Kim De Leeneer

Jan Hellemans

Joachim De Schrijver

Machteld Baetens

Bruce Poppe

Wim Van Criekinge

Anne Depaepe

Paul Coucke

Ph.D Kathleen B M Claes
email: kathleen.claes@ugent.be

Poppe Bruce

Anne De Paepe

Massive parallel amplicon sequencing

Keywords: massive parallel sequencing, BRCA1/2, multiplex, barcoding, amplicon sequencing

In the case of 454 sequencing, single DNA strands with 5' and 3' adaptor sequences are attached to beads and then clonally amplified by PCR in an oil-water emulsion. The beads are mixed with DNA polymerase and deposited in plates containing more than 1 million wells, with one bead per well. Nucleotides then flow sequentially over the wells and as each nucleotide is added to form complementary DNA strands, pyrophosphate is released and detected in a chemiluminescent flash (pyrosequencing chemistry).

Since the launch of MPS, an increasing number of applications have been published, amongst others de novo sequencing [START_REF] Pearson | The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828)[END_REF][START_REF] Pol | Methanotrophy below pH 1 by a new Verrucomicrobia species[END_REF][START_REF] Velasco | A high quality draft consensus sequence of the genome of a heterozygous grapevine variety[END_REF], whole genome re-sequencing [START_REF] Albert | Direct selection of human genomic loci by microarray hybridization[END_REF][START_REF] Korbel | Paired-end mapping reveals extensive structural variation in the human genome[END_REF], amplicon sequencing (e.g. exon re-sequencing, virus variant detection, DNA methylation) [START_REF] Dahl | Multigene amplification and massively parallel sequencing for cancer mutation discovery[END_REF][START_REF] Korshunova | Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA[END_REF][START_REF] Pettersson | Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads[END_REF][START_REF] Taylor | Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing[END_REF][START_REF] Thomas | Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing[END_REF] , miRNA and splice variant discovery [START_REF] Ruby | Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans[END_REF][START_REF] Yao | Cloning and characterization of microRNAs from wheat (Triticum aestivum L.)[END_REF]. However, the implementation of this high throughput sequencing in molecular diagnostics remains largely unexplored. Over the last decades, Sanger sequencing [START_REF] Sanger | DNA sequencing with chain-terminating inhibitors[END_REF] has been the dominant DNA sequencing technology and the "gold standard" for DNA-based mutation detection. However, due to cost limitations direct sequencing of large genes in diagnostics, is often preceded by a mutation scanning technique, followed by characterization of the variant with Sanger sequencing. Denaturing gradient gel electrophoresis (DGGE) [START_REF] Van Der Hout | A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting[END_REF], denaturing High-performance Liquid chromatography (dHPLC) [START_REF] Liu | Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations[END_REF] and High Resolution Melting Curve Analysis (HRMCA) [START_REF] De Leeneer | Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms[END_REF][START_REF] De Leeneer | Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden[END_REF] are well known examples. For these pre-screening techniques, sensitivities varying from 50-100% and specificities close to 100% were reported [START_REF] Gerhardus | Diagnostic accuracy of methods for the detection of BRCA1 and BRCA2 mutations: a systematic review[END_REF]. Some of these methods are laborious and cannot be fully automated since a sequencing step is required to define the nature of the variant.

We chose the BReast CAncer susceptibility genes BRCA1 (MIM +113705) and BRCA2 (MIM +600185) to optimize high throughput amplicon sequencing, because of their size, polymorphic character and lack of mutation hot spots. These genes require high throughput screening as an increasing number of samples need to be tested within shorter turnaround times. With the approval of targeted therapeutic agents like PARP inhibitors with selective toxicity for tumors derived from germline carriers of mutations in BRCA1&2, expectations for genetic testing keep increasing [START_REF] Curtin | PARP inhibitors for cancer therapy[END_REF]. To obtain a cost efficient MPS strategy, an easy set-up, and uniform distribution of coverage are required to maximize throughput. We explored the challenges of optimizing the clinical use of MPS with two different approaches and compared sensitivity and specificity of MPS with Sanger sequencing and prescreening techniques currently used in molecular diagnostics. For our studies we chose the Roche Genome Sequencer FLX (GS FLX) system because of the longer read lengths generated by this instrument compared to the other 2 major platforms.

DNA samples and sequence variants evaluated

In total 123 DNA samples isolated from blood were evaluated. To evaluate the specificity of our MPS set-up genomic DNA samples from 30 patients previously analysed for the complete coding region of BRCA1/2 with other mutation detection techniques were used. Eleven of these patients were completely Sanger sequenced. Nineteen were analysed with High resolution melting curve analysis (HRMCA) followed by Sanger sequencing of all aberrant melting curves [START_REF] De Leeneer | Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms[END_REF][START_REF] De Leeneer | Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden[END_REF]. For Sanger sequencing and HRMCA identical primers sets were used as for MPS.

