Low-Pass Genome Sequencing-Based Detection of Paternity: Validation in Clinical Cytogenetics.

Submission of a non-biological parent together with a proband for genetic diagnosis would cause a misattributed parentage (MP), possibly leading to misinterpretation of the pathogenicity of genomic variants. Therefore, a rapid and cost-effective paternity/maternity test is warranted before genetic testing. Although low-pass genome sequencing (GS) has been widely used for the clinical diagnosis of germline structural variants, it is limited in paternity/maternity tests due to the inadequate read coverage for genotyping. Herein, we developed rapid paternity/maternity testing based on low-pass GS with trio-based and duo-based analytical modes provided. The optimal read-depth was determined as 1-fold per case regardless of sequencing read lengths, modes, and library construction methods by using 10 trios with confirmed genetic relationships. In addition, low-pass GS with different library construction methods and 1-fold read-depths were performed for 120 prenatal trios prospectively collected, and 1 trio was identified as non-maternity, providing a rate of MP of 0.83% (1/120). All results were further confirmed via quantitative florescent PCR. Overall, we developed a rapid, cost-effective, and sequencing platform-neutral paternity/maternity test based on low-pass GS and demonstrated the feasibility of its clinical use in confirming the parentage for genetic diagnosis.

A pilot investigation of low-pass genome sequencing identifying site-specific variation in chromosomal mosaicisms by a multiple site sampling approach in first-trimester miscarriages.

STUDY QUESTION: Can multiple-site low-pass genome sequencing (GS) of products of conception (POCs) improve the detection of genetic abnormalities, especially heterogeneously distributed mosaicism and homogeneously distributed mosaicism in first-trimester miscarriage? SUMMARY ANSWER: Multiple-site sampling combined with low-pass GS significantly increased genetic diagnostic yield (77.0%, 127/165) of first-trimester miscarriages, with mosaicisms accounting for 17.0% (28/165), especially heterogeneously distributed mosaicisms (75%, 21/28) that are currently underappreciated. WHAT IS KNOWN ALREADY: Aneuploidies are well known to cause first-trimester miscarriage, which are detectable by conventional karyotyping and next-generation sequencing (NGS) on a single-site sampling basis. However, there are limited studies demonstrating the implications of mosaic genetic abnormalities in first-trimester miscarriages, especially when genetic heterogeneity is present in POCs. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional cohort study carried out at a university-affiliated public hospital. One hundred seventy-four patients diagnosed with first-trimester miscarriage from December 2018 to November 2021 were offered ultrasound-guided manual vacuum aspiration (USG-MVA) treatment. Products of conception were subjected to multiple-site low-pass GS for the detection of chromosomal imbalances. PARTICIPANTS/MATERIALS, SETTING, METHODS: For each POC, multiple sites of villi (three sites on average) were biopsied for low-pass GS. Samples with maternal cell contamination (MCC) and polyploidy were excluded based on the quantitative fluorescence polymerase chain reaction (QF-PCR) results. The spectrum of chromosomal abnormalities, including mosaicism (heterogeneously distributed and homogeneously distributed) and constitutional abnormalities was investigated. Chromosomal microarray analysis and additional DNA fingerprinting were used for validation and MCC exclusion. A cross-platform comparison between conventional karyotyping and our multiple-site approach was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred sixty-five POCs (corresponding to 490 DNA samples) were subjected to low-pass GS. Genetic abnormalities were detected in 77.0% (127/165) of POCs by our novel approach. Specifically, 17.0% (28/165) of cases had either heterogeneously distributed mosaicism (12.7%, 21/165) or homogeneously distributed mosaicism (6.1%, 10/165) (three cases had both types of mosaicism). The remaining 60.0% (99/165) of cases had constitutional abnormalities. In addition, in the 71 cases with karyotyping performed in parallel, 26.8% (19/71) of the results could be revised by our approach. LIMITATIONS, REASONS FOR CAUTION: Lack of a normal gestational week-matched cohort might hinder the establishment of a causative link between mosaicisms and first-trimester miscarriage. WIDER IMPLICATIONS OF THE FINDINGS: Low-pass GS with multiple-site sampling increased the detection of chromosomal mosaicisms in first-trimester miscarriage POCs. This innovative multiple-site low-pass GS approach enabled the novel discovery of heterogeneously distributed mosaicism, which was prevalent in first-trimester miscarriage POCs and frequently observed in preimplantation embryos, but is currently unappreciated by conventional single-site cytogenetic investigations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported partly by Research Grant Council Collaborative Research Fund (C4062-21GF to K.W.C), Science and Technology Projects in Guangzhou (202102010005 to K.W.C), Guangdong-Hong Kong Technology Cooperation Funding Scheme (TCFS), Innovation and Technology Fund (GHP/117/19GD to K.W.C), HKOG Direct Grant (2019.050 to J.P.W.C), and Hong Kong Health and Medical Research Fund (05160406 to J.P.W.C). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Mate-pair genome sequencing reveals structural variants for idiopathic male infertility.

