A Comprehensive Strategy for Accurate Mutation Detection of the ...

The Journal of Molecular Diagnostics, Vol. 17, No. 5, September 2015

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A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2 Jianli Li,* Hongzheng Dai,* Yanming Feng,* Jia Tang,* Stella Chen,* Xia Tian,* Elizabeth Gorman,* Eric S. Schmitt,* Terah A.A. Hansen,y Jing Wang,z Sharon E. Plon,x Victor Wei Zhang,z and Lee-Jun C. Wongz From the Baylor Miraca Genetics Laboratories,* Houston, Texas; the Central Washington Genetics Program,y Yakima Valley Memorial Hospital, Yakima, Washington; and the Departments of Molecular and Human Genetics,z and Pediatrics,x Baylor College of Medicine, Houston, Texas Accepted for publication April 29, 2015. Address correspondence to Lee-Jun C. Wong, Ph.D., or Victor Wei Zhang, M.D., Ph.D., Baylor College of Medicine, One Baylor Plaza, NAB 2015, Houston, TX 77030. E-mail: [email protected] or [email protected].

Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 30 region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. (J Mol Diagn 2015, 17: 545e553; http://dx.doi.org/10.1016/j.jmoldx.2015.04.001)

Heterozygous germline mutations in DNA mismatch repair genes MLH1, MSH2, MSH6, and PMS2 contribute to hereditary nonpolyposis colorectal cancer, or Lynch syndrome, which accounts for 2% to 4% of all colorectal cancers.1 Carrier individuals are at increased risk of developing cancers, including colorectal and endometrial cancers.2 In rare situations, biallelic mutations in PMS2 cause constitutional mismatch repair deficiency syndrome, which is characterized by childhood-onset malignancy, including brain cancers, hematological malignancies, colorectal cancers, and multiple intestinal polyposis.3e5 PMS2 is the most common cause of this recessive condition. Accurate molecular testing of these genes will improve the diagnosis and help with appropriate genetic counseling and medical management. It was originally thought that most of Lynch syndromee associated mutations were due to defective MLH1 and MSH2.6 PMS2, identified in 1994,7 was considered to play a minor Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2015.04.001

role in Lynch syndrome.6,8,9 This appears to be an underestimate because of the relatively lower penetrance for PMS2,10,11 causing kindreds to be less likely to fit the Amsterdam criteria for Lynch syndrome.11 In addition, the presence of a large number of highly homologous sequences in other genomic regions and the frequent sequence exchange between the active and pseudogenes hindered the accurate molecular analysis of Supported in part by a Muscular Dystrophy Association grant MDA 175369 (L.-J.W). Disclosures: J.L., H.D., Y.F., J.T., S.C., X.T., E.G., E.S.S., J.W., V.W.Z., and L.-J.W. are faculty members or employees in the Joint Venture of Baylor Miraca Genetics Laboratories and Department of Molecular and Human Genetics at Baylor College of Medicine. The Baylor Miraca Genetics Laboratories offers extensive fee-based genetic tests, including the use of massively parallel sequencing for molecular analyses of hereditary cancers. S.E.P. is on the scientific advisory board of Baylor Miraca Genetics Laboratories. T.A.A.H. has no conflict of interest.

Li et al PMS2.12e16 The high frequency of negative staining of PMS2 immunohistochemistry in colorectal cancer tissues without identifiable PMS2 mutations suggests that PMS2 mutations have been grossly underestimated as a result of these technical difficulties.11,13,17,18 Recent advancements in technologies and reagents have significantly improved the molecular diagnostic yield of PMS2 in cancer patients with tumor specimens that demonstrate negative PMS2 immunohistochemistry and normal MLH1 staining.19e21 However, a comprehensive approach to unequivocally identify PMS2 mutations remains to be developed. Molecular testing of PMS2 is especially challenging because of the presence of the PMS2CL pseudogene, which has more than 98% sequence identity with the 30 region of PMS2, including exons 9 and 11 to 15 (Supplemental Table S1).12,13 PMS2CL, a 100-kb inverted duplication of PMS2, is located about 700 kb centromeric of PMS2. Several studies suggest that extensive sequence exchange as a result of gene conversion is still ongoing between PMS2 and PMS2CL.14,22 Such gene conversion is one of the mechanisms introducing pathogenic alleles into the active gene.14e16 Multiplex ligation-dependent probe amplification (MLPA) assay using probes targeting paralogous sequence variants (PSVs) of PMS2 and PMS2CL has been designed to evaluate the copy number variation (CNV) of these two genes (MRC Holland, Amsterdam, the Netherlands). By combining gene-specific long-range PCR (LR-PCR) of the active gene with Sanger sequencing analysis, it is possible to differentiate CNVs at the 30 end of PMS2 from these of PMS2CL.11,16,23,24 However, CNVs cannot be unambiguously differentiated when PMS2 and PMS2CL share identical PSVs because of sequence exchange.24 Indeed, a previous study16 estimated that 22% of cases have identical PSVs because of sequence transfer events between PMS2 and PMS2CL. Here, we describe a novel approach that combines targeted capture/next-generation sequencing (NGS), MLPA, and LRPCR/NGS to unambiguously identify sequence variants and copy number changes of PMS2.

