Detection and characterization of new mutations in the human angiotensinogen gene (AGT).

As part of the multicenter project entitled "Pathobiological Determinants of Atherosclerosis in Youth (PDAY)," we are testing polymorphisms in candidate genes of atherosclerosis and hypertension for associations with arterial lesions in autopsied young persons. In this study, we used temperature gradient gel electrophoresis (TGGE) to type the Met235-->Thr polymorphism in exon 2 of the angiotensinogen gene (AGT) that is associated with essential hypertension in some human populations. In addition to Met235-->Thr, we detected and sequenced four other TGGE variants in exon 2 of AGT. These included two new amino acid substitutions (Thr209-->Ile and Leu211-->Arg) that were found only among black PDAY cases. The frequency of the Ile209 mutation was 0.002 and the frequency of the Arg211 was 0.006 in 260 black PDAY cases. The other two TGGE variants were Tyr248-->Cys and a T-->C substitution at nucleotide position 171 that had been identified in previous studies. We also developed restriction isotyping for rapid typing of each AGT variant using PCR amplification and digestion with diagnostic restriction enzymes.

Baboon apolipoprotein A-IV. Identification of Lys76-->Glu that distinguishes two common isoforms and detection of length polymorphisms at the carboxyl terminus.

Various protein isoforms have been identified for human apolipoprotein A-IV (apoA-IV). However, investigations of their physiological effects have been limited because of low frequencies for many of the apoA-IV variants. Recent discovery of extensive variation in baboon apoA-IV using isoelectric focusing (IEF) makes this primate species an excellent model for genetic studies of apoA-IV. In this study, the molecular basis for net charge differences between two common apoA-IV isoforms (I and E) was determined by cloning and sequencing of intestinal cDNAs from homozygous baboons. An A-->G substitution was found in the third amphipathic repeat of the E isoform. This substitution causes a Lys-->Glu substitution at amino acid position 76 (Lys76-->Glu), adding two negative charges to the E isoform compared to the I isoform, consistent with their relative mobilities on IEF gels. Restriction isotyping was used to identify the substitution in leukocyte DNA from 15 baboons that had been typed by IEF, thus verifying Lys76-->Glu as the basis for the charge differences between the I and E isoforms. Physiological effects of the Lys76-->Glu substitution on high density lipoprotein-C levels were investigated in 431 baboons carrying the E and I isoforms. These studies revealed that the I isoform was associated with higher levels of high density lipoprotein-C on a high cholesterol, saturated fat diet (p = 0.04). The cDNA sequences showed that the carboxyl terminus of baboon apoA-IV contains a region of hydrophilic repeats (Glu-Gln-X-Gln) that is the largest yet found in any species (nine repeats compared to three to five repeats in human, mouse, and rat). A common length polymorphism was identified that inserts a single amino acid to form a five amino acid repeat. This is the first report of this type of length variation (insertion of a single amino acid rather than insertion of an entire repeat) in this region. In addition, a rare variant was found that inserts an entire four-amino-acid repeat, similar to the human apoA-IV-0 isoform.

The human apolipoprotein B 3' hypervariable region: detection of eight new alleles and comparisons of allele frequencies in blacks and whites.

We investigated common length polymorphisms in the hypervariable region located 3' to the human gene encoding apolipoprotein B (APOB 3' HVR) as part of the "Pathobiological Determinants of Atherosclerosis in Youth (PDAY)" study. PDAY is a multicenter study of young persons who died of external causes (accident, homicide, and suicide). The APOB 3' HVR contains multiple copies of AT-rich tandem repeats (15bp) called hypervariable elements (HVE). Using polymerase chain reaction (PCR) to amplify APOB sequences in hepatic DNA samples, we identified 22 different HVR alleles among 232 PDAY cases. In addition to 14 previously identified alleles, we detected 8 new alleles that had not been observed in population surveys. Of these new alleles, 7 were present only in black cases. We also examined distributions of HVR allele frequencies for blacks and whites. The frequency distributions for whites did not differ from those from previous studies of French populations (P = 0.3811) and Austrian populations (P = 0.1885). In contrast, the allele frequency distribution for blacks differed from whites (P < 0.001). Blacks had higher frequencies of smaller alleles (< or = 33 repeats) and larger alleles (> or = 37 repeats) than whites. We also sequenced specific HVR alleles to identify differences responsible for size variation. The most frequent alleles were identical in sequence to HVR alleles described in previous studies. However, one allele was not identical in sequence to an equivalent-sized allele from a previous study. In all likelihood, detection of sequence substitutions in the APOB 3' HVR would result in an even greater amount of allelic variability than detected by size differences alone.

Restriction isotyping of human apolipoprotein A-IV: rapid typing of known isoforms and detection of a new isoform that deletes a conserved repeat.

Genetic polymorphisms of apolipoprotein A-IV (apoA-IV) have been detected by isoelectric focusing of serum proteins. Because genetic variation in apoA-IV has significant effects on lipid risk factors, we used restriction enzyme isoform genotyping (restriction isotyping) to determine apoA-IV isoform genotypes at the DNA level for a large population (n = 509). In contrast to isoelectric focusing methods, restriction isotyping relies on nucleotide differences, enabling unambiguous typing of known isoforms and detection of new alleles that mimic other isoforms with shared charge properties. To determine genotypes for the common A-IV-1 isoform (Gln at aa position 360) and A-IV-2 isoform (360His), we used a mismatched primer for polymerase chain reaction (PCR) to introduce a restriction site (PvuII) that distinguishes each isoform. Using a portion of the same PCR reaction, we used HinfI to distinguish isoforms with Thr at position 347 (347Thr) versus Ser (347Ser). In surveys for these common genotypes, we detected heterozygotes for an allele with an insertion of 12 bp. Nucleotide sequencing showed that this allele is identical to the A-IV-0 isoform that inserts a hydrophilic repeat (Glu Gln Gln Gln) in a conserved region near the carboxy terminus. In addition, we discovered a new allele with a 12 bp deletion that removes a repeat (Glu Gln Gln Gln) from the same region. Nucleotide sequencing showed that this allele removes an acidic charge relative to A-IV-1, so we have named this isoform A-IV-2*. This isoform has not been discovered at the protein level, perhaps due to shared charge properties with A-IV-2 isoforms.