The differential diagnosis of dysphagia with onset in CASE REPORT

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1 GASTROENTEROLOGY 2002;122: CASE REPORT Esophageal Stenosis in Childhood: Dystrophic Epidermolysis Bullosa Without Skin Blistering Due to Collagen VII Mutations KLAUS PETER ZIMMER,* HAUKE SCHUMANN, SABINE MECKLENBECK, and LEENA BRUCKNER TUDERMAN Departments of *Pediatrics and Dermatology, University of Münster, Münster, Germany We report a 9-year-old girl who experienced recurrent dysphagia since infancy. Crohn s disease was suspected because she had aphthous ulcers of the mouth and anal dermatitis with hematochezia. After bougienages of esophageal stenoses and medication for inflammatory bowel disease proved unsuccessful, interdisciplinary reexamination revealed the cause of the symptoms to be an extracutaneous form of dystrophic epidermolysis bullosa, a genetic skin fragility disorder. Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene encoding collagen VII, a protein of the epidermal attachment complex, and typically manifests with trauma-induced skin blistering, scarring, nail dystrophy, and, in some cases, mucosal involvement. The present proband never developed skin blisters but had nail dystrophy and erosions of the oral, esophageal, and genitoanal mucosa, which healed with slight scarring. Mutation analysis disclosed compound heterozygosity for recessive mutations in the COL7A1 gene. The paternal mutation 425 A3G caused abnormal splicing resulting in a premature stop codon. The maternal mutation G2775S led to the substitution of a glycine by a serine in the triple helical domain of collagen VII. This case shows that mucosal disease and esophageal strictures in childhood are not always acquired, but can also represent a genetic defect of dermal-epidermal adhesion, even in the absence of skin blistering. The differential diagnosis of dysphagia with onset in late infancy is broad, including both structural and functional causes. A rare condition underlying swallowing difficulties is dystrophic epidermolysis bullosa (DEB), an inherited mechanobullous disorder caused by abnormalities of the anchoring fibrils that attach the epidermis to the dermis. 1 In this disease, mild external shearing forces, such as mechanical pressure or rubbing, induce formation of vesicles and blisters in the skin or mucous membranes. The disorder can cause esophageal stenosis as a consequence of mechanically induced mucosal blistering during food intake and of subsequent scarring. Gastrointestinal tract involvement is observed in different forms of epidermolysis bullosa, e.g., esophageal involvement in DEB or pyloric stenosis in junctional epidermolysis bullosa. 2 More than 50% of patients with DEB experience oral blisters and erosions, accompanied by dysphagia and secondary chronic constipation. Esophageal webs, strictures, or stenosis are noted in about a third of patients with recessive forms of DEB. In general, severe recessive DEB is associated with more extensive involvement of oral and esophageal mucosa than the milder subtypes. However, a subset of patients with mild skin blistering exhibits surprisingly severe esophageal involvement. Despite rapid progress in understanding the molecular basis and etiology of DEB in the last years, the reasons for the different phenotypes are not yet fully understood. 2 The fibrils that connect the epidermal basement membrane with the underlying connective tissue, the anchoring fibrils, are absent or defective in DEB. 1,3 The major component of these fibrils is collagen VII, and mutations in the collagen VII gene (COL7A1) cause DEB. 3 Collagen VII is a product of epidermal keratinocytes and of the epithelial cells in the upper and lower gastrointestinal tract, i.e., oral, esophageal, and anal mucosa. The cells synthesize and secrete collagen VII into the extracellular matrix where the polymerization into anchoring fibrils takes place. 1,4 The fibrils are attached to the basement membrane through binding to laminin 5 and collagen IV. 5 Studies on genotype-phenotype correlations in DEB have revealed an unusual complexity of the gene defects Abbreviations used in this paper: COL7A1, collagen VII gene; DEB, dystrophic epidermolysis bullosa; EB, epidermis bullosa; PCR, polymerase chain reaction by the American Gastroenterological Association /02/$35.00 doi: /gast