Furthermore, 93 samples with previously characterized (Sanger sequencing) deletion or insertion variants were used as positive control samples to validate the detection capacities of MPS. These samples were only sequenced for the amplicon containing the mutation and other amplicons within the specific multiplex set. An overview of all variants evaluated is shown in Supp. Table 1. In total forty-three 1-3 bp deletions, fourteen 1-3 bp insertions (of which 13 duplications), three combined indels and twenty-two deletions and an insertion larger than 3bp were sequenced. Twenty of these deletions and insertions are located in a homopolymeric region longer than 3bp. Furthermore, 410 (40 unique) nucleotide substitutions were present in all samples analyzed.

The majority (406) were frequent SNPs and 4 were nonsense mutations.

PCR set-up

To cover all coding regions and splice sites, we used primer sets thoroughly validated by HRMCA [START_REF] De Leeneer | Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms[END_REF][START_REF] De Leeneer | Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden[END_REF] generated prior to sequencing. To fuse the amplicon-specific primers with the adaptor-MID (Multiplex Identifiers) barcoded primers, 2 consecutive PCR rounds were used with a universal M13 tail as linker (Hellemans J et al, under review). A schematic representation of the principle and workflow of both approaches is shown in Figure 1.

Run 1 & 2

Per sample 111 singleplex PCRs with amplicon specific primers were performed with identical reaction conditions and primers as published before [START_REF] De Leeneer | Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms[END_REF]. PCRs were performed on the CFX384 (Bio-Rad) and RFU data (endpoint fluorescence) were used for subsequent normalization to obtain 12 equimolar pools of PCR products per patient.

Run 3 & 4

Sixteen multiplex reactions were optimised to cover the complete coding and splice site regions of BRCA1 and BRCA2, except BRCA1 exon 2. As this amplicon amplified less efficiently compared to the other PCRs, it was optimized as an individual reaction.

The specifications of each multiplex reaction are given in Supplemental Table 2 &3 94°C for 2 minutes, followed by 12 cycles of denaturation at 94°C for 20 seconds, annealing starting at 60 (58) °C for 20 seconds (decreasing 1°C per cycle) and an extension at 72°C during 1 minute. This initial PCR is followed by 25 additional cycles of denaturation at 94°C for 40 seconds, annealing at 48 (46) °C during 40 seconds and extension at 72°C for 30 seconds. Final extension was accomplished at 72°C for 10 minutes.

Primer concentrations in one multiplex vary between 0.025µM and 0.8µM and were adjusted to obtain equimolar quantities of each amplicon in one reaction (Supp. Table 3).

PCR with MID barcoded primers

MID barcoded primers consist of (Figure 1A): (i) the required sequencing adaptor (A or B), (ii) a 10-nucleotide long MID tag or barcode to identify the patient (MID sequences provided by Roche/454, application note (CRF00104)) (iii) a universal M13-tail (forward primer: cacgacgttgtaaaacgac and reverse primer: caggaaacagctatgacc), identical with the M13 tail used in the first PCR round.

After the initial PCRs (singleplex or multiplex), all samples were diluted 1000 times and 1 µl product was used as a template for a second PCR with MID barcoded primers. Total volume of this reaction was 15µl. The amplification mixture included 1.5 mM MgCl 2 (Invitrogen), 1X PCR buffer (Invitrogen) and 1.5U Platinum Taq (Invitrogen), 3% DMSO (VWR International), 200µM of each dNTP (GE healthcare) and 0.2 µM of both forward and reverse primer. Temperature cycling protocol consists out of following steps: 4 minutes at 94°C, 15 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C during 50 seconds, and final extension at 72° for 10 minutes. PCRs were performed on a CFX384 instrument (Bio-Rad). During optimization FAM labeled MID primers were used to evaluate equimolarity between amplicons within one multiplex reaction and fluorescent peaks were separated on an ABI3730 capillary system.

Sequencing runs and data analysis

PCRs were normalized and equimolarly pooled in relation to the RFU data. This pool was purified on a High Pure PCR Cleanup Micro kit (Roche). Fragment length of this total amplicon pool was evaluated on the Bioanalyzer (Agilent) and compared to the theoretically predicted pattern (Figure 2).

Emulsion PCR and sequencing reactions on the GS-FLX (454-Roche) were performed according to the manufacturer's instructions. The FASTA files were analysed with in house developed variant interpretation pipeline (VIP) software version 1.3 (De Schrijver et al., 2010) and with the commercially available Nextgene software (Softgenetics) version 2.0.

