Genetic Characterization of 128 Chinese Individuals with Neurodevelopmental Disorders via Whole-Exome Sequencing

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

Specific copy number variants (CNVs) have been robustly associated with intellectual disability, autism, and schizophrenia. Most of the literature focus has been on documenting the existence of these phenomena. There are few data to guide therapeutic choices for these “orphan” diseases. We call for systematic and longitudinal case reports which, if carefully conducted, may provide crucial initial knowledge to guide therapeutics. We provide a step-by-step overview, a tailored set of consensus criteria for high-quality case reports, and a specific set of learning resources.

Copy number variants (CNVs) and single-nucleotide variations (SNVs) or insertions and deletions are key genetic contributors to neurodevelopmental disorders (NDDs). Traditionally, chromosome microarray and exome sequencing (ES) have been used to detect CNVs and single gene variants, respectively. To identify genetic variants causing NDDs and evaluate the diagnostic yield and clinical utility of ES by simultaneously analyzing CNVs and SNVs in patients with NDDs and their biologic parents (trios). This retrospective cohort study included pediatric patients with suspected NDDs who visited Shanghai Children's Hospital between January 1, 2018, and December 31, 2023. ES was used to investigate trios (trio-ES) including patients with NDDs who remained undiagnosed after phenotype identification and underwent gene panel testing, multiplex ligation-dependent probe amplification, or karyotyping. Comprehensive clinical and laboratory data were collected. Data were analyzed from July 2022 to December 2023. NDDs, characterized by global developmental delay or intellectual disability. The study measured the overall diagnostic yield of SNVs and CNVs in the NDD cohort as well as within NDD syndromic subtypes. Of the 1106 patients with NDDs, 731 (66.1%) were male. The mean (SD) age of patients at diagnosis was 3.80 (2.82) years. The overall diagnostic yield of trio-ES was 46.1% (510 diagnoses among 1106 patients), with 149 CNVs (13.5%), 355 SNVs (32.1%), and 4 cases of uniparental disomy (0.4%). Codiagnosis of SNVs and CNVs occurred in 2 cases (0.2%). Among the trios, 812 candidate germline variants were identified, including 634 SNVs (78.1%), 174 CNVs (21.4%), and 4 cases of uniparental disomy (0.5%). Of these, 423 SNVs (66.7%) and 157 CNVs (90.2%) were diagnostic variants, while 211 SNVs (33.3%) and 17 CNVs (9.8%) were variants of uncertain significance. Sixteen CNVs smaller than 20 kilobase were detected using ES. In this cohort study, trio-ES, by simultaneously detecting SNVs and CNVs, achieved a diagnostic yield of 46.1%. Trio-ES may be particularly applicable for identifying small CNVs and recessive genetic diseases involving both SNVs and CNVs. These findings suggest that in clinical practice, simultaneously analyzing SNVs and CNVs using trio-ES data has a favorable genetic diagnostic yield for children with NDDs.

To explore the application value of whole exome sequencing (WES) in the diagnosis of prenatal and postnatal neurodevelopmental disorders (NDDs). A total of 70 patients diagnosed with NDDs who underwent WES at the Medical Genetics Center of the Maternal and Child Health Hospital of Hubei Province between June 2020 and July 2021 were retrospectively analyzed. Genomic DNA was extracted from peripheral blood samples and amniotic fluid. WES-based copy number variant (CNV) analysis was integrated into the routine WES data analysis pipeline. The results showed that a molecular diagnosis rate could be made in 21/70 (30%) cases. Of 21 positive cases, 14 (23%) cases were detected by single-nucleotide variant/small insertion/deletion (SNV/Indel) analysis, of which 12 variants were novel, 6 (9.8%) cases were detected by WES-based CNV analysis, and 1 (1.6%) case was detected by a combination of both. The diagnostic yield of WES combined with CNV analysis was higher than that of SNV/Indel analysis alone (30%, 21/70 vs. 20%, 14/70). Of the 28 prenatally diagnosed cases, 6 cases were found to have inherited parental variation for NDDs, 10 cases were found not to have the same pathogenic variation as the proband, and the remaining 12 cases were found to have no pathogenic or likely pathogenic variation that could explain the NDDs phenotype. Clinical follow-up showed that 5 families opted for abortion and the remaining had no current abnormalities. In conclusion, WES may be an effective method to clarify the genetic etiology and prenatal diagnosis of NDDs, which is helpful in assessing the prognosis to aid clinical management and reproductive guidance.