Workflow of the Comprehensive Approach A comprehensive workflow incorporating MLPA, capture NGS, and LR-PCR/NGS was designed for sequence and CNV analyses. Three methods were performed in parallel (Figure 1). MLPA was used to detect CNVs in PMS2 and PMS2CL. Capture sequence detects PSVs and CNVs from both PMS2 and PMS2CL. To distinguish mutations in the active gene from changes in the pseudogene, the active gene was specifically enriched by LR-PCR, followed by NGS analysis using previously described protocols.25e27 Consistent results of four PSVs at the 30 region of PMS2 must be observed by all three different methods to avoid false positives (Supplemental Table S2).

MLPA Copy number analysis for PMS2 was performed using MCR Holland SALSA MLPA Kit P008-C1 according to the manufacturer’s recommendations.28 The P008-C1 commercial kit includes probes specific for PMS2 exons 1 to 11, exons 11 to 15, and PSVs in exons 11 to 15 targeting both PMS2 and PMS2CL. We selected a reference DNA (NA12878 from the Coriell Institute, Camden, NJ) that has five pairs of different PSVs in exons 11 to 15. The MLPA data are analyzed using Coffalyzer software version 140429.1057 (MRC Holland).

Target Capture-NGS and CNV Analysis

Eight positive samples with known PMS2 mutations and six negative samples were used as control specimens for validation. The validated method incorporating MLPA, capture NGS, and LR-PCR/NGS was then applied to 32 clinical samples for molecular diagnosis. These samples were sent to us for genetic testing of hereditary cancer panels. Total genomic DNA was extracted from blood using a commercially available DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. This study was performed in accordance with protocols approved by the institutional review board at the Baylor College of Medicine.

A probe library (Roche NimbleGen SeqCap; Roche NimbleGen, Madison, WI) targeting all coding exons and 50 bp of flanking intronic regions of 197 hereditary cancer genes, including PMS2, was custom designed followed by NGS analysis according to published procedures.25,26,29e32 The sample preparation was performed according to the manufacturer’s recommendation in the SeqCap EZ Choice Library User’s Guide (Roche NimbleGen). Equal molar ratios of 15 indexed samples were pooled to loaded to one lane of the flow cell for sequencing on a HiSeq2000 sequencer (Illumina, San Diego, CA) with 100-cycle single-end reads. For NGS data analysis, the raw data in base call files (.bcl format) were conveyed to qseq files before demultiplexing using CASAVA software version 1.7 (Illumina). Demultiplexed sequence reads were aligned to the PMS2 reference sequence NM_000535.5 using the NextGENe software version 2.3 (SoftGenetics, State College, PA). The mean coverage per base was extracted from the aligned data. The normalized average coverage depth per individual exon was used to detect exonic CNVs.31 The script for the detection of CNVs is deposited online at https:// sourceforge.net/projects/cnvanalysis (last accessed April 29, 2015). The identification of breakpoints is similar to previously published procedures.26 Variant annotation and copy number analyses using capture NGS data were performed.25,30e32 Short and long interspersed repeat sequences were identified using online software (RepeatMasker Open-4.0

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Materials and Methods Specimens and DNA Preparation

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Figure

1 Comprehensive workflow of sequence and copy number variation (CNV) analyses of PMS2. CNVs in PMS2 or PMS2CL are detected by both multiplex ligation-dependent probe amplification and capture-based nextgeneration sequencing (NGS). Long-range-PCR (LR-PCR)/NGS is used to differentiate the source of changes in PMS2 or PMS2CL. The LRPCR/NGS is used for both sequence analysis and confirmation of CNVs in the active gene. Capture-based NGS is also used to overcome allele dropout. del/dup, deletion/duplication.

