Mutations in CD96, a member of the immunoglobulin superfamily, cause a form of the C (Opitz trigonocephaly) syndrome

Abstract

The C syndrome is characterized by trigonocephaly and associated anomalies, such as unusual facies, psychomotor retardation, redundant skin, joint and limb abnormalities, and visceral anomalies. In an individual with the C syndrome who harbors a balanced chromosomal translocation, t(3;18)(q13.13;q12.1), we discovered that the TACTILE gene for CD96, a member of the immunoglobulin superfamily, was disrupted at the 3q13.3 breakpoint. In mutation analysis of nine karyotypically normal patients given diagnoses of the C or C-like syndrome, we identified a missense mutation (839C-->T, T280M) in exon 6 of the CD96 gene in one patient with the C-like syndrome. The missense mutation was not found among 420 unaffected Japanese individuals. Cells with mutated CD96 protein (T280M) lost adhesion and growth activities in vitro. These findings indicate that CD96 mutations may cause a form of the C syndrome by interfering with cell adhesion and growth.

Figure 1.

CD96 mutations in the C or C-like syndrome and its expression. A, BAC and cosmid/plasmid map spanning the 3q13.3 breakpoint of a patient with a translocation, t(3;18)(q13.13;q12.1). The red vertical line and dashed red line indicate the breakpoint. Blue bars for BACs or plasmids indicate clones covering the breakpoint (detected by FISH analysis). A red horizontal arrow indicates the CD96 gene. A red vertical arrow indicates the breakpoint in the CD96 gene. B, FISH analysis of the patient harboring the translocation. FISH signals for BAC RP11-159B11 are separated into two chromosomes, der(3) and der(18), whose signals are indicated by arrowheads. An arrow indicates signals on a normal chromosome 3. C, RT-PCR for CD96 expression in the B cells of an unaffected individual and the patient with C syndrome. The left, middle, and right lanes depict a 100-bp ladder, an unaffected control, and the patient with C syndrome with translocation, respectively. The lower panel indicates expression of GAPDH as the control. D, Direct DNA-sequence analysis of CD96 demonstrates a point mutation in a patient. E–H, Immunocytochemical analysis for CD96 localization in HT1080 cells with use of rabbit anti-CD96 antibody and FITC-conjugated (E) or HRP-conjugated (G–H) goat anti-rabbit IgG. E, Counterstained with 4′,6-diamidino-2-phenylindole (original magnification 400×). F–H, Counterstained with methyl green. F, Negative control. Original magnification was 100× (F and G) or 400× (H). White arrows (E) and black arrows (H) indicate regions adherent to the plastic dish.

Figure 2.

Expression of the CD96 gene in fetal and adult tissues. A, Expression in human tissues. An arrow indicates CD96 cDNA. An arrowhead indicates GAPDH cDNA as control. Lane M, size marker (100-bp ladder); 1, brain (whole); 2, cerebellum; 3, spinal cord; 4, heart; 5, kidney; 6, lung; 7, skeletal muscle; 8, spleen; 9, thymus; 10, trachea; 11, stomach; 12, small intestine; 13, colon; 14, salivary gland; 15, prostate; 16, testis; 17, uterus; 18, placenta; 19, fetal brain; 20, fetal liver; and 21, bone. Lanes 1–17 and 21, adult tissues. Lanes 19 and 20, fetal tissues. B–F, Whole mount in situ hybridization with Cd96 antisense RNA in 10-dpc mouse embryo, showing high expression in developing forehead (arrow in B) and in heart and blood vessels (arrowheads in B). D–F, Horizontal sections of the embryo. Cd96 is expressed in the pharynx (arrow in D); in cardiac jelly, endocardial cells, and blood cells (arrow in E); and in forebrain tissues (arrow in F). All sections are counterstained with nuclear fast red.

Figure 3.

Functional characterization of wild-type and mutated CD96 proteins. A and B, Cell-adhesion assay for tissue-culture plates. A, Images captured after exposure with vibration for 20 min and 70 min. HT1080 indicates untransfected cells (control); wCD96 indicates highly expressed clone for wild-type CD96 in HT1080; and mCD96 indicates highly expressed clone for mutated CD96 in HT1080. The arrow indicates attached cell. Arrowheads indicate nonattached cells. B, Quantitated adhesion activity in each transformant. Attached cells and nonattached cells are counted in more than five different fields under a microscope. A total of at least 500 cells were counted for each experiment. Error bars are mean±SD. Adhesion activities are indicated by percentages of attached cells per total cell number at 0 min, 20 min, 70 min, and 150 min after spreading cells. C, Cell proliferation assessed by a tetrazolium-based (MTS) assay. The ordinates show the cell number expressed as arbitrary units. Two wCD96s are clones highly expressing wild-type CD96, HT1080 is an untransfected control clone, and mCD96 is a clone expressing mutated CD96. Error bars are mean±SD. Data shown are from three independent experiments, each performed in quadruplicate (n=12).