Whole-exome sequencing increases the diagnostic rate for prenatal fetal structural anomalies

Exome sequencing (ES), originally developed to detect single nucleotide variants (SNVs), has been increasingly leveraged to detect copy number variants (CNVs) through read-depth analysis, enhancing diagnostic yield with minimal computational overhead. However, chromosomal microarray (CMA) testing remains widely used. To evaluate the utility of ES as a first-tier clinical diagnostic test, we compared the sensitivity of CNV detection by ES to that of CMA in individuals who underwent both tests, and developed triploidy screening based on ES data. ES identified most clinically relevant CNVs, with a 98.91% concordance for regions adequately captured. A retrospective analysis of CNVs detected from ES over a ~3-year period, comprising 1563 prenatal and 4884 postnatal cases, revealed CNVs in 3.8% of prenatal and 4.4% of postnatal samples. The relatively low percentage stems from the fact that most cases underwent CMA before ES. Pathogenic or likely pathogenic variants constituted 78.7% and 78.0% of these subgroups, with the remainder classified as variants of unknown significance. We highlight clinically relevant examples of CNVs across a range of sizes, including cases involving both CNVs and SNVs. The high consanguinity rate of the cohort allowed for systematic analysis of homozygous CNVs. Additionally, we demonstrate that ES can capture other diagnostic utilities traditionally associated with CMA, specifically uniparental disomy (UPD) and triploidy. Overall, our findings support ES as a robust, cost-effective alternative to CMA and advocate for its broader use as a first-tier diagnostic test for neurodevelopmental delay and congenital malformations, particularly until whole genome sequencing becomes more accessible and affordable.

Intellectual and developmental disabilities (IDD) are clinically and genetically heterogeneous disorders of global concern. While whole exome sequencing (WES) is used to identify single nucleotide variants (SNVs) and small insertions/deletions (Indels) in IDD patients, its detection rate is limited. This study evaluated the value of integrating copy number variation (CNV) analysis into traditional SNV/Indel analysis based on trio-WES. One hundred eighty sevenpatients with IDD in 140 families from southwest China were incorporated into the study cohort. The overall diagnostic rate was 40.11% (75/187), with 33.16% (62/187) from SNV/Indel analysis and 6.95% (13/187) from CNV analysis. SNV/Indel analysis identified 52 variants in 42 genes, including 30 novel and 22 reported variants; CNV analysis identified 11 CNVs, comprising 1 repeat and 10 deletions, with sizes ranging from 1313 to 55 184 kb. 39.29% (55/140) families benefited from this study for their clinical diagnosis, treatment, and reproduction. Furthermore, our strategy, with an incremental cost-effectiveness ratio (ICER) of $2546.22/diagnosis, had demonstrated significant advantages in terms of cost-effectiveness and detection speed compared to previous methods. In general, by incorporating SNV/Indel and CNV analysis based on trio-WES, a robust, cost-effective, and time-saving approach for diagnosing IDD has been developed.

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

Copy number variation is now recognized as an important class of risk factor for several child psychiatric disorders. In this article, we first explain what copy number variants (CNVs) are. We then consider key findings and what these have told us about the etiology of these conditions. Finally, we discuss whether these findings can yet translate into clinical practice.

