Tetranucleotide Repeat Sequences Research Articles

Characterization of perfect microsatellite based on genome-wide and chromosome level in Rhesus monkey (Macaca mulatta)

Microsatellite studies based on chromosomes level would contribute to the biometric correlation analysis of chromosome and microsatellite applications on the specific chromosome. In this study, the total microsatellite length of 1,141,024 loci was 21.8Mb, which covered about 0.74% of the male Rhesus monkey genome. Perfect mononucleotide SSRs were the most abundant, followed by the pattern: perfect di->tetra->tri->penta->hexanucleotide SSRs. The main range of repeat times focused on 12–32 times (mono-), 7–23 times (di-), 5–10 times (tri-), 4–14 times (tetra-), 4–9 times (penta-), 4–8 times (hexa-), respectively. The largest SSRs number was found in chromosome 1 with 94,347 loci, followed by chromosome 3, 2, 7 and 5, and the smallest number was in chromosome 18. The predominant repeat types in male Rhesus monkey genome and chromosome Y were basically A, AC, AG, AAT, AAC, AAAT, AAAC, AAAG, AAACA and AAACAA. SSRs number of all chromosomes was closely positively correlated with chromosome sequence size (r=0.969, p<0.01), and significantly negatively correlated with abundance (r=−0.24, 0.01<p<0.05). The lengths of all chromosomes were significantly negatively correlated with microsatellite density (r=−0.456, 0.01<p<0.05), and relative abundance and density of SSRs in all chromosomes were significantly negatively correlated with SSR GC content (r=−0.939/−0.928, p<0.01). The SSRs GC content on chromosome X (accounting for 16.71%) was found to be the highest in female Rhesus monkey, which might contributed to the DNA methylation of CpG islands for sex chromosome X inactivation and expression regulation. These results and exported tetranucleotide repeat sequences in each chromosome for primer design would facilitate the exploration of microsatellites structural function, composition mode and molecular markers development in Rhesus monkey genome.

Colorectal Cancer: Sailing with a T-Cell EMAST

During the past two decades, considerable progress has been made in understanding and defining the molecular basis of human colorectal cancer (CRC). The genetic heterogeneity of this disease is now well-established, and it has become clear that a distinct subgroup of CRC possesses a unique form of genetic instability, defined as microsatellite instability (MSI). The MSI phenotype is a hallmark of defective DNA mismatch repair (MMR), and inactivation of MMR genes permits increased instability and accumulation of genomic alterations in the sequences containing mono[e.g.(A)n] and di-nucleotide [e.g. (CA)n] microsatellite repeats. Approximately 12–15% of CRC exhibit high frequency MSI (MSI-H), which includes the majority of Lynch syndrome cancers with germline mutation of a MMR gene and about 12% of sporadic tumors due to methylation-induced transcriptional silencing of the MLH1 gene [1]. The remaining 85% of CRCs either possess very low levels of MSI (MSI-L) or are microsatellite stable (MSS); however, the molecular basis of these tumors remains poorly understood. Almost a decade ago, Sidransky and colleagues described a distinct form of genetic instability present in respiratory tumors, which was termed as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) [2]. In contrast to instability at the monoand di-nucleotide repeats seen in MSI CRCs, EMAST tumors are identified when the frameshift mutations occur at certain tetranucleotide repeat sequences [e.g. (AAAG)n or (ATAG)n]. Since its initial discovery in non-small cell lung cancers [2, 3], EMAST has subsequently been observed in several other human cancers, including skin [4], ovarian [5], endometrial [6], bladder [4, 7], prostate [8, 9] and colorectal [10–13]. Most microsatellite sequences (either mono-, dior tetra-nucleotide repeats) are present in the non-coding regions of DNA; however, small minorities of these tandem repeats exist within the coding sequences of growth regulatory genes that play a critical role in driving CRC pathogenesis. Frameshift mutations within this latter class of genes, such as observed with EMAST, have direct functional consequences for the growth and proliferation of tumor cells. The underlying molecular mechanism(s) causing EMAST remain unclear. The earliest reports in this regard proposed an association of EMAST with mutations in the p53 gene [3, 4]. It was suggested that exposure to environmental carcinogens may exacerbate this phenotype [14]. With the limited evidence at hand, EMAST appears to be distinct from the classic MSI signature that is frequently observed in Lynch syndrome and sporadic CRC. Whether EMAST contributes to tumor progression by specific geneinactivating mutations within repetitive coding sequences, as is the case with MSI-positive CRC, is a matter of ongoing investigation. Recently, it was shown that EMAST is present in the majority of sporadic CRCs [10]. Haugen et al. demonstrated that EMAST is associated with MSI-L and a deficiency in MSH3 protein expression in colorectal cell lines and tumor tissues [10]. Building upon this evidence, later on it was demonstrated that EMAST can be acquired during the adenoma to carcinoma transformation, as well as upon transformation of well-differentiated carcinomas to moderately and poorly differentiated CRCs [12]. Interestingly in this study, EMAST best correlated in CRCs with ulcerated features, and the authors suggested that inflammation may help drive EMAST [12]. A. Goel (&) Gastrointestinal Cancer Research Laboratory, Sammons Cancer Center and Baylor Research Institute, Baylor University Medical Center, 3500 Gaston Avenue, Suite H-250, Dallas, TX 75246, USA e-mail: ajay.goel@baylorhealth.edu