Currently, routine genetic investigation for male infertility includes karyotyping analysis and PCR for Y chromosomal microdeletions to provide prognostic information such as sperm retrieval success rate. However, over 85% of male infertility remain idiopathic. We assessed 101 male patients with primary infertility in a retrospective cohort analysis who have previously received negative results from standard-of-care tests. Mate-pair genome sequencing (large-insert size library), an alternative long-DNA sequencing method, was performed to detect clinically significant structural variants (SVs) and copy-number neutral absence of heterozygosity (AOH). Candidate SVs were filtered against our in-house cohort of 1077 fertile men. Genes disrupted by potentially clinically significant variants were correlated with single-cell gene expression profiles of human fetal and postnatal testicular developmental lineages and adult germ cells. Follow-up studies were conducted for each patient with clinically relevant finding(s). Molecular diagnoses were made in 11.1% (7/63) of patients with non-obstructive azoospermia and 13.2% (5/38) of patients with severe oligozoospermia. Among them, 12 clinically significant SVs were identified in 12 cases, including five known syndromes, one inversion, and six SVs with direct disruption of genes by intragenic rearrangements or complex insertions. Importantly, a genetic defect related to intracytoplasmic sperm injection (ICSI) failure was identified in a patient with non-obstructive azoospermia, illustrating the additional value of an etiologic diagnosis in addition to determining sperm retrieval rate. Our study reveals a landscape of various genomic variants in 101 males with idiopathic infertility, not only advancing understanding of the underlying mechanisms of male infertility, but also impacting clinical management.

TEDD: a database of temporal gene expression patterns during multiple developmental periods in human and model organisms.

Characterization of the specific expression and chromatin profiles of genes enables understanding how they contribute to tissue/organ development and the mechanisms leading to diseases. Whilst the number of single-cell sequencing studies is increasing dramatically; however, data mining and reanalysis remains challenging. Herein, we systematically curated the up-to-date and most comprehensive datasets of sequencing data originating from 2760 bulk samples and over 5.1 million single-cells from multiple developmental periods from humans and multiple model organisms. With unified and systematic analysis, we profiled the gene expression and chromatin accessibility among 481 cell-types, 79 tissue-types and 92 timepoints, and pinpointed cells with the co-expression of target genes. We also enabled the detection of gene(s) with a temporal and cell-type specific expression profile that is similar to or distinct from that of a target gene. Additionally, we illustrated the potential upstream and downstream gene-gene regulation interactions, particularly under the same biological process(es) or KEGG pathway(s). Thus, TEDD (Temporal Expression during Development Database), a value-added database with a user-friendly interface, not only enables researchers to identify cell-type/tissue-type specific and temporal gene expression and chromatin profiles but also facilitates the association of genes with undefined biological functions in development and diseases. The database URL is https://TEDD.obg.cuhk.edu.hk/.

