Chromosomal Copy Number Variation Analysis in Pregnancy Products from Recurrent and Sporadic Miscarriage Using Next-Generation Sequencing.

Chromosomal abnormality is one of the causes of fetal miscarriage. The potential differences of fetal chromosomal abnormalities in sporadic miscarriage (SM) and recurrent miscarriage (RM) remain unclear. The purpose of this study was to investigate copy number variations (CNVs) in SM and RM to provide useful genetic guidance for pregnancy and prenatal diagnosis. Four hundred eight samples of aborted fetuses were analyzed by CNV sequencing, and further functional enrichment analysis was performed. Chromosomal abnormalities were identified in 218 (53.4%) fetuses. There were 62 cases (15.2%) with structural chromosomal abnormalities, including 41 with VUS CNVs, 8 with pathogenic CNVs (pCNVs), and 5 with likely pCNVs. Duplications or deletions of 7p22, 8p22, 8p23, and Xp22.31 were significantly more common in RM cases and therefore believed to be related to RM. A total of 289 genes were identified, and 29 different functions were enriched as potential RM candidate genes and functions, which were mainly concentrated in 4 functional categories: chemokines and chemotaxis, protease activity and protein modification, defense response to bacterial and fungal infections, and immune response. The results of this study may improve our understanding of the etiology of RM and contribute to the establishment of a population-based genetic marker information for RM.

Prenatal diagnosis of 913 fetuses samples using copy number variation sequencing.

BACKGROUND: The present study aimed to explore the etiological relationship between fetal abnormalities and copy number variations (CNVs) with the aim of intervening and preventing the birth of children with birth defects in time. METHODS: Samples of 913 fetuses with puncture indications were collected from January 2017 to December 2019. Karyotype analysis and CNV sequencing (CNV-seq) testing was performed for fetuses with ultrasonic abnormalities, a high risk of Down's syndrome and an adverse birth history. All cases were followed up. RESULTS: In total, 123 cases (13.47%) had abnormal karyotypes, including 109 cases with chromosome number abnormalities and 14 cases of chromosomal structural abnormalities. Thirty-seven (4.05%) cases with pathogenic CNVs were detected. The detection rate of pathogenicity CNVs was 12.82% for mixed indications, followed by 7.5% for an adverse birth history, 5.88% at high risk of non-invasive prenatal testing, 5.00% with an abnormal ultrasonic marker, 1.89% at high risk of screening for Down's syndrome and 1.45% with advanced maternal age. There were 12 (1.31%) cases with microduplications and 25 (2.74%) cases with microdeletions. Trisomy 21 (39.02%), trisomy 18 (13.82%) and Turner syndrome (9.76%) were the top three chromosome abnormalities. There were 104, 746 and 63 cases in the 11-13 weeks, 14-27 weeks 28-38 weeks gestational ages, respectively. The abnormal rates of fetal chromosome aneuploidy and the rate of pathogenic CNVs were decreased and increased with the increase of gestational age (p < 0.05), respectively. CONCLUSIONS: Compared with karyotype analysis, CNV-seq can improve the detection rate of chromosomal abnormalities. CNV-seq combined karyotype analysis should be performed simultaneously in fetuses with puncture indications.

Analysis of copy number variation by sequencing in fetuses with nuchal translucency thickening.

OBJECTIVE: Copy number variation sequencing (CNV-seq) technique was used to analyze the genetic etiology of fetuses with increased nuchal translucency (NT). METHODS: A total of 139 women with gestational 11-14 weeks whose fetuses were detected with increased NT (NT ≥ 2.5 mm) in our hospital from July 2016 to December 2018 were selected. Fetal specimens were performed for karyotyping analysis and CNV sequencing. RESULTS: According to the nuchal translucency thickness, 2.5-3.4, 3.5-4.4, 4.5-5.4, and more than 5.5 mm, the rates of chromosomal abnormalities were 22.8% (13/57), 30.8% (12/39), 42.1% (8/19), and 62.5% (15/24), respectively. There was significant difference among the incidences of chromosomal abnormalities in four groups (χ2  = 37.69, P < .01) and the incidences increased with fetal NT thickness. Among 139 cases, there were 36 cases (25.9%) with abnormal chromosome karyotypes. Meanwhile, there were 45 cases (32.3%) with abnormal CNV. In the 12 cases with abnormal CNV and normal chromosome karyotypes, there were 2 cases of pathogenic CNV, 7 cases of CNV with unknown clinical significance, and 3 cases of possibly benign CNV. There was no significant difference in CNV between pregnant women in advanced maternal age and those in normal maternal age (χ2  = 1.389, P = .239). In the fetus who showed abnormalities in NT and ultrasonography (χ2  = 5.13, P < .05) and the fetus aborted (χ2  = 113.19, P < .05), the abnormal rate of CNV was higher with statistically significant difference. CONCLUSION: CNV-seq combined karyotype analysis should be performed simultaneously in fetuses with increased NT, providing a basis for genetic counseling, which is of great significance for prenatal diagnosis.

Association of methylenetetrahydrofolate reductase C677T polymorphism with the pre-eclampsia risk in Hakka pregnant women in Southern China.

