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

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

Linkage mapping of the highly informative DNA marker D21S156 to human chromosome 21 using a polymorphic GT dinucleotide repeat

A (GT)n repeat within the anonymous DNA sequence D21S156 was shown to be highly polymorphic in DNA from members of the 40 CEPH families. At least 12 alleles of this locus were recognized by electrophoresis on polyacrylamide gels of DNA amplified by the polymerase chain reaction (PCR) using primers flanking the (GT)n repeat. The polymorphism information content was 0.82. PCR amplification of DNA from somatic cell hybrid lines mapped D21S156 to human chromosome 21 and linkage analysis localized this marker close to the loci ETS2, D21S3, and HMG14 on chromosomal band 21q22.3. This polymorphism is highly informative and can serve as an anchor locus for human chromosome 21.     

D21S210: A highly polymorphic (GT)n marker closely linked to the β-amyloid protein precursor (APP) gene

We describe a highly polymorphic (GT)n repeat with 14 alleles that is closely linked to the amyloid precursor protein (APP) gene on human chromosome 21. This marker, D21S210, will be useful for studies of linkage of disorders such as Alzheimer disease to the APP gene.     

D21S215 is a (GT)n polymorphic marker close to centromeric alphoid sequences on chromosome 21.

A plasmid, AWZ1, that contained a dinucleotide (GT)n repeat was identified from a chromosome 21-specific genomic library. When amplified by PCR from human genomic DNA, the repeat length was highly polymorphic between individuals; its location, D21S215, was mapped in the CEPH pedigrees by linkage analysis to the pericentromeric region of chromosome 21. It is the closest polymorphic marker to alphoid sequences on this chromosome.     

Rapid identification of polymorphic CA-repeats in YAC clones

Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci. The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.     

Linkage analysis of the human HMG14 gene on chromosome 21 using a GT dinucleotide repeat as polymorphic marker

A (GT)n repeat in intron 4 of the functional human HMG14 gene on chromosome 21 was used as polymorphic marker to map this gene relative to the genetic linkage map of human chromosome 21. Variation in the length of the (GT)n repeat was detected by electrophoresis on polyacrylamide gels of DNA amplified by the polymerase chain reaction using primers flanking the repeat. The observed heterozygosity of this polymorphism in 40 CEPH families was 58% with six different alleles. Linkage analysis localized the HMG14 gene close to the ETS2 gene and locus D21S3 in chromosomal band 21q22.3.     

Three CA/GT repeat polymorphisms from loci D21S414 and D21S1234 on human chromosome 21

We report three new polymorphic CA repeat microsatellites (ABM-21, ABM-C37A, and ABM-C37B) in two different loci (D21S414 and D21S1234), located in bands q21 and q11.2 of human chromosome 21 (HC21) and that were isolated from a HC21 phage library (LA21NS01). Heterozygosities for ABM-21, ABM-C37A, and ABM-C37B were 0.74, 0.50, and 0.67 respectively. These three CA repeat markers should be useful in the construction of a high resolution genetic map of this region of HC21.     

Segments containing alternating purine and pyrimidine dinucleotides: patterns of polymorphism in humans and prevalence throughout phylogeny.

Tandem dinucleotide repeats of GT or AC [(GT)n/(AC)n] where n greater than or equal to 14 are highly polymorphic and other simple repeats such as (CT)n/(AG)n and (A)n(T)n are also polymorphic. The uniformity of these sequences precludes a mechanistic differentiation between recombination or polymerase slippage. Since (GT)n/(AC)n or (CT)n/(AG)n segments of desired size were not available in our gene of interest, we analyzed a 187+ bp segment in the factor IX gene with multiple short dinucleotide repeats. This sequence contains a melody of short dinucleotide repeats which includes a 142+ bp segment of alternating purines and pyrimidines. Amplification of this sequence in 167 individuals of different ethnicity and direct sequencing of 106 individuals (23 kb of sequence) failed to reveal simple variation in the number of tandem dinucleotide repeats. However, polymorphism in the 142+ alternating purine and pyrimidine segment (RY)n was detected due to the insertion of two related repeat units of 24 bp (A) and 26 bp (B). Two previously described alleles (AB, A2B2) and two novel presumptive recombinants were found (A2B, A3B2) for a total of four alleles. An analysis of (RY)n segments in GenBank revealed an extraordinary enrichment in the genome of mammals, invertebrates, and yeast and a marked reduction in bacteria. Rodent (RY)n were larger and substantially more frequent than those in primates. When a second (RY)n was examined in the exon 8 of human factor IX gene, it was polymorphic at short repeats of (GT)n/(AC)n (n = 3-6) in Western Europeans and Koreans. In addition, an (RY)n in the dystrophin gene had four polymorphic alleles involving AT and GT dinucleotides. Thus (RY)n segments appear to be abundant and highly polymorphic. The asymmetric patterns of polymorphism and the absence of simple dinucleotide variation in 23 kb of sequence are compatible with recombination or sister chromatid exchange, but not polymerase slippage. By inference, recombination should underlie the polymorphisms at (GT)n/(AC)n since they are a subset of (RY)n and they commonly occur in the context of longer (RY)n.     

