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Hemophilia B is due to multiple molecular defects in the factor IX gene. Over 80% of mutations are single base substitutions. By amplification and direct sequencing, 51 single base substitutions were found in the transcribed sequence of the factor IX genes of patients from 50 distinct families with hemophilia B. These include 30 mutations in 29 families not previously reported by us; of these, 12 are novel, i.e., not previously published in other series. Of the 51 substitutions in our overall series 23 (45%) occurred as C-to-T or G-to-A transitions at 11 sites within CG dinucleotides. It is estimated that CG transitions occur from one to two orders of magnitude more frequently than mutations in nucleotides that are not within a CG pair. More than one family had identical defects for 6 of the CG mutations. At 4 of these sites, most patients had different haplotypes compatible with distinct mutations. Non-CG-type mutations occurred throughout the coding regions with only one mutation in more than one family. The latter included 7 families with a 397 Ile-to-Thr defect that all share a rare haplotype, suggesting a common ancestor.

We have evaluated the structure of the CGG repeat within the FMR1 gene of an Asian population and found the most common size of the repeat to be 29 and 30 with a minor population of 36 repeats. We have isolated and sequenced DNA containing the 36 repeats and found the basis sequence to be (CGG)9AGG(CGG)9AGG-(CGG)6AGG(CGG)9; with a (CGG)6)AGG insertion, designated as 9A9A6A9. Of 144 Asian chromosomes, 11 (8%) had sequences with this insertion. Six different variations of the basic sequence were observed in the population: 9A9A6A2A9, 9A9A6A11, 9A9A16, 9A9A15, 8A9A6A6A9, and 11A6A6A9. All but one of the chromosomes with the insertion had the haplotype of DXS548/ FRAXAC1: 194/D suggesting that the sequences with the 6A insertion arose from a single ancestral allele. We have not observed the insertion in the FMR1 gene of Caucasians or Native Americans. The (CGG)6AGG insertion may be unique to Asians.

The gene frequencies of ADH 2 2 and ALDH 2 2 were lower in Tibetan and Mongolian populations than in Vietnamese, Han Chinese, and three Chinese minority populations.

The germ line of origin for 13 of 14 de novo hemophilia B mutations has been determined. When added to previous reports, the origin, assuming no mosaicism, occurred in 43 female and 33 male gametes. Mutation rate estimates are twofold higher in males than in females. The pooled data also indicate that male and female germ lines have different mutation rates depending upon the type of mutation.

The frequencies of the alleles encoding isozymes of alcohol dehydrogenase and aldehyde dehydrogenase were low in Northwest Coast Amerindians compared to Chinese subjects.

Direct sequencing of the regions of the factor IX gene of likely functional significance was performed in four patients with severe hemophilia B. In two of the individuals, a transition at the dinucleotide CpG caused a nonsense mutation at arginine 333. In the other two individuals, a transition at CpG caused a nonsense mutation at arginine 29. Since these patients are all unrelated, as shown by differing alleles of the TaqI polymorphism in intron four or extensive nonoverlapping pedigrees, the mutations arose independently. In addition, the origin of one arginine 333 mutation in one family has been traced to the germline of the maternal grandfather. The frequent occurrence of mutations at arginine codons that contain the sequence CGN can be explained by the dramatic elevation of transitions at CpG. As a result, approximately one in four individuals with hemophilia B is expected to have a mutation at arginine and nonsense mutations at one of six arginine residues should be common causes of severe hemophilia.

We have localized the position of the MspI polymorphic site that exists within the factor IX gene. The location of the MspI polymorphic site is within intron D, 1.9 kb upstream from the beginning of exon V and 4 kb downstream from a known polymorphic TaqI site. The use of a specific genomic probe simplifies the interpretation of the MspI polymorphism by reducing the number of non-overlaping DNA fragments to three bands; 2.4, 3.4, and 5.8 kb.

Phenotypes of five polymorphic enzymes: red cell acid phosphatase, phosphoglucomutase, esterase D, adenosine deaminase, and 6-phosphogluconate dehydrogenase were determined in extracts of 24 amniotic fluid cell cultures and in the corresponding maternal red cells. Twenty-one of the 24 fetus/mother pairs can be distinguished by at least one of the markers. Thus, polymorphic enzyme markers may be useful in affirmation of fetal origin of cultured cells and to avoid possible diagnostic errors.

The polymerase chain reaction (PCR) method was used to amplify a 356-bp DNA segment containing the suspected BamHI polymorphic site of the factor IX gene. Following the enzyme digestions and gel electrophoresis, polymorphic genotypes (-,+ and +/- types) were observed. The gene frequencies for the rare (+) allele are about 36% in blacks and 2% in Caucasians. The 356-bp DNA was further purified and sequenced. The sequencing gels revealed a single nucleotide substitution (T to G) at position -561 of the gene in blacks and Caucasians. The T-to-G transversion generated a new BamHI site (GGATCC,+type) from a nonenzymatic site, (TGATCC,-type).

Two probes, ALDH1 and ALDH2, for restriction fragment length polymorphisms (RFLPs) are described, with notes on their locations and the RFLPs found with them.