Haploinsufficiency for One COL3A1 Allele of Type III Procollagen Results in a Phenotype Similar to the Vascular Form of Ehlers-Danlos Syndrome, Ehlers-Danlos.

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Haploinsufficiency for One COL3A1 Allele of Type III Procollagen Results in a Phenotype Similar to the Vascular Form of Ehlers-Danlos Syndrome, Ehlers-Danlos Syndrome Type IV Ulrike Schwarze, Wouter I. Schievink, Elizabeth Petty, Michael R. Jaff, Dusica Babovic-Vuksanovic, Kenneth J. Cherry, Melanie Pepin, Peter H. Byers The American Journal of Human Genetics Volume 69, Issue 5, Pages 989-1001 (November 2001) DOI: 10.1086/324123 Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 1 SDS-PAGE of radiolabeled procollagens from medium and fibroblasts cell layer, analyzed under reducing conditions, from a control cell line (lanes C) and cell strains from four individuals with EDS type IV (P1–P4). In cells from P1 the diminution in the amount of proα1(III) is evident, but it is not clear in the other three individuals. FN = fibronectin. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 2 Demonstration of loss of expression of one COL3A1 allele in three of four individuals with EDS type IV. A, Heterozygosity for the exon 31 (2092G/A) polymorphism assayed as a SpeI site in genomic DNA (left) from P1 and P2. The cDNA from P1 contained only the SpeI(−) allele. In the cDNA from P2, only the SpeI (+) allele was stable. B, Amplification products for the AG insertion/deletion in the 3′UTR, separated on MDE gel. In genomic DNA from P3 (left lane) both alleles were detectable in equal amounts, whereas in cDNA (right lane) only the AG-insertion allele was present, which resulted in the disappearance of the heteroduplex (H). C, Heterozygosity for the exon 33 (2244C/T) polymorphism assayed as a HaeIII site in genomic DNA (left) from P4. In cDNA, after digestion with Fnu4HI, both alleles were present in P4, comparable to the heterozygous control. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 3 Amplification of COL3A1 exon 27 in genomic DNA from P1 and from a control individual. A, Apparent heteroduplex (H) in P1, owing to deletion of 2 bp in one COL3A1 allele. B, Nucleotide sequence in the normal (left four lanes) and mutant (right four lanes) alleles from P1. The two deleted nucleotides (AA) are in boldface in the letter code for the normal sequence. The consequence of the 2-bp deletion in P1 is a PTC (boxed triplet) immediately downstream of the deletion site. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 4 COL3A1 exon 4/5 genomic sequence around the 1-bp deletion (underlined) in P2. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 5 A, Amplification of COL3A1exons 6–13 in cDNA of a control individual and in P3. Complementary DNA was derived from either total cellular RNA, nuclear RNA, or nuclear RNA, after treatment with CHX (100 μg/ml). When nuclear RNA was used, in P3 a heteroduplex (H) became visible that was barely detectable in total RNA and that was not enhanced by treatment with CHX. B, Exon 7 genomic sequence around the 1-bp deletion (underlined) in P3, obtained by an antisense sequencing primer located in intron 7. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 6 Comparison of RNA stability in pre-mRNA and in nuclear and cytoplasmic RNA from P1. The two COL3A1 alleles were distinguishable after restriction digestion with AluI, owing to heterozygosity for 2092G/A. The AluI(−):AluI (+) allele ratio in pre-mRNA from P1 was similar to that seen in the heterozygous control. In nuclear (lanes Nu) RNA the AluI(−) allele was markedly reduced, and in cytoplasmic (lanes Cp) RNA it was barely detectable. The treatment of cells with CHX prior to RNA isolation resulted in increased appearance of the AluI(−) allele in both cellular compartments. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 7 Heterozygosity for a nonsense mutation identified in P4, in exon 52 of COL3A1. A, Sequence around the site of the mutation obtained by an antisense sequencing primer. The substitution of T for C resulted in the conversion of a codon for arginine (CGA) to a PTC (TGA). B, Restriction digestion, with SspI, of genomic-amplification products from a control individual and from P4. The mutation shown in panel A created a recognition sequence for SspI. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 8 Pulse-chase analysis of procollagen assembly. Cells from a control and from P4 were pulse-labeled, and the cell-layer proteins were harvested either immediately or after differing periods of chase and were separated by SDS-PAGE under nonreducing conditions. In control cells, proα1(III) chain monomers were detectable only at times 0 min and 20 min. In P4, proα1(III) chain monomers with increasingly delayed electrophoretic mobility were detectable throughout the entire chase period, whereas the proα1(I) chains that migrated directly below the proα1(III) chains had completely disappeared at 60 min, in both the control and P4, owing to incorporation into dimers and trimers of type I procollagen, which are denoted by “D(I)” and “T(I),” respectively]. FN = fibronectin. The American Journal of Human Genetics 2001 69, 989-1001DOI: (10.1086/324123) Copyright © 2001 The American Society of Human Genetics Terms and Conditions