Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR

• Published in: European journal of obstetrics & gynecology and reproductive biology Vol. 161; no. 1; pp. 34 - 37
• Main Authors: Kolialexi, Aggeliki, Tounta, Georgia, Apostolou, Paraskevi, Vrettou, Christina, Papantoniou, Nikos, Kanavakis, Emmanuel, Antsaklis, Aris, Mavrou, Ariadni
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 01.03.2012
• Subjects: Actins - genetics; Adult; Base Sequence; Cell-free fetal DNA; Chromosomes, Human, Y - chemistry; Chromosomes, Human, Y - genetics; Digestion; DNA methylation; Early Diagnosis; Edetic acid; Endonuclease; Female; Fetal marker; Fetal sex; Fetuses; Genetic Diseases, X-Linked - diagnosis; Genetic Diseases, X-Linked - genetics; Genitalia; Gestation; Gynecology; Humans; Multiplex Polymerase Chain Reaction - methods; Non-invasive prenatal diagnosis; Nucleotide sequence; Obstetrics; Obstetrics and Gynecology; Polymerase chain reaction; Pregnancy; Pregnancy Trimester, First; Prenatal diagnosis; Prenatal Diagnosis - methods; RASSF1A protein; Sex determination; Sex Determination Analysis - methods; SOXB1 Transcription Factors - genetics; Tumor Suppressor Proteins - genetics; Ultrasonography, Prenatal; Ultrasound; X chromosome; Y chromosome; Cell-free fetal DNA; Non-invasive prenatal diagnosis; Fetal sex; Fetal marker
• ISSN: 0301-2115; 1872-7654
• DOI: 10.1016/j.ejogrb.2011.12.025

Abstract Objective Clinical indications for fetal sex determination include risk of X-linked disorders, a family history of conditions associated with ambiguous development of the external genitalia, and some fetal ultrasound findings. It is usually performed in the first trimester from fetal material obtained through CVS and is associated with an approximately 1% risk of miscarriage. Ultrasound fetal sex determination is often performed after 11 weeks of gestation. This study aims to validate a reliable method for non-invasive prenatal diagnosis of fetal gender using maternal plasma cell-free fetal DNA (cffDNA) for fetal sex assessment in the first trimester of pregnancy and test its clinical utility in the diagnosis of potentially affected pregnancies in carriers of X-linked disorders. Study design In the validation study, blood samples from 100 pregnant women at 6–11 weeks of gestation were analysed. In the clinical study, 17 pregnancies at risk of having an affected fetus were tested. 7 ml of maternal blood in EDTA were obtained and cffDNA was extracted using a commercially available kit. DNA was enzymatically digested using a methylation sensitive endonuclease ( Aci I) to remove maternal unmethylated sequences of the RASSF1A gene. A multiplex PCR was performed for the simultaneous amplification of target sequences of SRY and DYS14 from chromosome Y, along with RASSF1A and ACTB sequences. Amplification of these loci indicates fetal gender, confirms the presence of cffDNA and allows assessment of digestion efficiency. Results After establishing the appropriate experimental conditions, validation studies were successful in all 100 cases tested with no false negative or false positive results. Y chromosome-specific sequences were detected in 68 samples, and 32 cases were diagnosed as female based on the amplification of RASFF1A sequences only, in the absence of ACTB . In the clinical studies, fetal sex was correctly diagnosed in 16 pregnancies, and one case was reported as inconclusive. Conclusions Fetal sex assessment by detecting Y chromosome sequences in maternal blood can be routinely used from the 6th week of gestation. Reliable fetal sex determination from maternal blood in the 1st trimester of gestation can avoid conventional invasive methods of prenatal diagnosis.