Human minisatellite MS32 (D1S8) displays somatic but not germline instability in transgenic mice

Human minisatellite MS32 (D1S8) shows instability both in the germline and, at much lower levels, in somatic DNA. To investigate factors that influence somatic and germline mutation, large cosmid-based constructs containing MS32 were introduced into mice, bred to homozygosity and tested for instability in blood and sperm. Analysis of single copy and multicopy transgenic lines revealed somatic mutants occurring at a frequency comparable with that seen in man. As in humans, these mutants arose mainly by simple intra-allelic duplications and deletions. In contrast, analysis of sperm DNA from four different transgenic lines showed no trace of the complex recombination-based germline instability seen in man, even using PCR-based approaches capable of detecting very rare mutants. These data provide further evidence that germline and somatic mutation at human minisatellite MS32 occur via distinct pathways, that a major barrier exists to the transfer of germline instability from humans to mice and that the mouse germline appears to be protected from mitotic instability of the type seen in blood.      

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.     

Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309)

Many tandemly repeated minisatellite loci display extreme levels of length variation as a consequence of high rates of spontaneous germline mutation altering repeat copy number. Direct screening for new allele lengths by small-pool PCR has shown that instability at the human minisatellite locus MS205 (D16S309) is largely germline specific and usually results in the gain or loss of just a few repeat units. Structural analysis of the order of variant repeats has shown that these events occur preferentially at one end of the tandem array and can result in complex rearrangements including the inter-allelic transfer of repeat units. In contrast, putative mutants recovered from somatic DNA occur at a substantially lower rate and are simple and non- polar in nature. Germline mutation rates vary considerably between alleles, consistent with regulation occurring in cis. Although examination of DNA sequence polymorphisms immediately flanking the minisatellite reveals no definitive associations with germline mutation rate variation, differences in rate may be paralleled by changes in mutation spectrum. These findings help to explain the diversity of MS205 allele structures in modern humans and suggest a common mutation pathway with some other minisatellites.      

Comparative sequence analysis of human minisatellites showing meiotic repeat instability

The highly variable human minisatellites MS32 (D1S8), MS31A (D7S21), and CEB1 (D2S90) all show recombination-based repeat instability restricted to the germline. Mutation usually results in polar interallelic conversion or occasionally in crossovers, which, at MS32 at least, extend into DNA flanking the repeat array, defining a localized recombination hotspot and suggesting that cis-acting elements in flanking DNA can influence repeat instability. Therefore, comparative sequence analysis was performed to search for common flanking elements associated with these unstable loci. All three minisatellites are located in GC-rich DNA abundant in dispersed and tandem repetitive elements. There were no significant sequence similarities between different loci upstream of the unstable end of the repeat array. Only one of the three loci showed clear evidence for putative coding sequences near the minisatellite. No consistent patterns of thermal stability or DNA secondary structure were shared by DNA flanking these loci. This work extends previous data on the genomic environment of minisatellites. In addition, this work suggests that recombinational activity is not controlled by primary or secondary characteristics of the DNA sequence flanking the repeat array and is not obviously associated with gene promoters as seen in yeast.     

The human minisatellites MS1, MS32, MS205 and CEB1 integrated into the yeast genome exhibit different degrees of mitotic instability but are all stabilised by RAD27

The yeast Rad27 protein is homologous to mammalian Fen1 and is involved in the processing of replication intermediates. Enhanced instability of various artificial repetitive DNA sequences in RAD27-deficient yeast strains has been observed previously and shown to involve preferentially expansion mutations. In the present investigation, we characterised the mitotic instability of alleles of the naturally occurring human minisatellites MS1, MS32, MS205 and CEB1 and the modified MS1 alleles containing more highly homogeneous repeat regions than the original alleles. These minisatellites demonstrated more pronounced instability in rad27 Delta strains, with increases in the frequencies of both expansion and contraction mutants. In RAD27 strains, MS32 and MS205 were relatively stable, while MS1 and CEB1 were unstable, indicating that the effect of RAD27 on stability is influenced by intrinsic properties of the repeat array. This conclusion received further support from the remarkably high frequency of length-mutants observed for the modified allele of MS1. Thus, our findings emphasise the importance of: (1) comparing results obtained with various naturally occurring minisatellites and (2) manipulating their sequences in attempts to understand the molecular basis for mitotic stability/instability of minisatellite DNA.     

Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome

Immortal human cells maintain telomere length by the expression of telomerase or through the alternative lengthening of telomeres (ALT). The ALT mechanism involves a recombination-like process that allows the rapid elongation of shortened telomeres. However, it is not known whether activation of the ALT pathway affects other sequences in the genome. To address this we have investigated, in ALT-expressing cell lines and tumours, the stability of tandem repeat sequences known to mutate via homologous recombination in the human germline. We have shown extraordinary somatic instability in the human minisatellite MS32 (D1S8) in ALT-expressing (ALT+) but not in normal or telomerase-expressing cell lines. The MS32 mutation frequency varied across 15 ALT+ cell lines and was on average 55-fold greater than in ALT- cell lines. The MS32 minisatellite was also highly unstable in three of eight ALT+ soft tissue sarcomas, indicating that somatic destabilization occurs in vivo. The MS32 mutation rates estimated for two ALT+ cell lines were similar to that seen in the germline. However, the internal structures of ALT and germline mutant alleles are very different, indicating differences in the underlying mutation mechanisms. Five other hypervariable minisatellites did not show elevated instability in ALT-expressing cell lines, indicating that minisatellite destabilization is not universal. The elevation of MS32 instability upon activation of the ALT pathway and telomere length maintenance suggests there is overlap between the underlying processes that may be tractable through analysis of the D1S8 locus.     

Allele diversity and germline mutation at the insulin minisatellite

Previous analysis of germline mutation at highly unstable GC-rich minisatellites with continuous allele size distributions revealed similar meiotic recombinational mechanisms operating at all loci investigated. The insulin minisatellite has been studied intensively due to its associations with diabetes, polycystic ovary syndrome, obesity and birth size. Its bimodal allele size distribution in Caucasians suggests a much lower mutation rate and possible differences in the mutation process compared with highly unstable minisatellites. Mutation at the insulin minisatellite therefore was studied both indirectly from allele diversity surveys and directly by recovering de novo mutants from sperm DNA. Structural analysis of variant repeat distributions in 876 alleles identified 189 different alleles, almost all of which could be assigned to one of three very distinct lineages. Variation within a lineage was minor and due mainly to the gain or loss of one or a few repeat units. These events most probably arise by mitotic replication slippage at a frequency of perhaps 10(-3)per gamete. Sperm DNA analysis revealed a second class of mutation occurring at a frequency of approximately 2 x 10(-5)that involved highly complex intra- and inter-allelic rearrangements very similar to those seen at unstable minisatellites. These complex rearrangements were not seen in somatic DNA and are probably meiotic in origin. Minisatellite homozygosity did not reduce the frequency of these mutants in sperm. The insulin minisatellite therefore appears to evolve by two distinct processes: one involving slippage-like events and the second resulting in complex recombinational turnover of allele structure.     

Somatic mutations in VNTR-Locus D1S7 in human colorectal carcinomas are associated with microsatellite instability

To elucidate mutation mechanisms in hypervariable VNTR loci, we have studied somatic mutation events with the minisatellite probe MSI (VNTR locus D1S7) in 224 colorectal carcinomas (CRC). The D¹ S7 locus consists of a 9-basepair (bp) repeat unit. The copy number varies from about 100 to 2000, and the germline mutation rate is high. Here we demonstrate a high D1S7 somatic mutation rate in CRC (37/224), higher than indicated earlier by others. We atso demonstrate that the most frequent mutational event by far (n = 34) involves small reductions in VNTR fragment size (median loss 22 repeat units, range 2-1.54), furthermore, in one-half of these cases, this event is biallelic. We wanted to test whether these somatic mutations mirror the same genetic instability as seen by RER (replication error), a phenomenon recently described in tumour DNA from both sporadic and familial cases of CRC. All blood/tumour DNA pairs displaying MS1 mutation (n = 37) as well as 37 randomly selected pairs without MS1 mutation were tested with four tetranucleotide short tandem repeats (STRs, microsatellites). There is a strong association between mutations at the D1S7 locus and the occurrence of new STR alleles (P<0.001). This is the first report of the existence of a minisatellite as a marker for genetic instability/RER in colorectal carcinomas. The findings may also cast light upon the mechanism for somatic mutations in this minisatellite. © 1995 Wiley-Liss, Inc.     