2 January 2002 ESOPHAGEAL STENOSIS DUE TO COL7A1 MUTATIONS 221 and their biological and clinical consequences. 6,7 In some cases, the mutation allows prediction of the clinical course of the disease. For example, COL7A1 null mutations are associated with severe phenotypes, whereas dominantly inherited mutations are often associated with minimal phenotypes. Extracutaneous abnormalities may be associated with DEB because the basement membrane zones of the mucous membranes of the orifices, the gastrointestinal, respiratory, and urogenital tracts, the eye, or developing teeth are similar to that in the skin. 8 Recent studies have shown that minimal symptoms in these tissues, such as nail dystrophy or dental anomalies, may occur in heterozygous mutation carriers. These signs are often not recognized to be part of a skin fragility syndrome and, therefore, remain undiagnosed. 9 Here we show that compound heterozygosity for collagen VII gene mutations can also cause complex extracutaneous symptoms without skin blistering. In the present case, gastroenterological symptoms, i.e., episodes of dysphagia, recurrent esophageal stenosis, and hematochezia caused by rectal erosions, anal dermatitis, and fissures were the presenting symptoms. Extensive esophageal stenosis without skin blisters has not been described as a DEB manifestation so far, but was diagnosed in the present case using molecular genetic analysis. Figure 1. Esophageal stenosis. Barium study showing tight stricture in the mid-esophagus. Case Report The 9-year-old girl was admitted with dysphagia caused by esophageal stenosis, chronic constipation, and anal dermatitis with fissures. There was no known family history of gastrointestinal or skin disorders. Her family included an older brother and nonconsanguineous parents who all were clinically unaffected. At birth, the proband was normal. Within the first year of life, dystrophy of toenails developed, but fingernails remained normal. She suffered from dysphagia already during infancy, clinically suggestive of congenital fibromuscular stenosis. Since the age of 2 years, she had recurrent anal fissures and subsequent painful defecation. Occasionally, the perianal skin was inflamed and hematochezia was observed. These symptoms were accompanied by chronic constipation. A few years later, erosions of the oral mucosa developed and were thought to represent recurrent oral aphthous ulcers. Odynophagia was a recurrent problem. Her condition worsened at the age of 8 years, when blisters of the oral mucosa were first observed. At this point also, vulvovaginal erosions and subsequent mild genital skin atrophy started to develop. Notably, she never had had skin blisters or showed any sign of skin fragility. Gastroduodenoscopy and coloscopy were performed because Crohn s disease had been suspected because of hematochezia, aphthous ulcera, and anal fissures. However, neither macroscopical nor microscopical findings in the bowel biopsy specimens nor the laboratory evaluation confirmed Crohn s disease, in agreement with the fact that a mesalazine treatment had not been effective in the past. The proband received 3 times a bougienage of the esophagus without complete relief of dysphagia and was then referred to our hospital. The growth and weight course of the patient was in the range of the fiftieth percentile. Radiography examination revealed a fixed short-segmental tight stricture of the mid-esophagus (Figure 1). During endoscopic dilatation of the stenosis, the mucosa sloughed off in its whole circumference in the area of dilatation. The symptoms recurred within 4 weeks, and the patient received further dilatations with gradually increasing volumes and supportive therapy with omeprazole and corticosteroids for 2 weeks after each dilation. Dermatologic examination revealed an intact integument, except for dystrophy of most toenails. Gingival mucosa showed localized enanthema and small erosions along the lower front teeth (Figure 2). The vulvovaginal mucosa was erythematous and the surrounding skin discretely atro-

3 222 ZIMMER ET AL. GASTROENTEROLOGY Vol. 122, No. 1 phic. A painful perianal fissure was present. The fingernails, hair, and teeth were normal. Clinical examination of the parents revealed no pathological changes of the skin, mucous membranes, or nails. Mucosal and skin biopsy specimens and blood samples of the family were obtained after an informed consent. The mucosal biopsy specimens were subjected to immunofluorescence staining with antibodies to structural proteins of the dermal-epidermal junction. The staining revealed dermoepidermal separation below the basement membrane, at the level of the anchoring fibrils.10 The staining with antibodies to collagen VII was clearly attenuated, suggesting abnormal expression of this epidermal adhesion protein (Figure 3). Therefore, mutations were sought in the collagen VII gene, COL7A1. Mutation analysis was performed with direct polymerase chain reaction (PCR) amplification of all COL7A1 exons from genomic DNA, followed by heteroduplex analysis and dideoxynucleotide sequencing In the proband s DNA, a heterozygous 425A3 G exchange at position 2 at donor splice site of exon 3 and a hetš Figure 2. Clinical phenotype of dystrophic epidermolysis bullosa in the proband. (A ) Erosions and occasional blistering in the oral mucosa occurred since school age. (B) Dystrophy of toenails was the only nonmucosal manifestation of DEB.

