Paradoxical contraction of pelvic floor muscles: clinical significance

Accurate quantification of relative allele frequencies in pooled DNA samples can be carried out for microsatellite markers having a dinucleotide repeat unit, conditional on the absence of overlapping "shadow" bands. This provides a basis for extending DNA pooling to this useful class of DNA marker. Expressions for the standard error of densitometric estimates of allele frequencies from pooled samples are presented, and their statistical application is illustrated in a variety of situations. This enables DNA pooling to be utilized in situations requiring the testing of statistical hypotheses concerning differences in allele frequencies between populations, or samples.     

Chromosome-specific panels of tri- and tetranucleotide microsatellite markers for multiplex fluorescent detection and automated genotyping: evaluation of their utility in pathology and forensics

A set of 391 microsatellite markers (Weber set 6), 85% of which consist of tri- and tetranucleotide repeat markers, were used to design chromosome-specific panels that allowed for a high degree of multiplexing with respect to the fragment size range and fluorophore (FAM, HEX, TET). This marker set has an average coverage of 10.5 cM, with the largest gap being 28.1 cM. The markers were divided into 49 panels, with a maximum degree of multiplexing of 15 markers per panel. The utility of the markers for analysis of DNA from blood, hair, and formalin-fixed archival tissue biopsies was evaluated with respect to amplification efficiency, product yield, and degree of preferential amplification of the shorter allele in heterozygotes. The amplification efficiency was inversely related to repeat length and amplicon length. Based on the analysis of DNA from formalin-fixed biopsies, 51 markers suitable for loss of heterozygosity (LOH) studies were identified. The utility of the marker set for genome scanning, LOH, and forensic analyses is discussed.     

Multiple levels of single-strand slippage at cetacean tri- and tetranucleotide repeat microsatellite loci

Between three and six tri- and tetranucleotide repeat microsatellite loci were analyzed in 3720 samples collected from four different species of baleen whales. Ten of the 18 species/locus combinations had imperfect allele arrays, i.e., some alleles differed in length by other than simple integer multiples of the basic repeat length. The estimate of the average number of alleles and heterozygosity was higher at loci with imperfect allele arrays relative to those with perfect allele arrays. Nucleotide sequences of 23 different alleles at one tetranucleotide repeat microsatellite locus in fin whales, Balaenoptera physalus, and humpback whales, Megaptera novaeangliae, revealed sequence changes including perfect repeats only, multiple repeats, and partial repeats. The relative rate of the latter two categories of mutation was estimated at 0.024 of the mutation rate involving perfect repeats only. It is hypothesized that single-strand slippage of partial repeats may provide a mechanism for counteracting the continuous expansion of microsatellite loci, which is the logical consequence of recent reports demonstrating directional mutations. Partial-repeat mutations introduce imperfections in the repeat array, which subsequently could reduce the rate of single-strand slippage. Limited computer simulations confirmed this predicted effect of partial-repeat mutations.     

Detection of loss of heterozygosityat microsatellite loci in esophageal squamous-cell carcinoma

Microsatellite alterations at 3 genetic loci (chromosomes 2p, 3p and 17p) were analyzed in 25 tumors (20 primary tumors and 5 metastatic lymph nodes) from 20 patients after surgical treatment for esophageal cancer. DNA samples from tumors were compared with control DNA from lymphocytes obtained from the peripheral blood of the individual patients. Microsatellite alterations [microsatellite instability (MSI) and loss of heterozygosity (LOH)] were detected in 15% of 20 primary tumors with marker D2S123 (chromosome 2p), 55% with marker D3S1067 (chromosome 3p) and 50% with marker TP53 (chromosome 17p). The 3-year disease-free survival rate of the 10 patients who had tumors without alterations or with an alteration at only 1 of 3 microsatellite loci was 75% and it was better than that of the 10 patients who had tumors with alterations at 2 or 3 microsatellite loci (48%, p = 0.049). This finding suggests that esophageal cancer with alterations at multiple microsatellite loci might have strong malignant potential. However, MSI was only detected in one of 20 patients, which suggests that MSI might not play an important role in the development of this cancer. Three of 5 metastatic lymph nodes showed no LOH even though primary tumors of these patients exhibited LOH with 1 or 2 markers, and 1 metastatic lymph node had LOH that was detected with D3S1067 even though the primary tumor of this patient had no LOH with all markers. Thus, clonal heterogeneity might exist in esophageal squamous-cell carcinomas.     

