The study protocol adhered to the tenets of the Declaration of Helsinki. Corneal dystrophies were diagnosed by clinical and/or histopathological evaluation, and clinical examination was performed by slit lamp biomicroscopy. Histopathological data were available for 11 patients with LCD and 10 patients with GCD who underwent corneal transplantation. For histopathology, corneal sections were processed by standard methods and stained with Congo red or Masson trichrome. The presence of amyloid was confirmed by birefringence under polarized light. Blood samples were collected from patients and available family members after obtaining informed consent and approval of the study by the Institutional Review Board of the L. V. Prasad Eye Institute. Among the patients studied, 7 with LCD and 13 with GCD had a family history of disease, and all patients were bilaterally affected. None of the patients had any systemic manifestations. The control population consisted of 100 unrelated individuals who had no history of the diseases studied.
Genomic DNA was isolated from blood leukocytes by standard procedures.
18 Individual exons of the
TGFBI gene were amplified with primers designed by us, specific for flanking intron sequences (primer sequences available on request). PCR-amplified products of all 17 exons were screened for sequence changes by the method of single-strand conformation polymorphism (SSCP), as previously described.
19 Fragments showing altered mobility relative to control subjects were sequenced bidirectionally. Sequencing of purified PCR products was performed (BigDye Terminator Kit on a model 310 Prism sequencer; Applied Biosystems, Inc. [ABI], Foster City, CA). Sequences were compared with the published sequence of
TGFBI (GenBank accession no. for genomic sequence, AY149344; mRNA sequence- NM_000358, version NM_000358.1; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). All samples that were negative for mutations on screening by SSCP, were sequenced directly in all exons to identify mutations. At least 100 normal unrelated control individuals as well as family members were screened for identified mutations to confirm pathogenicity. PCR-restriction fragment length polymorphism (RFLP) was used to test for the presence of various mutations in unrelated control subjects and family members. Restriction site changes are detailed in
Table 1 . PCR products of normal and mutant DNAs were digested with the relevant restriction enzyme and resolved on polyacrylamide or agarose gels. DNA was visualized by staining with ethidium bromide.