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Copy number variation characterization and possible candidate genes in miscarriage and stillbirth by next‐generation sequencing analysis

Abstract Background The present study aimed to explore the etiological relationship between miscarriage and stillbirth and copy number variations (CNVs), as well as provide useful genetic guidance for high‐risk pregnancy. Methods In total, 659 fetal samples were recruited and subjected to DNA extraction and CNV sequencing (CNV‐seq), relevant medical records were collected. Results There were 322 cases (48.86%) with chromosomal abnormalities, including 230 with numerical abnormalities and 92 with structural abnormalities. Chromosomal monosomy variations mainly occurred on sex chromosomes and trisomy variations mainly occurred on chromosomes 16, 22, 21, 18, 13 and 15. In total, 41 pathogenic CNVs (23 microdeletions and 18 microduplications) were detected in 27 fetal tissues. The rates of numerical chromosomal abnormalities were 29.30% (109/372), 32.39% (57/176) and 57.66% (64/111) in < 30‐year‐old, 30–34‐year‐old and ≥ 35‐year‐old age pregnant women, respectively, and increased with an increasing age (p < 0.001). There was statistically significant difference (χ2 = 7.595, p = 0.022) in the rates of structural chromosomal abnormalities in these groups (13.71%, 18.75% and 7.21%, respectively). The rates of numerical chromosomal abnormalities were 45.44% (219/482), 7.80% (11/141) and 0% (0/36) in the ≤ 13 gestational weeks, 14–27 weeks and ≥ 28 weeks groups, respectively, and decreased with respect to the increasing gestational age of the fetuses (p < 0.001). Conclusions The present study has obtained useful and accurate genetic etiology information that will provide useful genetic guidance for high‐risk pregnancies.


| INTRODUCTION
Miscarriage is the spontaneous loss of a pregnancy occurring before 28 weeks. The spontaneous loss of the fetus with a weight less than 1000 g occurring before 12 gestational weeks is called early miscarriage, and that occurring from 12 to 28 gestational weeks is called late miscarriage. 1 Stillbirth involves a fetus that dies in the uterus after 20 weeks of gestation. 2 The incidence of miscarriage is about 15-20% and there is an increasing trend year by year. 3 The incidence of stillbirth is about 0.5-0.6%. 4 The causes of miscarriage and stillbirth including environmental factors, endocrine diseases, immune diseases and genetic factors. A related study had shown that genetic factors play a leading role in early miscarriage and stillbirth. 5 Some 6-13% of stillbirths were associated with abnormal karyotype, 6 and 5-40% of stillbirths with an abnormal anatomical structure were associated with abnormal karyotype. 7 Genetic analysis of miscarriage and stillbirth tissue is of great value with respect to the analysis of the causes of miscarriage and stillbirth, assessing the risk of recurrence and prenatal diagnosis. 8 Chromosomal abnormalities include numerical chromosomal abnormalities (such as monosomy, trisomy and polyploidy) and structural chromosomal abnormalities (such as deletion, duplication, insertion, inversion, cross-displacement, ring chromosome and translocation). 9,10 Around the beginning of the 21st Century, scientists began to recognize an intermediate size variation. Copy number variants (CNVs) are copy number changes of the genome, with variants ranging in size from several dozens of bases (> 50 bp) to megabases. 11,12 CNVs have been shown to affect gene function by changing coding sequences and regulatory elements, and thus they have association with the susceptibility to diseases such as genetic diseases, cancer, infections and metabolic disorders. 13,14 An increasing number of studies had shown that pathogenic copy number variations (pCNVs) account for a certain percentage of the fetuses in older pregnant women and with abnormal ultrasound. 15,16 Karyotype analysis is one of the main detection techniques for chromosomal abnormality. However, karyotype analysis has some limitations. First, it has a long experimental period, high technical requirements and a high risk of failure. Second, it can only diagnose chromosomal deletion and duplication with more than 5-10 Mb, and minor structural changes may be missed. 17 With the rapid development of high-throughput sequencing technology, the advantage of sequencing technology in the detection of CNV is becoming more and more obvious. Copy number variation sequencing (CNV-seq) based on next generation sequencing (NGS) technology was used for sequencing analysis of samples, and the sequencing results were compared with the human reference genome and CNV was found through bioinformatics analysis. CNV-seq can detect chromosome aneuploidies, chromosome CNVs and polyploidies, and can also detect microdeletions and microduplications with < 5 Mb. [18][19][20] CNV-seq can detect DNA extracted directly from uncultured tissues, which greatly improves the success rate of detection. Second, it has high resolution and can detect CNVs that cannot be detected by karyotype analysis.
At the same time, it can detect unknown variations and reveal new genetic information related to the disease.
In recent years, studies using CNV-seq to analyze the relationship between CNV and miscarriage and stillbirth have been reported. The prevalence of aneuploidy and pathogenicity-associated CNV in aborted fetal tissue was associated with an increased risk of miscarriage in advanced maternal age pregnant women. [21][22][23] Numerical chromosomal abnormality was the most important reason for embryo termination in early and middle pregnancy, followed by pCNVs. 24 Dai et al. 25 found that the fetal chromosomal abnormality rate in firsttrimester spontaneous abortion was significantly higher than the second-trimester spontaneous abortion. Wang et al. 26 reported that 309 genes were identified as potential miscarriage candidate genes by analyzing 5,003 miscarriage specimens. These studies found that chromosomal aneuploidy was one of the main genetic factors for abortion and also that some pCNVs were associated with miscarriage and stillbirth. The number of cases with miscarriage and stillbirth studied so far is still too small to allow identification of specific variations or genes for miscarriage and stillbirth, and some of the relevant biological processes are not emerging.
To investigate the differences in the incidence and distribution of chromosomal abnormalities of miscarriage and stillbirth systematically and investigate the role of CNV with respect to genetic etiology in miscarriage and stillbirth, samples of miscarriage and stillbirth fetuses were analyzed by CNV-seq in the present study. We analyzed the genomic regions of detected CNVs aiming to identify potential miscarriage and stillbirth candidate genes, and analyzed gene functions using enrichment and signaling pathways analysis. The results of this study may help to establish population-based genetic markers for miscarriage and stillbirth screening and provide useful genetic guidance for high-risk pregnancy.   (4) likely benign CNVs; and (5) benign CNVs.