Reads were aligned against GenBank reference sequences, NC_000017.10 (41196313-41277467, complement BRCA1) and NC_000013.10 (32889617-32973809;BRCA2) from build 18 of the Human Genome assembly.

RESULTS

We selected crucial characteristics like uniformity of coverage, sensitivity, specificity and throughput, to evaluate the utility of MPS in diagnostics. Furthermore, MPS should outperform current methods used in molecular diagnostics in terms of cost efficiency to make the implementation worthwhile.

The performance evaluation of the selected approach is based on the results of 4 proof of concept (PoC) experiments (Figure 1). In the first two experiments an identical singleplex approach was used for the analysis of 22 different patient samples.

To reduce workload we started in PoC 3&4 from a multiplex set-up. For PoC4 a strong optimization was performed compared to PoC3: primer concentrations within several multiplex sets were adjusted for amplicons poorly covered in PoC3. Furthermore, the composition of a few multiplexes was changed to obtain more uniform coverage.

Evaluation of uniformity in coverage distribution: singleplex vs. multiplex approach

In a cost efficient test, the full capacity of the GS-FLX instrument is used. This can be achieved by pooling different patients and/or disorders in a single lane. To maximize sample size in a single experiment, a uniform distribution of coverage is required. This means that the difference in number of reads between the "less efficient" amplified fragments and the "best performing" fragments should be as small as possible. We evaluated a singleplex (PoC1&2) and a multiplex approach (PoC3&4). A summary on the number of amplicons sequenced, reads mapped and coverage distribution is shown for each PoC study in Table 1. By calculating the "fold difference to mean coverage", we showed that starting from strongly optimized multiplex sets results in a more uniform distribution of coverage compared to the 3). The "fold difference to mean coverage" can be used as "spread correction factor" to calculate the average coverage one needs to aim for to make sure that also for the less efficiently amplified fragments the minimum required coverage is obtained. The smaller the fold difference to mean coverage, the larger the number of samples that can be pooled in a single experiment, the more cost efficient a test will be.

Considering our best optimized multiplex sets (PoC4) we obtained a 3.16-fold difference to mean coverage for 95% of the amplicons. This value can be used to calculate the average coverage we need to aim for to obtain a predefined minimum coverage for at least 95% of the amplicons (see below).

Multiplex optimization

Optimizing the multiplex sets by fragment analysis using FAM labeled MID primers, turned out to be a good strategy: on average, we found a nearly linear correlation between peak height (=relative fluorescence) on capillary electrophoresis within sets and coverage obtained after 454 sequencing as shown for 2 multiplex sets in Figure 4. The better results for the multiplex approach indicate that the second PCR round attaching the MID barcodes, introduces inequimolarities between the amplicons within the pool of singleplex PCRs. These equimolarities are further increased during the emulsion PCR.

Calculation of the number of samples that can be pooled in a single run

Knowing the fold difference to mean coverage, allows calculating the number of patients that can be analyzed in a single standard GS-FLX run (calculation template available in supplemental files of Hellemans et al. (under review):

F o r P e e r R e v i e w
• With a fold difference to mean coverage of 3.16, the required average coverage to obtain a minimum coverage of 38 = 38*3.16= 120. We opted for a threshold of 38fold coverage based on statistical analyses made by Hellemans et al. who found that 38-fold coverage is required to detect a particular heterozygous variant with a probability of 99.9% when only variants present in at least 25% of the reads are considered as possible true variants. Reasons for the 25% variant frequency: see "Sensitivity".

• A standard GS-FLX run has 400 000 reads available; based on previous runs we found that the number of reads mapped is 95%= 400 000*95% = 380 000

• 111 amplicons are required to cover the complete coding region and splice sites of BRCA1&2 and considering a 5% safety margin to correct for possible run errors and differences in MID amplification efficiencies during sequencing: 111*120 + 5% = 13986 reads/patient are required

• therefore, 380 000/13986= 27 patients can be pooled in a standard GS-FLX run, with a maximum of 5% of the amplicons not meeting the 38x coverage threshold.

• With the Titanium chemistry (1,100,000 reads available), the number of patients can be increased to 74.

Figure 5 shows that for PoC4 on average 4 of 111 amplicons (96.4%) did not meet the 38X-treshold. However, based on the capillary electrophoresis panels, higher coverage was expected for these amplicons, indicating that some fragments are less efficiently amplified by the emulsion PCR or had reduced coverage due to experimental variation.