4 January 2002 ESOPHAGEAL STENOSIS DUE TO COL7A1 MUTATIONS 223 Figure 3. Antigen mapping of mucosal biopsy specimens. Immunofluorescence staining of biopsy specimens from the oral mucosa stained with antibodies to collagen VII. (A) The staining of the dermoepidermal junction is strongly positive in a biopsy specimen derived from oral mucosa of a healthy individual, whereas (B) the patient s biopsy specimen exhibited very weak staining. erozygous 8323G3 A transition in exon 112 were identified. The first mutation could be verified with digestion with the restriction enzyme StyI and the second with Msp I (Figure 4). The 425A3 G mutation is a common COL7A1 defect that leads to abnormal splice products with premature termination codons, 14,15 and the G2775S mutation substitutes serine for glycine within the triple helical domain of collagen VII. 16 The parents were heterozygous carriers of the respective mutations (Figure 4). These sequence variations were not present in 150 normal chromosomes, indicating that they did not represent neutral polymorphisms. Screening of the entire coding sequence and of all intron-exon borders of the COL7A1 gene did not reveal any other nucleotide variations predicting changes in collagen VII polypeptide sequence. Discussion Here we describe an unusual extracutaneous gastrointestinal manifestation of DEB. Typically, DEB presents with trauma-induced skin blistering that can be accompanied by extracutaneous abnormalities. Recent investigations have revealed some minimal and atypical clinical symptoms. 15,17,18 For example, toenail dystrophy can be a minimal sign in otherwise unaffected parents of DEB patients who were carriers of a heterozygous COL7A1 mutation, 15,17,19 but mucosal manifestations without skin involvement have not been known so far. Therefore, the present proband extends the spectrum of clinical DEB manifestations by a unique combination of nail dystrophy with mucosal fragility and scarring, but normal skin. In concert with the rare manifestation, the initial diagnostic procedures, endoscopy and histologic examination of esophagus biopsy specimens, yielded nonspecific findings indicative of mild inflammation and led to the treatment of a presumed inflammatory bowel disease. The symptoms, aphthous ulcers, anal fissures, and hematochezia, were suggestive of Crohn s disease with assumed esophageal involvement. However, the diagnosis of Crohn s disease or of other inflammatory disorders such as celiac disease, allergic (eosinophilic), or reflux esophagitis could not be confirmed by endoscopic or laboratory evaluation. The early onset of dysphagia was suggestive of congenital causes of esophageal stenosis, such as esophageal atresia and webs, but these disorders show distinct radiographic and clinical features and are not associated with aphthous ulcers, anal dermatitis, and hematochezia. This case shows that DEB can be the underlying cause of esophageal stenosis even in the absence of skin blistering, and that a careful clinical search for minimal yet pathognomonic signs of DEB is warranted. Statistical evaluation shows that gastrointestinal tract involvement is common in epidermolysis bullosa (EB). 2 Š Figure 4. Identification of mutations in COL7A1. (A) Pedigree. The unaffected mother and the unaffected father were heterozygous mutation carriers. The patient was compound heterozygous for 2 different mutations. (B) Automated dideoxynucleotide sequencing disclosed a heterozygous G3A transition in nucleotide position 8323 in exon 112 in the patient and her unaffected mother (upper panel). This sequence alteration led to the glycine substitution G2775S. The paternal mutation was a heterozygous A3G transition in nucleotide position 425 in exon 3, a relatively common mutation in DEB (lower panel). (C) The maternal mutation led to the loss of an Msp I site (upper panel). (C) The normal 400 base pair (bp) PCR product was cleaved into 3 fragments of 101, 125, and 174 bp. In contrast, the mutated PCR product of the patient (P) and the mother (M) showed an uncleaved 299-bp fragment, in addition to the 101-bp fragment (not visible on the gel). The father s samples (F) showed bands similar to the control. ND, control not digested; D, control digested. The paternal mutation 425 A3G eliminated a Sty I site (lower panel). The 422-bp PCR product of the normal control (C) and the unaffected mother (M) was cleaved by Sty I into two 211-bp fragments. Sty I digestion of the PCR product of the patient (P) and the father (F) produced the 211-bp fragment and the 422-bp uncleaved mutated fragment.

