Multiple Microsatellites Research Articles

Worldwide analysis of multiple microsatellites: Language diversity has a detectable influence on DNA diversity

Previous studies of the correlations between the languages spoken by human populations and the genes carried by the members of those populations have been limited by the small amount of genetic markers available and by approximations in the treatment of linguistic data. In this study we analyzed a large collection of polymorphic microsatellite loci (377), distributed on all autosomes, and used Ruhlen's linguistic classification, to investigate the relative roles of geography and language in shaping the distribution of human DNA diversity at a worldwide scale. For this purpose, we performed three different kinds of analysis: (i) we partitioned genetic variances at three hierarchical levels of population subdivision according to language group by means of a molecular analysis of variance (AMOVA); (ii) we quantified by a series of Mantel's tests the correlation between measures of genetic and linguistic differentiation; and (iii) we tested whether linguistic differences are increased across known zones of increased genetic change between populations. Genetic differences appear to more closely reflect geographic than linguistic differentiation. However, our analyses show that language differences also have a detectable effect on DNA diversity at the genomic level, above and beyond the effects of geographic distance.

Genome-Wide Single Nucleotide Polymorphism Analysis in Juvenile Myelomonocytic Leukemia Uncovers Long-Range Uniparental Disomy Surrounding the NF1 Locus in Cases Associated with Type 1 Neurofibromatosis but Not in Cases with Mutant RAS or PTPN11.

Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder of early childhood with myeloproliferative and myelodysplastic properties. The proliferative component is a result of RAS pathway deregulation caused by somatic mutation in the RAS or PTPN11 oncogenes (60% of cases) or by underlying neurofibromatosis type 1 (NF-1) with a germline NF1 gene defect (clinically 11% of cases). To search for potential collaborating genetic abnormalities, we used Affymetrix GeneChip Mapping 50K arrays to analyze over 116,000 single nucleotide polymorphisms (SNPs) across the genome using DNA from bone marrow or peripheral blood granulocytes from 16 children with JMML and normal karyotype (mutant RAS, n=4; mutant PTPN11, n=7; NF-1, n=5). Quantitative evaluation of hybridization intensities at each SNP locus failed to identify recurrent allelic gains or losses in the 16 cases. We were specifically interested in chromosome 7 because monosomy 7 or interstitial/terminal 7q deletions are found in about 30% of JMML cases and it is conceivable that submicroscopic 7q lesions occur in the other cases but remain undetected by standard techniques. However, at the resolution provided by the arrays used here, we saw no evidence for genomic copy number alterations on chromosome 7. Interestingly, evaluation of the SNP allelotypes identified large regions of copy-neutral loss of heterozygosity (LOH) on chromosome 17q, including the NF1 locus, in 4 of the 5 samples from patients with JMML and NF-1. The LOH region spanned a genomic range of approximately 55 Mbp in each case and included more than 1,400 contiguous SNPs. Allelic copy numbers were normal within the homozygous regions, indicating uniparental isodisomy (UPD). Compatible with isodisomy, 17q was normal in the corresponding conventional karyotypes. By contrast, the array data provided no evidence for 17q UPD in any of the 12 JMML cases without NF-1. In all four cases with 17q UPD, the recombination breakpoints appeared to be confined to a 400-kbp region on 17q11.1–17q11.2; however, lack of parental or nonleukemic DNA precluded definitive mapping of the breakpoints. Of note, the 17p chromosomal arm retained heterozygosity in all cases, indicating that the p53 tumor suppressor was not affected by the UPD event. We confirmed 17q disomy in the four NF-1 samples using matrix-based comparative genomic hybridization and are currently verifying homozygosity of multiple microsatellites spaced across chromosome 17. Furthermore, NF1 mutational analysis is under way to show that the individual lesion within this tumor suppressor gene is biallelic in leukemic cells from patients with JMML and NF-1. In summary, we assume that a mitotic recombination event in an early hematopoietic progenitor cell led to UPD involving the NF1 locus. Our observations provide strong confirmatory evidence that it is indeed the NF1 gene that is responsible for the increased incidence of JMML in NF-1 patients. In addition, our data support the emerging role of mitotic recombination as a second hit in leukemogenesis and corroborate the concept that RAS pathway deregulation is central to JMML pathogenesis.

