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.     

Molecular basis of five apolipoprotein B gene polymorphisms in noncoding regions

Restriction fragment length polymorphisms (RFLPs) are useful in linkage and clinical association studies of human diseases. In this report, we characterize the molecular basis and frequencies of two new RFLPs, AvaII and BalI, two previously reported RFLPs, HincII and PvuII, and one new sequence polymorphism in the human apolipoprotein B gene. For the AvaII RFLP, the two alleles yield either a 1 kb fragment or 0.7 and 0.3 kb fragments, and have frequencies of 20% and 80%, respectively. The polymorphic site is about 4 kb upstream of exon 1. For the BalI RFLP, the two alleles yield either a 4.9 or 6.2 kb fragment, and have about equal frequencies. The polymorphic site is within an Alu sequence in intron 20, 146 bp 5' to exon 21. The BalI recognition sequence TGGCCA is replaced by TAGCCA. For the HincII RFLP, the two alleles yield either a 1.7 or 1.3 kb fragment and have frequencies of 80% and 20%, respectively. The polymorphic site is in intron 4, 171 bp 3' to exon 4. The HincII recognition sequence GTTAAC, present in the minor allele, is replaced by GTTACC. HincII fragments of 7.4 and 7.0 kb, previously reported for this polymorphism, are the result of partial digestion at the invariant HincII site in intron 3, 334 bp 3' to exon 3. For the PvuII RFLP, the two alleles yield either a 7.5 or 5.5 kb fragment and have frequencies of 96% and 4%, respectively. The polymorphic site is within an Alu sequence in intron 4, 523 bp 5' to exon 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular evaluation of an Alu repeat including a polymorphic variable poly(dA) (AluVpA) in the vitamin D binding protein (DBP) gene

We investigated an Alu element at the end of intron 8 of the human vitamin D-binding protein (hDBP, group-specific component, GC) gene that shows a polymorphic poly(A) tail due to a variable number of tandem repeats (AluVpA) forming the 3 end of this member of the most abundant class of short interspersed repeated DNA element (SINES). The Alu element sequence in intron 8 of the GC gene was identical in all three common GC alleles (GC*1F, GC*1S, and GC*2) and could be classified as an Alu-Sa or Alu class-II sequence. The polymerase chain reaction was used to amplify selectively a fragment of about 200 bp containing the identified (TAAA)n repeat from genomic DNA of 188 unrelated human subjects. The size of the amplified products was determined by polyacrylamide gel electrophoresis. Four alleles (named GC-18*6, GC-I8*8, GC-I8*10, and GC-18*11) were found that differed in size by multiples of four nucleotides. The allele frequencies ranged from 0.0053 to 0.8511 and the observed heterozygosity was 26%. The stable inheritance of this polymorphic patterned poly(A) sequence was confirmed by a segregation study of a highly informative family with 19 members. Statistically significant linkage disequilibrium between the AluVpA and the GC isoelectric focusing (IEF) phenotypes was found in a sample of 188 unrelated individuals and delta values were calculated from the observed haplotype distribution.     

Serial Alu sequence transposition interrupting a human B creatine kinase pseudogene.

We have isolated, sequenced, and characterized a single-copy B creatine kinase pseudogene. The chromosomal assignment of this gene is 16p13 and a unique sequence probe from this locus detects EcoRI restriction fragment length polymorphisms of 7.8 and 5.4 kb. In 26 unrelated individuals, the frequencies for the 7.8- and 5.4-kb B creatine kinase pseudogene alleles were calculated to be 17.3 and 82.7%, respectively. The B creatine kinase pseudogene is interrupted by a 904-bp DNA insertion composed of three Alu repeat sequences in tandem flanked by an 18-bp direct repeat, derived from the pseudogene sequence. Nucleotide sequence analysis of the Alu elements suggests that the Alu sequences were incorporated into this locus in three separate integration events. Several complex clustered Alu repeat sequences without defined integration borders have been previously identified at different genomic loci. This is the first evidence that complex tandem Alu elements can integrate in an apparently serial manner in the human genome and supports the contention that Alu repeats integrate nonrandomly into the human genome.     

Polymorphic molecular markers from anonymous nuclear DNA for genetic analysis of populations

A simple method is presented for developing polymorphic, anonymous DNA markers suitable for population genetic studies. Anonymous DNA fragments are screened for sequence variability using a common mutation detection technique (single strand conformation polymorphism analysis; SSCP) and locus-specific PCR primers are designed for polymorphic DNA fragments. Detection of the markers by SSCP analysis coupled with sequence analysis of SSCP variants allows rapid screening while retaining information about the genealogical relationship among alleles. Variability detected for six markers was assessed in rainbow trout Oncorhynchus mykiss and was compared with variability detected by similar analysis of intron loci. Between three and 12 distinct alleles were observed at each marker locus, and average within-population heterozygosity ranged from 0.12 to 0.44. Advantages and limitations of the methodology for population genetic analysis are discussed.     

Genetic mapping of the human tryptophan hydroxylase gene on chromosome 11, using an intronic conformational polymorphism.