2013-2015, http://repeatmasker.org, last accessed April 29, 2015). The alignment of Alu sequences was completed using web tool ClustalW2 (http://www.ebi.ac.uk/Tools/ msa/clustalw2, last accessed April 29, 2015).

LR-PCR and NGS Previously published primers were used to amplify both PMS2 and PMS2CL genes.10,33 LR-PCR primer pairs were used to amplify three fragments, E1 to E5, E6 to E10, and E11 to E15, specific for the active PMS2 gene and the pseudo gene PMS2CL. The LR-PCR was performed using TaKaRa

LA Taq DNA polymerase Hot-Start Version (Takara Bio, Otsu, Japan) with 100 ng of total genomic DNA in a final volume of 50 mL. LR-PCR products of PMS2 were fragmented and indexed for the subsequent analysis by high-throughput sequencing on the HiSeq2000 sequencer (Illumina) as described above.25,30e32

Confirmation of Mutations by Sanger Sequencing Analysis All point mutations and breakpoints identified by NGS were confirmed by PCR-based Sanger sequencing analysis

Table 1

Summary of Point Mutations and CNVs Detected by Comprehensive Approach

Case number

Test results MLPA

Capture NGS

LR-PCR/NGS

Final results

S1 S2 S3 S4 S5 S6

E2eE9 deletion E5eE9 deletion E8 deletion E10 deletion E11eE12 duplication Deletion in PMS2/PMS2CL

S7 S8 C1 C2

E1eE15 deletion Deletion in PMS2/PMS2CL negative Deletion in PMS2/PMS2CL

E2eE9 deletion E5eE9 deletion E8 deletion E10 deletion E11eE12 duplication c.65C>A; deletion in PMS2/PMS2CL ND ND c.466A>G (p.T156A) Deletion in PMS2/PMS2CL

ND ND ND E10 deletion c.1145-95_c.2174þ1850dup c.65C>A (p.S22*) and c.2276-113_c.2445þ1596del ND c.2276-138_c.2445þ1571del c.466A>G (p.T156A) c.2276-113_c.2445þ1596del

E2eE9 deletion E5eE9 deletion E8 deletion E10 deletion, hom c.1145-95_c.2174þ1850dup, hom c.65C>A (p.S22*) and c.2276-113_c.2445þ1596del E1eE15 deletion c.2276-138_c.2445þ1571del c.466A>G (p.T156A) c.2276-113_c.2445þ1596del

Unless otherwise indicated, all point mutations and deletions are heterozygous. hom, homozygous; LR-PCR, long-range PCR; MLPA, multiplex ligation-dependent probe amplification; ND, not done; NGS, next-generation sequencing.

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Figure 2 Identification of deletions in exons 1 to 10. A: Heterozygous deletion involving exons 5 to 9 in patient S2 was identified by coverage analysis of capture-based next-generation sequencing (NGS). The normalized NGS coverage generated a ratio of approximately 0.5 for deleted exons 5 to 9. B: The plot of mean coverage depth of the test sample (y axis) versus mean coverage depth of 20 reference samples, reveals exons 5 to 9 deletion as outliers (oval) below the 45 line, suggesting a heterozygous deletion. C: Deletion of exons 5 to 9 is shown by multiplex ligation-dependent probe amplification (MLPA) test. The ratios of probes targeted to exons 5 to 9 of PMS2 are approximately 0.5 in the MLPA test compared to normal reference DNA (two copies). D: A homozygous deletion of exon 10 is revealed by the ratio of 0 generated by probes targeted to exon 10 in MLPA test of sample S4. All error bars represent 2 SD.

using BigDye terminator chemistry (Life Technologies, Carlsbad, CA) and Mutation Surveyor version 4 (SoftGenetics).34 The primers used for nested PCR to identify rearrangement of exons 11 and 12 are 50 -CAGAAAAATAATCTTGTAAAATGTCTG-30 (E12F) and 50 -CCGGTATCTTCCTGGTTTGAA-30 (E11R). The primers used for sequencing are 50 -ACTGGAATGTTTTACCCTGACT30 (forward) and 50 -TGTGACGAAGAGAAAAGGCC-30 (reverse).