STR markers for detecting heterogeneity in Indian population

Short tandem repeats are highly polymorphic sequences of nucleotides, which are abundant in eukaryotic genome. They form approximately 3% of the total human genome and occur on average in every 10, 000 nucleotides. Due to their small dimension, low mutation, and high level of polymorphism, these markers are intensely used as important genetic markers for mapping studies, disease diagnosis, and human identity testing. In the present study allelic distribution of four autosomal short tandem repeat markers (D21S2055, D21S11, D21S1435 and D21S1411) has been analyzed in Indian population. For determination of heterogeneity and their allelic frequency QF-PCR analysis have been done. All the loci were found highly polymorphic. Marker D21S1411 was the most informative (93.6%) and D21S1435 (70.1%) was the least informative marker in Indian population.     

Microsatellites in Malus X domestica (apple): abundance, polymorphism and cultivar identification

Screening of an apple genomic library with (GA)15 and (GT)15 probes demonstrated that these repeats are abundant, occurring about every 120 and 190 kb, respectively. Microsatellites isolated from a small insert library enriched for (GA) repeats contained numbers of repeats ranging from 7 to 39. Primers to these microsatellite loci were able to direct the amplification of the repeats in 21 different cultivars. The majority of markers were highly polymorphic, diploid, and showed simple Mendelian inheritance, although about 25% of markers generated complex banding patterns consistent with the amplification of more than one locus. As few as three microsatellite markers were sufficient to differentiate between all 21 cultivars. Received: 20 February 1996 / Accepted: 24 May 1996     

Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci.

The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.     

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT)n and (CA/GT)n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty-six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT)n motif was much more abundant than the (CA/GT)n motif in these clones. The (GA/CT)n repeats also showed longer average repeat length (mean n=10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.     

Maternal uniparental disomy for human chromosome 14, due to loss of a chromosome 14 from somatic cells with t(13;14) trisomy 14.

Uniparental disomy (UPD) for particular chromosomes is increasingly recognized as a cause of abnormal phenotypes in humans. We recently studied a 9-year-old female with a de novo Robertsonian translocation t(13;14), short stature, mild developmental delay, scoliosis, hyperextensible joints, hydrocephalus that resolved spontaneously during the first year of life, and hypercholesterolemia. To determine the parental origin of chromosomes 13 and 14 in the proband, we have studied the genotypes of DNA polymorphic markers due to (GT)n repeats in the patient and her parents' blood DNA. The genotypes of markers D14S43, D14S45, D14S49, and D14S54 indicated maternal UPD for chromosome 14. There was isodisomy for proximal markers and heterodisomy for distal markers, suggesting a recombination event on maternal chromosomes 14. In addition, DNA analysis first revealed--and subsequent cytogenetic analysis confirmed--that there was mosaic trisomy 14 in 5% of blood lymphocytes. There was normal (biparental) inheritance for chromosome 13, and there was no evidence of false paternity in genotypes of 11 highly polymorphic markers on human chromosome 21. Two cases of maternal UPD for chromosome 14 have previously been reported, one with a familial rob t(13;14) and the other with a t(14;14). There are several similarities among these patients, and a "maternal UPD chromosome 14 syndrome" is emerging; however, the contribution of the mosaic trisomy 14 to the phenotype cannot be evaluated. The study of de novo Robertsonian translocations of the type reported here should reveal both the extent of UPD in these events and the contribution of particular chromosomes involved in certain phenotypes.     