De novo mutations and allelic diversity at minisatellite locus D7S22 investigated by allele-specific four-state MVR-PCR analysis

We have studied the allelic diversity and de novo mutations at the hypervariable minisatellite locus D7S22. A four-state minisatellite variant repeat unit mapping by PCR (MVR-PCR) method was developed for this purpose, and a substitution polymorphism close to the repeat array was used to design allele-specific flanking primers to study individual haplotypes in genomic DNA. A total of 150 alleles from different allele size groups and flanking haplotypes were mapped. On average, MVR-codes extending 65 repeats (2.4 kb) into the repeat array were obtained. The interspersion patterns of variant repeats were highly polymorphic. However, subgroups of alleles close in size and with identical flanking haplotype revealed common MVR-code characteristics indicating a close evolutionary relationship. Unlike the situation in many other hypervariable minisatellites, no polarized variability was revealed at this minisatellite locus. Fifty four small families with D7S22 de novo mutations were analysed by MVR-PCR. The sites where the length change occurred were revealed in 22 cases, while in 32 cases the mutation obviously occurred further into the repeat array. In agreement with a non-polar distribution of the allelic variation, there was no evidence for a hypermutable hot spot for mutation within the repeat array. Comparison of MVR-codes in the mutant and progenitor in gain mutations indicated that at least one, possibly four cases, reflected inter- allelic events. Together with evidence from DNA sequencing of alleles of <2 kb, this indicates that as many as half of the gain mutations might be inter-allelic events in D7S22. Based on these results, different factors which might affect the mutation rate are discussed.      

Allelic diversity at minisatellite MS205 (D16S309): evidence for polarized variability

We have determined the allelic structures on 106 Caucasian chromosomesat the minisatellite locus MS205 (D16S309). In addition to theInternal structures deduced by minisatellite variant repeat(MVR) mapping, the genotypes at six flanking substitutionalpolymorphic sites have been analysed. The minisatellite structuresshow a polarity of variation at MS205, with most observed variationdue to differences at one extremity of the tandem repeat array.This locus, therefore, provides a further example of polarityof variation at human minisatellites. Analysis of haplotypesat flanking polymorphic sites suggests that there may be a higherfrequency of exchange near the highly unstable end of the minisatellite,and thus that exchanges of flanking polymorphisms may be dueto co-conversion or recombination events occurring during unequalexchanges between minisatellite alleles.     

Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy

To determine whether the measurement of repeat number mutations at a minisatellite locus could detect human germline mutations induced by chemotherapy, we performed a longitudinal study of the mutation frequencies in sperm from 10 patients treated for Hodgkin's disease. Polymerase chain reaction on small pools of DNA equivalent to 100 sperm and Southern blotting were used to screen at least 7900 sperm in each sample to quantify the mutation frequency at the minisatellite MS205 locus. Pretreatment and posttreatment semen samples were obtained at least 2 months after completion of therapy from 4 patients treated with a regimen (Novantrone, Oncovin, vinblastine and prednisone [NOVP]) that lacks alkylating agents and from three patients treated with regimens (Cytoxan, vinblastine, procarbazine and prednisone/Adriamycin, bleomycin, dacarbazine, lomustine, and prednisone [CVPP/ABDIC] or mechlorethamine, Oncovin, procarbazine and prednisone [MOPP]) containing alkylating agents. There were no effects of NOVP or CVPP/ABDIC on the mutation frequencies. In the 1 patient treated with MOPP, the treatment with the highest dose of gonadotoxic alkylating agents, there was a statistically significant increase in mutation frequency from 0.79% pretreatment to 1.14% posttreatment, indicating induction of mutations in stem spermatogonia. During-treatment semen samples obtained from 2 patients treated with ABVD, which does not contain gonadotoxic alkylating agents, and 1 with NOVP also did not show any increases above the baseline mutation frequencies, indicating no increase in the minisatellite mutation frequency in spermatocytes. Thus, measurement of repeat number changes at minisatellite MS205 appears to be able to detect induced germline mutations in human sperm. However, most chemotherapy regimens do not significantly increase this class of mutations.     

Asymmetry in chromosome pairing: a major factor in de novo mutation and the production of genetic disease in man.