The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes

Microsatellites can be highly unstable and show a high level of polymorphism between individuals. Here we present the analysis of the CAG trinucleotide repeat polymorphism at the SBMA locus in 57 phenotypically normal individuals rigorously assigned to the Spanish Basque population. Results are compared with 100 Spanish non-Basque individuals who were already analyzed by us (175 alleles). This is the first study undertaken in these populations for this marker. In addition, we compared our results with those published for other populations. Relative allele frequencies showed differences between the samples and no unimodal distribution. The expected heterozygosity in the Basque sample was slightly lower than in the non-Basque sample. Conformity with Hardy-Weinberg equilibrium was verified by three tests. When compared with published data, the predominant alleles appear to be the same in the various populations. There are more differences between Basques and other Caucasoid samples than between non-Basques and Caucasoid samples. Population relationships were also examined by dendrograms based on genetic distances. The results obtained showed some peculiarities in the Basque population. The high degree of similarity with other dendrograms based on different markers and the efficiency of this STR marker in differentiating closely related populations, support the potential usefulness of microsatellites as tools for human population studies.     

Human bladder cancers and normal bladder mucosa present the same hot spot of heterozygous chromosome-9 deletion

Loss of heterozygosity (LOH) on chromosome 9 is the most frequent genetic alteration in bladder cancer identified to date, suggesting the presence of key gene(s) for this pathology. In this study, we examined 44 bladder tumors and 21 normal bladder samples for LOH on both arms of chromosome 9. Sixteen microsatellite markers, 12 on the short arm (encompassing 9p21-22) and 4 on the long arm (encompassing 9q33-34), were chosen for their highly frequent alterations in bladder cancer. LOH for at least one marker was identified in 42 tumor samples (95.5%), and 14 tumors (32%) displayed LOH for all informative tested markers. Detailed analysis showed that 2 markers on chromosome 9p (D9S157 and D9S156) had the highest frequencies of allelic loss (about 70%), independent of tumor grade and stage. The same study was performed on the 21 normal bladder mucosa samples: 50% of informative cases presented a single specific LOH at the D9S156 locus. Normal samples showing LOH at this locus were therefore screened with 3 novel microsatellite markers in the 810-kb region incorporating D9S156. Using this marker, we found no further heterozygous loss in this region. This result allows different interpretations of the D9S156 loss in normal bladder mucosa, and suggests that D9S156 may be more an indicator of bladder epithelium impairment than a tumor-initiation marker. Similarly, this unexpected result calls in question the interpretation of LOH studies.     

Parentage and kinship studies in an obligate brood parasitic bird, the brown-headed cowbird (Molothrus ater), using microsatellite DNA markers

Recent studies suggest that single-locus microsatellite DNA markers have the potential to unambiguously resolve parentage among individuals in natural populations where maternity is known. However, their power for determining parentage when neither parent is known is unclear. Here we investigate the usefulness of microsatellite DNA markers to determine parentage in a brood parasitic bird, the brown-headed cowbird (Molothrus ater), where, for a given offspring, no a priori knowledge of either parent is available. Seven polymorphic microsatellite DNA markers isolated from brown-headed cowbirds and yellow warblers (Dendroica petechia) were used to genetically characterize an individually marked breeding population of male and female cowbirds at Delta Marsh, Manitoba. Forty-four males, 21 females, and 61 cowbird chicks were genotyped at seven loci using DNA amplified from blood and tissue samples. The mean exclusion probabilities pooled across all seven loci were 0.9964 for males and 0.9948 for females. Two null (non-amplifying) alleles at one locus were discovered and accounted for by constructing alternate nonoverlapping primer sets. Exclusion analyses performed using all individuals determined both paternity and maternity for 43 chicks and paternity only for 4 chicks. Another microsatellite locus was then used to determine paternity for three additional chicks. Relatedness analyses placed 12 of the 18 remaining chicks not assigned both maternity and paternity into four unique full sibling groups. Overall, 90.16% (55 of 61) of all offspring examined were placed into distinct parent/sibling groups, demonstrating that this marker set is extremely useful for parentage studies in this species.     