A Universal Microsatellite Multiplex Kit for Genetic Analysis of Great Apes

DNA profiling with microsatellite markers is a commonly used genetic method of studying the great apes. An efficient method of generating the genetic data is amplification of multiple microsatellites in a single PCR reaction. Here we describe a PCR multiplex in which 9 genetic markers can be amplified simultaneously, thereby saving time, expenses and DNA. This marker system can discriminate between all the great ape species except bonobos and chimpanzees. Furthermore, the cumulative probability of identity values were low for all 4 species tested.

Genetic Signatures of Coancestry within Surnames

Surnames are cultural markers of shared ancestry within human populations. The Y chromosome, like many surnames, is paternally inherited, so men sharing surnames might be expected to share similar Y chromosomes as a signature of coancestry. Such a relationship could be used to connect branches of family trees, to validate population genetic studies based on isonymy, and to predict surname from crime-scene samples in forensics. However, the link may be weak or absent due to multiple independent founders for many names, adoptions, name changes and nonpaternities, and mutation of Y haplotypes. Here, rather than focusing on a single name, we take a general approach by seeking evidence for a link in a sample of 150 randomly ascertained pairs of males who each share a British surname. We show that sharing a surname significantly elevates the probability of sharing a Y-chromosomal haplotype and that this probability increases as surname frequency decreases. Within our sample, we estimate that up to 24% of pairs share recent ancestry and that a large surname-based forensic database might contribute to the intelligence-led investigation of up to approximately 70 rapes and murders per year in the UK. This approach would be applicable to any society that uses patrilineal surnames of reasonable time-depth.

GCF2/LRRFIP1 Represses Tumor Necrosis Factor Alpha Expression

Tumor necrosis factor alpha (TNF-alpha) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-alpha -308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-alpha in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the -308 site in cells that do not make TNF-alpha. Cells competent to produce TNF-alpha have Ets-1 bound to the -308 promoter site. Active transcription is accompanied by NF-kappaB and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-alpha promoter, particularly at the -308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-alpha repressor identified.

Comparative Informativeness for Linkage of Multiple SNPs and Single Microsatellites

Single nucleotide polymorphisms (SNPs), or biallelic markers, are popular in genetic linkage studies due to their abundance in the genome, stability, and ease of scoring. We determined the ‘information ratio’ (IR) of closely spaced SNPs in simulated nuclear families and affected sib pairs (ASPs). (The IR is the ratio of actual average maximum lod score to the maximum lod score attainable if the marker were fully informative.) The nuclear families included parental information, whereas the ASPs did not. We analyzed these SNPs in two ways: (1) using multipoint analysis, and (2) treating the SNPs as ‘composite markers’ (i.e., haplotypes, as assigned by GENEHUNTER). (3) We also calculated the IR of a single microsatellite marker with multiple alleles and compared with the IR from the SNPs. For each set of input conditions, we simulated 1000 nuclear families, of 2, 3, 4, or 5 children each, as well as 1000 ASPs. We generated SNP marker data for strings of k = 1, 2, 3, 5, 7, and 10 SNP loci, with no recombination (Θ = 0) and no linkage disequilibrium among the SNPs. The MAF (minor allele frequency) was either 0.5 or 0.25, and allele frequencies were the same for all k loci in any analysis. We also generated marker data for one single-locus microsatellite marker, with m = 3, 4, 5, 6, 7, and 9 equally frequent alleles. In all simulations, the disease was fully penetrant dominant, and there was no recombination or linkage disequilibrium among markers or between marker and disease. When multipoint analysis was used, we found that 5–7 closely spaced SNPs were usually enough to yield an IR of ≈100%, for nuclear families of any size. However, for the ASPs, even 7–10 SNPs yielded an IR of only 70–80%. A microsatellite with 9 equally frequent alleles yielded about the same IR (86–88%) as a string of 4–5 SNPs, in nuclear families. SNPs analyzed as ‘composite markers’ analyses performed worse, due to the inherent ambiguity of SNP haplotyping.

