To understand the molecular basis of hemophilia A and to provide heterozygote detection and prenatal diagnosis by DNA analysis, we used cloned factor VIII:C DNA fragments to study 10 affected families. In four of these families, inhibitors of factor VIII:C had developed in affected persons. In one such family a deletion of approximately 80 kb within the factor VIII:C gene was identified. Carriers of the deletion were identified through detection of an abnormal DNA fragment located at the deletion end points. In another family a single nucleotide change in the coding region of the factor VIII:C gene produced a nonsense codon leading to premature termination of factor VIII:C synthesis. Carrier detection was performed in eight female members of this four-generation family. In a third family a small change in the size of a restriction-endonuclease fragment correlated with the presence of the mutant gene, and in the other seven families the molecular defect has not yet been identified. In addition, we used two common polymorphic sites in the factor VIII:C gene to differentiate the normal from the defective gene in four of six obligate female carriers from families with patients in whom inhibitors did not develop. Carrier detection was possible in other members of these families. These data suggest that DNA analysis of the factor VIII:C gene provides an accurate method of carrier detection and, potentially, of prenatal diagnosis in at least 50 per cent of the pedigrees affected by hemophilia A.

Gyrate atrophy of the choroid and retina (GA) is an inherited chorioretinal degeneration caused by deficiency of ornithine 8-aminotransferase (OAT; L-ornithine: 2-oxo-acid aminotransferase; EC 2.6.1.13). GA is one of the "Finnish genetic diseases," a group of several rare monogenic disorders that occur with increased frequency in the Finnish population. Using a combination of RNase A protection, genomic cloning, and polymerase chain reaction amplification of genomic DNA, we found one of two missense mutant OAT alleles to be present in each of 16 Finnish GA pedigrees. The first mutation R180T, in which arginine-180 is replaced by threonine, was present in homozygous form in patients from two pedigrees. The second mutation L402P, in which leucine-402 is replaced by proline, was present in homozygous form in patients from 14 pedigrees. Neither mutation was present in 19 Finnish controls. L402P was not present in 18 non-Finnish GA patients but R180T was found in an American GA patient. We constructed full-length mutant cDNAs by amplifying patient cDNA with the polymerase chain reaction and cloning a restriction fragment containing the mutation into an otherwise normal human OAT cDNA. These mutant cDNAs were then expressed in CHO-K1 cells, which lack endogenous OAT. Both R180T and L402P inactivate OAT. These results show molecular heterogeneity in GA alleles even in the Finnish population.Ornithine 8-aminotransferase (OAT; L-ornithine:2-oxo-acid aminotransferase; EC 2.6.1.13) is a mitochondrial matrix enzyme that catalyzes the reversible transamination of ornithine to Al-pyrroline 5-carboxylate (1). Deficiency of OAT in man causes the autosomal recessive chorioretinal degeneration, gyrate atrophy of the choroid and retina (GA). To study the cell biology of OAT and the mutations causing GA, we have cloned and sequenced a near full-length human liver OAT cDNA and have determined the structure of the human OAT structural gene, which has 11 exons and spans 22 kilobases (kb) (2). We (2, 3) and others (4, 5) have shown that the OAT structural gene is located on chromosome 10 and that there is a group of nonfunctional OAT-related sequences on the X chromosome.Three missense mutations of OAT causing GA have been reported. The first mutation, in which the initiation methionine was replaced with an isoleucine, was found in affected members of two unrelated pedigrees of Lebanese Maronite descent (6). GA probands from 17 pedigrees of a variety of other ethnic backgrounds, including Finnish, were shown to lack this mutation (6). The second mutation was the Val-332 -* Met change found in a pyridoxine-responsive patient, and the third was the Asn-54 --Lys change (7). The ethnic background of the latter two patients was not provided.The Finnish population, which expanded from -250,000 in 1700 A.D. to -5 million at present, has been isolated genetically by geographical, linguistic, and cultural barriers (8). For these reasons, the variety and frequency of monogenic disorders in Finns is quite different from other European pop...