SEARCH

SEARCH BY CITATION

Keywords:

  • Target capture;
  • Sequencing;
  • Mutation;
  • Copy number variation;
  • Genetic testing

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Purpose

Early onset epileptic encephalopathies (EOEEs) are heterogeneous epileptic disorders caused by various abnormalities in causative genes including point mutations and copy number variations (CNVs). In this study, we performed targeted capture and sequencing of a subset of genes to detect point mutations and CNVs simultaneously.

Methods

We designed complementary RNA oligonucleotide probes against the coding exons of 35 known and potential candidate genes. We tested 68 unrelated patients, including 15 patients with previously detected mutations as positive controls. In addition to mutation detection by the Genome Analysis Toolkit, CNVs were detected by the relative depth of coverage ratio. All detected events were confirmed by Sanger sequencing or genomic microarray analysis.

Key Findings

We detected all positive control mutations. In addition, in 53 patients with EOEEs, we detected 12 pathogenic mutations, including 9 point mutations (2 nonsense, 3 splice-site, and 4 missense mutations), 2 frameshift mutations, and one 3.7-Mb microdeletion. Ten of the 12 mutations occurred de novo; the other two had been previously reported as pathogenic. The entire process of targeted capture, sequencing, and analysis required 1 week for the testing of up to 24 patients.

Significance

Targeted capture and sequencing enables the identification of mutations of all classes causing EOEEs, highlighting its usefulness for rapid and comprehensive genetic testing.

Early onset epileptic encephalopathies (EOEEs), occurring before 1 year of age, are characterized by impairment of cognitive, sensory, and motor development by recurrent clinical seizures or prominent interictal epileptiform discharges (Berg et al., 2010). Ohtahara syndrome (OS), West syndrome (WS), early myoclonic encephalopathy (EME), migrating partial seizures in infancy (MPSI), and Dravet syndrome (DS) are the best known epileptic encephalopathies recognized by the International League Against Epilepsy (ILAE; Berg et al., 2010). However, many infants with similar features do not strictly fit the parameters of these syndromes.

To date, 11 genes have been shown to be associated with EOEEs (Mastrangelo & Leuzzi, 2012). The identification of causative mutations associated with EOEEs and their related phenotypes is useful for genetic counseling, and possibly for management of the patients; however, it is time-consuming and arduous to screen all known disease-causing genes one by one using Sanger sequencing or high-resolution melting curve analysis (Wittwer, 2009). In addition, copy number variations (CNVs) involving causative genes can also cause EOEEs (Saitsu et al., 2008; Mei et al., 2010; Saitsu et al., 2011, 2012b). Array comparative genomic hybridization (CGH) and multiplex ligation-dependent probe amplification (MLPA) are well established for the detection of CNVs; however, it is often difficult for array CGH to detect small CNVs such as a single-exon deletion and for MLPA to screen multiple genes at a time (Schouten et al., 2002; Dibbens et al., 2011; Mefford et al., 2011; Stuppia et al., 2012). Therefore, an integrated method that detects both point mutations and CNVs for multiple genes would be useful for comprehensive genetic testing in EOEEs.

Recent progress in massively parallel DNA sequencing in combination with target capturing has facilitated rapid mutation detection (Ng et al., 2009). It has been reported that CNVs involving disease-causing genes in patients with breast or ovarian cancer can be detected by target capture sequencing using the relative depth of coverage ratio (Walsh et al., 2010, 2011; Nord et al., 2011). Targeted capture and sequencing of patients with epileptic disorders has successfully identified potential disease-causing mutations in 16 of 33 patients (Lemke et al., 2012), revealing its efficacy for detecting mutations. However, the detection of both point mutations and CNVs has not been reported in patients with epilepsy.

