JOURNAL OF BONE AND MINERAL RESEARCH Volume 9, Number 6, 1994 Mary Ann Liebert, Inc., Publishers zyxwvutsrqp Mutation Analysis of Coding Sequences for Type I Procollagen in Individuals with Low Bone Density LORETTA D. SPOTILA, ALAIN COLIGElV2 LARISA SEREDA,' CONSTANTINOS D. CONSTANTINOU-DELTAS, MICHAEL P. WHYTE,4 B. LAWRENCE RIGGS,' JOSEPH L. SHAKER,6 TIMOTHY D. SPECTOR,' ERIC HUME,' NANCY OLSEN,* MAURICE IITTIE,~ ALAN TENENHOUSE," ELIZABETH SHANE," WALTER BRINEY," and DARWIN J. PROCKOP' ABSTRACT Mutations in one of the two genes encoding type I procollagen (COLlAl and COLlA2) are frequently the cause of osteogenesisimperfecta (01), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of 01 was considered by the clinician in some cases, the clinical criteria for 01 were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COLlAl and COLlA2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position a2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88:5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at a,(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon a2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COLlA2 gene, we determined that all patients expressed both alleles of the COLlA2 gene. The 12 patients who were heterozygous for a COLlAl neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal addition;d mutations in the COLlAl or COLlA2 genes. 'Department of Biochemistry and Molecular Biology, Jefferson Medical College, Philadelphia, Pennsylvania. *Present address: Centre Hospitalier Universitaire zyxwvutsr tle Liege, Litge, Belgium. 3Present address: Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus. 4Shriners Hospital for Crippled Children, St. Louis, Missouri. 5Mayo Clinic, Rochester, Minnesota. zyxwvutsrqp 'St. Luke's Medical Center, Milwaukee, Wisconsin. 'Medical College of St. Bartholomew's Hospital, London, England. 'Vanderbilt University, Nashville, Tennessee. 'University of Pennsylvania, Philadelphia, Pennsylvania. Deceased. "Montreal General Hospital, Montreal, Quebec, Canada. "Columbia University College of Physicians and Surgeons, New York, New York. "Denver Arthritis Clinic, Denver, Colorado. 923