Allelic variation adjacent to the human insulin and apolipoprotein C-II genes in different ethnic groups

Summary We have used DNA probes for the human insulin gene and apolipoprotein C-II (apo C-II) gene to determine the extent of allelic variation in different ethnic groups. The distribution of an apo C-II DNA polymorphism revealed by the restriction endonuclease Taq I showed no significant variation amongst racial groups; in contrast, an insulin gene-related DNA polymorphism showed marked variability. In Japanese, Chinese, and Asian Indian groups there was an increased frequency of homozygosity for the class 1 allele compared to Caucasian groups (P<0.001, P<0.01, and P<0.05, respectively). In Caucasian, Japanese, Chinese, and Asian Indian groups no class 2 allele was observed; but in the Negroid populations (African and West Indian) the class 2 allele frequencies were 0.23 and 0.25 respectively. Possible reasons for this variation in allele distribution are considered in relation to disease associations.     

Dinucleotide repeat in the 3′ flanking region provides a clue to the molecular evolution of the Duffy gene

The Duffy blood group system consists of three alleles, FYA, FYB, and FY. To study the molecular evolution of the three alleles, we established the polymorphism of a dinucleotide (GT) repeat sequence (designated FyGT/ C) in the 3′ flanking region of the Duffy gene, and studied the relationship between FyGT/C and Duffy polymorphism in Japanese, people of African origin, and chimpanzee. By single-strand conformation polymorphism and sequence analysis, five and two alleles were identified in Japanese and Africans, respectively. In 110 random Japanese, the FyGT/C genotypes observed were in agreement with Hardy-Weinberg law. From the sequence of the chimpanzee Duffy gene, including both flanking regions, FYB was identified as the ancestral gene of the human alleles. The FyGT/C sequences associated with the FY allele of Africans were distinct from those of Duffy positives, whereas the FYB and FYA alleles shared common FyGT/C sequences. Thus, it is suggested that the first split took place between the FYB and FY alleles, and the second between the FYB and FYA alleles. Received: 25 July 1996 / Revised: 10 October 1996     

The Apolipoprotein E (APOE) Gene Appears Functionally Monomorphic in Chimpanzees (Pan troglodytes)

Background

The human apolipoprotein E (APOE) gene is polymorphic, with three primary alleles (E2, E3, E4) that differ at two key non-synonymous sites. These alleles are functionally different in how they bind to lipoproteins, and this genetic variation is associated with phenotypic variation for several medical traits, including cholesterol levels, cardiovascular health, Alzheimer’s disease risk, and longevity. The relative frequencies of these alleles vary across human populations, and the evolution and maintenance of this diversity is much debated. Previous studies comparing human and chimpanzee APOE sequences found that the chimpanzee sequence is most similar to the human E4 allele, although the resulting chimpanzee protein might function like the protein coded for by the human E3 allele. However, these studies have used sequence data from a single chimpanzee and do not consider whether chimpanzees, like humans, show intra-specific and subspecific variation at this locus.

Methodology and Principal Findings

To examine potential intraspecific variation, we sequenced the APOE gene of 32 chimpanzees. This sample included 20 captive individuals representing the western subspecies (P. troglodytes verus) and 12 wild individuals representing the eastern subspecies (P. t. schweinfurthii). Variation in our resulting sequences was limited to one non-coding, intronic SNP, which showed fixed differences between the two subspecies. We also compared APOE sequences for all available ape genera and fossil hominins. The bonobo APOE protein is identical to that of the chimpanzee, and the Denisovan APOE exhibits all four human-specific, non-synonymous changes and appears functionally similar to the human E4 allele.

Conclusions

We found no coding variation within and between chimpanzee populations, suggesting that the maintenance of functionally diverse APOE polymorphisms is a unique feature of human evolution.     

The human preproapolipoprotein C-II gene. Complete nucleic acid sequence and genomic organization

The complete nucleic acid sequence of human preproapolipoprotein (apo) C-II has been determined from 2 apoC-II clones isolated from 2 different human genomic DNA libraries. The cloned fragments were approx. 14 and 18 kb long, and sequence analysis established that the apoC-II gene consists of 3338 nucleotides containing 3 intervening sequences of 2391, 167, and 298 bases. The first intron is located within the 5'-untranslated region of apoC-II and contains 4 Alu type sequences. The second intron interrupts the codon specifying amino acid - 11 of the apoC-II signal peptide. The last intron, which contains a 38 bp sequence which is repeated 6 times, interrupts the codon specifying for amino acid +44 of the mature apolipoprotein.     