The myelin basic protein gene in multiple sclerosis: identification of discrete alleles of a 1.3 kb tetranucleotide repeat sequence.

Myelin basic protein (MBP) is a potential autoantigen in multiple sclerosis (MS) and its gene therefore is an attractive candidate to confer genetic susceptibility to this disease. Linkage and association with certain alleles of a 1.2 kb tetranucleotide repeat region 5' to the MBP gene with MS have been reported in Finnish patients, and an association has been reported from Denmark. However, these findings have not been confirmed in subsequent analyses in other populations. A limitation of previous studies has been the low resolution of the typing procedure. We have investigated the same polymorphism in thirty-four Swedish nuclear families with 2 or 3 MS patients. and in 149 unrelated Swedish MS patients and 95 healthy controls using a fluorescence-based semi-automated technique which allowed the identification of discrete tetrarepeat numbers. Neither parametric two-point linkage analysis nor a nonparametric affected pedigree members analysis showed any sign of linkage. In addition, the distribution of alleles was similar in patients and controls. We conclude that the MBP gene does not influence susceptibility to MS in Swedish patients.

Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O‐acetyl groups

Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5'-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis.

Development of microsatellite markers in black tiger shrimp (Penaeus monodon Fabricius)

Abstract Microsatellites or Simple Sequence Repeats (SSRs) represent an abundant source for genetic markers in eukaryotic genomes. Nine synthesized repeat sequences labeled with biotin were used to create an enriched microsatellite library [(AG) 10 , (TG) 10 , (GAA) 10 , (GAC) 10 , (CAT) 10 , (TAC) 10 , (GACA) 8 , (GATA) 8 , and (TCAG) 8 ] for Penaeus monodon . From a total of 2417 clones, only 406 clones (16.8%) were positive after colony hybridization against the nine repeat sequences. Those clones with insert sizes ranging from 300 to 1000 bp were isolated and characterized. The most abundant repeat sequences in the P. monodon genome are (AG) n and (CAT) n making up to 22% and 21%, respectively. A total of 102 from the 129 primer pairs (designed for 129 clones chosen) were able to amplify P. monodon DNA. According to the sequences of the 102 clones, 27 loci, 17 loci, 4 loci, and 54 loci were dinucleotide, trinucleotide, tetranucleotide, and compound repeat sequences, respectively. Thirty microsatellite primer pairs were used to screen wild P. monodon germplasm and the result revealed PIC values ranged from 0.4275 to 0.9264. Therefore, this set of microsatellite primers would provide a useful tool in P. monodon breeding programs.

Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis

Prenatal diagnosis for chromosome abnormality is routinely undertaken by full karyotype analysis of chromosomes from cultured cells; pregnant women must wait on average 13-14 days for their results. Autosomal trisomies, which account for around 80% of significant abnormalities, can be detected by quantitative fluorescence (QF) PCR. We report on the development and implementation of this technique as the first such routine service within a diagnostic department of the UK National Health Service (NHS). We designed a "one-tube test" comprising four primer pairs for polymorphic tetranucleotide repeat sequences on chromosome 21, four primer pairs for sequences on chromosome 18, three primer pairs for sequences on chromosome 13, and one primer pair to identify the sex chromosomes. All prenatal samples received by our NHS diagnostic department between April, 2000, and April, 2001, were tested. After DNA extraction, PCR amplification was done and the products separated on a capillary-based genetic analyser; the results were interpreted with dedicated software. Follow-up karyotype analysis was done on all samples. 1148 amniotic fluid samples, 188 chorionic villus samples, and 37 fetal tissue samples were tested; the amplification failure rate was zero with our current protocol. QF-PCR results were obtained and reported on 1314 (98%) of the prenatal samples; the remaining 22 (2%) were uninformative because of maternal-cell contamination. One case of mosaicism in a chorionic villus sample, and two cases indicating somatic expansion of a tetranucleotide repeat were found. No false positive or false negative results were obtained. The mean reporting time for the last 4 months of data collection was 1.25 working days. QF-PCR aneuploidy testing is an efficient and accurate technique for the detection of autosomal trisomies in prenatal samples. Implementation of this service has led to the rapid diagnosis of abnormalities and early reassurance for women with normal results.

DXS10011: a hypervariable tetranucleotide STR polymorphism on the X chromosome.

The locus DXS10011 is a polymorphic system with a tetranucleotide repeat sequence located on the human X chromosome. The distribution of allele frequencies was examined in 334 Japanese and 171 German individuals and a total of 36 alleles was detected in the two population groups. This STR polymorphism will be a useful marker for linkage analysis.

Microsatellite instability of dinucleotide tandem repeat sequences is higher than trinucleotide, tetranucleotide and pentanucleotide repeat sequences in prostate cancer.

In order to investigate whether the change in length of simple repetitive genomic sequences (microsatellite instability) is associated with prostate cancer, we analyzed 40 prostate cancer samples with 44 microsatellite loci markers on chromosomes 1, 3, 5, 6, 8, 9, 11, 13, 16, 17 and X. DNA was extracted from normal and tumor cells of 40 microdissected cancer samples, amplified by PCR and analyzed for microsatellite instability using 44 primers for dinucleotide, trinucleotide, tetranucleotide and pentanucleotide repeat sequences. The results of this study demonstrate that 45% of the prostate cancer specimens (18 out of 40) showed microsatellite instability (MSI) at a minimum of one locus using dinucleotide repeat sequences. Two out of 40 samples (5%) showed MSI at a minimum of one locus using three different trinucleotide repeat primers (AR, SR and TBP). Ten out of 40 (25%) samples showed MSI at a minimum of one locus using five different tetranucleotide repeat primers (HPRT1, HPRTII, MYCL1, RB, REN). There were no MSI observed in samples using pentanucleotide repeat sequences. There were no MSI in benign prostatic hyperplasia samples (25 samples). These experiments suggest that the microsatellite instability of dinucleotide tandem repeat sequences is much higher than trinucleotide, tetranucleotide and pentanucleotide repeat sequences in prostate cancer. The MSI with different lengths of nucleotide repeat sequences did not correlate with the stage and grades of prostate cancer.

Diversity in a variable-number tandem repeat from Yersinia pestis.

We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.

A polymorphic tetranucleotide repeat in the CYP19 gene and male breast cancer.