In this study, we performed targeted capture and sequencing of a subset of 35 genes to detect mutations and CNVs simultaneously in 68 patients with EOEEs. By analyzing the relative depth of coverage ratio, we were able to detect microdeletions, in which the numbers of deleted exons varied from a single exon to all exons of two genes. In combination with rapid sequencing using a benchtop next-generation sequencer, our method provides a fast, comprehensive, and cost-effective method for genetic testing of patients with EOEE.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Patients

We examined 68 patients (36 male and 32 female) with EOEEs (20 patients with OS, 20 with WS, 3 with EME, 4 with MPSI, 2 with DS, and 19 with unclassified epileptic encephalopathy). Diagnoses were based on clinical features and characteristic patterns on electroencephalography. In 15 of 68 patients (10 male and five female), disease-causing mutations or CNVs had been previously identified in our laboratory, so these mutations were used as positive controls (Table 1) (Saitsu et al., 2008, 2010a,b, 2011, 2012b,c; Nonoda et al., 2013). Genomic DNA was isolated from blood leukocytes according to standard methods. Experimental protocols were approved by the Yokohama City University School of Medicine Institutional Review Board for Ethical Issues. Written informed consent for genetic testing was obtained from the guardians of all tested individuals prior to analysis.

Table 1. Known mutations and copy number variants used as positive controls
 CaseSexChrGenesReported mutations or copy number variants (positive controls)TypeDeletion size (kb)Refs
  1. SNVs, single nucleotide variants; Indels, insertion/deletions; CNVs, copy number variations.

SNVs27F9 STXBP1 c.1328T>G (p.Met443Arg)Missense Saitsu et al. (2008)
69MX CASK c.1A>GMissense Saitsu et al. (2012b)
241MX CDKL5 c.145G>A (p.Glu49Lys)Missense 
Indels95M9 STXBP1 c.388_389del (p.Leu130AspfsX11)Deletion Saitsu et al. (2010a)
313MX CASK c.227_228del (p.Glu76ValfsX6)Deletion 
26F9 SPTAN1 c.6619_6621del (p.Glu2207del)Deletion Saitsu et al. (2010b)
220M9 STXBP1 c.1381_1390del (p.Lys461GlyfsX82)Deletion 
16M9 SPTAN1 c.6923_6928dup (p.Arg2308_Met2309dup)Duplication Saitsu et al. (2010b)
309M9 SPTAN1 c.6908_6916dup (p.Asp2303_Leu2305dup)Duplication Nonoda et al. (2013)
CNVs12F9 STXBP1, SPTAN1 Del(9)(q33.33–q34.11)Microdeletion2150Saitsu et al. (2008)
22M9 STXBP1 STXBP1 Ex4 deletionMicrodeletion4.6Saitsu et al. (2011)
83MX CASK CASK Ex2 deletionMicrodeletion111Saitsu et al. (2012b)
102FX MECP2 Del(X)(q28)Microdeletion 
204M9 STXBP1, SPTAN1 Del(9)(q33.33–q34.11)Microdeletion2850Saitsu et al. (2011)
214FX CDKL5 Del(X)(q22.13)Microdeletion137Saitsu et al. (2011)

