Clinical application of PRINS

Antley‐Bixler syndrome (ABS) is a rare multiple anomaly syndrome comprising radiohumeral synostosis, bowed femora, fractures of the long bones, premature fusion of the calvarial sutures, severe midface hypoplasia, proptosis, choanal atresia, and, in some, ambiguous genitalia. Of fewer than 40 patients described to date, most have been sporadic, although reports of parental consanguinity and affected sibs of both sexes suggests autosomal recessive inheritance in some families. Known genetic causes among sporadic cases of ABS or ABS‐like syndromes are missense mutations in the IgII and IgIII regions of FGFR2, although the assignment of the diagnosis of ABS to such children has been disputed. A third cause of an ABS‐like phenotype is early in utero exposure to fluconazole, an inhibitor of lanosterol 14‐alpha‐demethylase. The fourth proposed cause of ABS is digenic inheritance combining heterozygosity or homozygosity for steroid 21‐hydroxylase deficiency with effects from a second gene at an unknown locus. Because fluconazole is a strong inhibitor of lanosterol 14‐alpha‐demethylase (CYP51), we evaluated sterol metabolism in lymphoblast cell lines from an ABS patient without a known FGFR2 mutation and from a patient with an FGFR2 mutation and ABS‐like manifestations. When grown in the absence of cholesterol to stimulate cholesterol biosynthesis, the cells from the ABS patient with ambiguous genitalia but without an FGFR2 mutation accumulated markedly increased levels of lanosterol and dihydrolanosterol. Although the abnormal sterol profile suggested a deficiency of lanosterol 14‐alpha‐demethylase, mutational analysis of its gene, CYP51, disclosed no obvious pathogenic mutation in any of its 10 exons or exon‐intron boundaries. Sterol metabolism in lymphoblasts from the phenotypically unaffected mother was normal. Our results suggest that ABS can occur in a patient with an intrinsic defect of cholesterol biosynthesis at the level of lanosterol 14‐alpha‐demethylase, although the genetic nature of the deficiency remains to be determined. © 2002 Wiley‐Liss, Inc.     

Using PRINS for gene mapping in polytene chromosomes

We have adapted the primed in situ labelling (PRINS) protocol for gene mapping in polytene chromosomes of two dipteran species. The method was used to localize the genes for the Balbiani ring (BR) 2.1 and the iron-regulatory protein 1A (IRP1A) in polytene salivary gland chromosomes of Chironomus tentans, and Drosophila melanogaster respectively. Two oligonucleotides, correspondin g to the BR 2.1 and IRP1A genes, were used as primers and the whole procedure was performed within 3–4 h. The strong labelling with low background revealed the localization of the BR 2.1 gene in polytene chromosome IV of C. tentans and the IRP1A gene in polytene chromosome 3R83 of D. melanogaster. The results demonstrated that PRINS is a fast, sensitive and suitable approach for physical gene mapping in polytene chromosomes.This revised version was published online in November 2005 with corrections to the Cover Date.     

Rapid chromosome detection by PRINS in human sperm

Primed in situ (PRINS) labeling may be used for direct estimation of disomy rate in human sperm. We combined the PRINS procedure with a rapid and efficient NaOH pretreatment allowing simultaneous decondensation and denaturation of sperm nuclei. This enabled double labeling of human sperm within a 2‐hr time span. Chromosome‐specific primers have been defined for most human chromosomes, including those most frequently involved in aneuploidy. Using the dual‐color PRINS method, we estimated incidences of disomy for 15 autosomes and the sex chromosomes in sperm samples from 6 normal fertile men. The frequency of disomy ranged from 0.28% to 0.36%. There were no significant interchromosomal differences in disomy rate, but chromosome 21 displayed a higher incidence of nondisjunction than did the other chromosomes. © 2001 Wiley‐Liss, Inc.     

Localisation of DNA sequences on plant chromosomes using PRINS and C-PRINS

Localisation of DNA sequences to plant chromosomes in situ has traditionally been accomplished using fluorescence in situ hybridisation (FISH). Although the method is suitable for most applications it is time-consuming and requires labelled probes. Recently, primed in situ labelling (PRINS) has been developed as an alternative to FISH. PRINS is based on annealing of unlabelled oligonucleotide primer(s) to chromosome DNA and its elongation by DNA polymerase in the presence of labelled nucleotide(s). The method was found useful to detect high-copy tandem repeats on plant chromosomes. Low copy repeats were detected after a more sensitive variant of PRINS called cycling PRINS (C-PRINS), which involves a sequence of thermal cycles analogous to polymerase chain reaction. This paper describes protocols of PRINS and C-PRINS, which have been optimised for chromosome spreads and for chromosomes purified using gradient centrifugation and/or flow sorting. The methods result in clear signals with negligible non-specific labelling. Further work is needed to improve the sensitivity to allow for reliable detection of single-copy DNA sequences.     

A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species

Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤5 parasites per μl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.     

Aneuploidy detection in human sperm nuclei using PRINS technique

Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a <2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28–0.34%. There were no significant interindividual or interchromosomal differences in disomy rates. © 1996 Wiley-Liss, Inc.     

Rapid chromosome detection by PRINS in human sperm.

Primed in situ (PRINS) labeling may be used for direct estimation of disomy rate in human sperm. We combined the PRINS procedure with a rapid and efficient NaOH pretreatment allowing simultaneous decondensation and denaturation of sperm nuclei. This enabled double labeling of human sperm within a 2-hr time span. Chromosome-specific primers have been defined for most human chromosomes, including those most frequently involved in aneuploidy. Using the dual-color PRINS method, we estimated incidences of disomy for 15 autosomes and the sex chromosomes in sperm samples from 6 normal fertile men. The frequency of disomy ranged from 0.28% to 0.36%. There were no significant interchromosomal differences in disomy rate, but chromosome 21 displayed a higher incidence of nondisjunction than did the other chromosomes.     

