Rapid detection of chromosome X,Y, 13, 18, and 21 aneuploidies by primed in situ labeling/synthesis technique

AIMS AND OBJECTIVE:

Primed in situ labeling/synthesis (PRINS) technique is an alternative to fluorescent in situ hybridization for chromosome analysis. This study was designed to evaluate the application of PRINS for rapid diagnosis of common chromosomal aneuploidy.

MATERIALS AND METHODS:

We have carried out PRINS using centromere specific oligonucleotide primers for chromosome X, Y, 13, 18 and 21 on lymphocyte metaphase and interphase cells spread. Specific primer was annealed in situ, followed by elongation of primer by Taq DNA polymerase in presence of labeled nucleotides. Finally, reaction was stopped and visualized directly under fluorescent microscope.

RESULTS:

Discrete centromere specific signals were observed with each primer.

CONCLUSION:

PRINS seems to be a rapid and reliable method to detect common chromosome aneuploidy in peripheral blood lymphocyte metaphase and interphase cells.     

Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y

The primed in situ labelling (PRINS) technique is an alternative to in situ hybridization for chromosomal screening. We have developed a semi-automatic PRINS protocol, using a programmable thermocycler. The method has been successfully tested with specific primers for chromosomes, 13, 16, 18, 21, X and Y. Specific chromosome detection has been obtained on both metaphases and interphase nuclei. This suggests that PRINS may be a reliable technique for detecting aneuploidies and some chromosomal aberrations.     

Analysis of aneuploidy for chromosomes 13, 21, X and Y by multicolour fluorescence in situ hybridisation (FISH) in a 47,XYY male.

The frequency of aneuploid sperm was assessed by fluorescence in situ hybridisation (FISH) in a 47,XYY male previously studied by sperm karyotyping. A total of 20,021 sperm were studied: 10,017 by two-colour FISH for chromosomes 13 and 21 and 10,002 by three-colour FISH for the sex chromosomes using chromosome 1 as an autosomal control for diploidy and lack of hybridisation. Results were compared with more than 500,000 sperm from 18 normal men. The frequencies of X-bearing (49.4%) and Y-bearing sperm (49.8%) were not significantly different from 50% as shown in our sperm karyotyping study. There was no significant increase in the frequency of diploid sperm compared with control donors. There was a significant increase in the frequency of disomy for chromosome 13 (p < 0.0001) and XY disomy (p = 0.0008) compared with control donors. However, since the frequency of disomy was 0.40% for chromosome 13 and 0.55% for XY disomy, it is not surprising that these increases were not discovered previously in our analysis of 75 sperm karyotypes. Our results suggest that the extra Y chromosome is eliminated during spermatogenesis in the majority of cells but that there may be a small but significant increase in the frequency of aneuploid sperm in these men.     

Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.     

Mouse telomere analysis using an optimized primed in situ (PRINS) labeling technique

Telomeres are chromosomal elements composed of variable numbers of a TTAGGG repeated DNA sequence required for genomic stability. Telomeric length is correlated with the number of copies of this repeated DNA sequence and is an important property relevant to telomeric function. Recently, it has been demonstrated that the length of the shortest telomere, not average telomeric length, is important for cell viability and chromosomal stability. Consequently, assays permitting assessment of telomeric length are important for the analysis of genomic instability disorders. The length of individual telomeres can be analyzed using the primed in situ (PRINS) labeling reaction, which produces a labeled copy of the telomeric DNA repeats in situ. In this study, we tested different variables to optimize the PRINS reaction to enable it to be applied to the detection of mouse telomeric DNA and the study of telomeric length. The specificity, efficiency and uniformity of staining were evaluated using digital fluorescence microscopy. Labeling efficiency is dependent upon the conditions used to denature the telomeric DNA and reaction duration. Staining uniformity is increased at higher annealing and elongation temperatures as well as when a fluorescently labeled nucleotide is incorporated during the elongation step. Our results also indicate that chromosomal background staining is observed when a fluorochrome-labeled nucleotide is used as opposed to a hapten-labeled nucleotide. From this study, we conclude that an optimized PRINS technique can be reliably employed to analyze mouse telomeres and, compared with the FISH (fluorescence in situ hybridization) technique, presents advantages including greater cost efficiency and reduced processing time. These advantages may encourage wider use of the PRINS technique for quantitative evaluation of the length of individual telomeres in situ.     