Chromosome-specific microsatellite sets for fluorescence-based, semi-automated genome mapping

To facilitate large-scale genetic mapping of the human genome, we have developed chromosome-specific sets of microsatellite marker loci suitable for use with a fluorescence-based automated DNA fragment analyser. We present 254 dinucleotide repeat marker loci (80% from the Généthon genetic linkage map) arranged into 39 sets, covering all 22 autosomes and the X chromosome. The average distance between adjacent markers is 13 centiMorgans, and less than 4% of the genome lies more than 20 cM from the nearest marker. Each set of microsatellites consists of up to nine marker loci, with allele size ranges that do not overlap. We selected marker loci on the basis of their reliability in the polymerase chain reaction, polymorphism content, map position and the accuracy with which alleles can be scored automatically by the Genotyper program.     

Estimation of numbers of malaria clones in blood samples

Methods are derived for estimating the mean number of clones of the haploid malaria parasite Plasmodium falciparum from samples of blood of infected hosts which have been tested for the presence of alleles at marker loci. For example, at a locus with three alleles the sample might contain only A1, or A1 and A2, or A1, A2 and A3, with multiple allele classes being more common at high infection rates. Assuming either a Poisson or negative binomial distribution of numbers of infections per host, formulae are derived for the frequency of different classes of blood samples, and maximum likelihood methods are used to estimate the mean number of clones and allele frequencies. Two data sets, each on two loci, are analysed. One data set was from the same locality in Tanzania from which oocysts of the parasite in mosquito vectors were tested for clonality (i.e. diploid unions of gametes from the same clone) using genetic markers. Good agreement was obtained between the observed clonality in oocysts and that expected from the number of infections per host (mean approximately three).     

Usefulness of molecular markers for detecting population bottlenecks via monitoring genetic change

It is important to detect population bottlenecks in threatened and managed species because bottlenecks can increase the risk of population extinction. Early detection is critical and can be facilitated by statistically powerful monitoring programs for detecting bottleneck-induced genetic change. We used Monte Carlo computer simulations to evaluate the power of the following tests for detecting genetic changes caused by a severe reduction in a population's effective size (Ne): a test for loss of heterozygosity, two tests for loss of alleles, two tests for change in the distribution of allele frequencies, and a test for small Ne based on variance in allele frequencies (the 'variance test'). The variance test was most powerful; it provided an 85% probability of detecting a bottleneck of size Ne = 10 when monitoring five microsatellite loci and sampling 30 individuals both before and one generation after the bottleneck. The variance test was almost 10-times more powerful than a commonly used test for loss of heterozygosity, and it allowed for detection of bottlenecks before 5% of a population's heterozygosity had been lost. The second most powerful tests were generally the tests for loss of alleles. However, these tests had reduced power for detecting genetic bottlenecks caused by skewed sex ratios. We provide guidelines for the number of loci and individuals needed to achieve high-power tests when monitoring via the variance test. We also illustrate how the variance test performs when monitoring loci that have widely different allele frequency distributions as observed in five wild populations of mountain sheep (Ovis canadensis).     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

A simple method for analyzing microsatellite allele image patterns generated from DNA pools and its application to allelic association studies

Allelic association studies provide the most powerful method for locating genes of small effect contributing to complex diseases and traits. However, in outbred populations, allelic association is usually maintained only over distances of <=1 cM. Therefore, systematic searches over large regions are costly. Here we present a method involving DNA pooling that can be used as a rapid preliminary screen for allelic association with the most common class of polymorphic markers, single-sequence repeats. Patient and control samples are pooled separately, and markers are typed in the two pools. By use of primers with fluorescent 5' ends, PCR products can be analyzed on an automated sequencing apparatus. Allele image patterns (AIPs) produced for the two groups are overlaid and differences in pattern area between pools computed. From this, a DeltaAIP statistic is calculated from the difference in areas between the two AIPs expressed as a fraction of the total shared and nonshared area. AIPs of a range of different-sized pools were generated by computer simulation for markers with a range of allele sizes and frequencies. DeltaAIPs from pools and chi2 values for individual genotypings were compared, with both simulated and real data from microsatellite markers. The results demonstrated a high correlation between DeltaAIP and chi2 values. DeltaAIP analysis of real microsatellite data indicated the feasibility of using this method in systematic searches for allelic association and generated a small number of false positives but few false negatives. We conclude that DeltaAIP analysis of DNA pools can be used effectively and efficiently as a rapid screen for allelic association in case-control studies.     

