Volume 138, Issue 7, Pages 2388-2398.e2 (June 2010) Mutations in TTC37 Cause Trichohepatoenteric Syndrome (Phenotypic Diarrhea of Infancy)  Jane Louise Hartley, Nicholas C. Zachos, Ban Dawood, Mark Donowitz, Julia Forman, Rodney J. Pollitt, Neil V. Morgan, Louise Tee, Paul Gissen, Walter H.A. Kahr, Alex S. Knisely, Steve Watson, David Chitayat, Ian W. Booth, Sue Protheroe, Stephen Murphy, Esther de Vries, Deirdre A. Kelly, Eamonn R. Maher  Gastroenterology  Volume 138, Issue 7, Pages 2388-2398.e2 (June 2010) DOI: 10.1053/j.gastro.2010.02.010 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Platelet abnormalities in patients with THES. Thin-section electron microscopy of a representative (A) normal and (B–D) 3 different THES patients. Black bar, 500 nm. Normal α-granules are observed in (A) control platelets (white arrowheads) and occasionally in some (B) THES platelets, but frequently (C and D) are absent in others. Although the membranous surface–connected canalicular system appears (A) normal in control platelets (white arrow), it was (B–D) disrupted with prominent tubules and smaller membranous vesicles in THES patient platelets (white arrows). (B) Lipid inclusions were observed frequently in THES platelets (black arrows). (C) Electron dense lysosomal bodies fused with an α-granule often were seen (large white arrowhead). Gastroenterology 2010 138, 2388-2398.e2DOI: (10.1053/j.gastro.2010.02.010) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 The fluorescence-activated cell sorting analysis of the platelets shows that in a control, stimulation by collagen-related peptide (crp) for 90 seconds induces a single, sharp peak in release of α-granule content from the platelet. In THES patients (n = 2) there are 2 peaks because of the absence of secretion in a subpopulation of platelets. Gastroenterology 2010 138, 2388-2398.e2DOI: (10.1053/j.gastro.2010.02.010) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 TTC37 is comprised of 43 exons. The position of each of the mutations is shown. The TPR domains in relation to the exons and mutations are shown in blue. Gastroenterology 2010 138, 2388-2398.e2DOI: (10.1053/j.gastro.2010.02.010) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 .(A) Microsatellite marker haplotype from the probands of Pakistani origin are shown (4a). The regions of homozygosity are highlighted in grey. At D5s2100 all the probands have an identical haplotype. (B) Gel electrophoresis of the reverse-transcriptase polymerase chain reaction products. (a) Small polymerase chain reaction products in patients 3C and 5C as compared with the other affected children (patients 4C and 6C) and the control samples. Patients 3C and 5C have c.2779-2G>A sequence change in intron 27. (b) Patient 6C, who has a c.751G>A mutation, has a smaller polymerase chain reaction product as compared with other affected children and controls. (C) Sequence of the reverse-transcriptase polymerase chain reactions. Exon 29 is skipped in patients 3C and 5C, which corroborates the gel electrophoresis. Exon 10 is skipped in patient 6C and corresponds to the smaller polymerase chain reaction product seen on gel electrophoresis. Gastroenterology 2010 138, 2388-2398.e2DOI: (10.1053/j.gastro.2010.02.010) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Expression and localization of apical and basolateral transport proteins in THES patients and age-matched controls. Paraffin-embedded jejunal samples were fixed and stained with antibodies against brush-border transport proteins, NHE2, NHE3, Aqp7, NIS, and the H/K ATPase, as well as the basolateral Na/K ATPase. Villin was used as a marker of the enterocyte brush border. Localization of NHE2, NHE3, NIS, and the H/K ATPase was altered in THES patient samples compared with age-matched controls. Alternatively, expression of Aqp7 appeared to be reduced significantly in THES patients vs controls. In contrast, expression and localization of the Na/K ATPase (basolateral) and villin (brush border) were not altered in any control or THES patient samples analyzed. Inserts show 6× zoom magnification of original images obtained with 63× water immersion objective. Arrowheads indicate localization of the brush border. Scale bar, 20 μm. Gastroenterology 2010 138, 2388-2398.e2DOI: (10.1053/j.gastro.2010.02.010) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 The amino acid composition of hair from 2 of the patients with THES shows a remarkable similarity to the amino acid composition of hair from children with trichothiodystrophy. Gastroenterology 2010 138, 2388-2398.e2DOI: (10.1053/j.gastro.2010.02.010) Copyright © 2010 AGA Institute Terms and Conditions