Minisatellite Variant Repeat Mapping Research Articles

Species-wide distribution of highly polymorphic minisatellite markers suggests past and present genetic exchanges among house mouse subspecies

Global analysis of four minisatellite loci in House Mouse reveals unexpected long-range gene flow between populations and subspecies.

Minisatellites and MVR-PCR for the individual identification of parasite isolates.

In recent years, a wide variety of biochemical and molecular typing systems have been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationships among different species and subspecies. Parasite sequence-specific polymerase chain reaction (PCR)-based genotyping systems are among the most useful tools employed to date, because they can be applied to very small quantities of host-contaminated parasite material and, using repeated loci such as mini- and microsatellites, allow the identification and tracking of individual strains as well as the determination of allele and genotype frequencies in populations. Although minisatellites have been used very successfully to study parasite populations, in particular Trypanosoma brucei populations, there are some technical problems involved in the use of these markers. For example, minisatellite alleles tend to vary in a quasi-continuous fashion, making unambiguous allele identification difficult. The development of minisatellite variant repeat (MVR) mapping by the polymerase chain reaction (MVR-PCR) as a digital approach to DNA typing has overcome many of the drawbacks of minisatellite length analysis. The system assays the dispersion patterns of MVRs within minisatellite alleles, producing an easily interpretable code for each allele. This technique not only allows unequivocal allele identification but also reveals cladistic information that can be used to determine the possible genetic relationships among the different strains and subspecies. The MVR mapping technique has been applied successfully to minisatellites in the parasite Plasmodium falciparum to uniquely identify strains, and more extensively in Trypansoma brucei, where it was used to determine population structure and to examine the relationships among T. brucei subspecies, providing evidence for multiple origins of human infectively. In this chapter, the methods for genotyping of T. brucei parasites using both minisatellite allele length and MVR mapping are described in full and can be easily adapted to apply to mini-satellites in other parasites.

Rapid Individual Identification by Minisatellite Variant Repeat (MVR)-PCR at D1S8 Locus Using "Exponential Law"

We describe an efficient and simple minisatellite variant repeat mapping by PCR (MVR-PCR) method based on the assignment of the tandem array of 29 bp repeating units into a-type, t-type and 0-type (a rarely appearing unamplified unit), from the first repeat unit position (code position 0) of 293 bp in D1S8 locus. After microchip electrophoresis of PCR product amplified from the target DNA of a human hair root, each rung of the ladder at the position of 293 + 29 n bp (n: code position) was detected and the type of repeating unit was determined, i.e., aa, a0, tt, t0, at and 00. The peak area of the rungs from PCR product decreased with increase in code position. We found for the first time that the logarithmic plots of the peak area against the code position showed a linear relationship, which implied that peak areas decrease exponentially. The present method was successfully applied to identify 37 individuals using only a hair root as a biological specimen. This "exponential law" is expected to be an effective tool in forensic science.

Haploid allele mapping of Y-chromosome minisatellite, MSY1 (DYF155S1), to a Japanese population

The present study analyses the human Y-chromosome minisatellite locus, MSY1 (DYF155S1), in 205 Japanese males of 191 pedigrees using the minisatellite variant repeat (MVR) mapping system. The internal haploid structures of the detected alleles considerably varied and consisted of three major repeat units: types 2, 3 and 4. A comparison of the haploid profiles of the MVR codes identified 185 distinct alleles, of which only five were shared. We did not detect a type 1 repeat unit, and variations were frequent at the 5′ end of the minisatellite locus. Within an analysis of 24 paternally linked DNA samples donated by ten families, no mutational events were identified even over two generation gaps. Furthermore, we applied this mapping system to a paternity test in which the alleged father was missing.

Evolution and population genetics of the H-ras minisatellite and cancer predisposition.