Target capture sequencing and variant detection

A custom-made SureSelect oligonucleotide probe library (Agilent Technologies, Santa Clara, CA, U.S.A.) was designed to capture the coding exons of 35 genes; 5 of them were potential candidates for EOEEs based on unpublished data (for a list of the 30 of 35 genes, see Table 2). We designed 120-bp capture probes with 3× centered probe-tiling, and avoiding 20-bp overlap to repeat region using the Agilent e-Array Web-based design tool. To cover regions where we could not design probes with the above settings, some probes from the SureSelect Human All Exon 50-Mb kit (Agilent Technologies) were added to the probe libraries. A total of 2,738 probes, covering 156 kb, were prepared. Exon capture, enrichment, and indexing were performed according to the manufacturer's instructions. Twenty-four captured libraries were mixed and sequenced on an Illumina MiSeq (Illumina, San Diego, CA, U.S.A.) with 150-bp paired-end reads. Image analysis and base calling were performed using the Illumina Real Time Analysis Pipeline version 1.13 and CASAVA software v.1.8 (Illumina) with default parameters. Sequence reads were aligned to the reference human genome (GRCh37: Genome Reference Consortium human build 37) with Novoalign (Novocraft Technologies, Selangor, Malaysia). After conversion of the SAM file to a BAM file with SAMtools (Li et al., 2009), duplicate reads were marked using Picard (http://picard.sourceforge.net/) and excluded from downstream analysis. Local realignment around insertion/deletions (indels) and base quality score recalibration were performed using the Genome Analysis Toolkit (DePristo et al., 2011). Single-nucleotide variants (SNVs) and indels were identified using the Genome Analysis Toolkit UnifiedGenotyper and filtered according to the Broad Institute's best-practice guidelines v.3 except for HaplotypeScore filtering. We excluded variants found in 147 exomes from healthy individuals previously sequenced in our laboratory. Variants were annotated using ANNOVAR (Wang et al., 2010). Candidate disease-causing mutations were confirmed by Sanger sequencing on a 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). The Human Gene Mutation Database professional 2012.3 (BIOBASE GmbH, Wolfenbuettel, Germany) was used to check whether the variants had been previously reported.

Table 2. Sequence performance for 30 target genes
GeneCytobandNo. of coding exonsMean read depth%bases above 5× depth (%)%bases above 10× depth (%)
ARHGEF9 Xq11.1–q11.210206100100
ARX Xp21.354459.4–94.438.7–90.6
CASK Xp11.42720195.9–10095.9–100
CDKL5 Xp22.1320238100100
COL4A1 13q345228798.3–10098.3–100
COL4A2 13q344719010099.1–100
FOXG1 14q12123186.5–10081.1–96.4
GABRG2 5q341130092.392.3
GRIN2A 16p13.213310100100
KCNQ2 20q13.331713510097.7–100
MAGI2 7q21.112225596–98.394.5–97.5
MAPK10 4q21.312304100100
MECP2 Xq28321796.296.2
MEF2C 5q14.310270100100
NTNG1 1p13.39298100100
PCDH19 Xq22.16212100100
PLCB1 20p12.332293100100
PNKP 19q13.331720810098.5–100
PNPO 17q21.327210100100
SCN1A 2q24.326345100100
SCN2A 2q24.326323100100
SLC25A22 11p15.59121100100
SLC2A1 1p34.21020910098.8–100
SNPH 20p134179100100
SPTAN1 9q34.1156277100100
SRGAP2 1q32.12032096.696.6
ST3GAL5 2p11.2830293.6–10093.6–99.9
STXBP1 9q34.1120306100100
SYN1 Xp11.231313193.4–10081–100
SYP Xp11.23614610099.1–100

Copy number analysis using target capture sequence data

Copy number changes were analyzed based on the relative depth of coverage ratios (Nord et al., 2011). Raw coverage on the target regions was calculated by SAMtools using BAM files, in which duplicate reads were excluded. Raw coverage was normalized and corrected for GC content and bait capture bias. Next, the ratios were calculated by comparing the sample-corrected coverage to the median-corrected coverage for the other 23 samples. A sliding window (20 bp) was used to identify CNVs for which the majority of bases had a ratio ≤0.6 (loss) or ≥1.4 (gain). We visually inspected the ratio data and judged whether the call was true or likely to be a false positive. A flow chart of our variant detection and copy number analysis scheme is illustrated in Fig. S1.

