Prenatal exclusion of UPD from cytogenetic slides: a simple method
✍ Scribed by F. Gualandi; A. Sensi; O. Calabrese; R. Gruppioni; M. C. Pittalis; E. Calzolari
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 128 KB
- Volume
- 19
- Category
- Article
- ISSN
- 0197-3851
No coin nor oath required. For personal study only.
✦ Synopsis
Prenatal Exclusion of UPD from Cytogenetic Slides: A Simple Method
The growing awareness of the effects of uniparental disomy (UPD) of several chromosomes on phenotype and the knowledge of many conditions at risk for UPD, such as Robertsonian translocations or confined placental mosaicism, has forced prenatal cytogenetic laboratories to face the problem.
Although the actual frequency of UPD in livebirth is unknown and probably low, the situations with an increased risk for UPD are not infrequent. When such conditions are met, a fast and reliable method of analysis that could be performed avoiding further fetal sampling would be useful.
Here we report on our experience with an improved method for prenatal diagnosis from chromosome spread slides.
We adapted, with slight modification, the method suggested by for cytogenetic suspensions.
Chromosome spread slides were obtained from a mesenchymal CVS culture of a de novo rob(13;14) fetus (case 1) and from amniocytes in a case of t(Y;15)pat (case 2). Both QFQ and GTG banded cytogenetic preparations were employed, as well as unstained and unbanded chromosome spreads.
Slides were washed with xylol (only when immersion oil had been employed), rinsed in absolute ethanol, then carefully scraped with a surgical knife. While scraping, slides were repeatedly rinsed with absolute ethanol so that chromosome spreads could be recovered and transferred into a 1•5 ml microtube. After sedimentation at 13 000 rpm for 30 minutes at 4 C in a microfuge, the pellet was resuspended in 30-50 l of distilled water.
4 l of suspension were directly put into the amplification tube together with the PCR mixture. The PCR reaction was performed in 25 l of a solution containing 2•5 l 10 PCR buffer (Boehringer Mannheim), 200 M of each dNTP, 0•8-1 M of each primer and 1 U of Taq polymerase (Boehringer Mannheim). Standard PCR conditions were as follows: 94 C for 3 minutes (1 cycle); 94 C for 45 seconds, 55 C for 30 seconds, 72 C for 45 seconds (25-30 cycles); 72 C for 3 minutes (1 cycle).
For case 1, chromosome 14 segregation was investigated with STS markers D14S267, D14S286, D14S77 and D14S301, while in case 2, for chromosome 15, GABRB3 and D15S113 polymorphic loci were sufficient for UPD exclusion. Primers for all markers were obtained from the Genome Data Base. Products obtained by PCR amplification were resolved on 12 per cent polyacrylamide gels and visualized by silver staining. Briefly, gels were fixed in 10 per cent ethanol-0•5 per cent acetic acid for 5 minutes, then incubated for 15 minutes in 0•1 per cent AgNO 3 . After two washes with distilled water, a solution containing 1•5 per cent NaOH, 0•15 per cent formaldehyde and 0•01 per cent NaBH 4 was added to the gels. Amplification products were completely visualized in 10-15 minutes.