Case-control studies have implicated rare length H-ras minisatellite alleles in cancer risk. In Europeans, this locus has four common alleles, and a larger number of rare alleles; possession of a rare allele has been identified as a risk factor responsible for 5-10% of some cancers. This unusual model of predisposition has been controversial in case-control studies, but also makes characteristic predictions about the population genetics of the locus, which we examine in this study. Using minisatellite variant repeat ("MVR") mapping, and compound haplotypes composed of the minisatellite and surrounding substitutional polymorphisms, we have reconstructed the main steps in the evolution of this locus in human populations. MVR-calibrated measurements of allele length yield rare allele frequencies significantly higher than most previous studies, and show that most other analyses have not distinguished two common alleles of 84 and 85 repeat units. Alleles classified as "rare" in European populations predominate (70%) in the African sample studied. Small-pool PCR (SP-PCR) analysis on common alleles in sperm DNA gives an estimate for the germline minisatellite mutation rate of about 0.05% (95% confidence upper limit 0.15%). Overall, our results do not reflect a locus subject to frequent mutation and strong selection, and are difficult to reconcile with the proposed cancer predisposition. Restricted variation at this locus is most simply explained by low mutation rate, and although definitive case-control studies can only be performed using methods capable of reproducibly distinguishing rare and common alleles, our work suggests that most studies to date have not resolved alleles adequately.

Minisatellites Show Rare and Simple Intra-allelic Instability in the Mouse Germ Line

Minisatellites provide very informative systems for analyzing processes of tandem repeat DNA turnover in humans. The mouse genome also contains authentic minisatellites, but none has yet been found to show high levels of instability. Indirect evidence using minisatellite variant repeat mapping by PCR in Mus musculus subspecies suggested that mouse minisatellites mutate at a rate below 10−3 per gamete and mainly by intra-allelic events. This is in sharp contrast to the complex interallelic mutations observed at high frequency at some human loci. To define more directly the turnover mechanisms and rates of instability at one of the most variable mouse minisatellites (MMS80), we used size-enrichment small-pool PCR (SESP-PCR) to recover de novo mutant alleles from sperm DNA from homozygous BALB/cJ mice and from strain DHA heterozygotes. The sperm mutation rate at MMS80 was extremely low, at or below 5×10−6 per sperm. Comparison of progenitor and mutant allele structures showed that these rare mutants had arisen by simple and primarily, if not exclusively, intra-allelic mutation events. These results suggest a fundamental difference in turnover mechanisms at minisatellites between mice and human.

Instability of the human minisatellite CEB1 in rad27Delta and dna2-1 replication-deficient yeast cells.

Convergent studies in human and yeast model systems have shown that some minisatellite loci are relatively stable in somatic cells but not in the germline, and little is known about the mechanism(s) that can destabilize them. Unlike microsatellite sequences, mini satellites are not destabilized by mismatch repair mutations. We report here that the absence of Rad27 and Dna2 functions but not RNase H(35) or Exo1, which play an essential role in the processing of Okazaki fragments during replication, destabilize the human minisatellite CEB1 in mitotically growing Saccharomyces cerevisiae cells, up to 14% per generation in rad27Delta cells. Analysis using minisatellite variant repeat mapping by polymerase chain reaction of the internal structure of 17 variants reveals that the majority of rearrangements in rad27Delta cells are extremely complex contraction events that contain deletions, often accompanied by duplications of motif unit. Altogether, these results suggest that the improperly processed 5' flap structures that accumulate when replication is impaired can act as a potent stimulator of minisatellite destabilization and can provoke an unexpectedly broad range of mutagenic events. This replication-dependent phenomenon differs from the recombination-induced instability in yeast meiotic cells.

Erratum to Evidence for Multiple Origins of Human Infectivity in Trypanosoma brucei Revealed by Minisatellite Variant Repeat Mapping

