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Targeted NGS: A Cost-Effective Approach to Molecular Diagnosis of PIDs.

Stoddard JL, Niemela JE, Fleisher TA, Rosenzweig SD - Front Immunol (2014)

Bottom Line: Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful.Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients.While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Clinical Center, National Institutes of Health , Bethesda, MD , USA.

ABSTRACT

Background: Primary immunodeficiencies (PIDs) are a diverse group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful. Next-generation sequencing (NGS) methods offer an unbiased genotype-based approach, which can facilitate molecular diagnostics.

Objective: To develop an efficient NGS method to identify variants in PID-related genes.

Methods: We performed HaloPlex custom target enrichment and NGS using the Ion Torrent PGM to screen 173 genes in 11 healthy controls, 13 PID patients previously evaluated with either an identified mutation or SNP, and 120 patients with undiagnosed PIDs. Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients. Run metrics and coverage analyses were done to identify systematic deficiencies.

Results: A molecular diagnosis was identified for 18 of 120 patients who previously lacked a genetic diagnosis, including 9 who had atypical presentations and extensive previous genetic and functional studies. Our NGS method detected variants with 98.1% sensitivity and >99.9% specificity. Uniformity was variable (72-89%), and we were not able to reliably sequence 45 regions (45/2455 or 1.8% of total regions) due to low (<20) average read depth or <90% region coverage; thus, we optimized probe hybridization conditions to improve read-depth and coverage for future analyses, and established criteria to help identify true positives.

Conclusion: While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs. This approach decreases time to diagnosis, increases diagnostic rate, and provides insight into the genotype-phenotype correlation of PIDs in a cost-effective way.

No MeSH data available.


Related in: MedlinePlus

Gene-by-gene coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol and ion torrent PGM.
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Figure 1: Gene-by-gene coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol and ion torrent PGM.

Mentions: The gene-by-gene coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol is shown in Table S3 in Supplementary Material and Figure 1. Although the coverage for PIK3CD was expected to be 100%, the actual average coverage was 94% (85–97%). Unfortunately, one of the poorly covered regions contains a known hotspot mutation (PIK3CD c.1573G > A, P.E525K). The coverage for following genes was expected to be less than 90% based on the probe design: HLA-DRB5 (82.95%) and NOTCH2 (89.43%). Notably, the Haloplex kit is guaranteed to provide >90% coverage for most target regions; however, the actual coverage for following genes was suboptimal (less than 90% and more than 5% lower than expected) (expected/actual): HLA-DRB5 (83/29), TNFRSF13C (100/80), UNC93B1 (95/84), CD79A (100/89), NCF4 (100/89). The region-by region coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol (Table S1 in Supplementary Material and Figure 2) demonstrates that multiple regions were systematically poorly covered, including 45 regions (45/2455 or 1.8% of total regions) with low (<20) average read depth and <90% region coverage.


Targeted NGS: A Cost-Effective Approach to Molecular Diagnosis of PIDs.

Stoddard JL, Niemela JE, Fleisher TA, Rosenzweig SD - Front Immunol (2014)

Gene-by-gene coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol and ion torrent PGM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217515&req=5

Figure 1: Gene-by-gene coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol and ion torrent PGM.
Mentions: The gene-by-gene coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol is shown in Table S3 in Supplementary Material and Figure 1. Although the coverage for PIK3CD was expected to be 100%, the actual average coverage was 94% (85–97%). Unfortunately, one of the poorly covered regions contains a known hotspot mutation (PIK3CD c.1573G > A, P.E525K). The coverage for following genes was expected to be less than 90% based on the probe design: HLA-DRB5 (82.95%) and NOTCH2 (89.43%). Notably, the Haloplex kit is guaranteed to provide >90% coverage for most target regions; however, the actual coverage for following genes was suboptimal (less than 90% and more than 5% lower than expected) (expected/actual): HLA-DRB5 (83/29), TNFRSF13C (100/80), UNC93B1 (95/84), CD79A (100/89), NCF4 (100/89). The region-by region coverage analysis for 33 samples run using the default HaloPlex probe hybridization protocol (Table S1 in Supplementary Material and Figure 2) demonstrates that multiple regions were systematically poorly covered, including 45 regions (45/2455 or 1.8% of total regions) with low (<20) average read depth and <90% region coverage.

Bottom Line: Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful.Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients.While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Clinical Center, National Institutes of Health , Bethesda, MD , USA.

ABSTRACT

Background: Primary immunodeficiencies (PIDs) are a diverse group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful. Next-generation sequencing (NGS) methods offer an unbiased genotype-based approach, which can facilitate molecular diagnostics.

Objective: To develop an efficient NGS method to identify variants in PID-related genes.

Methods: We performed HaloPlex custom target enrichment and NGS using the Ion Torrent PGM to screen 173 genes in 11 healthy controls, 13 PID patients previously evaluated with either an identified mutation or SNP, and 120 patients with undiagnosed PIDs. Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients. Run metrics and coverage analyses were done to identify systematic deficiencies.

Results: A molecular diagnosis was identified for 18 of 120 patients who previously lacked a genetic diagnosis, including 9 who had atypical presentations and extensive previous genetic and functional studies. Our NGS method detected variants with 98.1% sensitivity and >99.9% specificity. Uniformity was variable (72-89%), and we were not able to reliably sequence 45 regions (45/2455 or 1.8% of total regions) due to low (<20) average read depth or <90% region coverage; thus, we optimized probe hybridization conditions to improve read-depth and coverage for future analyses, and established criteria to help identify true positives.

Conclusion: While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs. This approach decreases time to diagnosis, increases diagnostic rate, and provides insight into the genotype-phenotype correlation of PIDs in a cost-effective way.

No MeSH data available.


Related in: MedlinePlus