| Statistical analysis
SPSS, version 21.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. Data were reported with the descriptive statistics method and measurement data are expressed as the mean ± SD. A chi-square test was used to analyze the difference among the groups. p < 0.05 was considered statistically significant.

| Functional enrichment analysis
The genes located in the pathogenic CNVs, likely pathogenic CNVs and VOUS regions were referred to in the DECIPHER database F I G U R E 1 Flow diagram of the CNV-seq of miscarriage and stillbirth, as well as the analytical strategies used in the present study

| Detection results of fetuses with chromosome number abnormality and pCNVs
In the present study, there were 230 cases with numerical chromo-

| Identification of miscarriage candidate genes
To identify the critical genes and related signaling pathways associated with miscarriage and stillbirth, the genes in the pCNVs and VOUS  showed that no significant signaling pathways were enriched.
According to the GO analysis results mentioned above, the gene functions were divided into seven functional categories: development of the brain and nervous system, heart formation and development, embryo development and organ formation, cell structure and function, respiratory system development, regulation of ion channels, and regulation of endopeptidase activity ( Figure 3B). These genes were mainly enriched in functional categories: development of the brain and nervous system (40 genes) and heart formation and development (9 genes).    increasing number studies on the pathogenesis of some diseases using CNV-seq. 19,34 Recently, CNVs have also been observed in miscarriage samples. 35 In the present study, the rates of chromosomal abnormalities and The causes of fetal chromosomal aneuploidy related to maternal age may involve some functional changes or degeneration of oocytes in elderly pregnant women, such as meiotic recombination failure, deterioration of chromosome cohesion, spindle assembly checkpoint dysfunction, altered post-translational modifications, and mitochondrial dysfunction. 50 In addition, the incidences of chromosomal abnormalities and numerical chromosomal abnormalities decreased with an increasing gestational age of fetuses, whereas the lowest incidence was in the individuals ≥ 28 weeks. These results are consistent with those of previous study. 17 The chromosomal abnormalities of aborted fetuses in early pregnancy may involve any one chromosome or more chromosomes, although the chromosomal abnormalities of aborted fetuses in middle and late pregnancy may be trisomy 13, 18 and 21, sex chromosome aneuploidy and CNVs, which are similar to the types of chromosomal aberrations detected in live births. 51 However another study has found that the incidences of fetal abnormality and growth restriction increase as gestation proceeds. 52 The inconsistency of these results may be a result of differences in population, sample size and detection methods in the various studies. congenital hypopituitarism in children. 53 Variants in the aldehyde dehydrogenase 1 family member A2 (ALDH1A2) gene cause lethal multiple congenital anomaly syndrome. 54 ATPase copper transporting alpha (ATP7A) is a critical copper transporter involved in some X linked genetic disorders, such as Menkes disease, occipital horn syndrome and type 3 X-linked distal spinal muscular atrophy. 55 Protocadherin related 15 (PCDH15) is associated with nonsyndromic deafness and type 1 Usher syndrome. 56 Muscle skeletal receptor tyrosine kinase (MUSK) is the pathogenic gene of congenital myasthenic syndrome. 57 Variants in the GATA binding protein 4 (GATA4) gene cause congenital heart disease. 58

| CONCLUSIONS
In conclusion, CNV-seq can be used as an effective method for chromosomal CNVs analysis of fetal tissues in miscarriage and stillbirth.
The results of the present study show that CNVs are a genetic etiological factor with respect to miscarriage and stillbirth. Some useful and accurate genetic etiological information regarding miscarriage and stillbirth has been obtained that provides useful genetic guidance for