Loss of heterozygosity on the long arm of human chromosome 7 in sporadic renal cell carcinomas

Cytogenetic and molecular analysis of DNA sequences with highly polymorphic microsatellite markers have implicated allele loss in several chromosomal regions including 3p, 6p, 6q, 8p, 9p, 9q, 11p and 14q in the pathogenesis of sporadic renal cell carcinomas (RCCs). Deletions involving the long arm of chromosome 7 have not been described in RCCs although they have been seen in several other tumor types. However, there have been no detailed analysis of loss of heterozygosity (LOH) of 7q sequences in sporadic RCCs. We therefore studied LOH for DNA sequences on 7q with 10 highly polymorphic markers in 92 matched normal/tumor samples representing sporadic RCCs including papillary, nonpapillary, and oncocytomas in order to determine whether allelic loss could be detected in a tumor type with no visible 7q rearrangements at the cytogenetic level. We found chromosome 7q allele loss in 59 of 92 cases (64%) involving one, two, or more microsatellite markers. The most common allele loss included loci D7S522 (24%) and D7S649 (30%) at 7q31.1-31.2, a region that contains one of the common fragile sites, FRA7G. By comparative multiplex PCR analysis, we detected a homozygous deletion of one marker in the 7q 31.1-31.2 region in one tumor, RC21. These results support the idea that a tumor suppressor gene in 7q31 is involved in the pathogenesis of sporadic renal cell carcinomas.     

Population variation at the polymorphic ApaLI restriction enzyme site in intron 5 of the WT1 gene

We examined 63 unrelated individuals from the United States for the Apa-LI polymorphism in intron 5 of the WT1 gene. Allele frequencies of 0.13 and 0.87 for the A and B alleles, respectively, and a heterozygosity index of 24% contrast sharply with previous data obtained in the Japanese population where allele frequencies of 0.55 and 0.45 for the A and B alleles and a heterozygosity index of 59% were reported. These data suggest genetic heterogeneity at the WT1 locus, which may contribute to the differences in the incidence of Wilms tumor between the two population groups.     

Allelic frequencies of twelve dinucleotide repeat marker loci on chromosome 13 in the normal Japanese population

To establish a genotypic database for dinucleotide repeat marker loci in the Japanese population, we determined allelic frequencies of 12 such markers on chromosome 13 and compared them with data from Caucasians in the GDB archive. The average heterozygosity (79%) for the 12 loci was the same for the two populations. However, allelic distributions at two of the marker loci were quite different. These data will be useful for disease studies in the Japanese population that involve linkage or sibship-pair analyses, or association studies.     

Historical analysis of genetic variation reveals low effective population size in a northern pike (Esox lucius) population

Effective population size (Ne) of a natural fish population was estimated from temporal changes in allele frequencies at seven microsatellite loci. Use of a historical collection of fish scales made it possible to increase the precision of estimates by increasing the time interval between samples and to use an equation developed for discrete generations without correcting for demographic parameters. Estimates of Ne for the time intervals 1961-1977 and 1977-1993 were 35 and 72, respectively. For the entire interval, 1961-1993, the estimate of Ne was 48 when based on a weighted mean derived from the above two estimates or 125 when calculated from 1961 and 1993 samples only. Corresponding ratios of effective size to adult census size ranged from 0.03 to 0.14. An Ne of 48 over a 32-year period would imply that this population lost as much as 8% of its heterozygosity in that time. Results suggest the potential for using genetic methods based on microsatellite loci data to compare historical trends in Ne with population dynamic parameters. Such comparisons will help to evaluate the relationship between genetic diversity and long-term persistence of natural populations.     

