Abstract
Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, short stature, brachydactyly, characteristic facial features, developmental delay and other highly variable systemic defects. Classic PPS is caused by loss-of-function mutations in the B3GLCT gene encoding for a β3-glucosyltransferase that catalyzes the attachment of glucose via a β1–3 glycosidic linkage to O-linked fucose on thrombospondin type 1 repeats (TSRs). B3GLCT was shown to participate in a non-canonical ER quality control mechanism; however, the exact molecular processes affected in PPS are not well understood. Here we report the identification and characterization of two zebrafish orthologs of the human B3GLCT gene, b3glcta and b3glctb. The b3glcta and b3glctb genes encode for 496-aa and 493-aa proteins with 65% and 57% identity to human B3GLCT, respectively. Expression studies demonstrate that both orthologs are widely expressed with strong presence in embryonic tissues affected in PPS. In vitro glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an O-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double b3glct knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous b3glct-/- embryos demonstrated complete loss of in vitro b3glct activity. Surprisingly, b3glct-/- homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of b3glct-/- 24-hpf embryos identified 483 shared differentially regulated transcripts that may be involved in compensation for b3glct function in these embryos. The presented data show that both sequence and function of B3GLCT/b3glct genes is conserved in vertebrates. At the same time, complete b3glct deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.
Highlights
Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, brachydactyly, short stature, characteristic facial features and developmental delay as well as other variable systemic defects affecting the skeletal, cardiovascular, genitourinary and central nervous systems [1,2,3]
B3GLCT mutations result in classic PPS which is characterized by a triad of features including anterior segment defects (100%; Peters anomaly in 85%), short stature
When the identified b3glcta and b3glctb sequences were aligned with the human B3GLCT, both zebrafish orthologs were found to likely be lacking several 50 exons encoding for a large portion of their N-terminus and appeared to be incomplete (Fig 1A)
Summary
Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, brachydactyly, short stature, characteristic facial features and developmental delay as well as other variable systemic defects affecting the skeletal, cardiovascular, genitourinary and central nervous systems [1,2,3]. The ocular defect most often associated with PPS is Peters anomaly (PA); other defects affecting the anterior segment of the eye have been reported [1,2,3]. B3GLCT mutations result in classic PPS which is characterized by a triad of features including anterior segment defects (100%; Peters anomaly in 85%), short stature (100%), and brachydactyly (95%), as well as variable other highly penetrant features such as developmental delay (84%), congenital heart defects (40%), cleft lip/palate (37%) and other anomalies [3]. PPS-like cases (often clinically diagnosed as PPS) which demonstrate overlapping phenotypes but lack one or more of the key triad of features seen in classic PPS were found to not be caused by mutations in B3GLCT [2, 3, 16,17,18]
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