The CYP19 gene codes for the aromatase enzyme that is involved in the synthesis of oestrogens. This case–control study examines the relationship between a tetranucleotide repeat sequence in the CYP19 gene and the development of male breast cancer. No significant differences were found between male breast cancer cases and controls. © 2000 Cancer Research Campaign

Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses

Simple-sequence repeats (SSRs) comprising three tetranucleotide repeat sequences with two-base ’anchors’, namely 5´-(AGAC)4GC, 5´-AC(GACA)4 and 5´-(GACA)4GT, were used in PCR reactions as primers to develop inter-SSR DNA fingerprints of the outbreeding grass species Lolium multiflorum, L. perenne, Festuca pratensis and F. arundinacea. Each species was represented by DNA samples from 3 to 6 varieties. In all four species distinctive species-specific DNA profiles were produced that were common across a number of varieties despite their diverse origin. While the fingerprints of the two ryegrasses, L. multiflorum and L. perenne, were the most similar, a number of inter-SSR DNA markers were generated that enabled them to be distinguished from each other. Some slight variations were found between varieties, which provided putative variety-specific markers for cultivar identification. In addition, variations in the DNA profiles of the genotypes of L. multiflorum and F. pratensis were examined, and the results showed that variety-specific fingerprints are integrated patterns made up from the profiles of individual genotypes. Amongst the primers used, AC(GACA)4 generated the best distinction between Lolium and Festuca individuals and provides an effective new tool for genome identification. A number of species-discriminating sequences, ranging in size between 550 bp and 1,600 bp, were cloned: three clones for F. pratensis, one clone for L. multiflorum and one clone for F. arundinacea. A F. pratensis fragment pFp 78H582 was sequenced. Southern hybridization confirmed the presence of this fragment in F. arundinacea (which contains one genome of F. pratensis), but no homology was found with L. multiflorum. However, a F. arundinacea clone amplified with (GACA)4GT, pFa 104H1350, was found to be unique to the F. arundinacea genome.

Relative stabilities of dinucleotide and tetranucleotide repeats in cultured mammalian cells.

The differences in rates of frameshift mutations between a dinucleotide repeat sequence [(CA)(17)] and a tetranucleotide repeat sequence [(GAAA)(17)] have been determined in immortalized, non-tumorigenic, mismatch repair-proficient mouse cells and in mismatch repair-defective human colorectal cancer cells. Clones with mutations were selected on the basis of restoration of activity of a bacterial neomycin resistance gene whose reading frame was disrupted by insertion of the microsatellite upstream of the translation initiation codon. This gene was introduced into the cells on a plasmid, which integrated into the genome of the host cells. Mutation rates of the tetra-nucleotide repeat were much lower than those of the dinucleotide repeat in both cell types. In addition, independent subclones of the colorectal cancer cell line were assayed by PCR for instability of endo-gen-ous tetranucleotide and dinucleotide repeat sequen-ces. In all cases, the mutation frequencies of the dinucleotide repeats were higher than those of the tetranucleotide repeats.

Microsatellite instability in medullary breast carcinomas.

Microsatellite instability (MSI) has been reported to occur in a wide variety of sporadic tumours, such as colorectal and gastric cancers. MSI positivity has been associated with a particular clinico-pathologic profile, including the presence of abundant lymphoid infiltration, poor differentiation and a relatively good outcome for the patients. Since medullary breast carcinomas (MBCs) share these clinico-pathologic features with the MSI-positive tumours described above, we evaluated MSI in this particular histologic type of breast cancer. DNA of 24 MBC cases was extracted from formalin-fixed, paraffin-embedded tissue. The presence of MSI was analysed using BAT-26. We also searched mutations in 2 target genes: TGF-beta RII and BAX. Five cases of the series were also analysed for 1 (CA) dinucleotide tandem repeat sequence (D1S158), 8 tetranucleotide repeat sequences (D3S1358, D5S818, D7S820, D8S1179, D13S317, D21S11, FGA and VWA) and 1 pentanucleotide repeat (dAAAAT), localized in intron 1 of p53 gene. We found 2 carcinomas (8.3%) with BAT-26 instability. None of the cases had mutations in the "target genes", TGF-beta RII and BAX, including the 2 cases with BAT-26 instability. No MSI was observed using the panel of tetra- and pentanucleotide markers. Loss of heterozygosity was found in some loci. No significant difference in mean MIB-1 index according to RER status was observed. The low frequency of MSI in MBC is similar to that of other histologic types of breast cancer. Although MBCs share some clinico-pathologic features with colorectal and gastric carcinomas, which exhibit a high frequency of MSI, the underlying genetic events leading to this breast tumour are different from those leading to tumours of the digestive tract.

