Premature Termination Mutations in FBN1: Distinct Effects on Differential Allelic Expression and on Protein and Clinical Phenotypes Iris Schrijver, Wanguo.

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Premature Termination Mutations in FBN1: Distinct Effects on Differential Allelic Expression and on Protein and Clinical Phenotypes Iris Schrijver, Wanguo Liu, Raanan Odom, Thomas Brenn, Peter Oefner, Heinz Furthmayr, Uta Francke The American Journal of Human Genetics Volume 71, Issue 2, Pages 223-237 (August 2002) DOI: 10.1086/341581 Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 1 Effect of splice-site mutation 8051+1G→A on the FBN1 transcript. Direct sequencing of RT-PCR products from fibroblast of a normal control (top) and proband FB1477 with 8051+1G→A (bottom) reveals skipping of exon 63. This event leads to deletion of the two most C-terminal cbEGF-like domains and to a frameshift, resulting in premature termination of translation at amino acid position 2751 in exon 65 in the unique C-terminal region. The American Journal of Human Genetics 2002 71, 223-237DOI: (10.1086/341581) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 2 Detection of allele-specific transcripts. Top, Mutation 3778G→T abolishes a BpmI site. Top left, Unlabeled RT-PCR of a region spanning nts 3506–3996 of the FBN1 cDNA sequence, digested with BpmI. Digestion products from both the proband (P) and a normal control (C) are shown. Top right,32P-labeled RT-PCR and digestion with BpmI of proband cDNA. Both digested (D) and undigested (U) products are shown. Middle, Mutation 6185insA generates new MseI site. Middle left,32P-labeled RT-PCR and digestion with MseI. Undigested (U) and digested (D) samples from proband are shown. Middle right, Genomic PCR of exon 50 from proband and digestion with MseI. Bottom, Mutation 7240C→T abolishes BslI site. Bottom left,32P labeled RT-PCR and digestion with BslI. Undigested (U) and digested (D) samples from proband are shown. Bottom right,32P labeled genomic PCR of exon 58 from proband and digestion with BslI. The American Journal of Human Genetics 2002 71, 223-237DOI: (10.1086/341581) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 3 Comparison of transcript levels for FBN1 alleles distinguished by the C6236T RsaI polymorphism in the 3′ UTR. All samples are from heterozygotes. RT-PCR products from fibroblast RNA samples were digested with RsaI and electrophoresed on an SSCA gel under denaturing conditions. The band labeled “T” represents the uncut T allele, the two bands labeled “C” represent the cut C allele, and the band marked by an asterisk (*) is constant. Samples 3 and 12 are normal controls. Unequal transcript levels are evident in four samples with PTC mutations. Lane 1, FB 1058 (E1260X); lane 4, FB 1103 (L1161X); lane 6, FB773 (R1192X); and lane 11, FB997 (R1523X). Samples 1 and 11 reveal predominant transcripts with the C allele, whereas in samples 4 and 6 only the T allele is detectable. In contrast, the sample in lane 2 is from FB774, with a genomic deletion of exons 42 and 43 (Liu et al. 2001), and the one in lane 8 is from FB808, with the cysteine substitution C637R (Schrijver et al. 1999). Both of these samples, as well as the ones in lanes 5, 9, and 10, generated the normal pattern of fragments, indicating equal transcripts of both alleles. Lane 7 shows slight skewing towards the C allele. For samples 5, 7, 9, and 10, no FBN1 mutations have been identified as yet. The American Journal of Human Genetics 2002 71, 223-237DOI: (10.1086/341581) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 4 Reduced fibrillin 1 synthesis and deposition in a proband with a premature termination mutation in the FBN1 gene. Radioactively labeled fibrillin (arrows) is visualized after pulse-chase analysis of cultured skin fibroblasts from a normal control subject and FB1533 (5240insACACT, leading to a PTC in exon 46). After a 30-min pulse with 35S-cysteine, FB1533 demonstrates reduced synthesis of normal-sized fibrillin (34% of the normal control sample). Protein deposition is assessed after a 20-h “chase” period in which labeled fibrillin is secreted from the cells and accumulates in the extracellular matrix. Matrix deposition is reduced in FB1533. At 20% of the normal control level, it is disproportionately low, suggesting a dominant-negative mechanism. These results place FB1533 into protein group II (Aoyama et al. 1994). The American Journal of Human Genetics 2002 71, 223-237DOI: (10.1086/341581) Copyright © 2002 The American Society of Human Genetics Terms and Conditions

Figure 5 Frequency of connective-tissue clinical features in study participants with PTC mutations, compared with those with cysteine substitutions (Schrijver et al. 1999). The percentage of all participants who are positive for the studied skeletal, ocular, cardiovascular, and other phenotypes in each of the two groups is demonstrated along the Y-axis. Individual manifestations are listed along the X-axis. Blackened columns represent PTC mutations, and shaded columns reflect cysteine substitutions. DCS = dolichostenomelia; PE/PC = pectus excavatum/pectus carinatum; SC = scoliosis; H/NP = high/narrow palate; DC = dental crowding; CTR = contractures; HSJ = hypermobile small joints; HLJ = hypermobile large joints; WS = wrist sign; TS = thumb sign; ARD = arachnodactyly; EL = ectopia lentis; RD = retinal detachment; AAA = ascending aortic aneurysm; AD = aortic dissection; ARR = aortic root replacement; MVP = mitral valve prolapse; MR = mitral regurgitation; and PMT = pneumothorax. Differences for HLJ, EL, and RD were statistically significant. The American Journal of Human Genetics 2002 71, 223-237DOI: (10.1086/341581) Copyright © 2002 The American Society of Human Genetics Terms and Conditions