Gluten sensitivity in monozygous twins: a long-term follow-up of five pairs

THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2000 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.

Vol. 95, No. 6, 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(00)00879-0

Gluten Sensitivity in Monozygous Twins: A Long-Term Follow-Up of Five Pairs Maria Teresa Bardella, M.D., Clara Fredella, M.D., Luigia Prampolini, M.D., Roberta Marino, M.D., Dario Conte, M.D., and Anna Maria Giunta, M.D. Cattedra di Gastroenterologia, IRCCS Ospedale Maggiore, and Clinica Pediatrica II, Universita` degli Studi, Milan, Italy

OBJECTIVE: The aim of this study was to investigate the role of genetic factors and the characteristics of five monozygous twin pairs with at least one member affected by gluten sensitivity. METHODS: Five pairs of monozygous female twins, of whom one or both were affected by gluten sensitivity (i.e., celiac disease or dermatitis herpetiformis), were followed-up for 11–23 yr. RESULTS: Three pairs were concordant for celiac disease: the onset was comparable and synchronous in two pairs; in the third, one member presented an overt malabsorption syndrome, and the other developed iron deficiency anemia 10 yr later. Discordance for gluten sensitivity was found in the remaining two pairs, one of whose members was diagnosed as having, respectively, celiac disease and dermatitis herpetiformis. CONCLUSIONS: As no environmental factors were found to affect the phenotypic expression of the disease, genetic factors seem to play a major role. The presence of overt or latent celiac disease in three of the four siblings of the three concordant twins, and the association with cystic fibrosis in all three siblings of one of these families, further supports this hypothesis. (Am J Gastroenterol 2000;95:1503–1505. © 2000 by Am. Coll. of Gastroenterology)

INTRODUCTION Gluten sensitivity is the term used to describe the spectrum of symptoms and serological and histological abnormalities present in subjects who are genetically predisposed to an abnormal response to gluten, a proteic component of wheat, barley, and rye (1). It includes celiac disease (CD), a systemic disorder with a wide range of symptoms and characteristic histological intestinal findings (2), and dermatitis herpetiformis (DH), a blistering skin disease with specific linear granular deposits of IgA in the dermal-papillary pits and (in about two-thirds of cases) intestinal lesions similar to those of CD (3). Moreover, the so-called silent, latent, and potential forms of CD are considered to form part of the gluten-sensitivity picture (4). Celiac disease and DH share a common genetic make-up,

but multiple HLA and non-HLA genes and other factors seem to influence the phenotypic expression of both diseases, as suggested by familial and twin studies (5, 6). In particular, concordance for gluten sensitivity in monozygous twins is not absolute but approaches 75% (7). We here describe the characteristics of five monozygous twin pairs with at least one member affected by gluten sensitivity whom we have followed-up for a long period of time.

MATERIALS AND METHODS The series includes five female twin pairs born between 1971 and 1987, and regularly followed-up after the diagnosis of gluten sensitivity. Monozygosity was supported by their same gender, the characteristics of the placenta and amniotic sac, similar phenotypes and anthropometric findings, and identical blood groups and HLA status. HLA class I antigens (HLA-A, B, and C) were serologically typed on peripheral blood lymphocytes separated by gradient centrifugation (8), which were placed in microplates and submitted to a standard two-stage microcytotoxicity test (9); the results were scored by means of a fluorescence microscope. HLA class II antigens were typed at genomic level using PCRSSO and SSP techniques: the technique, primers, and digoxigenin-labeled probes used in the PCR-SSO assay were those validated by the 12th International Histocompatibility Workshop (10, 11). Celiac disease and DH were diagnosed based on standard histological criteria (12, 13) and confirmed by the response to a gluten-free diet (GFD). Intraepithelial lymphocytes (IEL) were measured by a differential count of at least 500 cell nuclei (epithelial and lymphocyte) and the results expressed as IEL/100 epithelial cells (reference values ⬍40 IEL/100 enterocytes). At diagnosis and during the yearly follow-up examinations, all of the patients underwent the D-xylose test and the determination of total serum immunoglobulin levels (standard nephelometric method), serum IgA antigliadin antibodies (AGA) by means of an enzymelinked immunosorbent assay (ELISA; Gluten IgA EIA, Pharmacia, Uppsala, Sweden), and, more recently, antiendomysium antibodies (EmA) by means of indirect immu-

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Table 1. HLA Phenotype of Five Monozygous Female Twin Pairs With at Least One Member Affected by Gluten Sensitivity Twin pairs, Nos. 1a 1b 2a 2b 3a 3b 4a 4b 5a 5b

HLA A

HLA B

HLA Cw

HLA DRB1

HLA DQA1

HLA DQB1

2;32 2;32 2;26 2;26 1;24 1;24 2;23 2;23 2;23 2;23

14;61 14;61 18;13 18;13 8;44 8;44 18;⫺ 18;⫺ 51;44 51;44

2;⫺ 2;⫺ 6;7 6;7 7;⫺ 7;⫺ 2;⫺ 2;⫺ ⫺;⫺ ⫺;⫺

03011;1601 03011;1601 1101;0701 1101;0701 03011;04 03011;04 1101;0701 1101;0701 04;15 04;15

0102;0501 0102;0501 0201;0501 0201;0501 0501;03 0501;03 0201;0501 0201;0501 0102;03 0102;03

0201;0502 0201;0502 0201;0301 0201;0301 0201;0301 0201;0301 0201;0301 0201;0301 0602;0302 0602;0302

nofluorescence on monkey esophagus (Eurospital, Trieste, Italy). AGA and EmA were determined in both the affected and unaffected twins to check compliance to the GFD and detect a silent CD, respectively. A questionnaire concerning pregnancy, delivery, lactation, weaning, growth, kindergarten development, and a detailed medical history was completed by the mothers during the follow-up period to highlight any differences between the sisters.

