Analysis of Keratin Polypeptides 8 and 19 Variants in Inflammatory Bowel Disease  Guo–Zhong Tao, Pavel Strnad, Qin Zhou, Ahmad Kamal, Leilei Zhang, Nahid D. Madani, Subra Kugathasan, Steven R. Brant, Judy H. Cho, M. Bishr Omary, Richard H. Duerr  Clinical Gastroenterology and Hepatology  Volume 5, Issue 7, Pages 857-864 (July 2007) DOI: 10.1016/j.cgh.2007.02.017 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Scheme of mutation screening for KRT19 and KRT8. (A) All IF proteins, including keratins, consist of a central and relatively conserved coiled-coil α-helical “rod” domain that is flanked by less conserved non–α-helical N-terminal “head” and C-terminal “tail” domains. The schematic shows the exonic regions of K19, all of which were amplified for mutation screening. K19 amino acids that correspond to the amplified exonic regions are also shown. (B) PCR-amplified fragments of the KRT19 gene were obtained by using, as a template, genomic DNA isolated from human liver explants. The primers that were used for amplification are listed in Table 1, and the amplified fragments were analyzed with 1.5% agarose gels. (C) Schematic showing the exonic regions of K8 that were amplified for mutation screening and the amino acids that represent each amplified domain. The number of amino acids for K8 and K19 were counted after excluding the first methionine. Clinical Gastroenterology and Hepatology 2007 5, 857-864DOI: (10.1016/j.cgh.2007.02.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Keratin protein profiles in digestive organs. Endoscopic biopsies (colon, ileum, duodenum), a surgical specimen (liver), or a tumor cell line (HT29) were used for the purification of cytoskeletal keratin preparations as described in Materials and Methods. The purified keratins were separated by using 9% SDS-PAGE and then visualized by Coomassie staining (A) or by immunoblotting with Abs to K8 and K18 (B), K19 (C), K20 (D), or desmin (E). The identity of the band highlighted by a single asterisk in (A) is not known. The band highlighted by a double asterisk in (E) represents a desmin Ab cross-reactive species. Note that K19 and K20 are expressed in the small and large intestine but not in hepatocytes, and that K19 is a major type I keratin particularly in the colon and ileum. Clinical Gastroenterology and Hepatology 2007 5, 857-864DOI: (10.1016/j.cgh.2007.02.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Identification of KRT19 and KRT8 variants in IBD patients. (A) Control and patient genomic DNA fragments were amplified by PCR and screened as described in Materials and Methods. Samples with abnormal DNA elution patterns (a) were subjected to DNA sequencing (b) as exemplified by the variants: −99 G→C within the KRT19 promoter region and CGT→TGT in KRT8 exon 6 that alter Arg341 to Cys (Table 2). Asterisks highlight the mutation sites. (B) KRT8 variants were detected with pyrosequencing. The nucleotide dispensation order is indicated at the bottom, and the variant positions are highlighted. The light emission is proportional to the number of nucleotides incorporated into the sequence at each nucleotide dispensation step. Clinical Gastroenterology and Hepatology 2007 5, 857-864DOI: (10.1016/j.cgh.2007.02.017) Copyright © 2007 AGA Institute Terms and Conditions