The identification of polymorphic alleles at loci coding for functional genes is crucial for genetic association and linkage studies. Since the tryptophan hydroxylase (TPH) gene codes for the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, it would be advantageous to identify a polymorphism in this gene. By examining introns of the human TPH gene by PCR amplification and analysis by the single-strand conformational polymorphism (SSCP) technique, an SSCP was revealed with two alleles that occur with frequencies of .40 and .60 in unrelated Caucasians. DNAs from 24 informative CEPH families were typed for the TPH intron polymorphism and analyzed with respect to 10 linked markers on chromosome 11, between p13 and p15, with the result that TPH was placed between D11S151 and D11S134. This region contains loci for several important genes, including those for Beckwith-Wiedemann syndrome and tyrosine hydroxylase.     

Association of hY4 pseudogenes with Alu repeats and abundance of hY RNA-like sequences in the human genome.

Three loci having homology with the small human cytoplasmic RNA, hY4, were isolated from human genomic DNA libraries and sequenced. Each sequence contains dispersed mismatches as compared with hY4 RNA, is followed by an A-rich or A + T-rich sequence, and is bordered by direct repeats. Each of these loci, therefore, appears to constitute a small RNA class-III pseudogene. Surprisingly, two of the three loci are associated with Alu repeats. In the hY4.B7 locus, the hY4 sequence has integrated into the tail of an Alu element and in the hY4.F2 locus, an Alu sequence has inserted into the hY4 tail, confirming that A-rich tracts are preferential targets for retroposition. In addition, Southern blots with probes for each of the four hY RNAs indicate that hY RNA-like sequences are abundant in the human genome.     

Major histocompatibility complex variation at three class II loci in the northern elephant seal

Northern elephant seals were hunted to near extinction in the 19th century, yet have recovered remarkably and now number around 175,000. We surveyed 110 seals for single-strand conformation polymorphism (SSCP) and sequence variation at three major histocompatibility (MHC) class II loci (DQA, DQB and DRB) to evaluate the genetic consequences of the population bottleneck at these loci vs. other well-studied genes. We found very few alleles at each MHC locus, significant variation among breeding sites for the DQA locus, and linkage disequilibrium between the DQB and DRB loci. Northern elephant seals are evidently inbred, although there is as yet no evidence of correlative reductions in fitness.     

Allelic dimorphism in the human tissue-type plasminogen activator (TPA) gene as a result of an Alu insertion/deletion event

Summary Polymerase chain reaction and direct sequencing were used to investigate an amplified DNA fragment containing the suspected polymorphic site of all known intragenic restriction fragment length polymorphisms (RFLPs) within the human tissue-type plasminogen activator (TPA) gene. Sequence data obtained showed that these RFLPs were all generated by the presence or absence of one of the two Alu sequences located in intron h of the human TPA gene. Furthermore, one of the direct repeats flanking this Alu sequence was absent in the minor allele. In addition to indicating the presence of an Alu insertion in an ancestral human TPA gene, these findings suggest a slip-replication mechanism for the deletion of this Alu repeat, once inserted into the gene. As both alleles have been observed in similar frequencies among different ethnic groups, the insertion or subsequent deletion of this Alu sequence in the human TPA gene must have occurred early in human evolution.     

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.     

Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.     

Natural killer gene complex (Nkc) allelic variability in inbred mice: evidence for Nkc haplotypes

Allelic variability for mouse Chromosome 6 Nkc loci was assessed in 22 common laboratory strains of mice using selected natural killer gene complex (Nkc)-linked sequence tagged site markers. Most Nkc markers distinguished three or more alleles for a particular locus in the assessed mouse strains. Nkc locus alleles were highly conserved among genealogically related inbred strains, whereas far less similarity was observed among unrelated strains. Concurrent strain-to-strain comparisons for all Nkc-linked loci revealed common and uncommon Nkc haplotypes, including some that were likely recombinant. Nkc allele and haplotype assignments in inbred mouse strains and correlation with phenotypic traits should facilitate positional gene cloning strategies for unknown Nkc-linked trait modification loci.     

Molecular analyses of five new chimpanzee MHC class I alleles: implications for differences between evolutional mechanisms of HLA-A, -B, and -C loci.

In order to study the origin of the polymorphism of MHC class I molecules, we have cloned and sequenced five new Patr-A, -B, and -C loci alleles from two chimpanzees. Previous studies of sequence comparison between Patr and HLA class I alleles revealed that many of the sequence motifs were shared and the origin of class I molecules predated the divergence of chimpanzees and humans. These findings are confirmed by our current study. Additionally, our data suggest significant differences between mechanisms of evolution of the A, B, and C loci: (1) The B locus is characterized by frequent nucleotide substitutions, whereas the A and C loci are relatively more conserved; (2) However, unlike the A locus, the alpha2 domains of the C locus sequenced appear to produce MHC polymorphism between these species. These differences might imply the distinctive contributions of each locus during the evolutionary history.     

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.     