Genomic microarray analysis and cloning of deletion breakpoints

The microdeletion involving SCN1A and SCN2A was confirmed using a CytoScan HD Array (Affymetrix, Santa Clara, CA, U.S.A.) according to the manufacturer's protocol. Copy number alterations were analyzed using the Chromosome Analysis Suite (ChAS; Affymetrix) with NA32 (hg19) annotations. The junction fragment spanning the deletion was amplified by long polymerase chain reaction (PCR) using several primer sets based on putative breakpoints according to the microarray data. Long PCR was performed in a 20-μl volume, containing 30 ng genomic DNA, 1× buffer for KOD FX, 0.4 mm each dNTP, 0.3 μm each primer, and 0.3 U KOD FX polymerase (Toyobo, Osaka, Japan). The deletion junction fragments were obtained using the following primers: #409-F (5′-TCCACAGTTTACAAACATCTTTTCATGG-3′) and #409-R (5′-AGAAATTGGCTTGGTCAGTACCAGCA-3′) (1.6-kb amplicon). PCR products were electrophoresed on agarose gels stained with ethidium bromide, purified with ExoSAP (USB Technologies, Cleveland, OH, U.S.A.), and sequenced with BIGDYE TERMINATOR CHEMISTRY v.3 according to the manufacturer's protocol (Applied Biosystems).

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Target capture sequencing yielded an average of 26 Mb per sample (range 17–41 Mb per sample) on the target regions, resulting in an average read depth of 255 (range across all samples: 173–437). The coverage of the protein-coding sequences of the 30 target genes is shown in Table 2. Overall, 98.6% of targeted coding sequence bases were covered by 10 reads or more; however, some genes such as ARX and FOXG1 were less well covered because of embedded repeat sequences (Fig. S2). To validate the performance of target capture sequencing for detecting mutations and CNVs, we analyzed 15 samples in which disease-causing mutations or microdeletions had been identified previously in our laboratory (Saitsu et al., 2008, 2010a,b, 2011, 2012b; Nonoda et al., 2013). All nine control point mutations and six control microdeletions were detected (Table 1; Fig. 1). These data indicate that our target capture sequencing method was able to detect both point mutations and microdeletions, including deletion of a single exon.

image

Figure 1. Detection of three known microdeletions by target capture sequencing. (A) Relative depth of coverage ratio for patient 12. Coverage ratios for each target gene are indicated by different colors. A microdeletion including STXBP1 and SPTAN1 is clearly observed. (B, C) Relative depth of coverage ratio for patient 214 in the CDKL5 region and patient 22 in the STXBP1 region, respectively. Black vertical lines indicate exons and horizontal lines indicate introns (top). Red vertical lines show bait regions that were judged to be “deleted.” A number of exons of CDKL5 were deleted in patient 214 (bidirectional arrow in B), and a single exon of STXBP1 was deleted in patient 22 (arrow in C).

Download figure to PowerPoint

Examination of 53 previously unresolved EOEE patients by targeted capture and sequencing revealed mutations in 12 patients (Table 3). Every patient harbored a different mutation. Of these 12 mutations, 9 were single-nucleotide variants (2 nonsense, 3 splice-site, and 4 missense mutations) and two were small indels leading to frameshifts. The other mutation was a microdeletion. All these 11 point mutations were confirmed by Sanger sequencing. Four of the mutations (STXBP1 c.902+1G>A, SCN1A c.580G>A, SCN1A c.3714A>C, and CDKL5 c.533G>A) have been reported in individuals with EOEEs, so are recurrent (Mancardi et al., 2006; Harkin et al., 2007; Azmanov et al., 2010; Liang et al., 2011; Milh et al., 2011). Nine of the 11 mutations occurred de novo. The other two could not be tested because the paternal sample for one patient (SCN1A c.580G>A) and parental samples for another patient (SCN1A c.3714A>C) were unavailable.

Table 3. Mutations in 53 patients with EOEEs detected by targeted capture and sequencing
 CaseSexDiagnosisChrGeneMutationTypeDeletion size (kb)InheritanceReferences
  1. OS, Ohtahara syndrome; EME, early myoclonic encephalopathy; MAE, myoclonic astatic epilepsy; DS, Dravet syndrome; WS, West syndrome; PCH, pontocerebellar hypoplasia; MPSI, malignant migrating partial seizures in infancy; SNVs, single nucleotide variants; CNVs, copy number variations; EOEEs, early onset epileptic encephalopathies.

