ANALYTICAL PERFORMANCE EVALUATION OF WHOLE GENOME SEQUENCING AS FIRST-LINE GENETIC TESTING FOR INTELLECTUAL DEVELOPMENT DISORDERS AND CONGENITAL MALFORMATIONS
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
Chromosomal microarray (CMA) has been recognized as a first-tier clinical genetic test for individuals with intellectual development disorders (IDD) and congenital malformations. However, its technical limitations often trigger additional testing, such as whole exome sequencing (WES) and Fragile X testing, which can increase the overall cost of genetic testing. Whole genome sequencing (WGS) has increasingly become the standard of care in genetic testing and can potentially provide a comprehensive genomics assessment of a wider range of genetic variations associated with IDD and congenital malformations, including single nucleotide variants (SNV), insertions and deletions (indel), structural variants (SV), copy number variants (CNV), regions of homozygosity (ROH), and trinucleotide repeats. This collaborative study conducted by Quest Diagnostics and Broad Clinical Labs (BCL) aimed to demonstrate that WGS is technically equivalent to CMA and can additionally rule out Fragile X syndrome.
Methods:
The study used 100 de-identified DNA samples from blood, buccal, and saliva specimens referred for IDD and congenital malformations and previously tested using Quest’s ClariSure Oligo-SNP CMA assay; 16 of the 100 specimens were also previously tested with Quest’s XSense Fragile X assay, a PCR-based assay. The samples were sequenced using BCL’s clinically validated (CAP-, CLIA-, and NYS-approved) WGS test based on Illumina NovaSeqX. In the 100 clinical samples tested with CMA assay, 42% carried pathogenetic/likely pathogenic CNVs, 51% carried variant of unknown significance CNVs, 8% carried ROHs, and 1% carried full chromosome aneuploidy; all reportable CNVs ranged from 55 kb to 39 Mb. In the subset samples tested with PCR-based Fragile X assay, 15 (94%) carried ≤45 trinucleotide repeats, and 1 (6%) carried >45 repeats. In previous studies, BCL’s clinical WGS assay demonstrated 99.9% sensitivity and 99.8% precision for SNV, 99.8% sensitivity and 99.8% precision for indel <50 bp, and 85% sensitivity and 90% specificity for CNV <20 kb within callability regions of the exome.
Results:
WGS detected 100% of reportable CNVs > 25 kb, including aneuploidy. Additionally, WGS detected 97% of autosomal reportable ROHs >5 Mb. Furthermore, WGS detected trinucleotide repeats ≤45 with 100% sensitivity using ExpansionHunter, indicating the ability to rule out Fragile X syndrome. Results were 100% concordant with previous Fragile X results from the 16 samples tested using PCR-based Fragile X assay.
Conclusion:
The results from this study demonstrated that WGS is technically equivalent to CMA with additional ability to reliably detect trinucleotide repeats ≤45. In addition, based on previous studies, the same WGS assay has similar technical equivalence to WES but with a better genome coverage. Thus, WGS is a genetic solution that can measure most types of genetics/genomics alterations (CNV, SV, SNV, indel, LOH, repeats) at a whole genome level. Such a genetic solution would aid in shortening the diagnostic odyssey for patients with IDD and congenital malformations. Finally, our study, in combination with other previous publications, could be used in an evidence-based review by professional societies and payors to recommend prioritizing WGS over CMA in health care decision making which will yield positive expected outcomes.
Chromosomal microarray (CMA) has been recognized as a first-tier clinical genetic test for individuals with intellectual development disorders (IDD) and congenital malformations. However, its technical limitations often trigger additional testing, such as whole exome sequencing (WES) and Fragile X testing, which can increase the overall cost of genetic testing. Whole genome sequencing (WGS) has increasingly become the standard of care in genetic testing and can potentially provide a comprehensive genomics assessment of a wider range of genetic variations associated with IDD and congenital malformations, including single nucleotide variants (SNV), insertions and deletions (indel), structural variants (SV), copy number variants (CNV), regions of homozygosity (ROH), and trinucleotide repeats. This collaborative study conducted by Quest Diagnostics and Broad Clinical Labs (BCL) aimed to demonstrate that WGS is technically equivalent to CMA and can additionally rule out Fragile X syndrome.
Methods:
The study used 100 de-identified DNA samples from blood, buccal, and saliva specimens referred for IDD and congenital malformations and previously tested using Quest’s ClariSure Oligo-SNP CMA assay; 16 of the 100 specimens were also previously tested with Quest’s XSense Fragile X assay, a PCR-based assay. The samples were sequenced using BCL’s clinically validated (CAP-, CLIA-, and NYS-approved) WGS test based on Illumina NovaSeqX. In the 100 clinical samples tested with CMA assay, 42% carried pathogenetic/likely pathogenic CNVs, 51% carried variant of unknown significance CNVs, 8% carried ROHs, and 1% carried full chromosome aneuploidy; all reportable CNVs ranged from 55 kb to 39 Mb. In the subset samples tested with PCR-based Fragile X assay, 15 (94%) carried ≤45 trinucleotide repeats, and 1 (6%) carried >45 repeats. In previous studies, BCL’s clinical WGS assay demonstrated 99.9% sensitivity and 99.8% precision for SNV, 99.8% sensitivity and 99.8% precision for indel <50 bp, and 85% sensitivity and 90% specificity for CNV <20 kb within callability regions of the exome.
Results:
WGS detected 100% of reportable CNVs > 25 kb, including aneuploidy. Additionally, WGS detected 97% of autosomal reportable ROHs >5 Mb. Furthermore, WGS detected trinucleotide repeats ≤45 with 100% sensitivity using ExpansionHunter, indicating the ability to rule out Fragile X syndrome. Results were 100% concordant with previous Fragile X results from the 16 samples tested using PCR-based Fragile X assay.
Conclusion:
The results from this study demonstrated that WGS is technically equivalent to CMA with additional ability to reliably detect trinucleotide repeats ≤45. In addition, based on previous studies, the same WGS assay has similar technical equivalence to WES but with a better genome coverage. Thus, WGS is a genetic solution that can measure most types of genetics/genomics alterations (CNV, SV, SNV, indel, LOH, repeats) at a whole genome level. Such a genetic solution would aid in shortening the diagnostic odyssey for patients with IDD and congenital malformations. Finally, our study, in combination with other previous publications, could be used in an evidence-based review by professional societies and payors to recommend prioritizing WGS over CMA in health care decision making which will yield positive expected outcomes.