Primer concentrations in the multiplex reactions can still be increased for BRCA2 11-16, BRCA2 11-18, BRCA2 11-19 and BRCA2 18-1 to improve coverage for these amplicons and may provide a possible solution. All 40 unique substitutions (SNP's, missense, nonsense, splice site mutations) were easily detected. Detection of deletions or insertions is more challenging. MPS analysis software is based on mapping of single stranded reads on a reference sequence; hence, reads lacking one or more nucleotides or containing an insertion will complicate this process. Furthermore, pyrosequencing has its limitations for correct basecalling in homopolymeric regions [START_REF] Huse | Accuracy and quality of massively parallel DNA pyrosequencing[END_REF]. Therefore, we specifically selected 93 insertion-deletion (indel) mutations, of which 20 deletions and insertions were present in homopolymeric tracts longer than 3 bp, to thoroughly evaluate the limitations of the technology.

Of the 93 indels, 2 remained completely undetectable and 1 additional mutation was filtered out due to low quality scores (<30) and was present in less than 25 % of the reads. Table 2 shows an overview of all undetected variants and their flanking sequences. All undetected variants affect homopolymer stretches of 7 nucleotides.

In total 130/133 of all unique variants were detected with the VIP software resulting in a sensitivity of 98% (100% for substitutions). In general, sensitivity of MPS will be higher, since we introduced a sample bias by selecting variants in complex sequence regions. A priori, heterozygote variants are assumed to be present in 50% of the reads and homozygous mutant samples in 100%. On average the heterozygous variants were present in 48.4% (95%CI: 47.7-49.1%; range 27-77%) of the reads. Homozygous mutant variants were found with an average variant frequency of 99.0% (range: 93-100%). Based on these data we conclude that the reference bias in mapping is minimal. To filter out as many false positives as possible the following filters were applied in VIP:

Reference bias

Specificity

1. Min. 38X coverage (cov) is required (Filter 1, cov >38x)

Data analysis with a commercial available software package: Nextgene (Softgenetics)

The performance of our in house developed VIP software package was compared with the commercially available software Nextgene version 2.0 (Softgenetics). This software has intrinsic filters in terms of coverage and frequency of a particular variant being present in the reads. An overall variant score is calculated, which provides an empirical estimation of the likelihood that a given SNP is real and not an artifact of sequencing or alignment. This score is mainly based on the concept of Phred scores where quality scores are logarithmically linked to error probabilities. For example a quality score of 10, gives a chance of 1 out of 10 that the base is incorrectly called. Furthermore, sub scores are integrated in this general score, taking into account the allele frequency, forward and reverse balance and a homopolymeric score, which penalizes indels found in homopolymeric regions. The maximum value for this overall mutation score is 30. Filters and settings were defined based on our data obtained on the analysis of the positive controls, as we did for VIP.

We obtained an overall specificity of 84% (533 false positives in 30 patients), when all the variants present in 25% of the reads at least 38X coverage were taken into account. This specificity can be highly increased by applying an additional filter with the overall variant score.

Alle 93 indel variations and 40 substitutions were detected (100%), when no filter was set on an overall variant score. To improve specificity, we included an overall variant score > 15 requirement (according to software developers recommendations), this resulted in a specificity of 96%, but also in loss of detection of 3 variants present in homopolymeric regions (shown in Table 2). BRCA1 c.1010del was also not detected with the VIP software, the remaining 2 are different compared to those missed with VIP, but they are also present in homopolymeric regions >6. Hellemans et al. found that the vast majority of reads for homopolymers up to the length of 6 can be correctly base called. However, for homopolymers of 7 nucleotides or more, the number of correctly called reads decreases. Therefore, detection of mutations in homopolymer tracts of 7 is challenging in in both programs evaluated.

Non random sequencing errors

Currently Sanger sequencing is considered as the gold standard. Since MPS involves the sequencing of single clones, it can be more vulnerable to errors introduced by polymerases.

We did not use proofreading polymerase enzymes (except for the emulsion PCR). Since we perform 3 PCRs (amplicon specific, MID and emulsion PCR) in our workflow, non specific errors may occur. Performing our assays with proofreading Taq might lead to a reduction of For at least one variant we have strong evidence of a non random sequence error caused by the Taq polymerase: BRCA2 c.9502-44 G>T (exon 26) was found in about 36% (95% CI:30-40% range: 19-61%) of all reads of all patients sequenced for this amplicon with Titanium Taq polymerase. Sanger sequencing of exon 26 and splice sites for all of the patients with Platinum Taq polymerase revealed only the G allele. Sanger sequencing of samples with Titanium Taq polymerase clearly showed the G>T substitution in all samples (Figure 9). These data clearly point at a possible role of the polymerase used for these non-random sequencing errors.

Evaluation specificity of MID barcodes used

No other mutation detection technology allows pooling of several patients in a single lane.