In recent years a wide variety of biochemical and molecular typing systems has been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationship between differcnt species and subspecies. However, such methods have failed to differentiate between two of the classically defined subspecies of the protozoan parasite Trypanosoma brucei: the human infective, T. b. rhodesiense, which causes African sleeping sickness, and the non-human infective T. b. brucei. This has led to the hypothesis that T. b. rhodesiense is a host range variant of T. b. brucei. In this paper we test this hypothesis by examining highly polymorphic tandemly repeated regions of the trypanosome genome, i.e., minisatellite loci. We have employed the technique of minisatellite variant repeat mapping by PCR (MVR-PCR), which determines the distribution of variant repeat units along the tandem array of one minisatellite, MS42. The maps generated by this technique not only allow unequivocal allele identification but also contain within them cladistic information which we used to determine the possible genetic relationship between the different subspecies of T. brucei. Our findings revealed that human infective (T. b. rhodesiense) isolates from Uganda are more closely related to the local non-human infective isolates (T. b. brucei) than they are to other human infective stocks from different regions, suggesting that human infectivity has originated independently in these different geographical regions. This would infer that the separate classification of all human infective stocks from East Africa into the subspecies T. b. rhodesiense is genetically inappropriate and it would be better to consider geographically separate populations as host range variants of T. brucei brucei or perhaps as a series of different subspecies. Based on these data, it is clear that MVR mapping is a very useful tool for the analysis of zoonotic eukaryotic pathogens where delineation of the origins of outbreaks of disease and definition of human infective strains are key questions.

Allelic structures at hypervariable minisatellite B6.7 in Japanese show population specificity.

Human minisatellite B6.7 shows extensive allele length and structural variability in north Europeans. We analysed this locus in the Japanese population. Allele size distributions showed that Japanese retain extensive allele length variability but have significantly smaller alleles compared with north Europeans. In contrast, there is very little variation in flanking DNA, with only one single-nucleotide polymorphism (SNP) near the minisatellite. Ninety-two Japanese alleles were further characterised by minisatellite variant repeat mapping by polymerase chain reaction (MVR-PCR). These alleles showed a wide variety of internal MVR structures, despite their relative shortness, with most alleles observed only once in the sample. The true heterozygosity is estimated at 99.95%, with well in excess of 2000 different alleles existing in the Japanese population. Dot matrix analysis showed that groups of related alleles sharing structural motifs could be identified within Japanese and in north Europeans, and that these groups are population specific with no examples of significant similarity between any Japanese and north European alleles. Minisatellite B6.7 therefore shows huge allele variability and fast repeat turnover in Japanese as well as north European populations, and provides novel lineage markers for exploring very recent events in human population history.

Evidence for Multiple Origins of Human Infectivity in Trypanosoma brucei Revealed by Minisatellite Variant Repeat Mapping

In recent years a wide variety of biochemical and molecular typing systems has been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationship between different species and subspecies. However, such methods have failed to differentiate between two of the classically defined subspecies of the protozoan parasite Trypanosoma brucei: the human infective, T. b. rhodesiense, which causes African sleeping sickness, and the non-human infective T. b. brucei. This has led to the hypothesis that T. b. rhodesiense is a host range variant of T. b. brucei. In this paper we test this hypothesis by examining highly polymorphic tandemly repeated regions of the trypanosome genome, i.e., minisatellite loci. We have employed the technique of minisatellite variant repeat mapping by PCR (MVR-PCR), which determines the distribution of variant repeat units along the tandem array of one minisatellite, MS42. The maps generated by this technique not only allow unequivocal allele identification but also contain within them cladistic information which we used to determine the possible genetic relationship between the different subspecies of T. brucei. Our findings revealed that human infective (T. b. rhodesiense) isolates from Uganda are more closely related to the local non-human infective isolates (T. b. brucei) than they are to other human infective stocks from different regions, suggesting that human infectivity has originated independently in these different geographical regions. This would infer that the separate classification of all human infective stocks from East Africa into the subspecies T. b. rhodesiense is genetically inappropriate and it would be better to consider geographically separate populations as host range variants of T. brucei brucei or perhaps as a series of different subspecies. Based on these data, it is clear that MVR mapping is a very useful tool for the analysis of zoonotic eukaryotic pathogens where delineation of the origins of outbreaks of disease and definition of human infective strains are key questions.

The Application of Minisatellite Variant Repeat Mapping by PCR (MVR-PCR) in a Paternity Case Showing False Exclusion Due to STR Mutation

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged father's medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boy's allele 17 could have originated from either of the alleged father's allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.