SNVs329MOS/EME9 STXBP1 c.247-2A>GSplice site De novo
402MOS9 STXBP1 c.902+1G>ASplice site De novoMilh et al. (2011)
423FOS9 STXBP1 c.246+1G>ASplice site De novo
403FMAE or DS2 SCN1A c.580G>A (p.Asp194Asn)Missense Not found in the motherMancardi et al. (2006)
415FEOEE2 SCN1A c.3714A>C (p.Glu1238Asp)Missense Not determinedHarkin et al. (2007)
416MEOEEX CDKL5 c.533G>A (p.Arg178Gln)Missense De novoLiang et al. (2011)
418FWS, severe hypotonia2 SCN2A c.632G>A (p.Gly211Asp) in NM_001040143 (variant 3)Missense De novo
244FEpilepsy + PCHX CASK c.55G>T (p.Gly19X)Nonsense De novo
404FEOEEsX MECP2 c.844C>T (p.Arg282X)Nonsense De novo
Indels336FOS9 STXBP1 c.1056del (p.Asp353ThrfsX3)Deletion De novo
397FDS2 SCN1A c.342_344delinsAGGAGTT (p.Phe114LeufsX6)Deletion–insertion De novo
CNV409FMPSI2 SCN2A, SCN1A MicrodeletionMicrodeletion3,726De novo

CNV analysis of the 53 patients revealed a microdeletion involving SCN1A and SCN2A at 2q24.3 in patient 409 (Fig. 2A). To investigate this mutation further, we performed genomic microarray analysis and identified an approximately 3.7-Mb microdeletion (Fig. 2B). The deletion contained 13 RefSeq genes including SCN2A and SCN1A. Breakpoint-specific PCR analysis of the patient and her parents confirmed that the rearrangement occurred de novo (Fig. 2C). The sequence of the junction fragment confirmed a 3,726,029-bp deletion (chr2: 164,420,771–168,146,801) (Fig. 2D).

image

Figure 2. A 3.7-Mb microdeletion including SCN2A and SCN1A in patient 409. (A) Relative depth of coverage ratio for patient 409 indicates a microdeletion encompassing SCN2A and SCN1A. Different colors distinguish the target genes. (B) The array profile clearly shows a 3.7-Mb microdeletion at 2q24.3 in this patient. Thirteen RefSeq genes, including SCN2A and SCN1A, lie within the microdeletion (bottom). (C) Breakpoint-specific PCR analysis of the patient's family. Primers flanking the deletion were able to amplify a 1,607-bp product from the patient only, indicating that the translocation occurred de novo. (D) Deletion junction sequence. The top, middle, and bottom strands show the proximal, deleted, and distal sequences, respectively. A single inserted nucleotide (colored in red) was identified at the breakpoint.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Several bench-top high-throughput sequencing platforms are now available (Glenn, 2011; Loman et al., 2012; Quail et al., 2012). We selected Illumina MiSeq because it provides reasonable sequence throughput (1.6 Gb per run), a low error rate, a short run time (27 h), and sufficiently long reads (150 bp). We captured genomic DNA fragments of target genes by 3× tiling complementary RNA oligonucleotide probes (Nord et al., 2011) and sequenced 24 samples per MiSeq run, achieving sufficient coverage (a mean read depth of 255) over the target regions. This high coverage enabled us to detect point mutations and CNVs simultaneously, and long reads enabled us to detect small indels (Krawitz et al., 2010). Mapping by Novoalign, we were able to detect indels ranging in size from a 10-bp deletion to a 9-bp duplication.