Two Tetranucleotide Repeats within the Xq21.3/Yp11.2 Human Specific Region of Homology and Their Conservation in Primate Evolution

We locate two tetranucleotide repeat sequences (AT3 and T2C2) between the markers sY44 and sY46 within the Xq21.3/Yp block of homology that has been created since the separation of the chimpanzee and human lineages, and trace their origin in primate evolution. The T2C2 repeat is present only in hominoid primates. The sequence AT3 is present in Old World monkeys but not in New World monkeys, and has been lost in some gibbon species. In the bonobo, the AT3 repeat is the site of a new Alu insertion. These findings and their relationship to the conservation of other markers in this region cast light on the structure of a genomic region that has been subject to change in the course of primate evolution and may include one or more sites of instability.

Cloning and characterization of the mouse pituitary adenylate cyclase-activating polypeptide (PACAP) gene.

The gene encoding the mouse precursor of pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned, and its structural organization was determined. Using the 5'-rapid amplification of cDNA ends (RACE) procedure, three types of transcription initiation produced by alternative exon usage of two untranslated alternative exons (exons 1A and 1B) were defined. The PACAP gene spans 6.6kb of genomic DNA and is composed of six exons including the alternative exons. The signal peptide, PACAP-related peptide and mature 38-amino acid PACAP (PACAP-38) are encoded within exons 2, 4 and 5, respectively. The 5'-flanking region of the PACAP gene contains several sequence motifs homologous to cAMP response element, TPA response element, and growth hormone factor-1 binding site. A dinucleotide repeat sequence is present in an intron. In addition, there are di- and tetranucleotide repeat sequences 2.4kb and 3.2kb upstream to the translation start point, respectively. The overall intron-exon organization and the production of the alternate mRNAs of the PACAP gene are markedly similar to those of the growth hormone-releasing hormone (GHRH), supporting the hypothesis that the two genes encoding GHRH or PACAP were originated from a gene duplication. Promoter analysis of the 5'-flanking region of the PACAP gene using a luciferase gene reporter system revealed that the isolated 5'-flanking region has functional promoter activity and is responsible for inducible expression.

No association between the tyrosine hydroxylase microsatellite marker HUMTH01 and schizophrenia or bipolar I disorder.

Linkage and association studies have implicated the involvement of the tyrosine hydroxylase (TH) gene on chromosome 11p15 in schizophrenia and bipolar disorder (BPD). An association of BPD with a polymorphic tetranucleotide repeat, HUMTH01, located in the first intron of the human TH gene has been reported. Subsequently a rare allele, Ep ([TCAT]10) of this microsatellite marker has been found in French and Tunisian schizophrenic patients only. We have genotyped a different sample of unrelated French schizophrenic and BPD patients from Alsace and matched controls for this polymorphic tetranucleotide repeat sequence. The Ep allele was insignificantly more common in controls than in schizophrenic patients, thus not showing a particular association with schizophrenia. In addition, analysis of the segregation of the Ep allele in the family of one of the schizophrenic patients showed no transmission of this allele from the healthy mother to her schizophrenic daughter. Nevertheless, we did observe a non-significant trend towards an association between HUMTH01 allele D ([TCAT]9) and schizophrenia (Fisher's exact test, p = 0.053). No association was apparent between HUMTH01 and BPD Psychiatr Genet.

DNA repeats identify novel virulence genes in Haemophilus influenzae.