The Burden and Benefits of Knowledge: Ethical Considerations Surrounding Population-Based Newborn Genome Screening for Hearing.

Recent advances in genomic sequencing technologies have expanded practitioners' utilization of genetic information in a timely and efficient manner for an accurate diagnosis. With an ever-increasing resource of genomic data from progress in the interpretation of genome sequences, clinicians face decisions about how and when genomic information should be presented to families, and at what potential expense. Presently, there is limited knowledge or experience in establishing the value of implementing genome sequencing into newborn screening. Herein we provide insight into the complexities and the burden and benefits of knowledge resulting from genome sequencing of newborns.

Investigation of Chromosomal Structural Abnormalities in Patients With Undiagnosed Neurodevelopmental Disorders.

Background: Structural variations (SVs) are various types of the genomic rearrangements encompassing at least 50 nucleotides. These include unbalanced gains or losses of DNA segments (copy number changes, CNVs), balanced rearrangements (such as inversion or translocations), and complex combinations of several distinct rearrangements. SVs are known to play a significant role in contributing to human genomic disorders by disrupting the protein-coding genes or the interaction(s) with cis-regulatory elements. Recently, different types of genome sequencing-based tests have been introduced in detecting various types of SVs other than CNVs and regions with absence of heterozygosity (AOH) with clinical significance. Method: In this study, we applied the mate-pair low pass (∼4X) genome sequencing with large DNA-insert (∼5 kb) in a cohort of 100 patients with neurodevelopmental disorders who did not receive informative results from a routine CNV investigation. Read-depth-based CNV analysis and chimeric-read-pairs analysis were used for CNV and SV analyses. The region of AOH was indicated by a simultaneous decrease in the rate of heterozygous SNVs and increase in the rate of homozygous SNVs. Results: First, we reexamined the 25 previously reported CNVs among 24 cases in this cohort. The boundaries of these twenty-five CNVs including 15 duplications and 10 deletions detected were consistent with the ones indicated by the chimeric-read-pairs analysis, while the location and orientation were determined in 80% of duplications (12/15). Particularly, one duplication was involved in complex rearrangements. In addition, among all the 100 cases, 10% of them were detected with rare or complex SVs (>10 Kb), and 3% were with multiple AOH (≥5 Mb) locating in imprinting chromosomes identified. In particular, one patient with an overall value of 214.5 Mb of AOH identified on 13 autosomal chromosomes suspected parental consanguinity. Conclusion: In this study, mate-pair low-pass GS resolved a significant proportion of CNVs with inconclusive significance, and detected additional SVs and regions of AOH in patients with undiagnostic neurodevelopmental disorders. This approach complements the first-tier CNV analysis for NDDs, not only by increasing the resolution of CNV detection but also by enhancing the characterization of SVs and the discovery of potential causative regions (or genes) contributory to could be complex in composition NDDs.

Investigation of the genetic etiology in male infertility with apparently balanced chromosomal structural rearrangements by genome sequencing.

Apparently balanced chromosomal structural rearrangements are known to cause male infertility and account for approximately 1% of azoospermia or severe oligospermia. However, the underlying mechanisms of pathogenesis and etiologies are still largely unknown. Herein, we investigated apparently balanced interchromosomal structural rearrangements in six cases with azoospermia/severe oligospermia to comprehensively identify and delineate cryptic structural rearrangements and the related copy number variants. In addition, high read-depth genome sequencing (GS) (30-fold) was performed to investigate point mutations causative of male infertility. Mate-pair GS (4-fold) revealed additional structural rearrangements and/or copy number changes in 5 of 6 cases and detected a total of 48 rearrangements. Overall, the breakpoints caused truncations of 30 RefSeq genes, five of which were associated with spermatogenesis. Furthermore, the breakpoints disrupted 43 topological-associated domains. Direct disruptions or potential dysregulations of genes, which play potential roles in male germ cell development, apoptosis, and spermatogenesis, were found in all cases (n = 6). In addition, high read-depth GS detected dual molecular findings in case MI6, involving a complex rearrangement and two point mutations in the gene DNAH1. Overall, our study provided the molecular characteristics of apparently balanced interchromosomal structural rearrangements in patients with male infertility. We demonstrated the complexity of chromosomal structural rearrangements, potential gene disruptions/dysregulation and single-gene mutations could be the contributing mechanisms underlie male infertility.