5 224 ZIMMER ET AL. GASTROENTEROLOGY Vol. 122, No. 1 The highest frequency, about 50%, is found in junctional and dystrophic EB. Patients with these subtypes suffer from oral blistering (up to 90%), dysphagia (2% 55%), anal strictures (1% 5%), and secondary chronic constipation (17% 45%). Esophageal webs, strictures, or stenosis are noted in about 35% of patients with DEB, but rarely in EB simplex or junctional EB. A rare form of junctional EB that is caused by mutations in the genes for integrin 6 4 is associated with congenital pyloric atresia. 2 The risk for malignancies of the gastrointestinal tract is not increased in EB. 2 The management of gastrointestinal involvement in EB can be challenging. 2 The only form that is successfully treated with surgery is junctional EB with pyloric atresia. Often the patients exhibit only minimal skin lesions after the pyloric atresia has been surgically removed. 20,21 Different strategies exist for management of the esophageal stenosis in DEB, including endoscopic balloon dilatations, radiologic balloon dilatations, and supportive drug therapy with omeprazole and corticosteroids. 2,22,23 However, recurrence of the stenosis or strictures tends to impede long-term success of these therapeutic regimens. In individual cases, more radical surgical procedures, such as esophageal reconstruction with reversed gastric tube, resection and end-to-end esophageal anastomosis, or colonic autotransplant have been used Recently, gastrostomy, preferentially performed by the rush technique, has become a successful alternative to alleviate the consequences of gastrointestinal symptoms in EB. 27 Benefits of dietary and liquid supplementation via gastrostomy include significant weight gain, improved wound healing, prevention of constipation, and reduction of episodes of obstructive dysphagia and odynophagia. Compound heterozygosity for COL7A1 gene mutations underlies recurrent esophageal stenosis in the present proband. The mutations are clinically silent when combined with a normal allele, as seen in the parents, but cause an unusual set of symptoms when combined with each other. Because the splice site mutation leads to premature termination codons, the patient is functionally homozygous for the glycine substitution G2775S. Over 200 COL7A1 gene defects have been disclosed in recessive and dominant DEB, 3,9,11,12,14 18,28,29 and a current opinion prevails that mutations in the distal segments of the collagen VII triple helix may cause less severe functional impairment. The hypothesis is that the distal noncollagenous domains may stabilize the triple helix in their vicinity, and a recent collagen VII stability study of ours supports this assumption. We showed that glycine substitutions located centrally in the molecule cause more severe unfolding of the triple helix than substitutions in the peripheral subdomains and that the degree of unfolding correlated with the clinical severity of DEB (S. Mecklenbeck, L. Bruckner-Tuderman, unpublished results, October 2001). In agreement with this, the glycine substitution of the present proband, G2775S, which is located in the most distal carboxyterminus of the triple helical domain, does not cause extensive skin blistering. The molecular mechanisms leading to mucosal, but not cutaneous, fragility in DEB remain elusive at the present time. Careful studies on more probands and their genotype-phenotype correlations are required to elucidate these questions and to design targeted therapeutic strategies in the future. Meanwhile, it is important to recognize unusual and minimal clinical symptoms to uncover yet undiagnosed DEB as such and provide the patients with adequate counseling and management. References 1. Burgeson RE. Type VII collagen, anchoring fibrils, and epidermolysis bullosa. J Invest Dermatol 1993;101: Fine JD, Bauer EA, McGuire J, Moshell A. Epidermolysis bullosa: clinical, epidemiological, and laboratory advances and the findings of the national epidermolysis bullosa registry. Baltimore: The Johns Hopkins University Press, Christiano AM, Anhalt G, Gibbons S, Bauer EA, Uitto J. Premature termination codons in the type VII collagen gene (COL7A1) underlie severe, mutilating recessive dystrophic epidermolysis bullosa. Genomics 1994;21: Bruckner-Tuderman L, Höpfner B, Hammami-Hauasli N. Biology of anchoring fibrils: lessons from dystrophic epidermolysis bullosa. Matrix Biol 1999;18: Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE. Laminin 5 binds the NC-1 domain of type VII collagen. J Cell Biol 1997;138: Bruckner-Tuderman L. Hereditary skin diseases of anchoring fibrils. J Dermatol Sci 1999;20: McGrath JA, Ashton GH, Mellerio JE, Salas-Alanis JC, Swensson O, McMillan JR, Eady RA. Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations. 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6 January 2002 ESOPHAGEAL STENOSIS DUE TO COL7A1 MUTATIONS 225 dystrophic epidermolysis bullosa. Curr Opin Dermatol 1996;3: Christiano AM, Anhalt G, McGrath JA, Tan KC, Uitto J. Glycine substitutions in the triple-helical region of type VII collagen result in a spectrum of dystrophic epidermolysis bullosa phenotypes and patterns of inheritance. Am J Hum Genet 1996;58: Ganguly A, Rock MJ, Prockop DJ. Conformation-sensitive gel electrophoresis for rapid detection of single base differences in double stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A 1993;90: Gardella R, Belletti L, Zoppi N, Marini D, Barlati S, Colombi M. Identification of two splicing mutations in the collagen type VII gene (COL7A1) of a patient affected by the localisata variant of recessive dystrophic epidermolysis bullosa. Am J Hum Genet 1996;59: Hammami-Hauasli N, Schumann H, Raghunath M, Kilgus O, Lüthi U, Luger TA, Bruckner-Tuderman L. Some but not all glycine substitution mutations in COL7A1 result in intracellular accumulation of collagen VII, loss of anchoring fibrils and skin blistering. J Biol Chem 1998;273: Kon A, Pulkkinen L, Ishida-Yamamoto A, Hashimoto I, Uitto J. Novel COL7A1 mutations in dystrophic forms of epidermolysis bullosa. J Invest Dermatol 1998;111: Shimizu H, Hammami-Hauasli N, Hatta N, Nishikawa T, Bruckner- Tuderman L. Compound heterozygosity for silent and dominant glycine substitution mutations in COL7A1 leads to a marked transient intracytoplasmic retention of procollagen VII and a moderately severe dystrophic epidermolysis bullosa phenotype. J Invest Dermatol 1999;113: Terracina M, Posteraro P, Schubert M, Sonego G, Atzori F, Zambruno G, Bruckner-Tuderman L, Castiglia D. Compound heterozygosity for a glycine recessive substitution and a splice site mutation in the COL7A1 gene causes an unusually mild form of localized recessive dystrophic epidermolysis bullosa. J Invest Dermatol 1998;111: Gedde-Dahl T Jr, Anton-Lamprecht I. Epidermolysis bullosa. In: Rimoin DL, Connor JM, Pyeritz RE, eds. Emery and Rimoin s Principles and Practice of Medical Genetics. Volume 1. 3rd ed. New York: Churchill Livingstone, 1996: Pulkkinen L, Rouan F, Bruckner-Tuderman L, Wallerstein R, Garzon M, Brown T, Smith L, Carter W, Uitto J. Novel ITGB4 mutations in lethal and nonlethal variants of epidermolysis bullosa with pyloric atresia: missense versus nonsense. Am J Hum Genet 1998;63: Pulkkinen L, Bruckner-Tuderman L, August C, Uitto J. Compound heterozygosity for missense (L156P) and nonsense (R554X) mutations in the beta4 integrin gene (ITGB4) underlies mild, nonlethal phenotype of epidermolysis bullosa with pyloric atresia. Am J Pathol 1998;152: Feurle G, Weidauer H, Baldauf G, Schulte-Braucks T, Anton-Lamprecht I. Management of esophageal stenosis in recessive dystrophic epidermolysis bullosa. Gastroenterology 1984;87: Kern IB, Eisenberg M, Willis S. Management of esophageal stenosis in epidermolysis bullosa dystrophica. Arch Dis Child 1989; 64: Harmel RP. Esophageal replacement in tow siblings with epidermolysis bullosa. J Pediatr Surg 1986;21: Sehhat S, Amirie SA. Esophageal reconstruction for complete stenosis due to dystrophic epidermolysis bullosa. Thorax 1977; 32: Absolon KB, Finney LA, Waddill GM Jr, Hatchett C. Esophageal reconstruction colon transplant in two brothers with epidermolysis bullosa. Surgery 1969;65: Haynes L, Atherton DJ, Ade-Ajayi N, Wheeler R, Kiely EM. Gastrostomy and growth in dystrophic epidermolysis bullosa. Br J Dermatol 1996;134: Hovnanian A, Rochat A, Bodemer C, Petit E, Rivers CA, Prost C, Fraitag S, Christiano AM, Uitto J, Lathrop M, Barrandon Y, de Prost Y. Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation. Am J Hum Genet 1997;61: Kon A, McGrath JA, Pulkkinen Nomura K, Nakamura T, Maekawa Y, Christiano AM, Hashimoto I, Uitto J. Glycine substitution mutations in the type VII collagen gene (COL7A1) in dystrophic epidermolysis bullosa: implications for genetic counseling. J Invest Dermatol 1997;108: Received August 9, Accepted October 25, Address requests for reprints to: Leena Bruckner-Tuderman, M.D., Department of Dermatology, University of Münster, Von-Esmarch- Strasse 58, Münster, Germany. tuderma@uni-muenster.de; fax: (49) Supported by grant SFB492-A3 from the Deutsche Forschungsgemeinschaft (DFG) (to L.B.-T.) and by grants from DEBRA (Dystrophic Epidermolysis Bullosa Research Association). Drs. Zimmer and Schumann contributed equally to this work. The authors thank Henrike Pentrup, Margit Schubert, and Andrea Wissel for expert technical assistance.