Diagnostic validation of a familial hypercholesterolaemia cohort provides a model for using targeted next generation DNA sequencing in the clinical setting

Hinchcliffe, Marcus; Le, Hanh N. D.; Fimmel, A L; Molloy, L.; Freeman, Lucinda; Sullivan, David; Trent, Ronald J.

doi:10.1097/pat.0000000000000026

Cited by 16 publications

(12 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Currently, there is no clear consensus about the number of samples or the number and type of DNA variant that should be tested for an NGS-based technical validation, although it has been suggested that at least 60 unique variants be tested. 17 In practice, the number of variants analyzed for the technical validation of NGS LDTs reported in the literature varies widely, from <20 to >1000 variants. 17,19,20 We set the minimum number of analyzed variants required for a thorough understanding of the NEO1 panel's analytical sensitivity and specificity at 500 variants.…”

Section: Discussionmentioning

confidence: 99%

“…17 In practice, the number of variants analyzed for the technical validation of NGS LDTs reported in the literature varies widely, from <20 to >1000 variants. 17,19,20 We set the minimum number of analyzed variants required for a thorough understanding of the NEO1 panel's analytical sensitivity and specificity at 500 variants. In silico analysis of five whole-genome RMs revealed a sufficient number of variants across the RM for our validation.…”

Section: Discussionmentioning

confidence: 99%

“…CNV detection of large (multiexonic) gene deletions and duplications that are not called by GATK was performed via a relative depth of coverage method as previously described. 17 The following parameters were used to flag a region as a candidate CNV. Relative coverage <0.735 for large deletions and >1.280 for large duplications, with an SD for that region <0.225, for a minimum of two consecutive target regions.…”

Section: Sequencing Analysis and Variant Calling For Neo1 Validation mentioning

confidence: 99%

See 2 more Smart Citations

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test

Shum

Henner²,

Belluoccio

et al. 2017

The Journal of Molecular Diagnostics

View full text Add to dashboard Cite

The sensitivity and specificity of next-generation sequencing laboratory developed tests (LDTs) are typically determined by an analyte-specific approach. Analyte-specific validations use disease-specific controls to assess an LDT's ability to detect known pathogenic variants. Alternatively, a methods-based approach can be used for LDT technical validations. Methods-focused validations do not use disease-specific controls but use benchmark reference DNA that contains known variants (benign, variants of unknown significance, and pathogenic) to assess variant calling accuracy of a next-generation sequencing workflow. Recently, four whole-genome reference materials (RMs) from the National Institute of Standards and Technology (NIST) were released to standardize methods-based validations of next-generation sequencing panels across laboratories. We provide a practical method for using NIST RMs to validate multigene panels. We analyzed the utility of RMs in validating a novel newborn screening test that targets 70 genes, called NEO1. Despite the NIST RM variant truth set originating from multiple sequencing platforms, replicates, and library types, we discovered a 5.2% false-negative variant detection rate in the RM truth set genes that were assessed in our validation. We developed a strategy using complementary non-RM controls to demonstrate 99.6% sensitivity of the NEO1 test in detecting variants. Our findings have implications for laboratories or proficiency testing organizations using whole-genome NIST RMs for testing.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Sequencing Analysis and Variant Calling For Neo1 Validation mentioning

confidence: 99%

See 1 more Smart Citation

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test

Shum

Henner²,

Belluoccio

et al. 2017

The Journal of Molecular Diagnostics

View full text Add to dashboard Cite

show abstract

“…The wide spectrum of FHassociated mutations identified in the LDLR gene, which harbors the majority of disease-causing mutations, requires molecular methods suitable for comprehensive scanning of the nucleotide sequence of the candidate genes (19,20). Next-generation sequencing as a comprehensive genetic analysis method has demonstrated high levels of specificity and sensitivity (21)(22)(23). However, a significant drawback is the recognition that sequencing 4 genes is insufficient, and approximately 15% of people with FH do not have a mutation in LDLR, APOB, or PCSK9 genes (though estimates range from 12% to 48%) (24).…”

Section: Discussionmentioning

confidence: 99%

Cascade Screening for Familial Hypercholesterolemia: PCR Methods with Melting-Curve Genotyping for the Targeted Molecular Detection of Apolipoprotein B and LDL Receptor Gene Mutations to Identify Affected Relatives