By evaluating depth of coverage ratios (Nord et al., 2011), we detected six control microdeletions and one novel microdeletion, ranging in size from 4.6 kb to 3.7 Mb. To date, CNVs causing EOEEs have been analyzed by array CGH and MLPA (Mulley & Mefford, 2011). Array CGH can detect genome-wide CNVs, but its standard resolution is relatively low (>10 kb). On the other hand, MLPA can detect CNVs in specific genes, including single exon deletions; however, it is difficult to screen many genes at a time because MLPA is limited to 50 target exons per reaction (Stuppia et al., 2012). In addition, copy number analysis using MLPA can be affected by single nucleotide variants and indels in regions corresponding to the MLPA probes (Stuppia et al., 2012). In contrast, targeted capture and sequencing can analyze all targeted genes to detect mutations and CNVs simultaneously. CNVs as small as a single exon can be identified. Because all the procedures—from the capture of target genes to the detection of mutations and CNVs—can be done within a week, our workflow provides a fast, sensitive, and comprehensive genetic testing method for patients with epilepsy.

Whole-exome sequencing will reveal novel mutations in unexpected genes in patients with EOEEs. For example, KCNQ2 mutations, which cause benign familial neonatal seizures (Biervert et al., 1998; Charlier et al., 1998), were identified in patients with OS by whole exome sequencing (Saitsu et al., 2012a). Similarly, screening known and potential candidate genes in patients with EOEEs will reveal novel mutations in unexpected genes, in addition to mutations in well-known genes.

In our target capture analysis, some exons of genes such as ARX and FOXG1 were insufficiently sequenced because repeat sequences hampered the design of capture probes. Repeat sequences also interfere with appropriate mapping of sequence reads, resulting in low coverage. For these exons, Sanger sequencing should be added for complete analysis.

In conclusion, a rapid and efficient system of target capture sequencing can be applied to the comprehensive genetic analysis of EOEEs. Point mutations, small indels, and CNVs are all detected by this method, confirming the potential of this approach for efficient genetic testing.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

We thank the patients and their families for their participation in this study. We also thank Aya Narita and Nobuko Watanabe for their technical assistance. This work was Supported by the Ministry of Health, Labour and Welfare of Japan (24133701, 11103577, 11103580, 11103340, 10103235), a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (24591500), a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (10013428, 11001011, 12020465), the Takeda Science Foundation, the Japan Science and Technology Agency, the Strategic Research Program for Brain Sciences (11105137), and a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (12024421).