Results Comprehensive Mutation Detection

Deletion Mutations in Exons 1 to 10 Detection of heterozygous exonic deletions in exons 1 to 10 does not face challenge, because the degree of homology between the pseudogene and the active gene is relatively low (89% to 97%) (Supplemental Table S1) in these coding regions. Thus, heterozygous deletions in exons 1 to 10 can be confidently detected by analyses of normalized exonic coverage depth31 and further confirmed by MLPA. For example, heterozygous deletions of multiple exons in samples S1 and S2 are readily detected. The deletion of exons 5 to 9 of sample S2 was detected by the reduced normalized coverage depth to half (Figure 2A) and as outliers in Figure 2B based on capture NGS data. The deletion was confirmed by MLPA (Figure 2C). Heterozygous single-exon (E8) deletion in case S3 and homozygous single exon 10 deletion in case S4 (Figure 2D) were also reliably detected by MLPA or capture-based NGS.

Using the workflow described above, we were able to detect sequence variants and CNVs, as well as distinguish the mutations in PMS2 from its pseudogene PMS2CL. Table 1 lists the mutations detected in the positive validation samples and two clinical samples. The detection of point mutations by capture NGS is relatively straightforward. For example, a novel variant c.466A>G (p.T156A) was detected in patient C1 (Table 1) with colon and breast cancer diagnosed at age 52 and a positive family history of cancers. This variant is absent in the Exome Sequencing Project and 1000 Genomes Project. The computer-based algorithms SIFT (http://sift.jcvi.org, last accessed April 29, 2015) and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2, last accessed April 29, 2015) predict p.T156A to be deleterious. Because a pathogenic variant, c.466A>C (p.T156P), at the same amino acid position has been reported in a patient with colorectal cancer and a positive family history,35 the p.T156A variant is interpreted as likely pathogenic according to newly revised American College of Medical Genetics and Genomics standards and guidelines.36

Traditionally, detection of CNVs of the 30 exons 11 to 15 requires combined approaches of MLPA, and LR-PCR followed by Sanger sequencing analysis, because of the highly homologous sequences (99% homology in exons 12 to 15) between PMS2 and PMS2CL. MLPA probes targeted to PSVs of these two genes were used to detect the exonic deletions. By combination of gene-specific sequence analysis of these PSVs, it is possible to differentiate the source of CNVs. For example, our capture/NGS analysis detected a nonsense point mutation, c.65C>A (p.S22*) in sample S6 (Supplemental Figure S1, A and B). Because the patient had childhood-onset hematopoietic malignancies, a clinical phenotype consistent with

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Detection of CNVs at 30 Highly Homologous Region in a Child with Constitutional Mismatch Repair Deficiency Syndrome

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NGS-Based Molecular Diagnosis of PMS2 biallelic PMS2 mutations, a search for the second mutation was indicated and the possibility of an exonic deletion was suspected. Capture-/NGS-based CNV analysis detected a reduced normalized coverage depth of 0.75 of exon 14, suggesting a deletion of one copy (Supplemental Figure S1C). In addition, capture NGS showed a 1 to 2 ratio of A allele to G allele at the PSV c.2324 position (Supplemental Figure S1D). MLPA analysis also identified one copy of A allele (active gene) and two copies of G allele (Supplemental Figure S1D). Sanger sequencing using gene-specific primers for PMS2 and PMS2CL confirmed that allele A is from the active gene and allele G is from the pseudogene (Supplemental Figure S1B). This multistep confirmation strategy is necessary to avoid incorrect calling of the PSVs in clinical practice. Collective results of NGS analysis, CNV detection by NGS and MLPA, PSV ratio, and PCR/Sanger sequencing, determined and confirmed that the exon 14 deletion is from the active gene. Similarly, exon 14 deletion was detected and confirmed in sample S8. Sample S7 has a whole-gene (E1 to E15) deletion, which was detected by MLPA and confirmed by PSV results from gene-specific LR-PCR sequencing of E11 to E15.

