The routine assessment to determine the genetic etiology for fetal ultrasound anomalies follows a sequential approach, which usually takes about 6–8 weeks turnaround time (TAT). We evaluated the clinical utility of simultaneous detection of copy number variations (CNVs) and single nucleotide variants (SNVs)/small insertion-deletions (indels) in fetuses with a normal karyotype with ultrasound anomalies. We performed CNV detection by chromosomal microarray analysis (CMA) or low pass CNV-sequencing (CNV-seq), and in parallel SNVs/indels detection by trio-based clinical exome sequencing (CES) or whole exome sequencing (WES). Eight-three singleton pregnancies with a normal fetal karyotype were enrolled in this prospective observational study. Pathogenic or likely pathogenic variations were identified in 30 cases (CNVs in 3 cases, SNVs/indels in 27 cases), indicating an overall molecular diagnostic rate of 36.1% (30/83). Two cases had both a CNV of uncertain significance (VOUS) and likely pathogenic SNV, and one case carried both a VOUS CNV and an SNV. We demonstrated that simultaneous analysis of CNVs and SNVs/indels can improve the diagnostic yield of prenatal diagnosis with shortened reporting time, namely, 2–3 weeks. Due to the relatively long TAT for sequential procedure for prenatal genetic diagnosis, as well as recent sequencing technology advancements, it is clinically necessary to consider the simultaneous evaluation of CNVs and SNVs/indels to enhance the diagnostic yield and timely TAT, especially for cases in the late second trimester or third trimester.

Objective: Absence of homozygosity (AOH) is a genetic characteristic known to cause human diseases mainly through autosomal recessive or imprinting mechanisms. The importance and necessity of accurate AOH detection has become more clinically significant in recent years. However, it remains a challenging task for sequencing-based methods thus far. Methods: In this study, we developed and optimized a new bioinformatic algorithm based on the assessment of minimum sequencing coverage, optimal bin size, the Z-score threshold of four types of allele count and the frequency for accurate genotyping using 28 AOH negative samples, and redefined the AOH detection cutoff value. We showed the performance of chromosome analysis by five-fold coverage whole genome sequencing (CMA-seq) for AOH identification in 27 typical prenatal/postnatal AOH positive samples, which were previously confirmed by chromosomal microarray analysis with single nucleotide polymorphism array (CMA/SNP array). Results: The blinded study indicated that for all three forms of AOH, including whole genomic AOH, single chromosomal AOH and segmental AOH, and all kinds of sample types, including chorionic villus sampling, amniotic fluid, cord blood, peripheral blood and abortive tissue, CMA-seq showed equivalent detection power to that of routine CMA/SNP arrays (750K). The subtle difference between the two methods is that CMA-seq is prone to detect small inconsecutive AOHs, while CMA/SNP array reports it as a whole. Conclusion: Based on our newly developed bioinformatic algorithm, it is feasible to detect clinically significant AOH using CMA-seq in prenatal diagnosis.

Karyotyping is considered the gold standard for the genome-wide detection of genomic imbalances in prenatal diagnosis, but it has a number of inherent limitations, namely the time required to culture cell and the limited resolution(5 ~ 10 Mb). Although fluorescence in situ hybridization (FISH) can also be used as a rapid prenatal diagnosis for common aneuploidies, it is labor intensive, requires prior knowledge of the regions of interest, and can only be used to diagnose one or a few genomic regions simultaneously. Array comparative genomic hybridization (aCGH) can overcome the resolution, the locus-specific, and the time limitations of the karyotyping and FISH techniques and is currently the most powerful method for detecting chromosomal alterations in pre and postnatal clinical cases. Several investigations have suggested that the aCGH testing should be considered a first-tier test for the diagnosis of cytogenetic aberrations in the fetus.

Objective: Mosaicism is a common biological phenomenon in organisms and has been reported in many types of chromosome abnormalities, including the absence of heterozygosity (AOH). Due to the detection limitations of the sequencing approach, mosaic AOH events are rarely assessed in clinical cases. Herein, we report the performance of mosaic AOH identification using a low-pass (5~8-fold) WGS method (termed ‘CMA-seq’, an abbreviation for ‘Chromosome Analysis by Sequencing’) in fetal genetic diagnosis. Methods: Thirty AOH-negative, eleven constitutional AOH, and three mosaic AOH samples were collected as training data sets to develop the algorithm and evaluate the suitable thresholds for distinguishing mosaic AOH. Twenty-four new chromosomal aberrant cases, along with sixteen constitutional AOH samples, which were previously ascertained via the SNP-array-based method, were used as a validation data set to measure the performance in terms of sensitivity and specificity of this algorithm. Results: A new statistic, ‘D-value’, was implemented to identify and distinguish constitutional and mosaic AOH events. The reporting thresholds for constitutional and mosaic AOH were also established. In the validation set consisting of 24 new cases, seven constitutional AOH cases and 1 mosaic AOH case were successfully identified, indicating that the results were consistent with those of the SNP-array-based method. The results of all sixteen constitutional AOH validation samples also met the threshold requirements. Conclusions: In this study, we developed a new bioinformatic algorithm to accurately distinguish mosaic AOH from constitutional AOH by low-pass WGS. However, due to the small sample size of the training data set, the algorithm proposed in this manuscript still needs further refinements.

Objective: Congenital diaphragmatic hernia (CDH) is a rare abnormality with highly heterogeneous genetic causes. This study investigated chromosomal and monogenic abnormalities in fetal CDH patients and evaluated the efficacy of chromosomal microarray analysis (CMA) and whole‐exome sequencing (WES) for genetic diagnosis. The clinical features of the patients were also evaluated. Methods: We evaluated the genetic and clinical data of 51 prenatally diagnosed fetuses with CDH. CMA was performed for every patient. If CMA did not yield diagnostic results, the samples were subjected to WES. Results: Compared with fetuses with isolated CDH ( n = 42), those with non‐isolated CDH ( n = 9) presented a higher genetic diagnostic rate (22.2% vs. 2.4%). The overall diagnostic yield was 5.9%, comprising 3.9% from chromosomal microarray analysis (CMA) and an additional 2.0% from whole exome sequencing (WES). CMA identified (1) mosaic trisomy 18 in a patient with isolated CDH; and (2) 4q terminal deletion syndrome in a patient with non‐isolated CDH. WES identified a novel missense mutation, PLS3 c.1763A > G , associated with X‐linked CDH in a patient with non‐isolated CDH and a family history of recurrent CDH. Conclusion: Genetic testing should be offered for all fetuses with CDH, regardless of whether the cases are isolated or non‐isolated. WES should be considered if CMA fails to provide a diagnostic result, particularly in patients with non‐isolated CDH and a family history of recurrent CDH.