Trio-Based Low-Pass Genome Sequencing Reveals Characteristics and Significance of Rare Copy Number Variants in Prenatal Diagnosis.

Background: Low-pass genome sequencing (GS) detects clinically significant copy number variants (CNVs) in prenatal diagnosis. However, detection at improved resolutions leads to an increase in the number of CNVs identified, increasing the difficulty of clinical interpretation and management. Methods: Trio-based low-pass GS was performed in 315 pregnancies undergoing invasive testing. Rare CNVs detected in the fetuses were investigated. The characteristics of rare CNVs were described and compared to curated CNVs in other studies. Results: A total of 603 rare CNVs, namely, 597 constitutional and 6 mosaic CNVs, were detected in 272 fetuses (272/315, 86.3%), providing 1.9 rare CNVs per fetus (603/315). Most CNVs were smaller than 1 Mb (562/603, 93.2%), while 1% (6/603) were mosaic. Forty-six de novo (7.6%, 46/603) CNVs were detected in 11.4% (36/315) of the cases. Eighty-four CNVs (74 fetuses, 23.5%) involved disease-causing genes of which the mode of inheritance was crucial for interpretation and assessment of recurrence risk. Overall, 31 pathogenic/likely pathogenic CNVs were detected, among which 25.8% (8/31) were small (<100 kb; n = 3) or mosaic CNVs (n = 5). Conclusion: We examined the landscape of rare CNVs with parental inheritance assignment and demonstrated that they occur frequently in prenatal diagnosis. This information has clinical implications regarding genetic counseling and consideration for trio-based CNV analysis.

Low-pass genome sequencing-based detection of absence of heterozygosity: validation in clinical cytogenetics.

PURPOSE: Absence of heterozygosity (AOH) is a genetic characteristic known to cause human genetic disorders through autosomal recessive or imprinting mechanisms. However, the analysis of AOH via low-pass genome sequencing (GS) is not yet clinically available. METHODS: Low-pass GS (fourfold) with different types of libraries was performed on 17 clinical samples with previously ascertained AOH by chromosomal microarray analysis (CMA). In addition, AOH detection was performed with low-pass GS data in 1,639 cases that had both GS and high-probe density CMA data available from the 1000 Genomes Project. Cases with multiple AOHs (coefficient of inbreeding F ≥ 1/32) or terminal AOHs ≥5 Mb (suspected uniparental disomy [UPD]) were reported based on the guidelines of the American College of Medical Genetics and Genomics. RESULTS: Low-pass GS revealed suspected segmental UPD and multiple AOHs (F ≥ 1/32) in nine and eight clinical cases, respectively, consistent with CMA. Among the 1,639 samples, low-pass GS not only consistently detected multiple AOHs (F ≥ 1/32) in 18 cases, but also reported 60 terminal AOHs in 44 cases including four mosaic AOHs at a level ranging from 50% to 75%. CONCLUSION: Overall, our study demonstrates the feasibility of AOH analysis (≥5 Mb) with low-pass GS data and shows high concordance compared with CMA.

The role of chromosomal microarray and exome sequencing in prenatal diagnosis.