RESULTS HLA typing of the five pairs of twins (Table 1) confirmed their monozygosity and supported the existence of a susceptibility to gluten. The clinical data and duration of follow-up are summarized in Table 2. In three twin pairs (nos. 1, 2, and 3) both members had CD: two pairs showed an overt malabsorption syndrome and the onset was synchronous before the age of 2 yr (nos. 1 and 2); in the third pair (no. 3), one sister had diarrhea at the age of 1 yr and the other iron deficiency anemia at the age of 10 yr, and an associated common variable immunodeficiency was diagnosed at the age of 20 yr. Investigations of the family members of the affected twins revealed other relevant findings. In family 1, on the Table 2. Characteristics of Five Monozygous Female Twin Pairs With at Least One Member Affected by Gluten Sensitivity Age at Twin Pairs, Diagnosis Nos. (Yr) Onset 1a 1b 2a 2b 3a 3b 4a 4b 5a 5b

1 11⁄2 1 1 1 10 2 13 24

Diarrhea Diarrhea Diarrhea Diarrhea Diarrhea Anemia Diarrhea

Duodenal Follow-Up Histology (Yr) SVA SVA SVA SVA SVA SVA SVA

DH IIEL/MA Symptom-free Normal

21 21 11 11 23 23 22 22 14 14

SVA ⫽ subtotal villous atrophy; IIEL ⫽ increased intraepithelial lymphocytes; MA ⫽ mild atrophy; DH ⫽ dermatitis herpetiformis.

basis of the appearance of EmA positivity, the first sister, 3 yr older than the twins, underwent multiple endoscopic duodenal sampling at the age of 20 yr, the results of which were normal; 2 yr later, she is still symptom-free and her duodenal histology normal. The fourth sister, 3 yr younger than the twins, was diagnosed by screening procedures as having celiac disease at the age of 12 yr; the adoption of a GFD led to both serological and histological normalization within 18 months. In family 2, cystic fibrosis was diagnosed in all the children: at the age of 5 yr in the two CD twins, and at the age of 2 yr in their younger sister. In family 3, the first sister, 3 yr older than the twins, who had previously undergone AGA screening with negative results, showed EmA positivity at the age of 25 yr and so underwent a complete clinical evaluation. Her HLA type was identical to that of her CD sisters (see Table 1) but multiple duodenal specimens appeared normal, as did the number of intraepithelial lymphocytes. She is currently being followed-up for possible latent CD. Discordant gluten-sensitivity was found in twin pairs 4 and 5: subject 4a was diagnosed as having CD at the age of 2 yr, but her sister (4b) is still well after 22 yr of follow-up; subject 5a was diagnosed as having DH at the age of 13 yr, but her sister (5b), who was serologically negative and had normal skin and duodenal biopsies in 1996, remains symptomless after 14 yr of follow-up. Parent screening in these two families was negative and no associated diseases were diagnosed. The answers to the questionnaires did not reveal any differences in these two discordant twin pairs in terms of living habits and medical history.

DISCUSSION The long-term follow-up of these five monozygous twin pairs affected by gluten sensitivity leads to two observations. First, concordance for gluten sensitivity is not absolute, and both the time of onset and the severity of the clinical manifestations may differ. Second, nongenetic factors possibly influencing the phenotypic expression of gluten sensitivity were not identified. Until 1973, when Walker-Smith (7) described a well-

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documented case, the reports of discordance for celiac disease in monozygous twins were isolated, and monozygosity or even the actual presence of CD were incompletely searched for (14 –17). Interestingly, some initially discordant patients became concordant during follow-up (18), thus indicating the possibility of a later phenotypical expression of CD; furthermore, asynchronous intestinal involvement has also been found in occasional cases of monozygous twins with DH (14). As described in other reports involving monozygous twins (7, 19), our own series of five pairs of certainly identical twins showed discordance for gluten sensitivity in two cases (nos. 4 and 5). However, despite a follow-up of, respectively, 22 and 14 yr, the possibility of a later onset cannot be excluded, as indicated by previous reports (18) and by one of our concordant pairs (no. 3, in which subject 3b, who was regularly followed-up and initially serologically negative, developed iron deficiency anemia and was diagnosed as having celiac disease 10 yr after her sister). All of these findings indicate that environmental factors may play a role in triggering gluten sensitivity, but no supporting evidence is available in the literature and our own data do not show any etiological factor other than genetic factors; family members other than the twins have CD or can be considered latent celiacs. Of particular interest in this regard is the finding that all three children in family 2 (i.e., the CD twins and their sister) have cystic fibrosis. The association between CD and cystic fibrosis has already been reported (20, 21) but it seems to be particularly rare in Italy, where the frequency of CD and cystic fibrosis in the general population is, respectively, 1:200 and 1:2500 births (22, 23). Our data confirm that identical twins may be discordant in terms of the type and time of onset of gluten sensitivity, and that siblings with potential CD should be carefully followed-up. Although genetic factors play a major role in the induction and clinical manifestation of gluten sensitivity, the concomitant relevance of unrecognized environmental factors can not be ruled out.

ACKNOWLEDGMENTS We thank the Lombardy Section of the Associazione Italiana Celiachia for their support, and Prof. Raffaella Scorza and Dr. Maurizio Marchini for the genetic typing. Reprint requests and correspondence: Maria Teresa Bardella, M.D., Istituto di Scienze Mediche, Via F. Sforza, 35, 20122 Milano, Italy. Received Sep. 17, 1999; accepted Jan. 26, 2000.

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