Unambiguous Determination of Pathogenic CNVs When PSVs Are Uninformative We designed an approach for unequivocal detection of CNVs at the 30 end of PMS2 without complicated analysis and multistep confirmation of PSVs. LR-PCR using genespecific primers followed by NGS-based coverage analysis provides uniform coverage of all exons and introns in the same LR-PCR fragment of the normal reference sample (Figure 3A). The uniform deep coverage ensures the detection of gross deletions and duplications, especially in the 30 region of PMS2, and the identification of the breakpoints. Figure 3A shows the coverage profiles of the LRPCR products of samples S6, S8, C2 (exon 14 deletion), and S5 (exons 11 and 12 duplication) generated by PMS2 gene-specific primers for E11 to E15 followed by NGS analysis. Deletions or duplications are clearly demonstrated by decreased or increased normalized NGS coverage depth, respectively (Figure 3A). All deletions and duplications were confirmed by agarose gel electrophoresis (Figures 3B and 4D) of the LR-PCR products followed by Sanger sequence analysis of breakpoints. This approach is especially helpful when the exonic deletions cannot be differentiated by traditional MLPA and Sanger sequence analysis because of uninformative PSVs. We have noticed that 62.5% (20 of 32) of clinical samples had at least one exon with one identical PSV between PMS2 and PMS2CL, and 15.6% (5 of 32) of samples had at least one exon with two identical PSVs, similar to a previously reported observation.16 Therefore, PSVs of a specific target region may be uninformative because of frequent sequence exchanges between PMS2 and PMS2CL. Sample C2 was from a 46-year-old Hispanic woman with colon cancer diagnosed at age 38. Her mother had uterine

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cancer at the age of 45 to 50, and her grandmother had ovarian cancer in her 70s. She also had a maternal aunt with pancreatic cancer and a maternal uncle with lung cancer. This family history is highly suggestive of Lynch syndrome. MLPA detected a loss of one copy of exon 14 in PMS2/ PMS2CL. However, sequence analysis of PMS2- and PMS2CL-specific PCR products revealed uninformative PSVs. All three alleles at PSV position c.2324 (exon 14 of PMS2) and corresponding position of PMS2CL have an A genotype, which makes it impossible to distinguish the source of the deletion (Supplemental Figure S2). Agarose gel electrophoresis analysis of the gene-specific LR-PCR products showed that the deletion was in PMS2 and not in PMS2CL (Figure 3B). LR-PCR/NGS coverage depth analysis unequivocally assigned the exon 14 deletion to the active gene, and its breakpoints were determined by NGS and confirmed by Sanger sequencing (Figure 3, C and D).

Characterization of Deletion Breakpoints Uniformity of coverage generated by LR-PCR/NGS makes breakpoint analysis applicable for sample C2 (Figure 3, AeD). Similarly, breakpoints of samples S6 and S8 were also determined (Table 1 and Figure 3A). Further investigation of these three samples with exon 14 deletions revealed similar breakpoints in intron 13 and intron 14. These breakpoints, although not identical, all overlapped with and were located at the 50 end of two Alu sequences (Figure 3, C and E). Thus, the frequent exon 14 deletions are most likely mediated by recombination involving homologous Alu sequences. Several microhomologous regions can be possible at the breakpoints.

Identification of Complex Rearrangement Sample S5 was from a 49-year-old white European female. She had uterine cancer diagnosed at age 27, colon cancer diagnosed at age 43, and café au lait spots. Using the comprehensive approach described above, we identified an intragenic duplication flanking exons 11 to 12 in S5 (Figures 3A and 4). This duplication was homozygous (2 þ 2 copies) instead of heterozygous triplication (1 þ 3 copies) because both parents showed heterozygous duplication (1 þ 2 copies, data not shown) consistent with the phenotype. The 6.5-kb duplication was confirmed by gel electrophoresis of the PMS2 gene-specific LR-PCR product containing exons 11 to 15 (Figure 4D). The breakpoint was determined by NGS of the LR-PCR product to be c.1145-95_c.2174þ1850dup, followed by Sanger sequencing confirmation (Figure 4, AeC). A microhomologous sequence TGTG was identified at the duplication junction. In addition, the 50 duplicated region overlapped with a LINE1 sequence.