Detection of Klinefelter syndrome by G-banding analysis combined with primed in situ labeling technique

Primed in situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We employed triple-color PRINS technique to detect chromosomal abnormalities in Klinefelter syndrome patients diagnosed by G-banding karyotype analysis. Among 1034 infertile male patients, 134 were found to be cytogenetically abnormal, including 70 with chromosomal number abnormalities and 64 with chromosomal structure abnormalities. Among these cytogenetically abnormal patients, 56 were diagnosed as having Klinefelter syndrome. PRINS technique was used on cultured lymphocyte metaphase cells of the Klinefelter syndrome patients; the same result was obtained with G-banding karyotype analysis. PRINS proved to be a rapid and reliable method to detect numerical chromosome abnormalities in peripheral blood lymphocytes in metaphase.     

Determination of Y chromosome aneuploidy in human sperm nuclei by nonradioactive in situ hybridization.

Sperm nuclei from eight normal, healthy donors were hybridized in situ with the biotin-labeled Y-specific pHY2.1 DNA probe to evaluate the X:Y ratio, the location of the Y chromosome, and the frequency of Y aneuploidy in human sperm. The streptavidine-horseradish-peroxidase and DAB detection system used permitted the unequivocal identification of sperm heads with zero, one, or two hybridization signals and proved superior to either quinacrine staining or radioactive in situ hybridization. The low incidence of 0.27% of sperm nuclei with two Y chromosomes that was found is similar to the frequency of XYY males among newborns. The average proportions of X- and Y-bearing sperm nuclei were 50.3% and 49.4%, respectively, corresponding to the expected 1:1 ratio. The Y heterochromatin was located in the central part of the nucleus in 58% of the Y-carrying sperm cells.     

Primed in situ labeling: sensitivity and specificity for detection of alpha-satellite DNA in the centromere regions of chromosomes 13 and 21

The centromeric alpha-satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence. So far it has proven difficult to discriminate between sequence variations in the chromosome 13 and 21 alpha-satellite regions using in situ techniques. To analyze whether the method of modified single-color and double-color PRINS could be used to detect single nucleotide polymorphisms within this region, we used previously published primers D13Z and D21Z that differ in the terminal 3'-nucleotide and an additionally constructed primer "D13/21-test" lacking the final nucleotide at the 3' end. The results show that a one-base pair mismatch at the 3' end is sufficient to be detected by PRINS. Surprisingly, only about 35% of our samples exhibited the expected combination of two chromosomes 13 specifically labeled with only primer D13Z and two chromosomes 21 specifically labeled with only primer D21Z. The rest of the samples showed a polymorphic distribution of the target sequence for the primers, therefore these primers are not suited for routine detection of chromosomes 13 and 21 during interphase. Our data indicate that an interchromosomal exchange of alpha-satellite DNA takes place between chromosomes 13 and 21, possibly due to a concerted evolution process.     

Four distinct alpha satellite subfamilies shared by human chromosomes 13, 14 and 21.

We describe the characterisation of four alpha satellite sequences which are found on a subset of the human acrocentric chromosomes. Direct sequence study, and analysis of somatic cell hybrids carrying specific human chromosomes indicate a unique 'higher-order structure' for each of the four sequences, suggesting that they belong to different subfamilies of alpha DNA. Under very high stringency of Southern hybridisation conditions, all four subfamilies were detected on chromosomes 13, 14 and 21, with 13 and 21 showing a slightly greater sequence homology in comparison to chromosome 14. None of these subfamilies were detected on chromosomes 15 and 22. In addition, we report preliminary evidence for a new alphoid subfamily that is specific for human chromosome 14. These results, together with those of earlier published work, indicate that the centromeres of the five acrocentric chromosomes are characterised by a number of clearly defined alphoid subfamilies or microdomains (with at least 5, 7, 3, 5 and 2 different ones on chromosomes 13, 14, 15, 21 and 22, respectively). These microdomains must impose a relatively stringent subregional pairing of the centromeres of two homologous chromosomes. The different alphoid subfamilies reported should serve as useful markers to allow further 'dissection' of the structure of the human centromere as well as the investigation of how the different nonhomologous chromosomes may interact in the aetiology of aberrations involving these chromosomes.     