人猿超科和旧大陆猴7种高等灵长类FKN全基因序列的测定及进化分析

测定人猿超科(人、黑猩猩、大猩猩、红毛猩猩和长臂猿)和旧大陆猴(猕猴和叶猴)7种高等灵长类FKN全基因序列, 探讨其系统进化分析。用简并引物PCR(Degenerated PCR)法分别扩增FKN的3个外显子, 其产物经琼脂糖凝胶回收、纯化后测序, 然后用BioEdit软件剪切拼接FKN基因全序列, 用DNAStar比对后比较基因和氨基酸序列同源性, Mega软件重构FKN基因进化树, 应用Datamonkey分析FKN的负选择位点。序列分析发现人猿超科较旧大陆猴FKN基因除了有散在的点突变外, 还有一明显的30 bp的核苷酸缺失突变; 人FKN基因序列与黑猩猩、大猩猩、红毛猩猩、长臂猿、猕猴和叶猴的同源性分别是99.2%、98.4%、98.1%、96.5%、95.9%和93.8%, 由此推导的氨基酸序列同源性分别是98.5%、98.0%、97.7%、94.7%、93.7%和90.5%; FKN基因进化树表明人与黑猩猩关系更近, FKN基因进化和通常认为的物种进化一致; Datamonkey分析结果显示FKN存在3个负选择位点53Q、84D、239N。成功获得人、黑猩猩、大猩猩、红毛猩猩、长臂猿、猕猴和叶猴7种高等灵长类物种FKN全基因序列, 为后续探讨FKN在高等灵长类物种进化过程中免疫学功能演变及其结构与功能的关系奠定基础。     

Allele frequency distribution of the (TG)n(AG)m microsatellite in the apolipoprotein C-II gene.

The dinucleotide (TG)n interspersed repetitive sequences are the most abundant microsatellites in the human genome. Using the polymerase chain reaction to amplify a (TG)n(AG)m microsatellite in the first intron of the apo C-II gene, we have detected 15 different alleles in 242 unrelated individuals of French ancestry. The heterozygosity index was 0.85 and codominant Mendelian inheritance of the alleles was observed in individuals from 121 nuclear families. We report that polymorphism at this locus is attributable to length variation at both (TG)n and (AG)m motifs, although the (AG)m motif contains only two alleles differing by one repeat unit. A quadrimodal allele frequency distribution was observed at the (TG)n(AG)m locus. Each of the first three modes comprises one frequent allele and one very rare allele adjacent in size. No alleles of intermediate size were found between the three first modes. The fourth mode encompasses nine alleles that span from 27 to 35 repeat units. We suggest that this distribution reflects the molecular mechanisms by which alleles give rise to one another.     

High degree of genetic polymorphism in apolipoprotein(a) associated with plasma lipoprotein(a) levels in Japanese and Chinese populations

We have developed a sensitve, high-resolution method for the analysis of the apolipoprotein(a) [apo(a)] isoforms using sodium dodecyl sulfate (SDS)-agarose/ gradient polyacrylamide gel electrophoresis. In an analysis of the genetic polymorphism of apo(a) isoforms and their relationship with plasma lipoprotein(a) [Lp(a)] levels in Japanese and Chinese, this method identified 25 different apo(a) isoforms and detected one or two apo(a) isoforms in more than 99.5% of the individuals tested. The apparent molecular weights of the apo(a) isoforms ranged from 370 kDa to 950 kDa, and 22 of the 25 different apo(a) isoforns had a higher molecular weight than of apo B-100. Studies on Japanese families confirmed the autosomal codominant segregation of apo(a) isoforms and the existence of a null allele at the apo(a) locus. The observed frequency distribution of apo(a) isoform phenotypes fit the expectations of the Hardy-Weinberg equilibrium in both the Japanese and Chinese populations. Our data indicate the existence of at least 26 alleles, including a null allele, at the apo(a) locus. The frequency distribution patterns of the apo(a) isoform alleles in Japanese and Chinese were similar to each other and also similar to that of apo(a) gene sizes reported in Caucasian American individuals. The average heterozygosity at the apo(a) locus was 92% in Japanese and 93% in Chinese. A highly significant inverse correlation was observed between plasma Lp(a) levels and the size of apo(a) isoforms in both the Japanese (r=-0.677, P=0.0001) and the Chinese (r=-0.703, P=0.0001). A highly skewed distribution of Lp(a) concentrations towards lower levels in the Japanese population may be explained by high frequencies of alleles encoding large apo(a) isoforms and the null allele.     

Plant transposable elements generate the DNA sequence diversity needed in evolution

Two germinal and 16 somatic reversion events induced by the Enhancer (En) transposable element system at the wx-8::Spm-I8 allele of Zea mays were cloned and studied by sequence analysis. Excision of the Spm-I8 receptor element from the wx gene results in various mutant DNA sequences. This leads to altered gene products, some of which are still capable of restoring the wild-type phenotype. Possible 'foot-print' sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild-type (wx+) and mutant (wx-m8) alleles of the wx gene were compared. The sequence divergence generated by visitation of a locus by plant transposable elements is discussed with respect to the molecular evolution of the new gene functions.     