PURPOSE OF REVIEW: Advancements in technologies have revolutionized prenatal diagnosis. Chromosomal microarray analysis (CMA) became a proven method and was implemented to detect gains and losses of DNA and absence of heterozygosity across the genome. Next-generation sequencing technologies have brought opportunities and challenges to genetic testing. Exome sequencing detects single-nucleotide variants (SNVs) across the exome and its prenatal application is an emerging field. We reviewed the literature to define the role of CMA and exome sequencing in prenatal diagnosis. RECENT FINDING: The application of exome sequencing in genetic diagnosis shows increased diagnostic yield and could be potentially implemented for prenatal diagnosis of fetuses with one or more ultrasound structural abnormalities or suspected monogenetic conditions. Although CMA is a gold standard for copy number variant (CNV) detection, large clinical cohort studies emphasized integrated CNV and SNV analyses for precise molecular diagnosis. Recent studies also suggest low-pass genome sequencing-based CNV detection can identify genome-wide imbalances at higher resolutions. SUMMARY: Data suggest exome sequencing for SNVs and CMA for CNV detection are the most effective approach for prenatal genetic diagnosis. Emerging evidences show genome sequencing has the potential to replace CMA and even exome sequencing to become a comprehensive genetic test in the clinical diagnostic laboratory.

Deciphering the complexity of simple chromosomal insertions by genome sequencing.

Chromosomal insertions are thought to be rare structural rearrangements. The current understanding of the underlying mechanisms of their origin is still limited. In this study, we sequenced 16 cases with apparent simple insertions previously identified by karyotyping and/or chromosomal microarray analysis. Using mate-pair genome sequencing (GS), we identified all 16 insertions and revised previously designated karyotypes in 75.0% (12/16) of the cases. Additional cryptic rearrangements were identified in 68.8% of the cases (11/16). The incidence of additional cryptic rearrangements in chromosomal insertions was significantly higher compared to balanced translocations and inversions reported in other studies by GS. We characterized and classified the cryptic insertion rearrangements into four groups, which were not mutually exclusive: (1) insertion segments were fragmented and their subsegments rearranged and clustered at the insertion site (10/16, 62.5%); (2) one or more cryptic subsegments were not inserted into the insertion site (5/16, 31.3%); (3) segments of the acceptor chromosome were scattered and rejoined with the insertion segments (2/16, 12.5%); and (4) copy number gains were identified in the flanking regions of the insertion site (2/16, 12.5%). In addition to the observation of these chromothripsis- or chromoanasynthesis-like events, breakpoint sequence analysis revealed microhomology to be the predominant feature. However, no significant correlation was found between the number of cryptic rearrangements and the size of the insertion. Overall, our study provide molecular characterization of karyotypically apparent simple insertions, demonstrate previously underappreciated complexities, and evidence that chromosomal insertions are likely formed by nonhomologous end joining and/or microhomology-mediated replication-based DNA repair.

The role of chromosomal microarray analysis among fetuses with normal karyotype and single system anomaly or nonspecific sonographic findings.

INTRODUCTION: Chromosomal microarray analysis is recommended as the first-tier test for the evaluation of fetuses with structural anomalies. This study aims to investigate the incremental diagnostic yield of chromosomal microarray over conventional karyotyping analysis in fetuses with anomalies restricted to one anatomic system and those with nonspecific anomalies detected by sonography. MATERIAL AND METHODS: This is a retrospective cohort analysis of 749 fetuses undergoing prenatal diagnosis for abnormal ultrasound findings isolated to one anatomic system and normal karyotype, utilizing chromosomal microarray. Overall, 495 (66%) fetuses had anomalies confined to one anatomic system and 254 (34%) had other nonspecific anomalies including increased nuchal translucency (≥3.5 mm), cystic hygroma, intrauterine growth restriction and hydrops fetalis. RESULTS: Fetuses with ultrasound anomalies restricted to one anatomic system had a 3.0% risk of carrying a pathogenic copy number variant; the risk varied dependent on the anatomic system affected. Fetuses with confined anomalies of the cardiac system had the highest diagnostic yield at 4.6%, but there were none in the urogenital system. Fetuses with nonspecific ultrasound anomalies had the highest diagnostic yield in fetuses with an intrauterine growth restriction at 5.9%. Overall, fetuses with a nonspecific ultrasound anomaly were affected with pathogenic copy number variants in 1.6% in the cases. CONCLUSIONS: The diagnostic yield of chromosomal microarray in fetuses with normal karyotype and ultrasound abnormality confined to a single anatomic system was highest if it involved cardiac defects or intrauterine growth restriction. This diagnostic yield ranges from 0% to 4.6% depending on the anatomic system involved. Chromosomal microarray has considerable diagnostic value in these pregnancies.