Disclosure

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

None of the authors has any conflicts of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
  • Azmanov DN, Zhelyazkova S, Dimova PS, Radionova M, Bojinova V, Florez L, Smith SJ, Tournev I, Jablensky A, Mulley J, Scheffer I, Kalaydjieva L, Sander JW. (2010) Mosaicism of a missense SCN1A mutation and Dravet syndrome in a Roma/Gypsy family. Epileptic Disord 12:117124.
  • Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas W, Engel J, French J, Glauser TA, Mathern GW, Moshe SL, Nordli D, Plouin P, Scheffer IE. (2010) Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005–2009. Epilepsia 51:676685.
  • Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, Steinlein OK. (1998) A potassium channel mutation in neonatal human epilepsy. Science 279:403406.
  • Charlier C, Singh NA, Ryan SG, Lewis TB, Reus BE, Leach RJ, Leppert M. (1998) A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat Genet 18:5355.
  • DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ. (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491498.
  • Dibbens LM, Kneen R, Bayly MA, Heron SE, Arsov T, Damiano JA, Desai T, Gibbs J, McKenzie F, Mulley JC, Ronan A, Scheffer IE. (2011) Recurrence risk of epilepsy and mental retardation in females due to parental mosaicism of PCDH19 mutations. Neurology 76:15141519.
  • Glenn TC. (2011) Field guide to next-generation DNA sequencers. Mol Ecol Resour 11:759769.
  • Harkin LA, McMahon JM, Iona X, Dibbens L, Pelekanos JT, Zuberi SM, Sadleir LG, Andermann E, Gill D, Farrell K, Connolly M, Stanley T, Harbord M, Andermann F, Wang J, Batish SD, Jones JG, Seltzer WK, Gardner A, Sutherland G, Berkovic SF, Mulley JC, Scheffer IE. (2007) The spectrum of SCN1A-related infantile epileptic encephalopathies. Brain 130:843852.
  • Krawitz P, Rodelsperger C, Jager M, Jostins L, Bauer S, Robinson PN. (2010) Microindel detection in short-read sequence data. Bioinformatics 26:722729.
  • Lemke JR, Riesch E, Scheurenbrand T, Schubach M, Wilhelm C, Steiner I, Hansen J, Courage C, Gallati S, Burki S, Strozzi S, Simonetti BG, Grunt S, Steinlin M, Alber M, Wolff M, Klopstock T, Prott EC, Lorenz R, Spaich C, Rona S, Lakshminarasimhan M, Kroll J, Dorn T, Kramer G, Synofzik M, Becker F, Weber YG, Lerche H, Bohm D, Biskup S. (2012) Targeted next generation sequencing as a diagnostic tool in epileptic disorders. Epilepsia 53:13871398.
  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. (2009) The sequence Alignment/Map format and SAMtools. Bioinformatics 25:20782079.
  • Liang JS, Shimojima K, Takayama R, Natsume J, Shichiji M, Hirasawa K, Imai K, Okanishi T, Mizuno S, Okumura A, Sugawara M, Ito T, Ikeda H, Takahashi Y, Oguni H, Imai K, Osawa M, Yamamoto T. (2011) CDKL5 alterations lead to early epileptic encephalopathy in both genders. Epilepsia 52:18351842.
  • Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ. (2012) Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 30:434439.
  • Mancardi MM, Striano P, Gennaro E, Madia F, Paravidino R, Scapolan S, Dalla Bernardina B, Bertini E, Bianchi A, Capovilla G, Darra F, Elia M, Freri E, Gobbi G, Granata T, Guerrini R, Pantaleoni C, Parmeggiani A, Romeo A, Santucci M, Vecchi M, Veggiotti P, Vigevano F, Pistorio A, Gaggero R, Zara F. (2006) Familial occurrence of febrile seizures and epilepsy in severe myoclonic epilepsy of infancy (SMEI) patients with SCN1A mutations. Epilepsia 47:16291635.
  • Mastrangelo M, Leuzzi V. (2012) Genes of early-onset epileptic encephalopathies: from genotype to phenotype. Pediatr Neurol 46:2431.
  • Mefford HC, Yendle SC, Hsu C, Cook J, Geraghty E, McMahon JM, Eeg-Olofsson O, Sadleir LG, Gill D, Ben-Zeev B, Lerman-Sagie T, Mackay M, Freeman JL, Andermann E, Pelakanos JT, Andrews I, Wallace G, Eichler EE, Berkovic SF, Scheffer IE. (2011) Rare copy number variants are an important cause of epileptic encephalopathies. Ann Neurol 70:974985.
  • Mei D, Marini C, Novara F, Bernardina BD, Granata T, Fontana E, Parrini E, Ferrari AR, Murgia A, Zuffardi O, Guerrini R. (2010) Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy. Epilepsia 51:647654.