BackgroundDevelopmental delay (DD) and intellectual disability (ID) are prevalent in children and often have genetic causes, particularly copy number variations (CNVs). Chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) are key diagnostic tools for identifying genetic contributions to these disorders. This study assesses the prevalence and clinical impact of CNVs in pediatric DD and ID patients.MethodsNinety-nine pediatric patients with DD or ID underwent CMA or WES. Of these, 82 received SNP array analysis, while 17 had WES. CNV pathogenicity was assessed using established databases and ACMG guidelines, with inheritance patterns determined where possible.ResultsAcross the 99 patients, 43 CNVs were identified in 40 individuals, with 32 classified as clinically significant, resulting in a diagnostic rate of 30.3%. These findings included 24 deletions (75%), 7 duplications (22%), and 1 instance of loss of heterozygosity (3%). Of the CNVs with known inheritance, 65.2% were de novo. Recurrent CNVs made up 36.4% of the total, especially in regions 15q11.2-q13.1, 16p11.2, and 22q11.2. Additionally, 11 CNVs were categorized as variants of uncertain significance (VOUS).ConclusionThis study supports CMA as an effective diagnostic tool for DD and ID, highlighting the importance of family-based CNV testing for genetic counseling. The findings emphasize the need for comprehensive genetic testing to improve diagnostic accuracy, with future multi-omics approaches potentially clarifying VOUS mechanisms and CNV variability in neurodevelopmental disorders.

<h3>Objective</h3> To evaluate a new tool to aid interpretation of copy number variants (CNVs) in individuals with neurodevelopmental disabilities. <h3>Methods</h3> Critical exon indexing (CEI) was used to identify genes with critical exons (CEGs) from clinically reported CNVs, which may contribute to neurodevelopmental disorders (NDDs). The 742 pathogenic CNVs and 1,363 variants of unknown significance (VUS) identified by chromosomal microarray analysis in 5,487 individuals with NDDs were subjected to CEI to identify CEGs. CEGs identified in a subsequent random series of VUS were evaluated for relevance to CNV interpretation. <h3>Results</h3> CEI identified a total of 2,492 unique CEGs in pathogenic CNVs and 953 in VUS compared with 259 CEGs in 6,965 CNVs from 873 controls. These differences are highly significant (<i>p</i> &lt; 0.00001) whether compared as frequency, average, or normalized by CNV size. Twenty-one percent of VUS CEGs were not represented in Online Mendelian Inheritance in Man, highlighting limitations of existing resources for identifying potentially impactful genes within CNVs. CEGs were highly correlated with other indices and known pathways of relevance. Separately, 136 random VUS reports were reevaluated, and 76% of CEGs had not been commented on. In multiple cases, further investigation yielded additional relevant literature aiding interpretation. As one specific example, we discuss <i>GTF2I</i> as a CEG, which likely alters interpretation of several reported duplication VUS in the Williams-Beuren region. <h3>Conclusions</h3> Application of CEI to CNVs in individuals with NDDs can identify genes of potential clinical relevance, aid laboratories in effectively searching the clinical literature, and support the clinical reporting of poorly annotated VUS.

ObjectiveTo evaluate a new tool to aid interpretation of copy number variants (CNVs) in individuals with neurodevelopmental disabilities.MethodsCritical exon indexing (CEI) was used to identify genes with critical exons (CEGs) from clinically reported CNVs, which may contribute to neurodevelopmental disorders (NDDs). The 742 pathogenic CNVs and 1,363 variants of unknown significance (VUS) identified by chromosomal microarray analysis in 5,487 individuals with NDDs were subjected to CEI to identify CEGs. CEGs identified in a subsequent random series of VUS were evaluated for relevance to CNV interpretation.ResultsCEI identified a total of 2,492 unique CEGs in pathogenic CNVs and 953 in VUS compared with 259 CEGs in 6,965 CNVs from 873 controls. These differences are highly significant (p < 0.00001) whether compared as frequency, average, or normalized by CNV size. Twenty-one percent of VUS CEGs were not represented in Online Mendelian Inheritance in Man, highlighting limitations of existing resources for identifying potentially impactful genes within CNVs. CEGs were highly correlated with other indices and known pathways of relevance. Separately, 136 random VUS reports were reevaluated, and 76% of CEGs had not been commented on. In multiple cases, further investigation yielded additional relevant literature aiding interpretation. As one specific example, we discuss GTF2I as a CEG, which likely alters interpretation of several reported duplication VUS in the Williams-Beuren region.ConclusionsApplication of CEI to CNVs in individuals with NDDs can identify genes of potential clinical relevance, aid laboratories in effectively searching the clinical literature, and support the clinical reporting of poorly annotated VUS.