The aim of this study is to clarify the possible association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and pre-eclampsia in Hakka pregnant women in southern China. Pre-eclampsia and normal pregnant women were consecutively collected and MTHFR C677T genotypes were determined by the DNA sequencing method. One hundred and thirteen pre-eclampsia patients were CC homozygote (113 of 191, 59.2%), 68 of 191 (35.6%) were CT heterozygote, and 10 of 191 (5.2%) were TT homozygote, with the frequency of the T allele equal to 0.77. This is in comparison with the normal control group where 106 of 202 (52.5%) were CC homozygote, 83 of 202 (41.1%) were CT heterozygote, and 13 of 202 (6.4%) were TT homozygote, with the frequency of the T allele equal to 0.27. No statistically significant differences were observed in genotype or allele frequencies between the pre-eclampsia and normal control for the C677T polymorphism of MTHFR gene (p > .05). The findings of this study suggest that polymorphisms of MTHFR C677T genes were not associated with pre-eclampsia in Hakka pregnant women from southern China, but additional studies are necessary to explore the mechanisms involving it.

Invasive molecular prenatal diagnosis of alpha and beta thalassemia among Hakka pregnant women.

This study is a retrospective analysis of the prenatal genetic diagnosis results of fetuses with high risk of major thalassemia to provide information for clinical genetic counseling and to better control the birth of major thalassemia child in Hakka population. Totally, 467 fetuses in at-risk pregnancies were collected from Meizhou people's hospital from January 2014 to December 2017. Genomic DNAs were extracted from peripheral blood of the couples and villus, amniotic fluid or cord blood of the fetuses. DNA-based diagnosis was performed using polymerase chain reaction (PCR) and flow-through hybridization technique. Follow-up visits were done half a year after the fetuses were born. Around 467 fetus at-risk pregnancies were performed prenatal diagnosis. We detected 88 CVS samples, 375 amniocentesis fluid samples and, 4 cord blood samples. The 356 fetuses in α-thalassemia families consisted of 69 (19.38%) with Bart's hydrops syndrome, 20 (5.62%) fetuses with Hb H disease, and 184 (51.68%) fetuses with heterozygote. And the 111 fetuses in ß-thalassemia families consisted of 31 (27.93%) thalassemia major, 51 (45.95%) fetuses with heterozygote. There are 13 fetuses with α+ß-thalassemia, including 2 cases with severe ß-thalassemia. DNA-based testing prenatal diagnosis of thalassemia was found to be highly reliable. Our findings provide key information for clinical genetic counseling of prenatal diagnosis for major thalassemia in Hakka pregnant women. Our work plays an important role in the prevention and control of thalassemia in Hakka population. We will also combine other techniques to further improve our molecular prenatal diagnostic capabilities, including the next-generation sequencing (NGS), Sanger sequencing and MLPA.

Analysis of mutation spectrum of common deafness-causing genes in Hakka newborns in southern China by semiconductor sequencing.

Hearing loss is a common neurosensory disorder, approximately half of the cases are caused by genetic factors, and approximately 70% of hereditary hearing impairments are nonsyndromic hearing loss (NSHL). The mutations of GJB2 (gap junction beta-2 protein), GJB3 (gap junction beta-3 protein), SLC26A4 (solute carrier family 26 member 4), and MT-RNR1 (mitochondrially encoded 12S RNA) are the most common inherited causes of NSHL. Because of different genetic backgrounds, the mutation spectrum of these common deafness-causing genes varies among different regions in China. Because no data are known on these mutations among the Hakka population of Southern China, we aim to investigate the mutation spectrum to add these to neonatal screening and genetic counseling. A total of 1252 blood samples from newborns have been detected by semiconductor sequencing for 100 mutations loci of 18 deafness-causing genes. Of the participants, 95 subjects carried deafness-causing genes mutations with the carrier rate of 7.59%. The mutation frequencies of GJB2, SLC26A4, GJB3, and mitochondrial genes were 3.04%, 3.51%, 0.16%, and 0.88%, respectively. We followed up subjects with single-gene homozygous or compound heterozygous mutations. Our study firstly analyzed deafness-causing genes mutation spectrum in Hakka population, providing evidence for future neonatal screening and genetic counseling in this area.

Molecular prenatal diagnosis of alpha and beta thalassemia in pregnant Hakka women in southern China.

BACKGROUND: To date, there has been no systematic study of DNA-based prenatal diagnosis of thalassemia in pregnant Hakka women in southern China. METHODS: A total of 279 pregnant Hakka women with confirmed cases of thalassemia who had been treated at the Meizhou People's Hospital in China's Guangdong Province from January 2014 to December 2016 were here enrolled. Genomic DNA was extracted from peripheral blood of couples and villus, amniotic fluid, or fetal cord blood. DNA-based diagnosis was performed on the tissues of fetuses whose parents had tested positive for α- and ß-globin gene mutations were found using polymerase chain reaction (PCR) and flow-through hybridization technique. Follow-up visits were performed 6 months after the fetuses were born. Prenatal diagnosis was performed on 279 fetuses in at-risk pregnancies. RESULTS: Here, 211 α-thalassemia fetuses were confirmed, including 41 (19.43%) that tested positive for Bart's hydrops syndrome and 15 (7.11%) for Hb H disease. There were 103 (48.81%) heterozygotes. ß-thalassemia was confirmed in 68 fetuses, including 23 (33.82%) with severe thalassemia and 27 (39.71%) heterozygotes. Another 12 cases were confirmed with α+ß-thalassemia, including three cases of severe ß-thalassemia. DNA-based testing prenatal diagnosis of thalassemia was found to be highly reliable. CONCLUSIONS: Our findings provide key information for clinical genetic counseling of prenatal diagnosis for major thalassemia in pregnant Hakka women in southern China.