  • Milh M, Villeneuve N, Chouchane M, Kaminska A, Laroche C, Barthez MA, Gitiaux C, Bartoli C, Borges-Correia A, Cacciagli P, Mignon-Ravix C, Cuberos H, Chabrol B, Villard L. (2011) Epileptic and nonepileptic features in patients with early onset epileptic encephalopathy and STXBP1 mutations. Epilepsia. 52:18281834.
  • Mulley JC, Mefford HC. (2011) Epilepsy and the new cytogenetics. Epilepsia 52:423432.
  • Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, Bamshad M, Nickerson DA, Shendure J. (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272276.
  • Nonoda Y, Saito Y, Nagai S, Sasaki M, Iwasaki T, Matsumoto N, Ishii M, Saitsu H. (2013) Progressive diffuse brain atrophy in West syndrome with marked hypomyelination due to SPTAN1 gene mutation. Brain Dev 35:280283.
  • Nord AS, Lee M, King MC, Walsh T. (2011) Accurate and exact CNV identification from targeted high-throughput sequence data. BMC Genomics 12:184.
  • Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerdlow HP, Gu Y. (2012) A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13:341.
  • Saitsu H, Kato M, Mizuguchi T, Hamada K, Osaka H, Tohyama J, Uruno K, Kumada S, Nishiyama K, Nishimura A, Okada I, Yoshimura Y, Hirai S, Kumada T, Hayasaka K, Fukuda A, Ogata K, Matsumoto N. (2008) De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy. Nat Genet 40:782788.
  • Saitsu H, Kato M, Okada I, Orii KE, Higuchi T, Hoshino H, Kubota M, Arai H, Tagawa T, Kimura S, Sudo A, Miyama S, Takami Y, Watanabe T, Nishimura A, Nishiyama K, Miyake N, Wada T, Osaka H, Kondo N, Hayasaka K, Matsumoto N. (2010a) STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern. Epilepsia 51:23972405.
  • Saitsu H, Tohyama J, Kumada T, Egawa K, Hamada K, Okada I, Mizuguchi T, Osaka H, Miyata R, Furukawa T, Haginoya K, Hoshino H, Goto T, Hachiya Y, Yamagata T, Saitoh S, Nagai T, Nishiyama K, Nishimura A, Miyake N, Komada M, Hayashi K, Hirai S, Ogata K, Kato M, Fukuda A, Matsumoto N. (2010b) Dominant-negative mutations in alpha-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic quadriplegia, and developmental delay. Am J Hum Genet 86:881891.
  • Saitsu H, Kato M, Shimono M, Senju A, Tanabe S, Kimura T, Nishiyama K, Yoneda Y, Kondo Y, Tsurusaki Y, Doi H, Miyake N, Hayasaka K, Matsumoto N. (2011) Association of genomic deletions in the STXBP1 gene with Ohtahara syndrome. Clin Genet 81:399402.
  • Saitsu H, Kato M, Koide A, Goto T, Fujita T, Nishiyama K, Tsurusaki Y, Doi H, Miyake N, Hayasaka K, Matsumoto N. (2012a) Whole exome sequencing identifies KCNQ2 mutations in Ohtahara syndrome. Ann Neurol 72:298300.
  • Saitsu H, Kato M, Osaka H, Moriyama N, Horita H, Nishiyama K, Yoneda Y, Kondo Y, Tsurusaki Y, Doi H, Miyake N, Hayasaka K, Matsumoto N. (2012b) CASK aberrations in male patients with Ohtahara syndrome and cerebellar hypoplasia. Epilepsia 53:14411449.
  • Saitsu H, Osaka H, Nishiyama K, Tsurusaki Y, Doi H, Miyake N, Matsumoto N. (2012c) A girl with early-onset epileptic encephalopathy associated with microdeletion involving CDKL5. Brain Dev 34:364367.
  • Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30:e57.
  • Stuppia L, Antonucci I, Palka G, Gatta V. (2012) Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci 13:32453276.
  • Walsh T, Lee MK, Casadei S, Thornton AM, Stray SM, Pennil C, Nord AS, Mandell JB, Swisher EM, King MC. (2010) Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. Proc Natl Acad Sci USA 107:1262912633.
  • Walsh T, Casadei S, Lee MK, Pennil CC, Nord AS, Thornton AM, Roeb W, Agnew KJ, Stray SM, Wickramanayake A, Norquist B, Pennington KP, Garcia RL, King MC, Swisher EM. (2011) Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci USA 108:1803218037.
  • Wang K, Li M, Hakonarson H. (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164.
  • Wittwer CT. (2009) High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30:857859.

Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
epi12203-sup-0001-FigS1.tifimage/tif615KFigure S1. Flow chart of our variant detection and copy number analysis scheme.
epi12203-sup-0002-FigS2.tifimage/tif898KFigure S2. Insufficient coverage of reads in two genes rich in repetitive sequences.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.