MPS made this possible but the specificity of the MID barcodes used to distinguish individual patients is crucial. MID tags are designed in such a way that 2 sequencing errors may occur, without being defined as another MID. We analyzed the data for an experiment containing only MIDs 1-5 and verified whether reads for MIDs 6-60 were generated. For MID10 approximately 900 reads were mapped (2 mismatches allowed), compared to about 14,000 reads for MIDs included in the experiment. Since these reads are randomly scattered over all amplicons, chances on generating false positives are minimal, but not impossible for amplicons with low coverage. Therefore, MID10 should be excluded when 2 mismatches are allowed for mapping. MID10 is not present in the data when allowing only a single mismatch. Hereby, only a small fraction of the reads (maximum 2000 scattered over all amplicons and patients) are lost and the risk on false positives is strongly reduced. As this is of major importance for reliable analyses in diagnostic settings, this solution is preferred.

Evaluation of multi-exon deletion detection

We evaluated the capacity to detect multi-exon deletions in both approaches. In PoC 2, a patient sample with a heterozygous deletion of BRCA2 exon 1-4 was included and in PoC 4 a heterozygous deletion of BRCA1 exon 18-19 was evaluated. By calculating the dosage quotient (DQ) [START_REF] Goossens | Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing[END_REF], we obtained values not statistically different for the deleted exons compared to the non-deleted exons. To clearly distinguish a deleted exon, DQ values of 0.5 are expected. Normalizing the read counts by the average coverage of the reference patients for these amplicons, resulted in a DQ of 0.35 (BRCA2 exon 2), DQ of 1 (BRCA2 exon 3) and 0.72 (BRCA2 exon 4). For BRCA1 exon 18 and 19, we found a dosage quotient of 0.9 for both exons. Therefore, differences in coverage for the deleted exons were in our experiments not significant and we failed to detect this type of mutations.

DISCUSSION

In this study we evaluated whether massive parallel amplicon sequencing on the GS-FLX is suitable for implementation in a diagnostic setting. We used a multiplex bar coded amplicon sequencing approach for BRCA1 and BRCA2 as an example. For these genes a thoroughly optimized and efficient mutation detection strategy was already previously optimized in our laboratory [START_REF] De Leeneer | Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms[END_REF][START_REF] De Leeneer | Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden[END_REF]. Therefore, we critically evaluated the progress that could be made using amplicon sequencing on the GS-FLX.

Different critical aspects such as uniformity of coverage, sensitivity and specificity and reliability of primers and consumables were considered. Our experiences will be very useful for the optimization of other genes.

The recommended approach for amplicon sequencing of multiple sequences is based on the use of fusion primers which start with an A or B sequence on which the pyrosequencing reaction is initiated, followed by a patient specific barcode (MID) and a target specific sequence at the 3' end. Although this fusion approach is very simple, some impractical issues, in terms of primer management and set-up, will arise when the complexity of the experiment (i.e. number of amplicons and number of patients) increases. Primer costs and workload can be strongly reduced by attaching sequencing adaptors and barcodes to the PCR product by ligation [START_REF] Meyer | Targeted high-throughput sequencing of tagged nucleic acid samples[END_REF] or nested patch PCR [START_REF] Varley | Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes[END_REF]. In our study, a second PCR was used to attach the MIDs and sequencing adaptors to the amplicon. We preferred this approach because of its simplicity, lower cost and workload compared to some other workflows. Multiplexing PCR products allowed to further reduce workload and consumable cost, and to save patient material. We have chosen to multiplex about 10 amplicons in a single set: optimization of such sets can be obtained with a minimum of extra efforts and results in a reduction of the initial workload by tenfold. Higher degrees of multiplexing would require significantly more efforts and would result in a relatively small additional increase in efficiency. Furthermore, it would hamper the evaluation of pools by capillary electrophoresis prior to sequencing, which turned out to be a cost effective optimization method. Our results showed a good correlation between peak height (=relative fluorescence) on capillary electrophoresis within sets and coverage obtained after 454 sequencing, indicating a limited influence of the emulsion PCR. We currently have no other explanation than experimental variation for 4% of the PCR fragments that were less efficiently amplified by the emulsion PCR. A correlation between coverage and amplicon length, GC content or other sequence related characteristics could not be found. Although a homopolymeric tract is present in 2 of these amplicons (BRCA2 11.18 and 11.19), this cannot explain the lower coverage, since the presence of homopolymer stretches did not influence the amplification efficiency of other homopolymer-rich fragments.

By analysis of 30 patient samples for the complete BRCA1/2 coding sequence with the VIP software, we obtained a list of 5513 variants present in at least 10% of the reads, of which only 443 are true variants, leaving 5070 false positives (specificity less than 10%). We are the first group defining filters to cope with MPS sequencing "errors" in a diagnostic setting. With this program we succeeded to obtain an overall specificity of 92%.

