Additional file 1: Table S1. of Case report of homozygous deletion involving the first coding exons of GCNT2 isoforms A and B and part of the upstream region of TFAP2A in congenital cataract

Summary of PCR/qPCR reactions and copy number status in the affected family. (DOCX 21 kb

Exonic structure, genomic context and multiple species alignment of B3GLCT/b3glct.

<p>(A) The two zebrafish orthologs of B3GLCT show overall similar exonic arrangement. The number of each exon is located within each box and the size of the exon (in base pairs) is shown above each exon. The 5’ and 3’ UTRs are indicated preceding the first ATG and following the stop codon (TAA/TAG). White indicates the N-terminal signal sequence, light grey indicates the stem region and dark grey indicates the catalytic domain. The vertical black bar in exon 12 of each gene indicates the location of nucleotides encoding for the catalytic tri-aspartic acid residues. Horizontal lines underneath the zebrafish genes indicate previously annotated sequence and sequence identified in this study. (B) Schematic of genomic context for B3GLCT/b3glct. (C) Multiple species alignment of B3GLCT orthologs from human (NP_919299), mouse (NP_001074673), Xenopus (NP_001072551), and zebrafish. Blue bar indicates signal peptide, green indicates stem region and orange indicates catalytic core. Grey shading of amino acids indicates conservation. The DxD motif is boxed in red.</p

Embryonic expression of zebrafish <i>b3glct</i> genes.

<p>(A) RT-PCR analysis of <i>b3glct</i> expression demonstrates robust expression of both <i>b3glcta</i> (left panel) and <i>b3glctb</i> (middle panel) at different stages of development in whole embryos as well as various embryonic tissues at 48-hpf (right panel). Controls included <i>pitx2c</i> as negative control for 0-hpf, <i>rhodopsin</i> as negative control for the lens, <i>beta-actin</i> as positive control for all tissues and H₂O as negative contamination control for all reactions. (B) In-situ hybridization analysis of <i>b3glcta</i> and <i>b3glctb</i> expression demonstrates broad expression in 24-120-hpf embryos with enrichment in the developing eyes, fins, brain, craniofacial region and somites. aer–apical ectodermal ridge, ase–anterior segment of the eye, b–brain, cmz–ciliary marginal zone, crc–craniofacial cartilage, e–eye, f–fins, h–heart, le–lens, sm–skeletal muscles.</p

Summary of differentially regulated genes implicated in ER quality control, unfolded protein response or cell survival.

<p>Summary of differentially regulated genes implicated in ER quality control, unfolded protein response or cell survival.</p

Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase <i>B3GLCT</i> gene mutated in Peters Plus Syndrome

<div><p>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 <i>B3GLCT</i> gene encoding for a β3-glucosyltransferase that catalyzes the attachment of glucose via a β1–3 glycosidic linkage to <i>O</i>-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 <i>B3GLCT</i> gene, <i>b3glcta</i> and <i>b3glctb</i>. The <i>b3glcta</i> and <i>b3glctb</i> 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. <i>In vitro</i> glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an <i>O</i>-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double <i>b3glct</i> knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous <i>b3glct</i>^<i>-/-</i> embryos demonstrated complete loss of <i>in vitro</i> b3glct activity. Surprisingly, <i>b3glct</i>^<i>-/-</i> homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of <i>b3glct</i>^<i>-/-</i> 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 <i>B3GLCT/b3glct</i> genes is conserved in vertebrates. At the same time, complete <i>b3glct</i> deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.</p></div