Deletion of RBM and DAZ in azoospermia: Evaluation by PRINS

Molecular and cytogenetic studies from infertile men have shown that one or more genes controlling spermatogenesis are located in proximal Yq11.2 in interval 6 of the Y chromosome. Microdeletions within the azoospermia factor region (AZF) are often associated with azoospermia and severe oligospermia in men with idiopathic infertility. We evaluated cells from a normal‐appearing 27‐year‐old man with infertility and initial karyotype of 45,der(X)t(X;Y)(p22.3;p11.2)[8]/46,t(X;Y)(p22.3;p11.2)[12]. By fluorescence in situ hybridization with dual‐color whole chromosome paint probes for X and Y chromosomes, we confirmed the Xp‐Yp interchange. By primed in situ labeling, we identified translocation of the SRY gene from its original location on Yp to the patient's X chromosome at band Xp22. We also obtained evidence that the apparent marker was a der(Y) (possibly a ring) containing X and Y domains, and observed that the patient's genome was deleted for RBM and DAZ, two candidate genes for AZF. © 2001 Wiley‐Liss, Inc.     

PRINS Analysis of the Telomeric Sequence in Seven Lemurs

We examined the chromosomal localization of the telomeric sequence, (TTAGGG)_n, in seven species of the lemurs and one greater galago, as an outgroup, using the primed in-situ labeling (PRINS) technique. As expected, the telomeric sequence was identified at both ends of all chromosomes of the eight prosimians. However, six species showed a signal at some pericentromeric regions involving constitutive heterochromatin as well. The pericentromeric region of chromosome 1 of Verreaux's sifaka (Propithecus verreauxi verreauxi) was labeled with a large and intense signal. The range of the signal considerably exceeded the area of DAPI positive heterochromatin. On the other hand, in the five lemurs, a large signal was detected also in the short arm of acrocentric chromosomes. Acquisition of the large block of the telomeric sequence into the acrocentric short arm might be interpretable in terms of the tandem growth of the heterochromatic short arm and the reciprocal translocation between heterochromatic short arms involving the telomeric sequence. Subsequently, it was postulated that meta- or submetacentric chromosomes possessing the telomeric sequence at the pericentromeric region could be formed by centric fusion between such acrocentric chromosomes.     

Deletion of RBM and DAZ in azoospermia: evaluation by PRINS.

Molecular and cytogenetic studies from infertile men have shown that one or more genes controlling spermatogenesis are located in proximal Yq11.2 in interval 6 of the Y chromosome. Microdeletions within the azoospermia factor region (AZF) are often associated with azoospermia and severe oligospermia in men with idiopathic infertility. We evaluated cells from a normal-appearing 27-year-old man with infertility and initial karyotype of 45,der(X)t(X;Y)(p22.3;p11.2)[8]/46,t(X;Y)(p22.3;p11.2)[12]. By fluorescence in situ hybridization with dual-color whole chromosome paint probes for X and Y chromosomes, we confirmed the Xp-Yp interchange. By primed in situ labeling, we identified translocation of the SRY gene from its original location on Yp to the patient's X chromosome at band Xp22. We also obtained evidence that the apparent marker was a der(Y) (possibly a ring) containing X and Y domains, and observed that the patient's genome was deleted for RBM and DAZ, two candidate genes for AZF.     

Rapid in situ detection of chromosome 21 by PRINS technique

The “PRimed IN Situ labeling” (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and inter-phase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies. © 1995 Wiley-Liss, Inc.     

Ultra-rapid multicolor PRINS protocol for chromosome detection in human sperm

We report a new multicolor PRINS procedure for chromosome identification on human sperm. Based on the direct in-situ mixing of the colors of the fluorochromes (FITC, TRITC, Cascade Blue) incorporated in sequential PRINS reactions, this method facilitates rapid distinct labeling of 3 or 4 chromosomes. Each PRINS reaction consists of a unique 4 minute step for annealing and elongation. The method was successfully tested on lymphocytes and spermatozoa. Estimates of disomy were performed for chromosomes 7, 9 and 16 on sperm samples from 2 healthy donors. There was no significant difference between the disomy rates obtained with the conventional two-color PRINS technique and this new three-color procedure. By simplifying the multicolor PRINS protocol, this new protocol should facilitate the use and adaptation of PRINS to various cytogenetic applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Combined use of PRINS and FISH in the study of the dystrophin gene

The efficacy of fluorescence in situ hybridization (FISH) may be limited in specific applications by low‐resolution sensitivity. Primed in situ labeling (PRINS) is based on specific hybridization of an unlabeled oligonucleotide with a denatured template and synthesis of a single‐strand DNA in situ. This method may represent a powerful alternative to FISH for gene mapping because of its ability to generate multiple independent signals within the same gene segment. We investigated the specificity of signals produced by a modified PRINS protocol combining a centromeric probe for the X‐chromosome with specific primers for 3′‐ and 5′‐terminal regions of the dystrophin gene. In approximately 70% of nuclei from male and female subjects, we detected one or two large signals (X‐chromosome centromere) and two or four smaller signals (the two regions of the dystrophin gene). Specific hybridization of the oligonucleotides on Xp was demonstrated by localization of the smaller (dystrophin) and larger (X‐centromere) signals on the same chromosome. Simultaneous hybridization with a centromeric probe and gene‐specific oligonucleotides allowed localization of PRINS signals, and assessment of the specificity of the primers used for hybridization. This approach could facilitate identification of female carriers of small intragenic deletions in the dystrophin gene. © 2001 Wiley‐Liss, Inc.