Apolipoprotein B signal peptide polymorphism: distribution and influence on lipid parameters in Tunisian population

Apolipoprotein B (apo B) is the major protein component of LDL, VLDL and chylomicrons. Numerous polymorphisms of the apolipoprotein B gene have been described. Particularly, the insertion/deletion polymorphism located in the coding part of the signal peptide of apo B, associated with modification of lipid concentrations and the risk of cardiovascular disease, has been reported in the general population. No such study in the Tunisian population has been performed. The aim of our study was to assess the effect of insertion/deletion polymorphism of the apolipoprotein B gene on lipid levels in a sample of the Tunisian population. A total of 458 unrelated subjects (321 men and 137 women) were included. The insertion/deletion polymorphism was determined by electrophoresis on polyacrylamide gels after PCR amplification. The relative frequencies of the Ins and Del alleles were 0.74 and 0.26, respectively. These frequencies were similar to those found in other Caucasian populations. There was no significant difference in serum TC, TG, and HDL-C levels due to the influence of the genotypes. However, significant variation among the three genotypes was seen for LDL-cholesterol (p<0.001) and apo B (p<0.001) levels. Individuals homozygous for the Del allele had higher levels than individuals homozygous for the Ins allele, while individuals heterozygous for both alleles exhibited intermediate levels. When the data were analyzed in men and women separately, a similar effect was seen in both groups. Our results show that distribution of apo B insertion/deletion polymorphism in Tunisians is similar to other Caucasian population and confirm the reported association with serum LDL-cholesterol and apo B concentrations.     

Genetic studies of human apolipoproteins. III. Polymorphism of apolipoprotein C-II

Using a simple and rapid one-dimensional isoelectric focusing technique followed by immunoblotting, we have detected genetic polymorphism of human apolipoprotein C-II (APO C-II) in normal unfractionated plasma samples of individuals of black ancestry. Two common autosomal codominantly expressed alleles, designated APO C-II*1 and APO C-II*2, at the APO C-II structural locus have been observed with frequencies of 0.975 and 0.025 in US blacks and 0.943 and 0.049 in Nigerian blacks. In addition, the gene product of a rare allele designated APO C-II*3 was observed in a single Nigerian black. Apart from a single example of an APO C-II 2-1 phenotype in plasma samples from 187 whites, which was electrophoretically identical to the 2-1 phenotype observed in blacks, it appears that APO C-II*2 is a unique black marker of potential importance in anthropogenetic and atherosclerosis studies.     

Rare variant of apolipoprotein E (Arg136 -->Ser) in two normolipidemic individuals

Through the analysis of the common apolipoprotein (apo) E gene polymorphism in large Caucasian population study with the PCR and subsequent restriction analysis, we have identified carriers of mutant allele Arg136-->Ser. Both of them (71-years-old female and her 43-years-old son) have normal lipid parameters. We suggest that Arg136-->Ser mutation in apoE is not necessarily connected with elevated lipid levels in all cases. Furthermore, so far unidentified factors (environmental and/or genetic) are important for the development of lipid metabolism disorders in apoE Arg136-->Ser mutation carriers.     

Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.     

Phenylketonuria: detection of a frequent haplotype 4 allele mutation

Summary By sequence analysis of 94 phenylketonuria (PKU) alleles using polymerase chain reaction (PCR) based techniques, we identified a G to A transition in exon 5 of the human phenylalanine hydroxylase gene. This base substitution predicts an Arg¹⁵⁸Glu¹⁵⁸ amino acid exchange and is strongly associated with the mutant haplotype 4 PKU allele.     

Rabbit apolipoprotein A-I mRNA and gene. Evidence that rabbit apolipoprotein A-I is synthesized in the intestine but not in the liver

In order to study the tissue-specific expression of rabbit apolipoprotein (apo) A-I, a 923-base-pair clone, pRBA-502, complementary to rabbit apo A-I mRNA was identified from a rabbit intestinal cDNA library by hybrid-select translation and immunoprecipitation methods. Northern blot and dot-blot hybridization, utilizing 32P-labeled pRBA-502, revealed that the rabbit apo A-I gene is expressed in the intestine, not in the liver and that rabbit apo A-I mRNA is about 950 nucleotides in length. The entire nucleotide sequence of pRBA-502 has been determined and the complete amino acid sequence of the corresponding apo A-I has been deduced. The mRNA codes for a protein comprising 265 amino acids. Amino acids 1-18 and 19-24 of the primary translation product represent the presegment and prosegment, respectively, of apo A-I. Matured rabbit apo A-I contains 241 amino acids and has a molecular mass of 27612 Da. Using pRBA-502 as a probe, a 15.5-kb genomic fragment, which contains the entire apo A-I gene, was isolated from a rabbit liver genomic library. Sequence analysis of the gene shows that the 200 base pairs of the 5' upstream flanking region of the rabbit and human apo A-I genes showed 78% sequence homology. Like the human apo A-I gene, the rabbit apo A-I gene is interrupted by three intervening sequences. Except for two nucleotides in the fourth exon, the coding sequence of the rabbit liver apo A-I gene is identical to that of pRBA-502. Our data showed that the lack of expression of apo A-I gene in rabbit liver is not due to the alternation of rabbit liver apo A-I gene sequence and suggest that the expression of apo A-I gene in rabbit liver is regulated by a trans-acting regulating element(s).     