Pandey

Leider

Khan

et al. 2016

The Journal of Applied Laboratory Medicine

View full text Add to dashboard Cite

Background: A key objective of the UK National Institute for Health and Care Excellence (NICE) pathway for diagnosis of familial hypercholesterolemia (FH) is the identification of affected relatives of index cases through cascade screening. At present, there is no systematic appraisal of available methodological options to identify the appropriate diagnostic testing protocol that would allow cost-effective cascade genetic screening. The majority of FH-causing mutations identified in the LDL receptor (LDLR) or apolipoprotein B (APOB) genes are single-nucleotide changes. This pattern of mutations suggests that PCR methods using melting curve-based genotyping might offer a convenient methodological approach for screening relatives. Methods: We developed and validated one-tube PCR methods for the mutations APOB c.10580G>A (p.Arg3527Gln), LDLR c.1474G>A (p.Asp492Asn), and c.2054C>T (p.Pro685Leu) and 3 novel LDLR mutations identified in the Coventry and Warwickshire population: LDLR c.1567G>C (p.Val523Leu), c.487dupC (p.Gln163Profs17), and c.647G>C (p.Cys216Ser). Results: These methods successfully amplified target sequence from genomic DNA extracted from either peripheral blood or saliva. They also demonstrated acceptable analytical performance characteristics (specificity of amplification, repeatability, and reproducibility) over a wide range of DNA concentrations and purity. This approach was used for cascade testing of relatives of index FH cases with confirmed mutations and identified family members with high plasma LDL cholesterol as heterozygous for disruptive alleles. Conclusions: Our study generates proof-of-concept evidence of methods suitable for detecting single nucleotide substitutions and insertions that can deliver reliable, easy, low-cost, and rapid family screening of FH patients and can be adopted by nonspecialist molecular diagnostic laboratories.

show abstract

“…Next-generation sequencing techniques including pyrosequencing allow the fast and reliable investigation of virtually every nucleotide in the whole genome or in specific loci of interest [ 13 , 14 ]. Recently, the use of targeted next-generation sequencing techniques has been validated for FH diagnosis [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

Genetic diagnosis of familial hypercholesterolaemia by targeted next‐generation sequencing

et al. 2014

View full text Add to dashboard Cite

Maglio C., Mancina R. M., Motta B. M., Stef M., Pirazzi C., Palacios L., Askaryar N., Borén J., Wiklund O., Romeo S. (University of Gothenburg, Gothenburg, Sweden; University Magna Graecia of Catanzaro, Italy; University of Milan, Italy; Progenika Biopharma SA, Derio, Spain). Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing.ObjectivesThe aim of this study was to combine clinical criteria and next-generation sequencing (pyrosequencing) to establish a diagnosis of familial hypercholesterolaemia (FH).Design, setting and subjectsA total of 77 subjects with a Dutch Lipid Clinic Network score of ≥3 (possible, probable or definite FH clinical diagnosis) were recruited from the Lipid Clinic at Sahlgrenska Hospital, Gothenburg, Sweden. Next-generation sequencing was performed in all subjects using SEQPRO LIPO RS, a kit that detects mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLR adapter protein 1 (LDLRAP1) genes; copy-number variations in the LDLR gene were also examined.ResultsA total of 26 mutations were detected in 50 subjects (65% success rate). Amongst these, 23 mutations were in the LDLR gene, two in the APOB gene and one in the PCSK9 gene. Four mutations with unknown pathogenicity were detected in LDLR. Of these, three mutations (Gly505Asp, Ile585Thr and Gln660Arg) have been previously reported in subjects with FH, but their pathogenicity has not been proved. The fourth, a mutation in LDLR affecting a splicing site (exon 6–intron 6) has not previously been reported; it was found to segregate with high cholesterol levels in the family of the proband.ConclusionsUsing a combination of clinical criteria and targeted next-generation sequencing, we have achieved FH diagnosis with a high success rate. Furthermore, we identified a new splicing-site mutation in the LDLR gene.

show abstract

Diagnostic validation of a familial hypercholesterolaemia cohort provides a model for using targeted next generation DNA sequencing in the clinical setting

Cited by 16 publications

References 11 publications

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test

Cascade Screening for Familial Hypercholesterolemia: PCR Methods with Melting-Curve Genotyping for the Targeted Molecular Detection of Apolipoprotein B and LDL Receptor Gene Mutations to Identify Affected Relatives

Genetic diagnosis of familial hypercholesterolaemia by targeted next‐generation sequencing

Contact Info

Product

Resources

About