At the outset of the meiotic pairing process in man, trial and error mismatching and misalignment, both within homologous pairs and between heterologues, can be observed cytologically. Pairing starts at early zygotene principally within subtelomeric regions where the synaptonemal complex initiates. In the present paper, evidence for the primary role in synaptic initiation of a GC rich minisatellite in the human XY pseudoautosomal segment is presented, and circumstantial evidence is provided to support the view that GC rich sequences (minisatellites and Alu repeats) function to promote pairing within autosomes. The known sequence hypervariability of proterminal human minisatellites, it is suggested, arises as a secondary consequence of unequal exchange after misalignment between tandem repeats at the outset of the pairing process. Unequal exchange within misaligned repeat sequences at early prophase of meiosis could make a major contribution to de novo germinal mutation (conversion, duplication, deficiency, inversion, translocation), with serious consequences in man for the production of hereditary disease. For somatic tissues, rare mispairing between G rich repeats followed by unequal exchange could be a key step in cancer progression. It might also explain somatic mosaicism in some non-neoplastic clinical conditions.     

Analysis of germline variation at the FMR1 CGG repeat shows variation in the normal-premutated borderline range

In order to characterize the dynamics of CGG repeat instability at the fragile X syndrome locus (FMR1 gene), we have used small pool PCR to estimate the mutation rate within germline (sperm) and somatic tissue (leukocytes) of two normal males, one carrying the most common 29 CGG repeats allele, the other carrying a borderline normal-premutated allele of 55 repeats. Large contractions and moderate expansions of the repeat were found in sperm and blood for the 55 repeat allele while almost no variation was found in sperm or blood with the 29 repeat allele. Somatic blood DNA exhibited fewer expansions and contractions than sperm. Contractions were more frequent than expansions, and all the expansions were found in the +4 to +10 repeats range, while most of the contractions were found in the -10 to -30 range, suggesting that a subset of contractions results from a distinct mechanism. These results also suggest that the dynamics of the CGG repeat could be partly due to germline instability within the high normal or premutated ranges.      

Gametic but not somatic instability of CAG repeat length in Huntington''s disease.

Instability of a CAG repeat in 4p16.3 has been found in Huntington's disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.     

Distribution of tandem repeat polymorphism within minisatellite MS621 (D5S110)

The minisatellite MS621 (D5S110) is a highly polymorphic tandem repeat locus which maps to the distal region of human chromosome 5p. Major repeat unit variants at D5S110 differ not by base substitutions but by differences in the repetition of an 11 bp sequence motif found within each repeat. The two major classes of repeat unit thus contain two (' dimeric '= D-type) or three (' trimeric '= T-type) copies of this short motif. Mapping the distribution of these D- and T-type repeat units within alleles has allowed the analysis of the structural basis of allelic variation and of one de novo mutation. In contrast to previous studies of some other highly polymorphic minisatellites, this analysis provided no clear evidence for polarized variability at D5S110.     

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.     

Polychlorinated biphenyls induce meiotic length mutations at the human minisatellite MS32 in yeast

Polychlorinated biphenyls (PCBs) are lipophilic compounds, several of which are toxic and carcinogenic. Complex mixtures of PCBs (e.g., Aroclor) have been widely used in the industry. The persistence of PCBs, in combination with poor waste management, has led to a large-scale distribution of PCBs in the biosphere. The toxic and carcinogenic effects of PCBs are poorly understood, but are suggested to be associated with Ah receptor binding and induction of the Ah-gene battery. We have previously shown that a higher-chlorinated PCB mixture, Aroclor 1254, significantly increased the germline mutation rate at the mouse minisatellite PC-1. We have recently developed an in vitro model system to study and characterize spontaneous and induced meiotic mutations in human minisatellites integrated in yeast. Here, for the first time, we have used this model system to show that chemicals, in this case Aroclor 1254, can induce meiotic length mutations at the human minisatellite MS32 in a yeast strain harboring 38- and 42-repeat-unit alleles. The results also show that the size distribution of mutant MS32 alleles differs between PCB and the control, with a larger proportion of mutant allele sizes below 29 repeat units in the PCB series. These alleles were not structurally different from the alleles of the same size in the control. We conclude that PCBs induce minisatellite mutations in meiosis and have recombinogenic properties, and that the mutations are induced in an Ah receptor-independent manner. The induction of minisatellite mutations in meiosis as an indication of genomic damage must be taken into account in the risk assessment of PCBs and other environmental contaminants.     