Filter 2 (AF >25%) is based on the minimal allele frequency found (27%) by analysis of 93 distinct indel variants and should avoid random PCR errors. The majority of false positives were generated by sequencing of homopolymeric regions, a known complication for pyrosequencing, resulting in undercalls and overcalls in homopolymeric stretches (i.e. one nucleotide missing or one nucleotide added compared to the reference sequence). Filter 3 (Q>30) and 4 (Hp>6) were applied to reduce these numbers, since homopolymeric stretches influenced the Q value assigned to a specific read. These results were compared to those obtained with the commercially available software Nextgene version 2.0. Application of filters 1 and 2 (AF >25% and coverage >38 x), resulted in a specificity of 84%. Using an overall mutation score filter of >15, allowed to increase the specificity to 96% but resulted in loss of detection of some mutations present in homopolymeric regions of >6 nucleotides.

Our results provide evidence that some Taq polymerases introduce non random sequencing errors. For example BRCA2 c.9502-44 G>T was found in almost all the patients analysed for the relevant amplicon when amplified with Titanium Taq polymerase (ClonTech), but not with Platinum Taq polymerase (Invitrogen). Since these errors will be reproducible in every run in almost all patients, they can be considered as true false positives and can be ignored in the long term.

Furthermore, the specificity of the barcodes used to identify patients was evaluated to confirm that false positives could not have been generated by aligning reads to a wrong MID. Despite the fact that no patients with MID10 were included in one of our experiments, reads for MID10 were detected when allowing 2 mismatches for the MIDs. To avoid possible false positives for amplicons with lower coverage we excluded MID10 from further experiments, since 10 misaligned reads for a given amplicon are sufficient to generate a variant with at a frequency of > 25% if only 38 fold coverage is obtained. In future experiments, data will be mapped allowing only a single mismatch as hereby the fraction of reads lost is minimal and misalignments to an incorrect MID will be avoided.

Homopolymeric stretches turned out to be major sources for false positive and false negative variants. For an efficient workflow a high specificity is required and our study shows that the However, it was impossible to define adequate filters in both software programs evaluated without loss of detection of some variants in homopolymeric regions longer than 6 nucleotides. The coding region of BRCA1/2 contains 7 homopolymer regions with 7 and 3 homopolymer regions with 8 nucleotides, in 11 of our amplicons. Until homopolymer analysis improves, these eleven amplicons are currently analysed with HRMCA in our setting, increasing detection capacity to 100% for all mutations evaluated [START_REF] De Leeneer | Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms[END_REF].

Sequencing technologies not based on pyrosequencing may outperform 454 sequencing for detection of variants in homopolymer regions.

Breast cancer diagnostics was earlier this year evaluated by deep sequencing on the GAIIx instrument (Illumina) by Morgan, et al. (starting from long-range PCRs) and by Walsh, et al. (DNA capturing by hybridization in solution to custom-designed cRNA oligonucleotide baits). Both reported detection of all variants/mutations evaluated, but the number of variants evaluated was much smaller and insertions/deletions were maximum 19bp in length. The deletion c.5503_5564del62 evaluated in our study may have remained undetected with this sequencing technology with read lengths of 2 × 76-bp paired-end reads (Walsh, et al.) or 51 bp. (Morgan, et al.). Walsh, et al. did not report any false positives after filtering out variants present in less than 15% of the reads. With an average coverage of 1286 (range 781-1854) a reduction in false positives is indeed expected, however, such high coverage largely increases the cost per sample. We calculated consumable costs and labor time for our MPS approach, and found that our MPS set-up by pooling 74 patients in a single run costs about 345 EUR per sample (consumable cost: 232 EUR), which becomes cost competitive with our HRMCA approach and is much smaller than Sanger sequencing. The current development of commercial user- Turnaround times for genetic testing need to be strongly reduced to meet increasing expectations. Pooling large numbers of patients in a single run will only be useful in a diagnostic setting if different disorders can be pooled in a single run. This requires the development of uniform workflows for different genetic tests.

In some populations, large intragenic founder deletions represent an important fraction of the BRCA1/2 mutation spectrum. Our MPS set-up allowed to detect point mutations with high sensitivity but turned out to be unreliable for the identification of large exon (or multi-exon) deletions. The application of 3 consecutive PCR rounds prior to sequencing most likely explains why deviations from diploidy remained undetected. Additionally we aimed for an average coverage of 120 (to obtain minimally 38), to allow a cost efficient test by pooling of a large number of samples in a single lane. Studies successfully reporting the detection of large intragenic rearrangements worked with much higher read depths, allowing more reliable quantifications of copy numbers [START_REF] Goossens | Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing[END_REF][START_REF] Walsh | Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing[END_REF] For a sensitive analysis an additional technique for the detection of copy number variations needs to be included in the mutation detection strategy.