Distinguishing minisatellite mutation from non-paternity by MVR-PCR

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was devised to map the interspersion pattern of subtle variant repeats along minisatellite tandem arrays. MVR-PCR has revealed enormous diversity of allele structures at several loci, far more than can be resolved by allele length analysis. We have reported the application of MVR-PCR at minisatellite MS32 (D1S8) and MS31A (D7S21) in a paternity case lacking a mother and showed that it resulted in higher paternity probabilities than for a set of 12 other DNA markers including six STRs. Hypervariable minisatellites like MS32 and MS3lA can however, show significant germline mutation rates to new length alleles which can generate false exclusions in paternity cases although paternity cases showing mutant paternal alleles at more than one locus will be rare when several MVR loci are examined. Detailed knowledge of mutation processes coupled with MVR analysis of allele structure can help distinguish mutation from non-paternity. We now show how similar mutant alleles are to their progenitors using both real and simulated data, and demonstrate how MVR-PCR can be used to identify mutant paternal allele in paternity cases showing apparent exclusions.

Sequencing and four-state minisatellite variant repeat mapping of the D1S7 locus (MS1) by fluorescence detection.

Minisatellite variant repeat mapping by polymerase chain reaction (MVR-PCR) reveals an enormous degree of variation in the human minisatellite regions. The original approach involved the use of 32P-labelled probes to detect the MVR-PCR products generated. To date, the loci mapped include D1S8, D7S21 and D16S309. However, the most polymorphic locus, D1S7 (MS1), which has been used in forensic analysis, has presented technical difficulties, initially due to its short 9 bp repeats that are much shorter than any conventional primer sequences. This was overcome by using the method of "wrapping around" primers employing inosine at the redundancy position. The difficulty of cloning highly repetitive DNA was overcome by utilising specialised competent SURE cells. We report the cloning and sequencing of selected short MS1 alleles to determine the variety of repeat types. This survey revealed nine types, four of which represented greater than 80% of the sequenced repeats. The reported MVR-PCR system maps the MS1 locus for these four common repeat types by fluorescence detection.

The Potential Contribution of MVR-PCR to Paternity Probabilities in a Case Lacking a Mother

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.

Extremely complex repeat shuffling during germline mutation at human minisatellite B6.7.

Human minisatellite B6.7 is a highly variable locus showing extensive heterozygosity with alleles ranging from six to >500 repeat units. Paternal and maternal mutation rates to new length alleles were estimated from pedigrees at 7.0 and 3.9% per gamete, respectively, indicating that B6.7 is one of the most unstable minisatellites isolated to date. Mutation at this locus was also analysed by small pool PCR of sperm and blood DNA. Male germline instability varied from <0.8 to 14% per allele and increased with tandem array size. In contrast, the frequency of mutants in somatic (blood) DNA was far lower (<0.5%), consistent with a meiotic origin of germline mutants. Sperm mutants were further characterized by minisatellite variant repeat mapping using four major polymorphic sites within the B6.7 repeats. This highly informative system revealed a wide variety of changes in allele structure, including simple intra-allelic duplications and deletions and more complicated inter- and intra-allelic transfers of repeat blocks, as seen at other human minisatellites. The main mode of sperm mutation, however, resulted in extremely complex allele reorganization with evidence of inter-allelic transfer plus the generation of novel repeats by rearrangement at the sub-repeat level, suggesting that recombinational instability at B6.7 is a complex multistep process.

Evaluation of the paternity probability on an application of minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) to paternity testing

Minisatellite variant repeat (MVR) mapping using polymerase chain reaction (PCR) was applied to a practical case of paternity testing to evaluate the paternity probability. In order to obtain single allele mapping by allele-specific MVR-PCR, three flanking polymorphic sites for each of the MS31A and MS32 loci were investigated and all three individuals were typed as heterozygous for at least one flanking polymorphic site at each locus. Allele-specific MVR-PCR was then performed using genomic DNA. It was confirmed that one allele in the child was identical to that from the mother and the other one in the child was identical to that from the alleged father. Mapped allele codes were also compared with those in the database by dot-matrix analysis, and no identical allele was found although some motifs were shared with Japanese alleles. The paternity index and the probability of paternity exclusion in the case at these two MVR loci were calculated using the presumed values of the allele frequencies. These studies seem to illustrate the practical value of MVR mapping of MS31A and MS32 loci in paternity testing.