Predicting human minisatellite polymorphism

We seek to define sequence-based predictive criteria to identify polymorphic and hypermutable minisatellites in the human genome. Polymorphism of a representative pool of minisatellites, selected from human chromosomes 21 and 22, was experimentally measured by PCR typing in a population of unrelated individuals. Two predictive approaches were tested. One uses simple repeat characteristics (e.g., unit length, copy number, nucleotide bias) and a more complex measure, termed HistoryR, based on the presence of variant motifs in the tandem array. We find that HistoryR and percentage of GC are strongly correlated with polymorphism and, as predictive criteria, reduce by half the number of repeats to type while enriching the proportion with heterozygosity >/=0.5, from a background level of 43% to 59%. The second approach uses length differences between minisatellites in the two releases of the human genome sequence (from the public consortium and Celera). As a predictor, this similarly enriches the number of polymorphic minisatellites, but fails to identify an unexpectedly large number of these. Finally, typing of the highly polymorphic minisatellites in large families identified one new hypermutable minisatellite, located in a predicted coding sequence. This may represent the first coding human hypermutable minisatellite.     

Short rare MUC6 minisatellites‐5 alleles influence susceptibility to gastric carcinoma by regulating gene

The human MUC6 gene, which is reported to be expressed in the stomach and gall bladder, is clustered on chromosome 11p15.5 with other secreted mucins. In this study, the genomic structure of MUC6 has been analyzed and five VNTR (minisatellites; MS1–MS5) were identified. These minisatellites were analyzed in genomic DNA extracted from 1,103 controls, 470 gastric cancer patients, and multigenerational families. Five novel minisatellites were found to be polymorphic and transmitted through meiosis by Mendelian inheritance in families. We evaluated allelic variation in these minisatellites to determine if such variation affected the susceptibility to gastric cancer. A significant association (odds ratio [OR]=7.08) between short rare MUC6–MS5 alleles and relative risks were observed for gastric cancer (95% confidence interval [CI], 1.43–35.19; P=0.005). To investigate the function of minisatellite alleles of MUC6–MS5, we examined the effects on gene expression from luciferase reporters when inserted with minisatellites. Interestingly, when the shortest allele (7TR) was inserted in the promoter, the expression level decreased over 20‐fold (P<0.001) in normal and cancer cell lines. Furthermore, the cancer‐specific rare allele (TR8) also showed decreased expression levels in cancer cells. Therefore, we suggest that the short rare MUC6–MS5 alleles may be related to cancer development by the regulation of MUC6 expression. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Finding new human minisatellite sequences in the vicinity of long CA-rich sequences.

Microsatellites and minisatellites are two classes of tandem repeat sequences differing in their size, mutation processes, and chromosomal distribution. The boundary between the two classes is not defined. We have developed a convenient, hybridization-based human library screening procedure able to detect long CA-rich sequences. Analysis of cosmid clones derived from a chromosome 1 library show that cross-hybridizing sequences tested are imperfect CA-rich sequences, some of them showing a minisatellite organization. All but one of the 13 positive chromosome 1 clones studied are localized in chromosomal bands to which minisatellites have previously been assigned, such as the 1pter cluster. To test the applicability of the procedure to minisatellite detection on a larger scale, we then used a large-insert whole-genome PAC library. Altogether, 22 new minisatellites have been identified in positive PAC and cosmid clones and 20 of them are telomeric. Among the 42 positive PAC clones localized within the human genome by FISH and/or linkage analysis, 25 (60%) are assigned to a terminal band of the karyotype, 4 (9%) are juxtacentromeric, and 13 (31%) are interstitial. The localization of at least two of the interstitial PAC clones corresponds to previously characterized minisatellite-containing regions and/or ancestrally telomeric bands, in agreement with this minisatellite-like distribution. The data obtained are in close agreement with the parallel investigation of human genome sequence data and suggest that long human (CA)s are imperfect CA repeats belonging to the minisatellite class of sequences. This approach provides a new tool to efficiently target genomic clones originating from subtelomeric domains, from which minisatellite sequences can readily be obtained. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ000377-AJ000383.]