Clinical Significance of Non-Invasive Prenatal Screening for Trisomy 7: Cohort Study and Literature Review.

Trisomy 7 is the most frequently observed type of rare autosomal trisomies in genome-wide non-invasive prenatal screening (NIPS). Currently, the clinical significance of trisomy 7 NIPS-positive results is still unknown. We reviewed two independent cohorts from two laboratories where similar NIPS metrics were applied. A total of 70,441 singleton cases who underwent genome-wide NIPS were analyzed, among which 39 pregnancies were positive for trisomy 7, yielding a screen-positive rate of 0.055% (39/70,441). There were 28 cases with invasive testing results available; the positive predictive value (PPV) was 3.6% (1/28). We then searched the published NIPS studies to generate a large cohort of 437,873 pregnancies and identified 247 cases (0.056%) that were screened positive for trisomy 7. The overall PPV was 3.4% (4/118) in the combined data. The presence of uniparental disomy 7 was not detected in the NIPS trisomy 7-positive pregnancies with normal fetal karyotype. Among the 85 cases with pregnancy outcome available in combined data, 88.2% were normal live births, 14.1% had intrauterine growth restriction, preterm birth or low birth weight, 3.5% presented with ultrasound abnormality, and no fetal loss was observed. Our data provide valuable information for counseling and management of trisomy 7-positive NIPS pregnancies.

The utility of genome-wide cell-free DNA screening in the prenatal diagnosis of Pallister-Killian syndrome.

OBJECTIVE: To report genome-wide cell-free DNA (cfDNA) screening facilitating the diagnosis of Pallister-Killian syndrome (PKS). METHODS: This is a retrospective cohort analysis of positive genome-wide cfDNA screening results showing increased signal from chromosome 12 and the detection of PKS. The genome-wide cfDNA screening results and the subsequent investigations were reviewed. RESULTS: Three singleton pregnancies (3/29007) from 2016 to 2017 yielded positive results indicating large gains on the entire p-arm of chromosome 12. In two cases, multiple structural abnormalities were detected by prenatal ultrasound and the couples opted for termination of pregnancy. Chromosomal microarray performed on fetal skin tissues of the two abortuses detected mosaic tetrasomy 12p, consistent with PKS. In the third case, karyotype and chromosomal microarray performed on an amniotic fluid sample also showed mosaic tetrasomy 12p. In each of the three cases, genome-wide cfDNA screening revealed a large gain on chromosome 12p; subsequent prenatal or postnatal diagnostic testing confirmed the diagnosis of PKS. CONCLUSION: We report the ability of genome-wide cfDNA screening to provide early suspicion and facilitate the subsequent genetic diagnosis of PKS. As genome-wide cfDNA screening becomes increasingly available, incidental diagnosis of partial aneuploidies is expected to increase.

Low-pass genome sequencing: a validated method in clinical cytogenetics.

