.6 cmmedullary thyroid carcinoma was detected. The tumor showed all the pathologic features of a sporadic tumour; it was unifocal and was not associatedwith C-cell hyperplasia. Genomic DNA from two areas of the tumour and two areas of normal thyroid tissue was extracted from formalin-fixed,paraffin-embedded tissue blocks with a precise microdissection under microscopic control. PCR-based, single-strand conformation polymorphism(SSCP) analysis using published oligonucleotide primers for exon 11 demonstrated the presence of additional bands with aberrant migration intumour DNA. These bands were not detected in normal thyroid tissue or in constitutional DNA. SSCP analysis of exon 10, and restrictionanalysis of exon 16 did not show germline or somatic mutations. Sequence analysis demonstrated a RET missense somatic point mutation (TGC ÃCGC, Cys à Arg) at codon 630 of exon 11, which was identified in tumour DNA but not in DNA extracted from peripheral blood.

Several authors including ourselves have demonstrated the presence of exon 16 somatic mutations in 23–86% of SMTC and the absence ofexon 10 and 11 somatic point mutations in the vast majority of the reported cases, which account for almost 150. However, the results of thepresent study confirm that somatic mutations may occur also in exon 11, a location that was thought to be exclusive for familial tumours.Similarly to the germline missense point mutations described in familial medullary thyroid carcinoma and MEN IIA patients, the somatic muta-tion removed one of the cysteine residues found in the extracellular domain of the RET protein, the same cysteine residue as in the case noted byDonis-Keller et al. (1993). It seems that codon 630 may be an additional locus for sporadic medullary thyroid carcinoma, since no germlinemutations involving such codon have been reported.

REFERENCESDonis-Keller H, Dou S, Chi D, et al. (1993) Mutations in the RET proto-oncogene are associated with MEN 2A and familial medullary thyroid carcinoma. Hum Mol

Genet 2:851–856.Eng Ch, Smith DP, Mulligan LM, et al. (1994) Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B

and related sporadic tumors. Hum Mol Genet 3:237–241.Mulligan LM, Kwork JB, Healey CS, et al. (1993) Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A: Nature 363:458–460.Romei C, Elisei R, Pinchera A, et al. (1996) Somatic mutations of the ret protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and

are associated with tumor recurrence. J Clin Endocrinol Metab 81:1619–1622.