Usher Syndrome Type III: Revised Genomic Structure of the USH3 Gene and Identification of Novel Mutations Randall R. Fields, Guimei Zhou, Dali Huang,

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Usher Syndrome Type III: Revised Genomic Structure of the USH3 Gene and Identification of Novel Mutations Randall R. Fields, Guimei Zhou, Dali Huang, Jack R. Davis, Claes Möller, Samuel G. Jacobson, William J. Kimberling, Janos Sumegi The American Journal of Human Genetics Volume 71, Issue 3, Pages 607-617 (September 2002) DOI: 10.1086/342098 Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 1 PCR analysis of the USH3Joensuu transcript. Primers based on the published sequence of USH3Joensuu were used to amplify DNA fragments between the first and second (e1f:e2r), first and third (e1f:e3r), first and fourth (e1f:e4r), second and third (e2f:e3r), and second and fourth (e2f:e4r) exons, with 35 cycles of 30 s at 96°C, 30 s at 58°C, and 4 min at 72°C. Marathon Ready human retina cDNA at two times the recommended concentration from Clontech (lane a) and human genomic DNA at 5 ng/μL (lane b) were used as template. Only the primer pair e2f:e3r produced a robust product of the expected size on retina-specific cDNA. Faint bands are visible in the cDNA amplifications for e1f:e2r and e1f:e4r, at approximately the expected sizes but at orders of magnitude below the intensity of e2f:e3r. Expected sizes for USH3Joensuu are 412, 574, 780, 186, and 392 bp for lane a, with an additional 87 nucleotides between exons 1 and 2 in the case of isoform bJoensuu. Evidence for an additional splice variant was observed in lane a, for e2f:e4r, and reamplification followed by sequencing showed that the band at about 165 bp represents a direct splice between exons 2 and 4, skipping exon 3. The American Journal of Human Genetics 2002 71, 607-617DOI: (10.1086/342098) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 2 Structure of the USH3revised gene (AF482697) and protein. A, Schematic structure of the USH3revised gene and its mRNA and protein products. The TMPred program predicted two inside-outside (green) and two outside-inside (red) TM domains, arranged in tandems in the USH3 protein (232 aa). There may also be a signal peptide (SignalP Server prediction; data not shown), which, if removed, could result in a mature protein with three TM domains, 204 aa in length. This signal peptide is grey in this figure, including the first inside-outside TM domain. No other previously characterized conserved domains were identified. B, PCR analysis of the transcript, performed using primers based on the sequence of USH3revised cDNA. The expected sizes of the amplified products were E1F/E2R, 337 bp; E1F/E3R, 499 bp; E2F/E3R, 187 bp; and E1F/ER3, 912 bp. M = molecular weight marker (Invitrogen catalogue number 15628019). The American Journal of Human Genetics 2002 71, 607-617DOI: (10.1086/342098) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 3 Alignment of mouse EST and Ensembl-Arachne data used to predict the mouse USH3revised orthologue. The original alignment was accomplished using DNASTAR's Seqman program in the Lasergene version 5.0 software package, and formatting was provided by Multalin (Corpet et al. 1988), with default settings and manual adjustment. The American Journal of Human Genetics 2002 71, 607-617DOI: (10.1086/342098) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 3 Alignment of mouse EST and Ensembl-Arachne data used to predict the mouse USH3revised orthologue. The original alignment was accomplished using DNASTAR's Seqman program in the Lasergene version 5.0 software package, and formatting was provided by Multalin (Corpet et al. 1988), with default settings and manual adjustment. The American Journal of Human Genetics 2002 71, 607-617DOI: (10.1086/342098) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 4 Alignment of human, mouse, and rat USH3revised proteins and two human USH3revised protein paralogues. “X” signifies sites of missense mutations found in the present study; a red background indicates identity of 3/5 residues at that position; blue indicates similarity of 3/5. Alignment of a putative USH3-like gene from human chromosomes 4 and 10 and the putative USH3 orthologues from rat and mouse show that Leu150 can be replaced by isoleucine but is otherwise conserved. Secondary-structure predictions indicate that the L150P mutation would disrupt a helical region (DNAStar's Protean program in the Lasergene version 5.0 software package; data not shown). The alignment also shows that Asn48 is conserved, except in the putative USH3-like gene on human chromosome 4, where it is replaced by an aspartate. Lysine, with its longer alkyl chain and lack of a carboxyl moiety, would be a radical substitution (N48K) in this region, which is predicted to form an extended loop. The original alignment was accomplished using DNAStar's Megalign program in the Lasergene version 5.0 software package; formatting was provided by The Sequence Manipulation Suite: Multiple Align Show. The American Journal of Human Genetics 2002 71, 607-617DOI: (10.1086/342098) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 5 Expression profile of USH3revised. Single-stranded cDNA from the indicated tissues—with primer pair E1F:E2R, as in figure 2—was used to amplify a 337-bp product corresponding to the first and second exons of USH3revised. The experiment was performed three times for the Clontech MTC panel 2, and one representative result is shown. M = molecular weight marker (Invitrogen catalogue number 15628019). The American Journal of Human Genetics 2002 71, 607-617DOI: (10.1086/342098) Copyright © 2002 The American Society of Human Genetics Terms and Conditions