Application of Restriction Site-Associated DNA Sequencing (RAD-Seq) for Copy Number Variation and Triploidy Detection in Human.

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

[Alleles dropout patterns of Y-short tandem repeats in infertile males with Y chromosome microdeletions].

Y chromosomal short tandem repeat (Y-STR) typing is the most commonly used genetic technique in forensic studies. However, there may be a limit to the application of Y-STR in forensic science as Y-STR loci are subject to loss or variation caused by the higher chromosomal structures' spontaneous mutation rate. Located in the long arm of the Y chromosome, azoospermia factor (AZF) have been shown to participate in spermatogenesis and its deletion could cause infertility. However, little is known about the Y-STR dropout pattern in individuals with Y chromosome microdeletions. In this study, 85 infertile males with Y chromosome interstitial deletion were identified and special Y-STR allele dropout patterns were analyzed by employing a Y-STR Commercial Kit and a Y chromosome Deletion Kit. Results demonstrate that AZF a region deletion are related to DYS439-DYS389I-DYS389II alleles dropout, while AZF b region or c region deletions correlate to DYS448 allele dropout. Null DYS385-DYS392-DYS448 alleles were observed in AZF b+c+d region deletion individuals. While null DYS390-Y-GATA-H4-DYS385-DYS392-DYS448 alleles were observed in AZF a+b+c+d large region deletion individuals. Our data suggest that Y chromosome microdeletions may indicate specific Y-STR locus dropout patterns.

[Clinical value of MLPA in the prenatal gene diagnosis of Duchenne muscular dystrophy].

OBJECTIVE: To investigate the clinical value of multiplex ligation-dependent probe amplification (MLPA) in the prenatal gene diagnosis of high risk pregnant women from Duchenne muscular dystrophy (DMD) families. METHODS: The 155 high risk pregnant women from DMD families were recruited from 2005 to 2012 in 4 hospitals in Guangzhou, such as Southern Hospital of Southern Medical University and the Third Affiliated Hospital of Guangzhou Medical University. Among all the samples, 7 were chorionic villus samples taken from early-stage pregnancy and 148 were amniotic fluid samples from mid-stage pregnancy. After the maternal contamination was eliminated, the fetal DMD gene screening was carried out by using MLPA. The mutation rates in DMD exons were calculated in all the 155 families. RESULTS: (1) Among the 155 fetuses of the DMD high risk pregnant women, there were 72 male fetuses and 83 female fetuses. In the male fetuses, there were 27 sufferers (38%). In the female fetuses, there were 28 carriers (34%). And there were 100 normal fetuses. (2) Among the 27 DMD sufferers, 22 cases were DMD exon homozygous deletions (14.2%, 22/155) and 5 cases were DMD exon duplications (3.2%, 5/155). Among the 28 carriers, 25 cases were gene heterozygous deletions (16.1%, 25/155) and 3 cases were gene heterozygous duplications (1.9%, 3/155). In the 155 families, the DMD mutations mainly occurred in exons 45-52, and the exon 49 had the highest mutation rates of 22 times. (3) Among the 7 cases of prenatal gene diagnosis using chorionic villus samples, 2 fetuses had the identical DMD genotypes with their mothers and probands. One was a DMD sufferer and the other was a carrier. Termination or continuation of pregnancy was suggested based on the genotype of the fetus. CONCLUSIONS: MLPA provides an accurate method in the prenatal diagnosis of DMD. It could be used to distinguish DMD gene homozygous deletions from heterozygous deletions and duplications. Therefore, it is valuable for DMD prenatal diagnosis in high-risk women. Chorionic villus sampling can be applied to the early prenatal diagnosis for DMD disease.