Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation

Abstract

Purpose Congenital disorders of glycosylation (CDG) are a heterogeneous group of disorders caused by deficient glycosylation, primarily affecting the N-linked pathway. It is estimated that over 40% of CDG patients lack a confirmatory molecular diagnosis. The purpose of this study was to improve molecular diagnosis for CDG by developing and validating a next generation sequencing (NGS) panel for comprehensive mutation detection in 24 genes known to cause CDG. Methods NGS validation was performed on 12 positive control CDG patients. These samples were blinded as to the disease causing mutations. Both RainDance and Fluidigm platforms were used for sequence enrichment and targeted amplification. The SOLiD platform was used for sequencing the amplified products. Bioinformatic analysis was performed using NextGENe® software. Results The disease causing mutations were identified by NGS for all 12 positive controls. Additional variants were also detected in three controls that are known or predicted to impair gene function and may contribute to the clinical phenotype. Conclusions We conclude that development of NGS panels in the diagnostic laboratory where multiple genes are implicated in a disorder is more cost-effective and will result in improved and faster patient diagnosis compared with a gene-by-gene approach. Recommendations are also provided for data analysis from the NGS-derived data in the clinical laboratory, which will be important for the widespread use of this technology.

Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin

“…In such cases, MPS-based target gene analysis has been proven to be an efficient and cost-effective approach. 11,24,25 This study demonstrated that 65% (11/17) of the patients with no previous molecular diagnosis of GSD did indeed carry mutations in one of the GSD-related genes, thereby confirming the presumptive clinical diagnosis ( Table 1). Of course, the detection rate is highly dependent on an accurate clinical evaluation.…”

“…Currently, the reported MPSbased analysis of target genes relies on multiplex PCR enrichment of the coding sequences, requiring routine confirmation with additional Sanger sequencing for genes harboring low-or no-coverage exons. 11,[21][22][23] The MPS-based sequence analysis of the genes involved in glycogen metabolism presented here relies on in-solution probe hybridization for the capture of target sequences under one uniform condition, followed by MPS with deep coverage. This strategy provides an average base-by-base coverage of >600X in all target regions.…”

“…[8][9][10] For clinically and genetically heterogeneous diseases caused by a group of genes involving a common metabolic pathway, MPS can also be used for simultaneous sequencing of the group of candidate genes. [11][12][13] To facilitate Table S1 online). This approach eliminates the hurdle of having to prioritize multiple candidate genes for step-wise sequencing.…”

Molecular diagnosis of glycogen storage disease and disorders with overlapping clinical symptoms by massive parallel sequencing

“…4 This very cost-effective technology is particularly appropriate for screening for mutations in disorders with highly heterogeneous genetic backgrounds, such as GSD, congenital disorder of glycosylation, lysosomal disorders, and mitochondrial disorders [5][6][7][8] In addition, its ability to detect mutations in large genes and to identify copy number variations is very advantageous. The implementation of massive parallel sequencing has begun to revolutionize the field of genetic diagnosis.…”

Assessing Copy Number Alterations in Targeted, Amplicon-Based Next-Generation Sequencing Data

“…1e9 Two widely used enrichment strategies for targeted sequencing are hybridization-capture, in which oligonucleotide baits complementary to the regions of interest are hybridized with fragmented genomic DNA, 3 and PCR, in which a pool of primers is used to generate target-specific amplicons. 4,10 Both of these approaches work well on DNA purified from formalin-fixed, paraffin-embedded (FFPE) tumor tissue, and they require only small amounts of input DNA (10 to 100 ng).…”