1 - Direct FISH, Cultured Chromosome Analysis, and Direct SNP Microarray Analysis in Identifying Ring Chromosomes on Prenatal Analysis: A Case of 45,X/46,X,r(X)

Abstract

We report the results of an amniotic fluid analysis on a 41.5 year old female presenting with a circulating cell free DNA (ccfDNA) abnormal for monosomy X. Complete ultrasound at 16 weeks 3 days revealed appropriately sized fetus without evidence of fetal abnormalities or aneuploidy markers within limitations of early gestational age. Ultrasound for nuchal translucency was not performed. Amniocentesis was performed same day at 16 weeks 3 days gestation. Fluorescent in situ hybridization analysis performed on direct amniotic fluid showed 22% of nuclei positive for one copy of the X chromosome using the X centromere probe. The remaining 78% of cells showed two centromere X hybridization signals by FISH and was reported as positive for monosomy X mosaicism. However, initial G-banded chromosome analysis of in situ cultures showed only a 45,X karyotype. No structurally abnormal X or two copies of an X was observed. Additional FISH analysis, using a centromere specific probe performed on a de-stained in situ cultured slide (previously used for G-banded chromosome analysis) showed two metaphase cells out of ten metaphase cells with two X centromere signals. A review of the DAPI FISH images of the two metaphases showed that one X chromosome appeared to be a ring chromosome. In addition 14 of 100 interphase nuclei showed two chromosome X centromere signals while the remaining cells showed a single X chromosome signal. An additional two slides from a sub-culture were also analyzed and showed one G-banded metaphase cell with a ring X chromosome with the remaining 27 cells showing a 45,X count. The mosaic ring X detection discrepancy between the cultured cells in vitro and the aneuploidy FISH on direct amniotic fluid in vivo cells (previously reported with two X hybridization signals in 78% of interphase cells) is likely due to the ring X chromosome poorly dividing and preferentially selected against in the in vitro cultures.