Restriction fragment length polymorphism caused by a deletion involving Alu sequences within the human alpha 2-plasmin inhibitor gene

A restriction fragment length polymorphism within the human alpha 2-plasmin inhibitor gene has been detected by Southern blot hybridization using an alpha 2-plasmin inhibitor cDNA probe. This restriction fragment length polymorphism can be attributed to the presence of two alleles, A and B, that are distributed in Hardy-Weinberg equilibrium with frequencies of 73.5% and 2.65%, respectively, in 66 unrelated Caucasian individuals or with frequencies of 51.0% and 49.0%, respectively, in 50 unrelated Japanese individuals. The minor allele, B, is due to a deletion of about 720 base pairs in intron 8 of the alpha 2-plasmin inhibitor gene. Sequence analysis of the deletion junction in allele B and the corresponding regions of allele A demonstrated the presence of oppositely oriented Alu sequences at the 5' and 3' deletion boundaries. These data suggest that this restriction fragment length polymorphism was caused by intrastrand recombination between Alu sequences.     

Length and sequence variation in the apolipoprotein B intron 20 Alu repeat.

We have developed a single-stranded conformation polymorphism (SSCP) protocol for typing both sequence and length variations in an Alu element located in intron 20 of the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction (PCR), we simultaneously amplified and isotopically labeled the apo B intron 20 Alu. The Alu tail, which is composed of two arrays of variable numbers of tandem repeats, (TTTX)y (X = A or G) and (T)z, was separated from the rest of the PCR product by restriction enzyme digestion with PstI. Length variation in the Alu tail (IN20-REP) was thus separated from sequence variation in the Alu body (IN20-SEQ), rendering the SSCP patterns both eaiser to interpret and more informative. In a sample of 242 unrelated individuals from Nancy, France, we observed 11 SSCP alleles at the IN20-SEQ locus that differed only in sequence. At the IN20-REP locus, we observed 7 alleles that differed in both sequence and length. All alleles at both loci were subcloned and sequenced. One additional allele that did not undergo a detectable mobility shift in SSCP gels was uncovered at each locus during sequencing of the SSCP alleles. The additional IN20-SEQ allele was typed by restriction enzyme digestion. Although the number of IN20-SEQ and IN20-REP alleles was large, most were uncommon; the three most common alleles at each locus represented more than 94% of those sampled. We also typed the children of the 242 unrelated French individuals, enabling verification of the Mendelian segregation of the two loci and construction of haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

HLA DNA sequence variation among human populations: molecular signatures of demographic and selective events

Molecular differences between HLA alleles vary up to 57 nucleotides within the peptide binding coding region of human Major Histocompatibility Complex (MHC) genes, but it is still unclear whether this variation results from a stochastic process or from selective constraints related to functional differences among HLA molecules. Although HLA alleles are generally treated as equidistant molecular units in population genetic studies, DNA sequence diversity among populations is also crucial to interpret the observed HLA polymorphism. In this study, we used a large dataset of 2,062 DNA sequences defined for the different HLA alleles to analyze nucleotide diversity of seven HLA genes in 23,500 individuals of about 200 populations spread worldwide. We first analyzed the HLA molecular structure and diversity of these populations in relation to geographic variation and we further investigated possible departures from selective neutrality through Tajima's tests and mismatch distributions. All results were compared to those obtained by classical approaches applied to HLA allele frequencies.Our study shows that the global patterns of HLA nucleotide diversity among populations are significantly correlated to geography, although in some specific cases the molecular information reveals unexpected genetic relationships. At all loci except HLA-DPB1, populations have accumulated a high proportion of very divergent alleles, suggesting an advantage of heterozygotes expressing molecularly distant HLA molecules (asymmetric overdominant selection model). However, both different intensities of selection and unequal levels of gene conversion may explain the heterogeneous mismatch distributions observed among the loci. Also, distinctive patterns of sequence divergence observed at the HLA-DPB1 locus suggest current neutrality but old selective pressures on this gene. We conclude that HLA DNA sequences advantageously complement HLA allele frequencies as a source of data used to explore the genetic history of human populations, and that their analysis allows a more thorough investigation of human MHC molecular evolution.