Microsatellites in starch-synthesizing genes in relation to starch physicochemical properties in waxy rice ( Oryza sativa L.).

Rice starch is composed of amylose and amylopectin. Amylose content, an important determinant of rice starch quality, is primarily controlled by the waxy gene, encoding granule-bound starch synthase (GBSS). The starch branching enzyme (SBE) and soluble starch synthase (SSS) play major roles in the synthesis of amylopectin. Microsatellite polymorphisms in the three genes, the wx gene encoding granule-bound starch synthase I, the SBE gene encoding starch branching enzyme I and the SSS gene encoding soluble starch synthase I, were studied for 56 accessions of waxy rice ( Oryza sativa L.). Four (CT)(n) microsatellite alleles, (CT)(16), (CT)(17), (CT)(18) and (CT)(19), at the wx locus were detected in this set of waxy rice, of which (CT)(17) was the most frequent. Three (CT)(n) microsatellite allele classes were found at the SBE locus, (CT)(8) or (CT)(10) together with an insertion sequence of CTCTCGGGCGA, and (CT)(8) alone without the insertion. There were multiple microsatellites clustered at the SSS locus. However, these alleles can also be grouped into three classes, i.e. the allele class SSS-A = (AC)(2) em leader TCC(TC)(11) em leader (TC)(5)C(ACC)(11), the allele class SSS-B = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(9), and the allele class SSS-C = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(8). The analyses of starch physicochemical properties among different microsatellite genotypes indicated that the waxy rice group with the (CT)(19) allele, the SBE-A allele and the SSS-B allele was quite different from other groups. Nine out of 15 accessions with a high gelatinization temperature (GT) belonged to the wx (CT)(19) group, all of them belonged to the SBE-A group and 13 of them belonged to the SSS-B group. These microsatellites might be useful in marker-assisted breeding for the improvement of rice grain quality.

Livestock variation of linked microsatellite markers in diverse swine breeds

A panel of nine framework microsatellites (MS) linked to the Calcium Release Channel (CRC) locus on swine chromosome 6 (SSC6) was developed from the consensus genetic map. MS were screened across groups of unrelated animals from Yorkshire, Hampshire, Duroc, Landrace and Meishan swine breeds. Unique MS alleles for Yorkshire, Duroc, Landrace and Meishan breeds, and statistically significant (P<.05) associations between breeds and allele frequencies were found for each MS. Although breed marker heterozygosities ranged from 0.0 (S0035 in Duroc) to 0.92 (S0087 in Meishan), Correspondence Analysis identified MS alleles uniquely associated with either the Meishan breed, western breeds or alleles common to all breeds. Furthermore, an overall marker heterozygosity of <0.70 demonstrates the need for multiple MS panels to accommodate reduced within‐breed differences for identification of quantitative trait loci (QTL), marker assisted selection (MAS) programs or parental identification in commercial breeds.

New microsatellite markers in chicken optimized for automated fluorescent genotyping.

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4.0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).

A DNA fingerprinting system for the ectoparasite Sarcoptes scabiei

We describe multiple hypervariable microsatellites that will provide a highly informative genetic marker system for the sarcoptid mite Sarcoptes scabiei. Eighteen positive clones containing the highly repetitive sequence (GA) n were isolated from a partial genomic library of S. scabiei. Ten of these clones were characterised by sequencing and primers were designed from the unique sequences flanking eight microsatellite loci. Genomic DNA was subsequently extracted from individual mites and the repeat blocks were amplified by way of [γ 33P] ATP end-labelled polymerase chain reaction. Fragment length polymorphisms were revealed in three of the loci when resolved on polyacrylamide sequencing gels. The high levels of allelic variability demonstrated between individual mites enable these three loci to form a DNA fingerprinting system that will be suitable for epidemiological and taxonomic studies both within and between host species.