Anne Girardet

Selection of chromosome-specific primers and their use in simple and double PRINS techniques for rapid in situ identification of human chromosomes

The PRimed IN Situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We have defined and tested new specific oligonucleotide primers for alpha-satellite DNA of several chromosomes. When using a semiautomatic PRINS protocol, specific labeling was obtained in both metaphase cells and interphase nuclei in a 1-h reaction. PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.

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.

A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS).

The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.

FISH and PRINS, a strategy for rapid chromosome screening : application to the assessment of aneuploidy in human sperm

The co-utilization of FISH and PRINS techniques for in situ chromosome screening was tested on human sperm nuclei. We used a centromeric repeat probe specific for chromosome 4 for FISH. PRINS reactions were performed with alpha-satellite primers specific for either chromosomes 9 or chromosome 18. Double labeling was obtained and estimates of disomy rates were carried out for the three chromosomes.

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Zmax=2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291–D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Zmax=3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.

Direct detection of disomy in human sperm by the PRINS technique.

We report the use of the PRimedIN Situ (PRINS) labelling technique for the direct estimation of disomy rates for various chromosomes in human sperm. The PRINS technique provides a rapid and reliable method for chromosome screening since specific labelling may be obtained in less than 2 h. In the present study, the disomy rates of chromosomes 2, 5, 9, 12 and 18 were investigated. The incidences of disomy are similar for all these chromosomes, ranging from 0.27% to 0.33%. These findings suggest an equal distribution of aneuploidies among autosomes in human sperm.

Cytogenetic analysis of meiotic segregation in sperm from two males heterozygous for reciprocal translocations using PRINS and humster techniques

The meiotic segregation patterns of 2 reciprocal translocations t(7;9)(q33;p21) and t(7;18)(q35;q11) were analyzed in sperm of 2 heterozygote carriers. Both sperm karyotyping and in situ PRINS labeling of sperm nuclei were performed on a sperm sample from each subject. Using the humster technique, 54 and 72 sperm chromosome complements were successfully analyzed for the t(7;9) and the t(7;18) respectively. The frequencies of alternate, adjacent 1, adjacent 2 and 3:1 segregations were 44.44%, 37.04%, 12.96% and 5.56% for the t(7;9) and 33.33%, 43.05%, 19.45% and 4.17% for the t(7;18). The PRINS procedure allowed the rapid screening of large samples of spermatozoa. However, alternate and adjacent 1 segregants were not discriminated because of the generation of centromeric signals. The segregation pattern was determined on 10,658 spermatozoa for the t(7;9) and 10,462 for the t(7;18). The distributions of segregants were similar to those obtained by sperm karyotyping. These data were pooled with results from 37 reciprocal translocations previously studied by sperm karyotyping and 6 recently investigated by FISH. The analysis of these compiled data demonstrates the particularity of the production of imbalances in male gametes; independent of the predisposition for a type of imbalance at term, there is a preferential production of adjacent 1 imbalance in sperm.

Evidence of Somatic and Germinal Mosaicism in Pseudo–Low-Penetrant Hereditary Retinoblastoma, by Constitutional and Single-Sperm Mutation Analysis

This study was supported by the Ligue Suisse contre le Cancer (grant SKL 443-2-1997) and the Recherche Suisse contre le Cancer (grant AKT 621). We thank Dr. Phil Shaw for his discussions.

Double and triple in situ chromosomal labeling of human spermatozoa by PRINS

The PRimed IN Situ (PRINS) labeling method allows rapid, specific detection of human chromosomes in situ. We have adapted the PRINS protocol to mature human sperm in combination with a 3 M NaOH protocol for simultaneous in situ decondensation and denaturation of sperm nuclei. Using fluorochrome-labeled dNTPs in a sequential PRINS reaction, the direct detection of two or three distinct chromosomes must be performed within a timespan of 3 h. The method was tested with primers specific for chromosomes 8, 9, 12, 13, 16, 18, and 21 and the X. The frequencies of disomy ranged from 0.11% to 0.34%. Chromosome-specific primers have been defined for most of the human chromosomes, including some that are indistinguishable by fluorescence in situ hybridization (FISH) with centromeric probes. Consequently, this new strategy constitutes a rapid and efficient alternative to FISH for detecting nondisjunction in human sperm.

Meiotic Segregation Analysis of RB1 Alleles in Retinoblastoma Pedigrees by Use of Single-Sperm Typing

In hereditary retinoblastoma, different epidemiological studies have indicated a preferential paternal transmission of mutant retinoblastoma alleles to offspring, suggesting the occurrence of a meiotic drive. To investigate this mechanism, we analyzed sperm samples from six individuals from five unrelated families affected with hereditary retinoblastoma. Single-sperm typing techniques were performed for each sample by study of two informative short tandem repeats located either in or close to the retinoblastoma gene (RB1). The segregation probability of mutant RB1 alleles in sperm samples was assessed by use of the SPERMSEG program, which includes experimental parameters, recombination fractions between the markers, and segregation parameters. A total of 2,952 single sperm from the six donors were analyzed. We detected a significant segregation distortion in the data as a whole (P=.0099) and a significant heterogeneity in the segregation rate across donors (.0092). Further analysis shows that this result can be explained by segregation distortion in favor of the normal allele in one donor only and that it does not provide evidence of a significant segregation distortion in the other donors. The segregation distortion favoring the mutant RB1 allele does not seem to occur during spermatogenesis, and, thus, meiotic drive may result either from various mechanisms, including a fertilization advantage or a better mobility in sperm bearing a mutant RB1 gene, or from the existence of a defectively imprinted gene located on the human X chromosome.