Exon Skipping in IVD RNA Processing in Isovaleric Acidemia Caused by Point Mutations in the Coding Region of the IVD Gene Jerry Vockley, Peter K. Rogan,

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Exon Skipping in IVD RNA Processing in Isovaleric Acidemia Caused by Point Mutations in the Coding Region of the IVD Gene Jerry Vockley, Peter K. Rogan, Bambi D. Anderson, Jan Willard, Ratnam S. Seelan, David I. Smith, Wanguo Liu The American Journal of Human Genetics Volume 66, Issue 2, Pages 356-367 (February 2000) DOI: 10.1086/302751 Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 1 Summary of mutations in the IVD gene leading to abnormal splicing of IVD RNA. IVD sequences, including intron and exon boundaries, were amplified from genomic DNA isolated from cell lines from patients with IVA and shown to be deficient in IVD enzymatic activity. Mutations identified are listed. For FB102, the effect of the intron 7 mutation on cDNA sequence is also shown. FB118 and FB145 were homozygous for their respective mutations. Only one mutation could be identified in FB143, despite complete sequencing of all exons and extensive sequencing of the intron/exon boundaries. The remaining mutation in the IVD gene responsible for this patient’s IVA is unknown. The American Journal of Human Genetics 2000 66, 356-367DOI: (10.1086/302751) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 2 Sequencing of DNA fragments amplified from IVD genomic DNA from IVA fibroblasts. A, Direct sequence of amplified genomic DNA from FB834, showing a 148C→T mutation. The patient is heterozygous at this position. B, 149G→C mutation in FB118 in products amplified from genomic DNA and sequenced directly. The patient is homozygous for this mutation, which was also identified in FB136. Control experiments confirmed that this was not related to contamination of PCR reactions with previously amplified template. C, Insertion of a G residue in genomic DNA from FB136 in an area that normally contains six Gs from cDNA positions 865–870. Direct sequence from amplified genomic DNA is shown. Because the patient is heterozygous for this mutation, a double sequencing pattern appears after the inserted nucleotide. This finding was confirmed in the reverse direction and by sequencing of subclones of the PCR amplified genomic DNA from this region (not shown). D, G→A point mutation of the intron 7 splice acceptor site in FB102 genomic DNA, identified by direct sequencing of amplified DNA. The patient is heterozygous at this location. The mutant A allele is easily visualized in the forward direction shown, whereas the wild-type G signal is more evident in the reverse direction (not shown). The American Journal of Human Genetics 2000 66, 356-367DOI: (10.1086/302751) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 3 Identification of exon skipping in mRNA from IVA fibroblasts. A, Agarose gel of DNA fragments amplified from cDNA from FB118. The 5′ amplification primer was in the 5′ untranslated region of IVD (position −20 to −1) and the 3′ amplification primer was from position 265–286, spanning exons 1–3. Arrows mark the predicted size of a normal fragment (306 bp) and one with a deletion of exon 2 (90 bp). The migration positions of 100 bp molecular mass standards are shown. B, Manual DNA sequencing of a subcloned DNA fragment amplified from FB102 cDNA, including exons 7 and 8. The arrows show the position of the g→a mutation in the intron 7 splice acceptor site. Sequences corresponding to exon 7, intron 7, and exon 8 are marked. C and D, Analysis of subclones of amplified products from FB136 cDNA, spanning exons 3–11 (5′ primer, IVD coding nucleotides 318–347; 3′ primer, IVD coding nucleotides 1122–1138; anticipated size 820 bp). Two different-sized inserts are obtained after subcloning, as seen on agarose gel electrophoresis (C). The sequence of the insert from lane 1 is normal (not shown). The sequence of the insert from lane 2 shows a deletion of exon 8 (D). The American Journal of Human Genetics 2000 66, 356-367DOI: (10.1086/302751) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 4 Use of an exon-trapping system to examine the effects of IVD exon mutations on RNA splicing. IVD genomic DNA fragments, containing control or mutation sequences, spanning exon 1 to intron 3 (A–D) or intron 7 to intron 9 (E–F) were cloned into the exon-trapping vector pSPL3. Inserts for all clones were sequenced to confirm that no PCR errors were present. The vector was transiently transfected into COS-7 cells, and total RNA from transfected cells was used as template for RT-PCR, with vector-specific primers. Amplified products were separated by electrophoresis on agarose gels and visualized with ethidium bromide staining. The number of products listed below the vector maps correspond to the numbering of the bands on the gel photo. Lanes containing PCR products amplified from cDNA of cells transfected with control and mutant IVD sequences are marked as “C” and “M,” respectively. The mutation being analyzed is listed underneath, and the migration of molecular mass markers is indicated to the left of each gel photograph. A, 148C→T; B, 149G→C; C, 205G→A. D and F, Map of amplified products identified by sequencing subcloned products obtained with normal and mutant IVD exon-trapping experiments. The test IVD exon-trapping vector plus insert is depicted on the top line. IVD exons are shown as white boxes and exon sizes are as labeled. Vector exon sequences (V) are denoted by black boxes and intron sequences by a line. Donor and acceptor sites are indicated by D and A, respectively. The horizontal arrows indicate the position of PCR primers. D and F, Exon 2 splicing test vector. Cryptic acceptor site in vector is indicated by “a.”E, iG870 = insertion of G at position 870 in the IVD coding region. F, Exon 8 splicing test vector. The American Journal of Human Genetics 2000 66, 356-367DOI: (10.1086/302751) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 5 Walker diagrams of IVD exon 2 splice acceptor sites. Splice sites are shown by walkers (Schneider 1997b), in which the height of each letter is the contribution of that base to the total conservation of the site (in bits). The upper bound of the vertical rectangles is at +2 bits, and their lower bound is at −3 bits. Letters that are upside down represent negative contributions. The top panel depicts the natural splice site one nucleotide upstream of exon 2 and a cryptic acceptor site at position 153. Neither cryptic site is used. The vertical arrows indicate the locations of mutations in the sequence. The middle panel corresponds to the G→C change at position 149, and the lower panel to C→T at position 148. The mutations alter the Ri value of the cryptic acceptor at position 153. The American Journal of Human Genetics 2000 66, 356-367DOI: (10.1086/302751) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 6 Polymorphic repeats in intron 1 of the IVD gene. A, Sequence of three, two, and one repeat alleles in intron 1, beginning 25 bp from the junction of exon 1 and intron 1. B, Amplification of intron 1 repeats from fibroblast cDNA. Fragments corresponding to each number of repeats are shown. The American Journal of Human Genetics 2000 66, 356-367DOI: (10.1086/302751) Copyright © 2000 The American Society of Human Genetics Terms and Conditions