Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing
Open accessAbstract
Massive parallel sequencing has revolutionized the search for pathogenic variants in the human genome, but for routine diagnosis, re-sequencing of the complete human genome in a large cohort of patients is still far too expensive. Recently, novel genome partitioning methods have been developed that allow to target re-sequencing to specific genomic compartments, but practical experience with these methods is still limited. In this study, we have combined a novel droplet-based multiplex PCR method and next generation sequencing to screen patients with X-linked mental retardation (XLMR) for mutations in 86 previously identified XLMR genes. In total, affected males from 24 large XLMR families were analyzed, including three in whom the mutations were already known. Amplicons corresponding to functionally relevant regions of these genes were sequenced on an Illumina/Solexa Genome Analyzer II platform. Highly specific and uniform enrichment was achieved: on average, 67.9% unambiguously mapped reads were derived from amplicons, and for 88.5% of the targeted bases, the sequencing depth was sufficient to reliably detect variations. Potentially disease-causing sequence variants were identified in 10 out of 24 patients, including the three mutations that were already known, and all of these could be confirmed by Sanger sequencing. The robust performance of this approach demonstrates the general utility of droplet-based multiplex PCR for parallel mutation screening in hundreds of genes, which is a prerequisite for the diagnosis of mental retardation and other disorders that may be due to defects of a wide variety of genes.
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Microdroplet PCR should theoretically achieve a sensitivity of about 99%, albeit at higher cost.
“Alternatively, microdroplet PCR should theoretically achieve a sensitivity of ~99%, albeit at higher cost (16, 47).”
From Carrier testing for severe childhood recessive diseases by next-generation sequencing (2011) · cites this paper as [47]
The Discussion (page unavailable) reports correct variant calls at up to 99.5% of uniquely mappable positions in the targeted regions, supporting a near-99% figure for this method. It does not describe that figure as a theoretical sensitivity or state that microdroplet PCR costs more; searches for sensitivity and cost found no such comparison. (LLM, conf=0.86)
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- Authors
- Hu, H., Wrogemann, K., Kalscheuer, V., Tzschach, A., Richard, H., Haas, S. A., Menzel, C., Bienek, M., Froyen, G., Raynaud, M., Van Bokhoven, H., Chelly, J., Ropers, H., & Chen, W.
- Affiliations
- Max Planck Institute for Molecular Genetics (DE)
- KU Leuven (BE)
- Inserm (FR)
- Max Delbrück Center (DE)
- Corresponding author
- Chen, Wei
- ORCID
- Venue
- The HUGO Journal
- Published
- 2009-12
- Volume
- 3
- Issue
- 1-4
- Pages
- 41-49
- ISSN
- 1877-6558 (ISSN-L)
- 1877-6566
- PMC
- PMC2882650
- OpenAlex
- W4251132686
- Cited by
- 49
- References
- 26
- Citations by year
- 2025: 2 · 2024: 1 · 2020: 2 · 2019: 2 · 2018: 1 · 2017: 2 · 2016: 8 · 2015: 3 · 2014: 6 · 2013: 13 · 2012: 6
- FWCI
- 1.74
- Type
- journal article
- Language
- en
- Update notices
- erratum (2010-04-11)
- Open access
- hybrid
- doi.org
- Licence
- Locations
- The HUGO Journal (publishedVersion, cc-by)
- MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
- HAL (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomic variations and chromosomal abnormalities
- Keywords
- X-linked intellectual disability, mutation detection, targeted re-sequencing, genome partitioning, pathogenic variants, molecular diagnosis
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