In conclusion, we developed an efficient workflow for high throughput BRCA1/2 amplicon sequencing. Sensitivity and specificity of MPS amplicon sequencing is high and can be further increased by supplementing MPS assays to overcome issues related to homopolymeric regions. In terms of throughput, diagnostic testing can be highly accelerated and MPS facilitates offering genetic analyses to more at-risk patients. Considering cost efficiency MPS outperforms all other mutation screening techniques, but there is a shift from "wet" lab work Left panel: Theoretical profile of the amplicon length of the total amplicon pool for one patient when all amplicons are equimolarly pooled together. Amplicon length range is 233bp to 437 bp, with an average amplicon length of 335bp.

Right panel: Amplicon length profile obtained with the Bioanalyzer (Agilent) after column purification of the pool. Comparison of the two patterns is used as a quality control prior to emulsion PCR. In total we performed 4 GS-FLX runs. In our first two experiments (green and orange curve), equimolar pooling of simplex PCR reactions was used. The third (blue) and the fourth (purple) experiment were prepared according to the multiplexing protocol.

Panel A: A distribution plot of the coverage is shown. The experiments with the multiplex approach(PoC 3&4) clearly show a more uniform distribution of coverage compared to the pooled simplex runs (PoC 1&2). 38 fold coverage threshold is depicted by the dotted line. Failure rate in PoC 3 was higher because of poor amplification of some multiplex sets.

Panel B: Fold difference to the mean coverage is shown in function of the fraction of amplicons. The dotted lines depict the factor where 90 or 95% fraction of all amplicons is F o r P e e r R e v i e w considered. Our last experiment (PoC4-purple curve) clearly shows the best result where the smallest difference to mean coverage was obtained. 95% of all amplicons are sequenced with a spread correction factor of 3.16 (Table 1) (X-axis is shown in log10 scale).

Figure 4: Correlation between relative fluorescence and coverage within multiplex sets

The correlation between the relative fluorescence seen on capillary electrophoresis and coverage is shown.

Equimolarity of amplicons within a multiplex set was verified on capillary electrophoresis. Coverage for each BRCA1 and BRCA2 amplicon is shown, with the line indicating the threshold of 38 fold coverage. In total 111 amplicons are needed to cover the complete coding region of both genes. In BRCA2, 4 amplicons did on average not reach the 38x coverage threshold; further optimisation for these amplicons is required.

BRCA2_10_4 BRCA1_21 BRCA1_23 BRCA1_10 BRCA1_15_2 BRCA2_27_3 BRCA1_11_1 BRCA1_11_14 BRCA2 9 BRCA1_22 BRCA1_17 BRCA2_14_3 BRCA2_23_2 BRCA1_18 BRCA1_19 BRCA2_27_2 BRCA1_11_12 BRCA2_14_2 BRCA1_3 BRCA1_5 BRCA1_6 BRCA2_18_2 BRCA1_15_1 BRCA1_11_8 BRCA2_11_15 BRCA1 11_2 BRCA2_11_10 BRCA1_24 BRCA2_7 BRCA2_11_12 BRCA2_22_1 BRCA2_15 BRCA1_8 BRCA1_9 BRCA1_11_15 BRCA2_11_3 BRCA2_25_1 BRCA1_11_18 BRCA2_10_3 BRCA2_11_24 BRCA2_22_2 BRCA1_11_4 BRCA2_25_2 BRCA2_10_2 BRCA1_11_6 BRCA1_11_13 BRCA2_11_28 BRCA1_16_1 BRCA1_16_2 BRCA1_13 BRCA2_11_11 BRCA1_12 AMPLICONS BRCA2_11_4 BRCA2_11_21 BRCA1_11_16 BRCA1_11_21 BRCA2_11_17 BRCA2_27_4 BRCA2_20 BRCA2_11_1 BRCA2_10_6 PCR program Touch60 Touch60 Touch58 Touch58 Touch60 Touch58 Touch58 Touch60
Taq polymerase

  high throughput or massive parallel sequencing (MPS) has opened many new research opportunities. Several platforms are released, of which the three major are the Genome Sequencer from Roche/454 Life Sciences, the Genome Analyzer from Illumina/Solexa and the SOLiD System from Applied Biosystems. These platforms differ in several ways, such as the technology applied, read length and the number of DNA molecules sequenced.

