Efficient Detection of Alport Syndrome COL4A5 Mutations With Multiplex Genomic PCR-SSCP David F. Barker, 1 * Joyce C. Denison, 1 Curtis L. Atkin, 2,3† and Martin C. Gregory 3 1 Department of Physiology, the University of Utah Health Sciences Center, Salt Lake City, Utah 2 Department of Biochemistry, the University of Utah Health Sciences Center, Salt Lake City, Utah 3 Department of Medicine, the University of Utah Health Sciences Center, Salt Lake City, Utah We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer condition and PCR profile. Application of the method to a consecutive series of 46 United States pa- tients with diverse indications of Alport syndrome resulted in detection of muta- tions in 31 cases and of five previously un- reported polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sensitivity is greater than 79%. The test examines 52 segments, in- cluding the COL4A6/COL4A5 intergenic pro- moter region, all 51 of the previously recog- nized exons and two newly detected exons between exons 41 and 42 that encode an al- ternatively spliced mRNA segment. New ge- nomic sequence information was generated and used to design primer pairs that span substantial intron sequences on each side of all 53 exons. For SSCP screening, 16 multi- plex PCR combinations (15 4-plex and 1 3-plex) were used to provide complete, par- tially redundant coverage of the gene. The selected combinations allow clear resolu- tion of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missense change in exon 32 but was found to cause exon skipping. An- other missense variant may mark a novel functional site located in the collagenous domain. © 2001 Wiley-Liss, Inc. KEY WORDS: COL4A5; multiplex PCR; SSCP; mutation detection; Alport syndrome INTRODUCTION Defects in the COL4A5 basement membrane (Type IV) collagen gene, located at Xq22, cause about 85% of Alport syndrome (AS), a glomerular membranopathy that progresses to end-stage renal disease (ESRD) and is usually associated with progressive hearing loss [Al- port, 1927; Gregory and Atkin, 1997; Kashtan, 1998]. The most deleterious COL4A5 mutations cause ESRD for males in their second or third decade, whereas milder variants may be associated with ESRD onset in middle age or later as well as tardive onset of hearing loss [Barker et al., 1996, 1997]. For female carriers of COL4A5 mutations, ESRD at ages similar to that oc- curring in male relatives is rare, but the lifetime inci- dence is about 15%. The general pattern of phenotypic expression in females is consistent with typical varia- tion in X-inactivation, although the role of X- inactivation has not been confirmed by experimental observation [Vetrie et al., 1992]. Nearly all COL4A5 defects are unique and a wide spectrum of mutation types have been presented in previous publications [Barker et al., 1990; Boye et al., 1995; Heiskari et al., 1996; Kawai et al., 1996; Knebelmann et al., 1996; Re- nieri et al., 1996; Plant et al., 1999]. Mutations in the COL4A3 or COL4A4 basement membrane collagen genes, located at 2q35–2q37, cause a recessive form of Alport syndrome with expression in homozygotes of ei- ther gender that is similar to that in males with COL4A5 mutations [Lemmink et al., 1994b; Mochizuki et al., 1994]. The recessive form is thought to account for about 15% of cases. Phenotypic expression in het- erozygous carriers of COL4A3 or COL4A4 mutations is variably present and may include mild hematuria, mild proteinuria, hearing loss and, in rare cases, late-age onset renal failure [Lemmink et al., 1996; Boye et al., 1998; Smeets et al., 1998] that may be construed as dominant expression in some instances [Jefferson et al., 1997]. All six Type IV collagen genes are large, with mRNAs of 6 to 10 kb, encoded by ∼50 exons that are dis- † Curtis L. Atkin is deceased. Grant sponsor: NIH; Grant numbers: DK43761, CA42014. *Correspondence to: David F. Barker, Department of Physiol- ogy, Room 156, 410 Chipeta Way, University of Utah Research Park, Salt Lake City, Utah 84108. E-mail: david.f.barker@m.cc.utah.edu Received 21 February 2000; Accepted 15 September 2000 Published online 29 December 2000 American Journal of Medical Genetics 98:148–160 (2001) © 2001 Wiley-Liss, Inc.