Clinically significant copy-number variants (CNVs) known to cause human diseases are routinely detected by chromosomal microarray analysis (CMA). Recently, genome sequencing (GS) has been introduced for CNV analysis; however, sequencing depth (determined by sequencing read-length and read-amount) is a variable parameter across different laboratories. Variating sequencing depths affect the CNV detection resolution and also make it difficult for cross-laboratory referencing or comparison. In this study, by using data from 50 samples with high read-depth GS (30×) and the reported clinically significant CNVs, we first demonstrated the optimal read-amount and the most cost-effective read-length for CNV analysis to be 15 million reads and single-end 50 bp (equivalent to a read-depth of 0.25-fold), respectively. In addition, we showed that CNVs at mosaic levels as low as 30% are readily detected, furthermore, CNVs larger than 2.5 Mb are also detectable at mosaic levels as low as 20%. Herein, by conducting a retrospective back-to-back comparison study of low-pass GS versus routine CMA for 532 prenatal, miscarriage, and postnatal cases, the overall diagnostic yield was 22.4% (119/532) for CMA and 23.1% (123/532) for low-pass GS. Thus, the overall relative improvement of the diagnostic yield by low-pass GS versus CMA was ~ 3.4% (4/119). Identification of cryptic and clinically significant CNVs among prenatal, miscarriage, and postnatal cases demonstrated that CNV detection at higher resolutions is warranted for clinical diagnosis regardless of referral indications. Overall, our study supports low-pass GS as the first-tier genetic test for molecular cytogenetic testing.

Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis.

PURPOSE: Emerging studies suggest that low-pass genome sequencing (GS) provides additional diagnostic yield of clinically significant copy-number variants (CNVs) compared with chromosomal microarray analysis (CMA). However, a prospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of low-pass GS compared with CMA is warranted. METHODS: A total of 1023 women undergoing prenatal diagnosis were enrolled. Each sample was subjected to low-pass GS and CMA for CNV analysis in parallel. CNVs were classified according to guidelines of the American College of Medical Genetics and Genomics. RESULTS: Low-pass GS not only identified all 124 numerical disorders or pathogenic or likely pathogenic (P/LP) CNVs detected by CMA in 121 cases (11.8%, 121/1023), but also defined 17 additional and clinically relevant P/LP CNVs in 17 cases (1.7%, 17/1023). In addition, low-pass GS significantly reduced the technical repeat rate from 4.6% (47/1023) for CMA to 0.5% (5/1023) and required less DNA (50 ng) as input. CONCLUSION: In the context of prenatal diagnosis, low-pass GS identified additional and clinically significant information with enhanced resolution and increased sensitivity of detecting mosaicism as compared with the CMA platform used. This study provides strong evidence for applying low-pass GS as an alternative prenatal diagnostic test.

Prenatal Diagnosis of Fetuses With Increased Nuchal Translucency by Genome Sequencing Analysis.

Background: Increased nuchal translucency (NT) is an important biomarker associated with increased risk of fetal structural anomalies. It is known to be contributed by a wide range of genetic etiologies from single-nucleotide variants to those affecting millions of base pairs. Currently, prenatal diagnosis is routinely performed by karyotyping and chromosomal microarray analysis (CMA); however, both of them have limited resolution. The diversity of the genetic etiologies warrants an integrated assay such as genome sequencing (GS) for comprehensive detection of genomic variants. Herein, we aim to evaluate the feasibility of applying GS in prenatal diagnosis for the fetuses with increased NT. Methods: We retrospectively applied GS (> 30-fold) for fetuses with increased NT (≥3.5 mm) who underwent routine prenatal diagnosis. Detection of single-nucleotide variants, copy number variants, and structural rearrangements was performed simultaneously, and the results were integrated for interpretation in accordance with the guidelines of the American College of Medical Genetics and Genomics. Pathogenic or likely pathogenic (P/LP) variants were selected for validation and parental confirmation, when available. Results: Overall, 50 fetuses were enrolled, including 34 cases with isolated increased NT and 16 cases with other fetal structural malformations. Routine CMA and karyotyping reported eight P/LP CNVs, yielding a diagnostic rate of 16.0% (8/50). In comparison, GS provided a twofold increase in diagnostic yield (32.0%, 16/50), including one mosaic turner syndrome, eight cases with microdeletions/microduplications, and seven cases with P/LP point mutations. Moreover, GS identified two cryptic insertions and two inversions. Follow-up study further demonstrated the potential pathogenicity of an apparently balanced insertion that disrupted an OMIM autosomal dominant disease-causing gene at the insertion site. Conclusions: Our study demonstrates that applying GS in fetuses with increased NT can comprehensively detect and delineate the various genomic variants that are causative to the diseases. Importantly, prenatal diagnosis by GS doubled the diagnostic yield compared with routine protocols. Given a comparable turnaround time and less DNA required, our study provides strong evidence to facilitate GS in prenatal diagnosis, particularly in fetuses with increased NT.