Gene Conversion between Active Gene and Pseudogene Results of MLPA and sequence analysis suggested that 20 of 32 cases demonstrated sequence exchange between

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Figure 3 Next-generation sequencing (NGS) coverage analysis of deletion/duplications and breakpoints. A: NGS coverage profiles of reference sample (R), three samples with exon 14 deletion (S6, S8, and C2), and one sample with duplication flanking exons 11 and 12 (S5). B: All deletions of exon 14 are confirmed by long-range PCR and gel electrophoresis. The deletion molecule of PMS2 has a band size of approximately 15 kb (bright band), whereas the nondeleted PMS2 allele and PMS2CL have a band size of approximately 16.7 kb (light band). C: Breakpoints of exon 14 deletion in sample C2 overlap with two Alu sequences. Blue and red arrows indicate the direction of Alu sequences. D: The sequence of breakpoints in patient C2 contains a 12-bp microhomology marked by solid black lines, and breakpoints are marked by red arrows. E: The breakpoints are marked by a red arrow in the alignment of two Alu sequences. The homologous sequenceemediated recombination in patient C2 and S6 is marked by a solid back line, and is marked by a dashed black line in patient S8.

PMS2 and PMS2CL. In 10 cases, PMS2 sequences were introduced to PMS2CL, whereas 6 cases had PMS2CL sequences introduced to PMS2, and 4 cases had reciprocal sequence exchanges. Nevertheless, none of the six healthy controls had sequence flow from PMS2CL to PMS2. The sequence exchange in exons and deep intronic regions revealed by LR-PCR/NGS provided direct evidence for gene conversion between PMS2 and PMS2CL. Whether multiple sequence flow into the intronic regions of the active gene, as

shown in one example in Supplemental Figure S3, would affect transcription or splicing remains to be investigated.

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Discussion Unambiguous Identification of Mutations in PMS2 The main advantage of our current approach is the ability to simultaneously detect point mutations and copy number

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Figure 4 Breakpoint analysis of case S5 with duplication involving exons 11 and 12. A: The sequence of the breakpoints is revealed by the adjacent mismatched sequences from next-generation sequencing. B: The breakpoint is confirmed by Sanger sequencing analysis. C: The primers used in nested PCR are marked with black arrowheads, and primers used for Sanger sequencing are marked with red arrowheads. The dashed box marks the region shown in A and B. D: The duplication is confirmed by long-range PCR followed by gel electrophoresis. F, forward; R, reverse.

changes, as well as unequivocally differentiate PMS2 from its pseudogenes. The results from the three methods serve as cross validation for enhanced accuracy and reduced turnaround time. The novelty of our approach is the incorporation of LRPCR/NGS to simultaneously and unambiguously detect single nucleotide variants and CNVs in PMS2. This approach generates long-range, contiguous uniform coverage of the genespecific LR-PCR fragments containing multiple exons and introns, which is important for CNV differentiation among highly homologous regions. Our approach is especially useful in the detection of 30 deletion/duplication in PMS2. The previous publications11,16,21,24,33,35 using Sanger sequencing only analyzed the coding exons and flanking intron regions individually, which did not provide CNV information. In addition, because of the high degree of sequence identity and frequent exchange between PMS2 and PMS2CL,12e16 the correct interpretation of MLPA ratios depends on PSVs. In particular, if there is a deletion detected by MLPA and PSVs in these exons or flanking intron regions are uninformative, the previously published methods cannot determine the origin of deletion unequivocally as in our clinical case C2, whereas our gene-specific LR-PCR/NGS resolves the case without complicated sequence analysis in the 30 region of PMS2. The advantages of gene specific LR-PCR followed by NGS in the identification of the source gene for mutations and CNVs cannot be overemphasized. This approach allows the detection of sequence and copy number changes in both exonic and intronic regions as well as the determination of breakpoints. One caveat could be that PCR preferentially amplified shorter fragments of the deletion products. Therefore, NGS coverage depth cannot be used to quantify the percentage of deletion molecules.26 However, percentage of copy number changes can be detected by MLPA and

