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

Abstract 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.


| INTRODUC TI ON
Nicolaides et al 1 first reported in 1992 that NT measurements were used as an ultrasound indicator for chromosome screening in early pregnancy. Increased NT is one of the most sensitive indicators of fetal chromosomal abnormalities in ultrasound screening in early pregnancy. NT refers to the anechoic zone under the skin of the fetal neck, that is, the hydronephrosis between the fetal neck and the skin. 2 Increased NT is associated with chromosomal abnormalities, genetic syndromes, structural abnormalities, and fetuses or perinatal death and other adverse pregnancy outcomes in early pregnancy.
Moreover, the degree of increased NT was directly proportional to the incidence of fetal abnormalities and adverse pregnancy outcomes. [3][4][5] The prevalence of congenital heart defects (CHD) is in the order of six times higher in fetuses with a NT ≥ 99th percentile than in an unselected population. [6][7][8] The association of Noonan syndrome with increased NT is clear. 9, 10 What has not been clearly defined is the incidence of other genetic syndromes, congenital defects, and adverse pregnancy and pediatric outcomes in the presence of increased nuchal translucency. Increased NT defined by The Fetal Medicine Foundation as NT ≥ 3.5 mm, as this measurement corresponds to the 99th percentile in the general population (https://fetal medic ine.org).
At present, the most common method for detection chromosomal abnormalities is karyotype analysis. Karyotype analysis is the "gold standard" of cytogenetic detection, but it has a long detection time and low resolution and cannot detect copy number variations (CNVs) no more than 5 Mb. Chromosome microarray analysis (CMA) is a technique for the detection of CNVs in human genome, and it can detect micro-deletions or micro-duplications that cannot be detected by traditional karyotype analysis. In recent years, CMA technology has been widely used in prenatal diagnosis. [11][12][13][14][15][16][17][18][19] However, its high cost and low throughput limit its large-scale application in prenatal diagnosis. In addition, due to the limited chip probe coverage, some CNVs may not be detected. 20,21 Due to its high resolution, high throughput, and relatively low cost, the genome copy number variation sequencing (CNV-seq) technology based on next generation sequencing (NGS) has been gradually developed. [22][23][24] Meizhou is located in eastern Guangdong Province with a resident population of 5.28 million and an annual birth rate of 12.45‰ There are no reports about the analysis of copy number variation by sequencing in fetuses with increased NT of Hakka population in Meizhou, China. The purpose of this study is to analyze the chromosomes in fetuses with increased NT by CNV-seq in order to evaluate its application in prenatal diagnosis.

| Subjects
From July 2016 to December 2018, fetuses underwent early pregnancy ultrasound screening in Meizhou People's Hospital. Among them, ultrasound screening indicated fetuses with NT ≥ 2.5 mm.
Increased NT defined by The Fetal Medicine Foundation as NT ≥ 3.5 mm, as this measurement corresponds to the 99th percentile in the general population (https://fetal medic ine.org). Different studies on the definition of NT thickening mostly use 3.5 mm for cutoff value criticality, but the risk of fetal abnormalities may increase when NT value within 2.5-2.9 mm, therefore, these fetuses with NT is greater than or equal to 2.5 mm were included in this study.
139 fetuses with prenatal diagnosis (chromosome karyotype analysis and CNV analysis) were finally included in the analysis.
All the 139 fetuses were single fetus. The average age of preg-

| Statistical analysis
SPSS statistical software version 21.0 was used for data analysis.
The abnormal degree of NT, the age of pregnant women, the presence of other abnormalities, and other CNV abnormalities were analyzed. The data were expressed as the means ± SD. Mann-Whitney U test was used to compare values between groups. Chi-square and ANOVA tests were used to analyze the differences among the two groups. A value of P < .05 was considered as statistically significant.

| The relationship between NT thickness and abnormal CNV detection
Among the 139 samples, 45 were abnormal with CNVs (32.37%).
We divided the included patients into four groups with NT value of 1 mm as an interval. 139 fetuses with NT ≥ 2.5 mm at 11-14 weeks of gestation were divided into four groups, including 2.5-3.4 mm, 3.5-4.4 mm, 4.5-5.4 mm, and ≥5.5 mm groups. With the increase in NT thickness, the ratio of live birth is decreasing, which is statistically significant (χ 2 = 54.03, P < .01). The detection rate of the corresponding abnormal CNV also increases with the increase, statistically significant (χ 2 = 37.69, P < .01), as shown in Table 1.

| CNV-seq and karyotype analysis
Among the 139 samples, 36 were abnormal with chromosome karyotype. There were 12 cases with abnormal CNVs and normal karyotype, 2 of which were pathogenic CNVs ( Table 2). The detected pathogenic CNVs is known as 22q11 micro-duplication syndrome and 15q11.

| Statistical analysis of CNV results under different factors
There was no statistically significant difference between elderly and non-elderly pregnant women with abnormal CNV (χ 2 = 1.389, P = .239). The difference between NT abnormality alone and NT with other ultrasound abnormalities was statistically significance (χ 2 = 5.128, P = .024). There was statistically significant difference in different pregnancy outcomes (χ 2 = 113.187, P < .01) ( Table 4).

| D ISCUSS I ON
As early as 2004, the British fetal foundation recommended that prenatal screening in early pregnancy include age, serological screening, and NT, which could lead to a screening rate of 80%-90% for Down syndrome. In recent years, the role of NT examination in chromosome aneuploidy screening has also been affirmed. Meanwhile, NT values above 99th percentile for gestational age seem to be associated with increased rates of chromosomal/structural abnormalities and adverse perinatal outcomes. 26 In this study, 139 cases of NT thickened fetus were studied and 45 fetal abnormal CNVs were detected. According to the thickness of NT, subjects were divided into 4 groups, and it was found that with the increase of thickness, the proportion of abnormal CNV also increased, and the induction rate also in-  The cost of CNV-seq for one case is only a few hundred yuan (RMB).
And it is time-efficient and prospective in the detection of copy number variation. In addition, the initial amount of DNA required by CNV-seq in clinical application is as low as 10 ng, which is 5% of the amount of DNA required by CMA, and the detection sensitivity is higher. Therefore, in prenatal diagnosis, CNV-seq technology has the advantages of rapid, accurate, high resolution, high throughput, and relatively low cost.
There are two cases with normal CNVs but abnormal karyo-

| CON CLUS ION
In conclusion, NT is an effective means of prenatal diagnosis and screening. For the prenatal diagnosis of NT thickened fetus, the chromosomal karyotype analysis plus CNV-seq detection mode will be able to detect chromosomal micro-deletions and micro-duplications at the whole genome level, which is of great significance for prenatal diagnosis.