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Next generation sequencing for molecular diagnosis of neuromuscular diseases.

Vasli N, Böhm J, Le Gras S, Muller J, Pizot C, Jost B, Echaniz-Laguna A, Laugel V, Tranchant C, Bernard R, Plewniak F, Vicaire S, Levy N, Chelly J, Mandel JL, Biancalana V, Laporte J - Acta Neuropathol. (2012)

Bottom Line: We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis.The cost was less than conventional testing for a single large gene.With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations.

View Article: PubMed Central - PubMed

Affiliation: IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), 1, rue Laurent Fries, BP10142, 67404, Illkirch, France.

ABSTRACT
Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN, RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.

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Detection of copy number and mapping of a deletion in patient C with DMD. a, b Gender determination: comparison of sequence reads mapping to the X chromosome between two female DNAs in (a) and a female (black) and a male (red) in (b). In b a deletion of several exons is detected on the X chromosome for the male (squared). c Next generation sequencing data showing the detection of a 27 exons deletion in patient C with DMD (middle panel) compared to two other DNAs (top and bottom panels). Random off-target reads allow a more precise mapping of the deletion breakpoints. Off-target reads varied between two different experiments. d CGH-array results showing the 5′ and 3′ breakpoints map between 32,538,435 and 32,538,443 and between 32,187,417 and 32,187,427, respectively
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Fig2: Detection of copy number and mapping of a deletion in patient C with DMD. a, b Gender determination: comparison of sequence reads mapping to the X chromosome between two female DNAs in (a) and a female (black) and a male (red) in (b). In b a deletion of several exons is detected on the X chromosome for the male (squared). c Next generation sequencing data showing the detection of a 27 exons deletion in patient C with DMD (middle panel) compared to two other DNAs (top and bottom panels). Random off-target reads allow a more precise mapping of the deletion breakpoints. Off-target reads varied between two different experiments. d CGH-array results showing the 5′ and 3′ breakpoints map between 32,538,435 and 32,538,443 and between 32,187,417 and 32,187,427, respectively

Mentions: Following DNA barcoding and pooling by group of 4, targeted sequencing of the 267 known NMD genes (online resource Table 1) was performed in 8 individuals (A to H) with different neuromuscular disorders and known mutations. After alignment with the human reference genome, mean coverage of the targeted exons was 138× and the percentage of nucleotides with at least 10× coverage was 94 (Table 1, online resource Table 2). Average enrichment for targeted exons was 1,410 fold. More than 97 % of the targeted exons were fully covered, while 168 targeted exons were covered <3× in at least half of the patients (online resource Fig. 1 a and online resource Table 2). Most low-covered exons were similar between patients and coverage decreased with increasing GC content (online resource Fig. 1b). Similar findings were obtained in an independent experiment with six of these DNA samples sequenced individually, validating the multiplexing approach (data not shown). DNAs prepared with different extraction protocols gave similar results, supporting the use of these different protocols on a routine diagnosis basis. Reproducibility between different DNAs treated in the same experiment was such that the coverage was similar for given targeted exons (online resource Table 2 and online resource Fig. 2; narrow distribution of the 95th percentile close to the median). This allowed the detection of the copy number to unambiguously determine the gender of patients as a control for the experiment (Fig. 2a, b), and the mapping of a large deletion (see below).Table 1


Next generation sequencing for molecular diagnosis of neuromuscular diseases.

Vasli N, Böhm J, Le Gras S, Muller J, Pizot C, Jost B, Echaniz-Laguna A, Laugel V, Tranchant C, Bernard R, Plewniak F, Vicaire S, Levy N, Chelly J, Mandel JL, Biancalana V, Laporte J - Acta Neuropathol. (2012)

Detection of copy number and mapping of a deletion in patient C with DMD. a, b Gender determination: comparison of sequence reads mapping to the X chromosome between two female DNAs in (a) and a female (black) and a male (red) in (b). In b a deletion of several exons is detected on the X chromosome for the male (squared). c Next generation sequencing data showing the detection of a 27 exons deletion in patient C with DMD (middle panel) compared to two other DNAs (top and bottom panels). Random off-target reads allow a more precise mapping of the deletion breakpoints. Off-target reads varied between two different experiments. d CGH-array results showing the 5′ and 3′ breakpoints map between 32,538,435 and 32,538,443 and between 32,187,417 and 32,187,427, respectively
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3400754&req=5

Fig2: Detection of copy number and mapping of a deletion in patient C with DMD. a, b Gender determination: comparison of sequence reads mapping to the X chromosome between two female DNAs in (a) and a female (black) and a male (red) in (b). In b a deletion of several exons is detected on the X chromosome for the male (squared). c Next generation sequencing data showing the detection of a 27 exons deletion in patient C with DMD (middle panel) compared to two other DNAs (top and bottom panels). Random off-target reads allow a more precise mapping of the deletion breakpoints. Off-target reads varied between two different experiments. d CGH-array results showing the 5′ and 3′ breakpoints map between 32,538,435 and 32,538,443 and between 32,187,417 and 32,187,427, respectively
Mentions: Following DNA barcoding and pooling by group of 4, targeted sequencing of the 267 known NMD genes (online resource Table 1) was performed in 8 individuals (A to H) with different neuromuscular disorders and known mutations. After alignment with the human reference genome, mean coverage of the targeted exons was 138× and the percentage of nucleotides with at least 10× coverage was 94 (Table 1, online resource Table 2). Average enrichment for targeted exons was 1,410 fold. More than 97 % of the targeted exons were fully covered, while 168 targeted exons were covered <3× in at least half of the patients (online resource Fig. 1 a and online resource Table 2). Most low-covered exons were similar between patients and coverage decreased with increasing GC content (online resource Fig. 1b). Similar findings were obtained in an independent experiment with six of these DNA samples sequenced individually, validating the multiplexing approach (data not shown). DNAs prepared with different extraction protocols gave similar results, supporting the use of these different protocols on a routine diagnosis basis. Reproducibility between different DNAs treated in the same experiment was such that the coverage was similar for given targeted exons (online resource Table 2 and online resource Fig. 2; narrow distribution of the 95th percentile close to the median). This allowed the detection of the copy number to unambiguously determine the gender of patients as a control for the experiment (Fig. 2a, b), and the mapping of a large deletion (see below).Table 1

Bottom Line: We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis.The cost was less than conventional testing for a single large gene.With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations.

View Article: PubMed Central - PubMed

Affiliation: IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), 1, rue Laurent Fries, BP10142, 67404, Illkirch, France.

ABSTRACT
Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN, RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.

Show MeSH
Related in: MedlinePlus