Genotypic Characterization of an MHC Class II Locus in Lake Trout (Salvelinus namaycush) from Lake Superior by Single-Stranded Conformational Polymorphism Analysis and Reference Strand–Mediated Conformational Analysis

This study compares the genotypic information provided by reference strand–mediated conformational analysis and single-stranded confirmational polymorphism (SSCP) analysis for the major histocompatibility complex (MHC) II locus in lake trout. For this study 80 wild-caught animals from the Apostle Islands of Lake Superior were genotyped using both RSCA and SSCP analysis. Their genotypes were recorded using both methods and compared. The genotypic information provided by the 2 methods was essentially the same although some inconsistencies were observed. Both methods detected approximately 65 genotypes, and both were able to distinguish heterozygous and homozygous animals. The analyses determined that only approximately 20% of alleles were shared between 2 morphologically different populations within the sample set, and identified the dominant alleles. SSCP analysis was quicker, simple, and more robust than RSCA. SSCP analysis using fluorescence technologies could be the method of choice for future genotypic analysis of the MHC II locus in salmonids.     

Relationships between DNA and protein polymorphisms of apolipoprotein B

Summary The associations between four restriction fragment length polymorphisms (RFLPs) of the gene for human apolipoprotein B (apo B) and five antigen group (Ag) protein-polymorphisms of apo B have been investigated in 24 unrelated Finnish individuals. In this sample a complete correlation exists between the EcoRI RFLP and the Ag(t/z) polymorphism. There is strong association between the alleles of the XbaI RFLP and Ag(c/g) and a weaker one of the same XbaI site with Ag(x/y). Linkage disequilibrium is observed between the PvuII RFLP and the Ag(a₁/d) polymorphism. These associations confirm that the Ag variants are true protein sequence polymorphisms of apo B.     

Two partial deletion mutations involving the same Alu sequence within intron 8 of the LDL receptor gene in Korean patients with familial hypercholesterolemia

Twenty-eight unrelated persons heterozygous for familial hypercholesterolemia (FH) were screened to assess the frequency and nature of major structural rearrangements at the low-density lipoprotein (LDL) receptor gene in Korean FH patients. Genomic DNA was analyzed by Southern blot hybridization with probes encompassing exons 1–18 of the LDL receptor gene. Two different deletion mutations (FH29 and FH110) were detected in three FH patients (10.7%). Each of the mutations was characterized by the use of exon-specific probes and detailed restriction mapping mediated by long-PCR (polymerase chain reaction). Mutation FH29 was a 3.83-kb deletion extending from intron 6 to intron 8 and FH110 was a 5.71-kb deletion extending from intron 8 to intron 12. In FH29, the translational reading frame was preserved and the deducible result was a cysteine-rich A and B repeat truncated protein that might be unable to bind LDL but would continue to bind β-VLDL. FH110 is presumed to be a null allele, since the deletion shifts the reading frame and results in a truncated protein that terminates in exon 13. Sequence analysis revealed that both deletions have occurred between two Alu-repetitive sequences that are in the same orientation. This suggested that in these patients the deletions were caused by an unequal crossing over event following mispairing of two Alu sequences on different chromatids during meiosis. Moreover, in both deletions, the recombinations were related to an Alu sequence in intron 8 and the deletion breakpoints are found within a specific sequence, 27 bp in length. This supports the hypothesis that this region might have some intrinsic instability, and act as one of the important factors in large recombinational rearrangements. Received: 3 April 1996 / Revised: 19 August 1996     

Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.     

Molecular analysis of human, porcine, and poultry Enterococcus faecium isolates and their erm(B) genes

Fifty-nine erm(B)-positive Enterococcus faecium strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the erm(B) gene was determined. Identical erm(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different erm(B) alleles. This may suggest that horizontal exchange of the erm(B) gene between animal and human E. faecium strains or the existence of a common reservoir of erm(B) genes might be more important than direct transmission of resistant strains.     

Exclusion of linkage between the human apolipoprotein B gene and abetalipoproteinemia

Abetalipoproteinemia (ABLP) is a rare autosomal recessive disease characterized by a lack of plasma apolipoprotein B (apo B). In this report, the hypothesis that ABLP is due to rare mutations in the apo B gene was tested. A total of eight ABLP families were studied. Apo B gene RFLPs were used to establish the haplotypes of the apo B alleles in family members. LOD score analysis was used to study the linkage between the apo B alleles and ABLP. These families were categorized arbitrarily as class I, II, III, or IV because of differences in the results derived from both haplotyping and LOD score analysis. In a class I family, affected siblings, who on the basis of the hypothesis would be expected to have the same apo B alleles, had different ones. LOD score analysis of this family gave an infinite negative number at a recombination fraction (theta) of zero. In two class II families, probands who were the result of consanguineous marriages and who, on the basis of the hypothesis, should be homozygotes for a defective apo B allele, were heterozygotes at this locus. The sum of the LOD scores from these two families was -1.7 at theta = 0. In one class III family, a parent was apparently homozygous for a particular apo B allele and yet not affected. This also contributed negatively to the LOD score. In four class IV families, disease inheritance was compatible with segregation of the apo B alleles. This, however, was not statistically significant (LOD score = 0.97 at theta = 0).(ABSTRACT TRUNCATED AT 250 WORDS)