This review is devoted to a comprehensive analysis of DNA copy number variations (CNVs) identified in patients with neurodevelopmental disorders (NDDs) from the literature. The selection of publications was conducted using specifically developed criteria. CNVs were characterized based on their clinical significance, type of copy number alteration (microdeletion/microduplication), size, origin, and gene content. The study sample comprised 3,375 patients with NDDs, among whom pathogenic and likely pathogenic CNVs, as well as variants of uncertain clinical significance, were identified in 395 individuals (12%). Chromosomal variations from each category were identified in 89 (3%), 56 (2%), and 241 (7%) patients, respectively. Nine individuals exhibited combinations of CNVs with varying clinical significance. The number of microduplications slightly exceeded the number of deletions (250 and 204, respectively). The size of most CNVs ranged from 193 bp to 400 kb and from 1 to 3 Mb (237 and 96, respectively). Seventy-two variants originated de novo, while 165 were inherited. Eighty-six CNVs were associated with 33 known microdeletion/microduplication syndromes. The most prevalent syndromes included 1q21.1 microduplication (7/395, 1.8%) (OMIM: 612475), 2p16.3 microdeletion (9/395, 2.3%) (OMIM: 614332), 15q13.3 microdeletion (7/395, 1.8%) (OMIM: 612001), 16p11.2 microdeletion (9/395, 2.3%) (OMIM: 611913), and 22q11.2 microduplications (7/395, 1.8%) (OMIM: 608363). Enrichment analysis revealed that pathogenic CNVs, as well as variants of uncertain clinical significance, are enriched in genes associated with abnormal behavioral/neurological phenotypes. Likely pathogenic CNVs included genes linked to disorders of the nervous system and homeostasis/metabolism.

Neurodevelopmental disorders (NDDs) are a genetically heterogeneous group of diseases, affecting 1%-3% of children. Whole-exome sequencing (WES) has been widely used as a first-tier tool for identifying genetic causes of rare diseases. Trio-WES was performed in a cohort of 74 pedigrees with NDDs. Exome-based copy number variant (CNV) calling was incorporated into the traditional single-nucleotide variant (SNV) and small insertion/deletion (Indel) analysis pipeline for WES data. An overall positive diagnostic yield of 54.05% (40/74) was obtained in the pipeline of combinational SNV/Indel and CNV analysis, including 35.13% (26/74) from SNV/Indel analysis and 18.92% (14/74) from exome-based CNV analysis, respectively. In total, SNV/Indel analysis identified 38 variants in 28 different genes, of which 24 variants were novel; exome-based CNV analysis identified 14 CNVs, including 2 duplications and 12 deletions, which ranged from 440 bp (single exon) to 16.86 Mb (large fragment) in size. In particular, a hemizygous deletion of exon 1 in the SLC16A2 gene was detected. Based on the diagnostic results, two families underwent prenatal diagnosis and had unaffected babies. The incorporation of exome-based CNV detection into conventional SNV/Indel analysis for a single trio-WES test significantly improved the diagnostic rate, making WES a more powerful, practical, and cost-effective tool in the clinical diagnosis of NDDs.

Prenatal exome sequencing for fetuses with structural abnormalities: the next step.