Minisatellite variant repeat (MVR) analysis of the HRAS1 minisatellite locus.

Two alternative electrophoretic strategies were used to study the internal variation of the HRAS1 minisatellite after minisatellite variant repeat mapping (MVR-PCR) was carried out. While the use of automated sequencers with fluorescent based technology is ideal for analyzing fragment size, and therefore, for analyzing the repeat number, the use of polyacrylamide gels and silver staining is more appropriate for the analysis of internal variation. Thirteen different fragments ranging from 27 to 80 repeats were found in a sample from 80 healthy Caucasian individuals. By using MVR mapping we were able to detect heterozygotes which appear as homozygotes when fragment length analysis was used. As a result of this, the 13 alleles, which we had detected, increased to 16 alleles when MVR sequences were analyzed. The extremely conservative arrays of repeats allow us to infer the theoretical origin of rare alleles from a major group of specific alleles. The HRAS1 minisatellite has been extensively studied due to its association with cancer. However, the methodology used up to now has limited the scope of previous research. Our approach permits the identification of alleles in a fast and reliable way using their MVR codes, thus allowing association studies with cancer.

Different ancestor alleles: a reason for the bimodal fragment size distribution in the minisatellite D2S44 (YNH24)

Three conserved repeat order motifs found in the D2S44 (YNH24) minisatellite by minisatellite variant repeat mapping by PCR (MVR-PCR) might be the remnants of three different ancestor alleles. One of the motifs is found in the group of alleles around the 2.8 kbp fragment size and the two others in the group of alleles around the 4.1 kbp fragment size in the bimodal frequency distribution of HinfI fragments characteristic for the D2S44 minisatellite. The conserved repeat-order motifs were found at the 3'-end of the alleles, whilst the 5'-end was highly polymorphic. This is the first time a minisatellite is shown to have repeat-order motifs that are associated with fragment length. These results were obtained by MVR-PCR mapping of 49 alleles with primers specific for the core nucleotide sequence variation ...(G)5TGTT, ...(G)4TTGTT or ...(G)4TGTT.

Isolation and Characterization of Mouse Minisatellites

Minisatellites provide the most informative system for analyzing processes of tandem repeat turnover in humans. However, little is known about minisatellites and the mechanisms by which they mutate in other species. To this end, we have isolated and characterized 76 endogenous mouse VNTRs. Fifty-one loci have been localized on mouse chromosomes and, unlike in humans, show no clustering in proterminal regions. Sequence analysis of 25 loci revealed the majority to be authentic minisatellites with GC-rich repeat units ranging from 14 to 47 bp in length. We have further characterized 3 of the most polymorphic loci both inMus musculussubspecies and in inbred strains by using minisatellite variant repeat mapping (MVR) by PCR to gain insight into allelic diversity and turnover processes. MVR data suggest that mouse minisatellites mutate mainly by intra-allelic nonpolar events at a rate well below 10−3per gamete, in contrast to the high-frequency complex meiotic gene conversion-like events seen in humans. These results may indicate a fundamental difference in mechanisms of minisatellite mutation and genome turnover between mice and humans.

Analysis of allelic structures at the D7S21 (MS31A) locus in the Japanese, using minisatellite variant repeat mapping by PCR (MVR-PCR).

To sample the diversity of allelic structures at the D7821 (MS31 A) locus in the Japanese, allele-specific minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) was performed on genomic DNA from a number of Japanese individuals. Three polymorphic positions in the MS31A 5' flanking DNA were typed from 214 un related Japanese, and the distribution of haplotypes was analysed. Allele-specific MVR-PCR using primers that discriminate between different alleles at these polymorphic positions in heterozygous individuals, allows single alleles to be mapped from genomic DNA in approximately 80% of Japanese. 149 Japanese alleles have been mapped to date and all of them, except for two pairs of indistinguishable alleles, have different internal structures. More than half of the mapped alleles showed similar regions of internal structure to other alleles and were classified into groups on this basis.