Length Polymorphisms of Simple Sequence Repeat DNA in Soybean

The objective of this work was to ascertain the presence and degree of simple sequence repeat (SSR) DNA length polymorphism in the soybean [Glycine max (L.) Merr.]. A search of GenBank revealed no (CA)n or (GT)n SSRs with n greater than 8 in soybean. In contrast, 5 (AT)n and 1 (ATT)n SSRs with n ranging from 14 to 27 were detected. Polymerase chain reaction (PCR) primers to regions flanking the six SSR loci were used in PCR amplification of DNA from 43 homozygous soybean genotypes. At three loci, amplification produced one PCR product per genotype and revealed 6, 7 and 8 product length variants (alleles) at the three loci, respectively. F1 hybrids between parents carrying different alleles produced two PCR products identical to the two parents. Codominant segregation of alleles among F2 progeny was demonstrated at each locus. A soybean DNA library was screened for the presence of (CA/GT)n SSRs. Sequencing of positive clones revealed that the longest such SSR was (CA)9. Thus, (CA)n SSRs with n of 15 or more are apparently much less common in soybean than in the human genome. In contrast to humans, (CA)n SSRs will probably not provide an abundant source of genetic markers in soybean. However, the apparent abundance of long (AT)n sequences should allow this SSR to serve as a source of highly polymorphic genetic markers in soybean.     

Haplotype analysis in Australian hemochromatosis patients: evidence for a predominant ancestral haplotype exclusively associated with hemochromatosis.

Hemochromatosis (HC), an inherited disorder of iron metabolism, shows a very strong founder effect in Australia, with the majority of patients being of Celtic (Scots/Irish) origin. Australian HC patients thus provide an ideal group in which to examine HC-gene-region haplotypes, to analyze the extent of linkage disequilibrium and genetic heterogeneity in HC. We have analyzed chromosomes from 26 multiply affected HC pedigrees, and we were able to assign HC status unambiguously to 107 chromosomes--64 as affected and 43 as unaffected. The haplotypes examined comprise the following highly polymorphic markers: the serological marker HLA-A and the microsatellites D6S248, D6S265, HLA-F, and D6S105. All show highly significant allelic association with HC and no evidence of separation from the disease locus by recombination. Analysis identified a predominant ancestral haplotype comprising alleles 5-1-3-2-8 (marker order: D6S248-D6S265-HLA-A-HLA-F-D6S105), present in 21 (33%) of 64 affected chromosomes, and exclusively associated with HC (haplotype relative risk 903). No other common haplotype was significantly associated with HC. Haplotype analysis in Australian HC patients thus provides strong evidence for (a) the introduction of HC into this population on an ancestral haplotype, (b) a common mutation associated with HC in Australian patients, and (c) a candidate HC-gene region extending between and including D6S248 and D6S105.     

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides (CT)10 and (GT)10 served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.     

Characterization of 42 highly polymorphic bovine microsatellite markers

We have isolated 42 highly polymorphic microsatellite (GT/CA)_n markers from Japanese black cattle Wagyu ( Bos taurus ). Forty-one of the markers were assigned to bovine autosomes with lod scores > 6, through linkage analyses performed on the International Bovine Reference Family Panel (IBRP). The remaining marker showed X-linked inheritance. These markers exhibited an average heterozygosity value of 0.67 with between four and 17 alleles on the IBRP.     

A microsatellite polymorphism in intron 2 of human Toll-like receptor 2 gene: functional implications and racial differences

The human Toll-like receptor 2 (TLR2) mediates responses of both innate and adaptive immunity to Gram-positive bacteria, including mycobacteria. We sought functional polymorphisms in the 5'-untranslated region (UTR) of TLR2. We found a highly polymorphic (GT)n dinucleotide repeat 100 bp upstream of the TLR2 translational start site in intron 2. The numbers of GT repeats varied from 12 to 28. There were significant differences in allele distribution between African Americans and Caucasians (P=0.008) and between African Americans and Koreans (P=0.0003). The promoter activities of recombinant promoter-intron2/reporter constructs including the shortest [GT)n=12] or longest [(GT)n=28] alleles were significantly more stimulated when exposed to 200 IU ml(-1) of interferon-gamma than when exposed to 100 IU ml(-1) of GM-CSF (P<==0.03). Since TLR2 plays a critical role in the human innate immune response, this functional microsatellite polymorphism may be important in the pathogenesis of infectious and inflammatory diseases.     

Characterization of a highly polymorphic region near the first exon of the human MAOA gene containing a GT dinucleotide and a novel VNTR motif.

The genes encoding the A and B forms of the human monoamine oxidase enzymes (MAOA and MAOB) are localized at Xp11.23-Xp11.4. We report the characterization of a highly informative polymorphic region within a 2.9-kb cloned fragment containing the first exon of the MAOA gene. The polymorphic region consists of a GT microsatellite directly adjacent to an imperfectly duplicated novel 23-bp VNTR motif. DNA sequencing within and flanking the repeated segment allowed the design of specific amplification primers. In 56 unrelated females, 15 different alleles were identified with sizes ranging from 285 to 388 bp. The alleles differed in both the number of dinucleotide and the number of VNTR repeats, yielding a highly informative polymorphic marker locus with a calculated heterozygosity value of 75%.