Localization of repetitive DNA sequences on in vitro Xenopus laevis chromosomes by primed in situ labeling (PRINS)

Xenopus laevis is an important reference model organism used in many vertebrate studies. Gene mapping in X. laevis, in comparison to other reference organisms, is in its early stages. Few studies have been conducted to localize DNA sequences on X. laevis chromosomes. Primed in situ labeling (PRINS) is a recently developed innovative tool that has been used to locate specific DNA sequences in various organisms. PRINS has been reported to have increased sensitivity compared to other in situ hybridization techniques. In the present study, PRINS was first used to label the location of telomeres at the ends of in vitro X. laevis chromosomes. The terminal location was as expected from in vivo reports, however, the overall amount seemed to decrease in the in vitro chromosomes. Once the PRINS technique was optimized, this technique was used to determine the chromosomal location of the satellite 1 repetitive sequence, which is an important sequence in X. laevis development. The sequence was observed on the interstitial regions of the majority of the chromosomes similar to the in vivo locations reported. In contrast to the telomeric sequence, the amount of sequence appeared to increase in the satellite 1 sequence. PRINS was found to be useful in the localization of repetitive DNA sequences in the X. laevis genome.     

Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.     

The frequency of aneuploidy among individual chromosomes in 6,821 human sperm chromosome complements

The human sperm/hamster egg fusion technique has been used to analyse 6,821 human sperm chromosome complements from 98 men to determine if all chromosomes are equally likely to be involved in aneuploid events or if some chromosomes are particularly susceptible to nondisjunction. The frequency of hypohaploidy and hyperhaploidy was compared among different chromosome groups and individual chromosomes. In general, hypohaploid sperm complements were more frequent than hyperhaploid complements. The distribution of chromosome loss in the hypohaploid complements indicated that significantly fewer of the large chromosomes and significantly more of the small chromosomes were lost, suggesting that technical loss predominantly affects small chromosomes. Among the autosomes, the observed frequency of hyperhaploid sperm equalled the expected frequency (assuming an equal frequency of nondisjunction for all chromosomes) for all chromosome groups. Among individual autosomes, only chromosome 9 showed an increased frequency of hyperhaploidy. The sex chromosomes also showed a significant increase in the frequency of hyperhaploidy. These results are consistent with studies of spontaneous abortions and liveborns demonstrating that aneuploidy for the sex chromosomes is caused by paternal meiotic error more commonly than aneuploidy for the autosomes.     

Classification of qh regions in human chromosomes 1, 9, and 16 by C-banding

Summary We present a classification for secondary constriction (qh) regions with C-banding technique in chromosomes 1,9, and 16 by means of comparing them to the short arm of chromosome 16. It is simple and convenient and can be used routinely. It can be incorporated into the modified Paris nomenclature system.     

Folate deficiency induces aneuploidy in human lymphocytes in vitro-evidence using cytokinesis-blocked cells and probes specific for chromosomes 17 and 21

Folate plays a critical role in the prevention of chromosome breakage and hypomethylation of DNA. Deficiency in this vitamin may lead to demethylation of heterochromatin causing structural centromere defects that could induce abnormal distribution of replicated chromosomes during nuclear division. Because aneuploidy of chromosomes 17 and 21 is often observed in breast cancer and leukaemia and increased risk for these cancers is associated with folate deficiency, we hypothesized that folate deficiency may lead to aneuploidy of chromosomes 17 and 21. To test these hypotheses we cultured lymphocytes from eight female volunteers (aged 40-48 years) in RPMI 1640 medium containing 12 or 120nM of folic acid (FA) or 5-methyltetrahydrofolate (MF) for 9 days. Chromosomes 17 and 21 aneuploidies induced by folate deficiency were measured in mononucleated (MONO) and cytokinesis-blocked binucleated (BN) lymphocytes after dual-color fluorescent in situ hybridization (FISH) with a digoxigenin-labeled probe for the alphoid satellite sequence of chromosome 17 and a biotin-labeled probe for the pericentric region of chromosome 21. The results showed that 12nm of MF or FA caused a significant 26-35% increment in frequency of aneuploidy of chromosome 17 (P = 0.0017) and aneupoidy of chromosome 21 (P = 0.0008) relative to 120nM MF or FA. The pattern of aneuploidy in binucleated cells was significantly correlated with that observed in mononucleated cells (R = 0.51-0.75, P < 0.0004) and was consistent with a model based on chromosome loss or partial aneusomy rescue as the cause rather than non-disjunction, although the latter mechanism could not be excluded. MF was not more efficient than FA in preventing aneuploidy in this in vitro system. We conclude that folate deficiency is a risk factor for chromosomes 17 and 21 aneuploidy.     

Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy.

Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed.     

Chromosomal localization of zebrafish AluI repeats by primed in situ (PRINS) labeling.

Two zebrafish AluI repeats were localized in metaphase chromosomes by means of the primed in situ (PRINS) labeling technique, using oligonucleotide primers based on published sequences. An AT-rich, tandemly repeated, long AluI restriction fragment (RFAL1) labeled the (peri)centromeric regions of all chromosomes. The GC-rich short fragment (RFAS) was found to be localized in the paracentromeric regions of 17 chromosome pairs, which were mostly subtelocentric. The RFAS labeling pattern generally fits the previously described chromomycin A3 (CMA3) staining pattern. The differential composition of heterochromatin in zebrafish chromosomes is discussed.     

An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure.

An improved primed in situ labeling (PRINS) procedure that provides fast, highly sensitive, and nonradioactive cytogenetic localization of chromosome-specific tandem repeat sequences is presented. The PRINS technique is based on the sequence-specific annealing in situ of unlabeled DNA. This DNA then serves as primer for chain elongation in situ catalyzed by a DNA polymerase. If biotin-labeled nucleotides are used as substrate for the chain elongation, the hybridization site becomes labeled with biotin. The biotin is subsequently made visible through the binding of FITC-labeled avidin. Tandem repeat sequences may be detected in a few hours with synthetic oligonucleotides as primers, but specific labeling of single chromosomes is not easily obtained. This may be achieved, however, if denatured double-stranded DNA fragments from polymerase-chain-reaction products or cloned probes are used as primers. In the latter case, single chromosome pairs are stained with a speed and ease (1 h reaction and no probe labeling) that are superior to traditional in situ hybridization. Subsequent high-quality Q banding of the chromosomes is also possible. The developments described here extends the range of applications of the PRINS technique, so that it now can operate with any type of probe that is available for traditional in situ hybridization.     

Aneuploidy in pig sperm: multicolor fluorescence in situ hybridization using probes for chromosomes 1, 10, and Y.

The objective of this research was to develop chromosome-specific probes for use in evaluating aneuploidy in boar spermatozoa through the application of fluorescence in situ hybridization (FISH) technology. A multicolor FISH method was developed to detect aneuploidy in the sperm of boars using DNA probes specific for small regions of chromosomes 1, 10, and Y. The average frequencies of sperm with disomy for chromosomes 1, 10, and Y were 0.075%, 0.067%, and 0.094%, respectively. The incidence of disomy did not differ significantly by chromosome. The average frequencies of diploidy were 0.177% for 1-1-10-10 and 0.022% for Y-Y-10-10. Thus, the incidence of overall diploidy (1-1-10-10) was significantly higher than that of disomy for the chromosomes examined (P < 0.01 for disomy of the autosomes and P < 0.05 for disomy of the Y chromosome). No significant age or breed effects on disomy and diploidy rates and no significant interindividual variations in disomy or diploidy were found. The observed level of numerical chromosome aberrations in pig sperm appear to be within the range of the baseline frequencies reported so far in men.     

Evaluation of aneuploidy frequency for chromosomes 6 and 17 in eyelid tumours using the FISH technique

Although basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are very common skin tumours, the incidence of chromosome aneuploidy with regard to the eyelid has not been investigated. We aimed to find the frequency of chromosome 6 and 17 aneuploidies in eyelid tumours' interphase nuclei with fluorescence in situ hybridization (I-FISH) with chromosome specific DNA probes. I-FISH with chromosome 6 and 17 centromere specific DNA probes was used in the eyelids of 10 patients with BCC or SCC and the peripheral blood cells of 10 healthy donors as controls. The frequency of chromosome 6 and 17 aneuploidies was significantly higher in 7 out of 10 patients and 5 out of 10 patients, respectively, than in controls, indicating a higher frequency of aneuploidy in BCC than in SCC of the eyelid. Distribution of hybridization signals for chromosome 6 and 17 was wide ranging, indicating heterogeneity of cell populations with aneuploidy between patients. These findings indicate that acquisition of chromosome aneuploidies in eyelid tumours may have an important pathogenic role in both BCC and SCC of the eyelid area.