Definition and distribution analysis of glycoprotein B gene alleles of human herpesvirus 7

As for other herpesviruses, glycoprotein B (gB) of human herpesvirus 7 (HHV-7) is believed to play a major role in virus infection and as a target of the host immunogenic response. Using nested PCR, we amplified the whole HHV-7 gB gene from 108 human peripheral blood mononuclear cell samples and studied its variability. By means of restriction fragment length polymorphism (RFLP) analysis, three distinct patterns, designated I, II, and III, were defined and detected at frequencies of 93, 5, and 2%, respectively. Determination of the nucleotide sequence allowed us to recognize five critical positions in the gB gene with six specific combinations of point changes at these positions. These combinations were gB alleles A, B, C, D, E, and F. Alleles D and E corresponded to RFLP patterns II and III, respectively, while the other four alleles corresponded to RFLP pattern I. Identical gB alleles were detected in serial samples as well as in paired samples of blood and saliva from the same individuals, except for one case. In contrast, the distribution of gB alleles differed according to the geographical origin of the human samples: C was the most frequent allele in both African and Caribbean samples, whereas F was the most frequent allele in European ones. Although none of the allele-specific nucleotide changes induced any modification at the protein level, the definition of gB alleles provided convenient viral markers for the study of both HHV-7 infections and human population genetics.     

Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients

We performed microsatellite analysis and tested telomerase activity in paired tissue and urine of bladder cancer patients from frozen archived samples. DNA obtained from microdissected tumor and urine sediment was analyzed and compared to peripheral lymphocytes for microsatellite alterations (loss of heterozygosity [LOH] or instability) using a panel of 20 microsatellite markers in 15 patients with transitional or squamous cell carcinoma of the urinary tract. Additionally, telomerase activity was determined in 12 microdissected tumor specimens and corresponding frozen urine pellets. Tumor cell DNA was detected by microsatellite analysis (LOH or shift) in at least one marker in 14/15 microdissected tumor specimens and in 13/15 DNA samples obtained from urine sediments. Telomerase activity was present in 11/12 tumor samples but could not be detected in any of the corresponding urine sediments. Frozen archived urine samples are useful for retrospective studies utilizing microsatellite analysis or other PCR-based approaches after DNA extraction. However, the evaluation of telomerase protein activity in stored urine samples appears to be unsuitable.     

Characterization of ancestral and derived Y-chromosome haplotypes of New World native populations

We analyze the allelic polymorphisms in seven Y-specific microsatellite loci and a Y-specific alphoid system with 27 variants (alphah I-XXVII), in a total of 89 Y chromosomes carrying the DYS199T allele and belonging to populations representing Amerindian and Na-Dene linguistic groups. Since there are no indications of recurrence for the DYS199C-->T transition, it is assumed that all DYS199T haplotypes derive from a single individual in whom the C-->T mutation occurred for the first time. We identified both the ancestral founder haplotype, 0A, of the DYS199T lineage and seven derived haplogroups diverging from the ancestral one by one to seven mutational steps. The 0A haplotype (5.7% of Native American chromosomes) had the following constitution: DYS199T, alphah II, DYS19/13, DYS389a/10, DYS389b/27, DYS390/24, DYS391/10, DYS392/14, and DYS393/13 (microsatellite alleles are indicated as number of repeats). We analyzed the Y-specific microsatellite mutation rate in 1,743 father-son transmissions, and we pooled our data with data in the literature, to obtain an average mutation rate of.0012. We estimated that the 0A haplotype has an average age of 22,770 years (minimum 13,500 years, maximum 58,700 years). Since the DYS199T allele is found with high frequency in Native American chromosomes, we propose that 0A is one of the most prevalent founder paternal lineages of New World aborigines.     

Misidentification rate in the Israeli dairy cattle population and its implications for genetic improvement

The DNA microsatellites can be efficiently used to determine incorrect paternity attribution of cattle without genotyping of dams. Allelic frequencies of the population were determined for 12 microsatellites using the maternal alleles of 102 AI sires. The frequency of the most common microsatellite allele ranged from 0.27 to 0.58. Most loci had at least one allele that was present in only a single individual. Paternity of 9 of 173 cows (5.2%) and 3 of 102 bulls (2.9%) was excluded because putative paternal alleles were not present in progeny for at least one locus. For 4 of the 9 cows and all 3 bulls, exclusion was based on at least two loci. Mean probability of exclusion was 0.85 for cows and 0.99 for bulls. With an assumed cost of US $5 per genotype, a misidentification rate of 5%, and a discount rate of 0.05, additional profit for the Israeli-Holstein breeding program from genotyping 100 test daughters of each young sire becomes positive within 10 yr and reaches nearly US $2.4 million after 20 yr.