Characteristics and mode of inheritance of pathogenic copy number variants in prenatal diagnosis.

BACKGROUND: Microdeletions and microduplications can occur in any pregnancy independent of maternal age. The spectrum and features of pathogenic copy number variants including the size, genomic distribution, and mode of inheritance are not well studied. These characteristics have important clinical implications regarding expanding noninvasive prenatal screening for microdeletions and microduplications. OBJECTIVES: The aim was to investigate the spectrum and characteristics of pathogenic copy number variants in prenatal genetic diagnosis and to provide recommendations for expanding the scope of noninvasive prenatal screening for microdeletions and microduplications. STUDY DESIGN: This was a retrospective study of 1510 pregnant women who underwent invasive prenatal diagnostic testing by chromosomal microarray analysis. Prenatal samples were retrieved by amniocentesis or chorionic villus sampling and sent to our prenatal genetic diagnosis laboratory for chromosomal microarray analysis. The risk of carrying a fetus with pathogenic copy number variants is stratified by the patients' primary indication for invasive testing. We searched the literature for published prenatal chromosomal microarray data to generate a large cohort of 23,865 fetuses. The characteristics and spectrum of pathogenic copy number variants including the type of aberrations (gains or losses), genomic loci, sizes, and the mode of inheritance were studied. RESULTS: Overall, 375 of 23,865 fetuses (1.6%) carried pathogenic copy number variants for any indication for invasive testing, and 44 of them (11.7%) involve 2 or more pathogenic copy number variants. A total of 428 pathogenic copy number variants were detected in these fetuses, of which 280 were deletions and 148 were duplications. Three hundred sixty (84.1%) were less than 5 Mb in size and 68 (15.9%) were between 5 and 10 Mb. The incidence of carrying a pathogenic copy number variant in the high-risk group is 1 in 36 and the low-risk group is 1 in 125. Parental inheritance study results were available for 311 pathogenic copy number variants, 71 (22.8%) were maternally inherited, 36 (11.6%) were paternally inherited, and 204 (65.6%) occurred de novo. CONCLUSION: Collectively, pathogenic copy number variants are common in pregnancies. High-risk pregnancies should be offered invasive testing with chromosomal microarray analysis for the most comprehensive investigation. Detection limits on size, parental inheritance, and genomic distribution should be carefully considered before implementing copy number variant screening in expanded noninvasive prenatal screening.

Chromosome copy number variants in fetuses with syndromic malformations.

Chromosome copy number variants (CNVs; gains and losses of DNA sequences >1 kb) are wide-spread throughout the genome of healthy individuals. Laboratory studies show that a subset of CNVs are pathogenic, and not only can be responsible for the pathogenesis of major birth defects and cancer, but are also associated with neurodevelopmental disorders at birth. The characteristics of the pathogenic microdeletions and microduplications are important for both clinical implications and genetic counselling regarding test selection for prenatal screening and diagnosis. Unfortunately, our knowledge of the phenotypic effects of most CNV is still minimal, leading to the classification of many CNVs as "genomic imbalances of unknown clinical significance". Microdeletions and microduplications can occur in all pregnancies and the spectrum of pathogenic CNVs in fetuses with syndromic malformations is not well studied. This review summarizes our current understanding of CNVs, the common detection methods, and the characteristics of pathogenic CNVs identified in fetuses with syndromic malformations. Birth Defects Research 109:725-733, 2017. © 2017 Wiley Periodicals, Inc.