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capture-based NGS. This is exemplified in sample C2 described above in Results. To exclude allele dropout because of single-nucleotide polymorphisms at the PCR primer sites, variant calls from LR-PCR/NGS are compared with those obtained from capture based NGS. Variant allele frequencies from NGS data (capture and LR-PCR) are compared with copy number ratios generated from the MLPA test (Supplemental Table S2). This quality control analysis ensures the clinical accuracy by avoiding falsepositive results. Furthermore, the ability to use the high-quality NGS results for CNV detection has greatly facilitated the molecular diagnoses of PMS2-related diseases.31

Breakpoint Analysis Alu-mediated genomic rearrangements have been previously reported in the DNA mismatch repair genes. MLH1, MSH2, and MSH68 and PMS221 with Alu repeat contents of 33.8%, 19.9%, 33.9%, and 44.5%, respectively; calculated by RepeatMasker. However, although the density is highest at PMS2, genomic deletions in hereditary nonpolyposis colorectal cancer patients are most often detected in MSH2.8,37 This could be due to the extreme difficulties in detecting these events at PMS2 from its highly homologous PMS2CL. With the improved combined LR-PCR NGS, which can provide both PSV and CNV information, and MLPA, it may be worthwhile to re-evaluate prior negative samples, particularly the cases with isolated loss of PMS2 on immunohistochemistry staining. With the ability to simultaneously detect point mutations and CNVs, and to differentiate the active gene from pseudogene by LR-PCR NGS, the diagnostic rate is expected to be further improved. Although the current deletion of exon 14 is seen with high frequency, the homozygous duplication involving exon 11 and

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Li et al 12 is a novel finding. The molecular findings in this patient were consistent with the clinical features of patients with constitutional mismatch repair deficiency syndrome including café au lait spots and very early diagnosis of uterine cancer compared to an average onset at age 59 in heterozygous PMS2 mutation carriers.11 The MLPA analysis detected a duplication of two copies (total of four copies) of exons 11 and 12 in the active gene, which is confirmed by the LR-PCR/NGS, gel electrophoresis of the LR-PCR product, and subsequent parental studies. Although there are numerous reports of gross duplications in other DNA mismatch repair genes such as MLH1 and MSH2 in the HGMD Human Genome Mutation Database (BIOBASE, Waltham, MA), there are only two reports on the duplication of the PMS2 gene in the literature.9,20,24 The first case of PMS2 duplication is a 2-kb insertion into intron 7 that is associated with colorectal, breast, and ovarian cancers at ages of 46, 54, and 59.9 The second case is a duplication involving exons 1 to 12 without being diagnosed with a cancer by age 70. The breakpoint analysis suggests potential LINE1 or microhomology-mediated intragenic duplications. The exact mechanism underlying the intragenic duplication remains to be further investigated. Complex genomic rearrangements in the human genome are being slowly resolved by longer-read sequencing technology such as single-molecule sequencing.38 Our method provides a practical approach for resolving some of the complicated rearrangements until single-molecule sequencing can be routinely used in clinical practice. One advantage of the LR-PCR/NGS is to specifically enrich and sequence large regions containing sequences with high homology in off-target regions, which prevents effective target capture. Allele dropout that is due to single-nucleotide polymorphisms or mutations at primer binding sites can be compensated by the advantage of hybridization capture-based NGS. Detection of biallelic mutations is often challenging because of allele dropout. The combination of LR-PCR/NGS and target capture NGS resolves both allele dropout and homology problems. Thus, this approach would be particularly advantageous in testing patients with early-onset cancers with suspicion of PMS2 mutations. Indeed, questions were raised recently about those PMS2 monoallelic carriers diagnosed with colorectal cancer under the age of 30.39 It would be interesting to see whether our combined approach could uncover a second PMS2 mutation in these PMS2 carriers with very early onset. In conclusion, using the comprehensive approach described here incorporating MLPA, capture-NGS, and gene-specific LR-PCR NGS, we have unequivocally identified a wide spectrum of mutations in all coding exons of the PMS2 gene, including point mutations, exonic deletions, exonic duplications, and a whole-gene deletion, as well as the determination of breakpoints. This approach can be applied to molecular analysis of genes containing multiple copies of highly homologous sequences such as PMS2.

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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2015.04.001.

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