The whole genome sequence (1.83 Mbp) of Haemophilus influenzae strain Rd was searched to identify tandem oligonucleotide repeat sequences. Loss or gain of one or more nucleotide repeats through a recombination-independent slippage mechanism is known to mediate phase variation of surface molecules of pathogenic bacteria, including H. influenzae. This facilitates evasion of host defenses and adaptation to the varying microenvironments of the host. We reasoned that iterative nucleotides could identify novel genes relevant to microbe-host interactions. Our search of the Rd genome sequence identified 9 novel loci with multiple (range 6-36, mean 22) tandem tetranucleotide repeats. All were found to be located within putative open reading frames and included homologues of hemoglobin-binding proteins of Neisseria, a glycosyltransferase (lgtC gene product) of Neisseria, and an adhesin of Yersinia. These tetranucleotide repeat sequences were also shown to be present in two other epidemiologically different H. influenzae type b strains, although the number and distribution of repeats was different. Further characterization of the lgtC gene showed that it was involved in phenotypic switching of a lipopolysaccharide epitope and that this variable expression was associated with changes in the number of tetranucleotide repeats. Mutation of lgtC resulted in attenuated virulence of H. influenzae in an infant rat model of invasive infection. These data indicate the rapidity, economy, and completeness with which whole genome sequences can be used to investigate the biology of pathogenic bacteria.

Genetic analysis of children of atomic bomb survivors.

Studies are under way for the detection of potential genetic effects of atomic bomb radiation at the DNA level in the children of survivors. In a pilot study, we have examined six minisatellites and five microsatellites in DNA derived from 100 families including 124 children. We detected a total of 28 mutations in three minisatellite loci. The mean mutation rates per locus per gamete in the six minisatellite loci were 1.5% for 65 exposed gametes for which mean parental gonadal dose was 1.9 Sv and 2.0% for 183 unexposed gametes. We detected four mutations in two tetranucleotide repeat sequences but no mutations in three trinucleotide repeat sequences. The mean mutation rate per locus per gamete was o% for the exposed gametes and 0.5% for the unexposed gametes in the five microsatellite loci. No significant differences in the mutation rates between the exposed and the unexposed gametes were detected in these repetitive sequences. Additional loci are being analyzed to increase the power of our study to observe a significant difference in the mutation rates at the 0.05 level of significance.

A class of highly polymorphic tetranucleotide repeats for canine genetic mapping.

We have identified and characterized a new class of polymorphic markers for the canine genome from a simple tetranucleotide repeat sequence, (GAAA)n. Genetic markers derived from this repeat are highly polymorphic compared with other canine microsatellites, yet are stable enough to be useful for following Mendelian inheritance in multigeneration pedigrees. We show further that (GAAA)n repeats are distributed throughout the canine genome and occur with sufficient frequency to be useful in the development of a framework map of the canine genome.

Acquired homozygosity (isodisomy) of chromosome 3 during clonal evolution of a uveal melanoma: Association with morphologic heterogeneity

Cytogenetic investigation of untreated uveal melanoma has shown that the most frequent abnormality is monosomy 3, which occurs in approximately 60% of cases. One of our cases showed distinct pigmented and nonpigmented areas at gross dissection; representative tissue was collected separately from each area, cultured, and harvested using standard cytogenetic techniques. On histologic examination, the pigmented area was found to be composed of small epithelioid cells, whereas the nonpigmented area contained large, pleomorphic epithelioid cells. The karyotype of the pigmented tumor revealed monosomy 3, whereas the nonpigmented tumor showed two apparently normal chromosomes 3. Our purpose in the present study was to investigate the two tumor areas by molecular techniques to determine whether the karyotype of the nonpigmented tumor evolved directly from the pigmented tumor with duplication of the remaining chromosome 3 or whether the two sublines evolved in a divergent fashion from a common precursor stemline. DNA was extracted from normal lymphocytes and separately from both areas of the tumor. The DNA was analyzed using the polymerase chain reaction for polymorphic dinucleotide and tetranucleotide repeat sequences on chromosome 3. Both pigmented and nonpigmented areas of the tumor showed loss of heterozygosity at all informative loci on chromosome 3 that were tested. These results support our hypothesis that an abnormality on chromosome 3 plays a central role in the molecular pathogenesis of uveal melanoma and that some melanomas develop acquired homozygosity (isodisomy) by loss and duplication of the remaining, presumably abnormal, chromosome 3.