  .Multiplex PCR was performed in 20µl total volume. For sets 1,2,5,8-15 the amplification mixture included 2X Titanium buffer (Clontech), 3% DMSO (VWR International), 200µM of each dNTP (GE Healthcare), 1x Titanium Taq Polymerase (Clontech) and approximately 100ng of DNA. In 5 reactions (set 3,4,6,7 and 16), 3 mM MgCl 2 (Invitrogen), 2X PCR buffer (Invitrogen) and 1.5U Platinum Taq polymerase was used instead of Titanium buffer and polymerase. Two touchdown PCR programs were used (abbreviated as Touch46 and Touch48 in Supp.

  (133 distinct) sequence variants, previously identified with Sanger sequencing were evaluated in our sample set (30 patients and 93 control samples) with VIP v1.3. (The sensitivity data obtained with NextGene v2.0 are described below.)

  MPS is more sensitive than Sanger sequencing in terms of random sequencing errors and errors introduced by Taq polymerases, since the technology is based on sequencing of single, clonally amplified molecules. It is challenging to filter out these errors in the data analysis.The VIP software ((version 1.3)(DeSchrijver et al., 2010)) generates a list of variants detected in more than 10% of mapped reads. By analyzing 30 patient samples for the complete BRCA1/2 coding sequence (3330 amplicons), this program generated a list of 5513 variants, of which only 443 are true variants, leaving 5070 false positives. Based on the analysis of the positive controls, criteria were defined to distinguish false positive from true variants. Reads defining a variant have to fulfill all of them, before being accepted as a possible true variant that requires confirmation with Sanger sequencing.

  2. The variant needs to be present in at least 25% of the reads (Filter 2, AF > 25%) 3. At least in 1 direction (forward (F) or reverse (R)) a high quality score is required (Filter 3 Quality (Q) >30) in homopolymer stretches longer than 6 basepairs are non reliable calls (Filter 4, Homopolymer (Hp) >6) An overview of the results is shown in Supplemental Table3 & Figure 6. Application of these filters allowed obtaining a specificity of ~92 %. Specificity for each PoC separately is shown in Figure7. Application of filters 1 and 2 resulted in the largest reduction of false positives: 1432 data points were filtered out because of <38X coverage and another 2628 because of being present in less than 25% of the reads, resulting in a specificity of 70% (filter 1&2). Fine tuning occurred by applying filters 3 and 4 decreasing the remaining 1010 false positives to 276. ln total, 104 distinct false positive variants remained. The recurrence of some false positives can be attributed to the Taq polymerase used or pseudogene amplification (Figure8). 89% (244/276) of the false positives found are indel variations in the close neighborhood of a homopolymer region.

  . Only 11% (32) of all false positives were single nucleotide substitutions, most of them can be explained by random sequencing or PCR artefacts and are only seen in a single direction.

  positives is strongly reduced by the application of predefined filters.

  data analysis, allows MPS to outperform prescreening techniques used in combination with Sanger sequencing. Automation of the workflow will even further decrease the workload.

Figure 1 :

 1 FIGURE LEGENDS

Figure 2 :

 2 Figure 2: Comparison of fragment length of the total amplicon pool in theory and in practice.

Figure 3 :

 3 Figure 3: Evolution of distribution of coverage uniformity in the 4 PoC experiments.

Figure 5 :

 5 Figure 5: Distribution of coverage for each amplicon in BRCA1/2 (PoC 4)

Figure 6 :

 6 Figure 6: Overview of data generated Data were analysed with an in house developed software program (VIP). Combined forward and reverse allele frequencies are plotted against coverage. Variants detected in 30 patients and 93 positive control samples are shown. Grey data points are the false positives filtered out when filters are applied. Green data points are true variants (503). The green outlier at almost 80% AF is deletion of 62 nucleotides, probably preferential amplification of the shorter allele has occurred. 80% of the true variants have an allele frequency in the 40-60 range. Red data points are the remaining false positives (276).

Figure 7 :

 7 Figure 7: Specificity of 4 PoC studies analysed with VIP software

Figure 8 :

 8 Figure 8: Pseudogene amplification by MPS

Figure 9 :

 9 Figure 9: Sanger sequencing results for BRCA2 c.9502-44G>T

  and Sanger sequencing. In run 1&2 we started from equimolar pools of singleplex PCRs, in run 3&4 multiplex PCRs were

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 )

 2 . The temperature cycling protocol consists of an initial denaturation step at

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	7

Table 1 :

 1 Overview of characteristics of the 4 PoC experiments

	Singleplex pooling	Multiplex pooling
	PoC 1	PoC 2	PoC 3	PoC 4

Table 2 : Examples of variants analysed in homopolymeric regions ≥5

 2

					Page 40 of 54
	Variant (c.)	flanking sequences	Change of homopolymer length coverage of	Quality VF (%)
				nucleotide
	esults with VIP software
	ndetected variants				F
	BRCA1 c.1016dup	ACTCCCAGCACAGAAAAAAAAGGTAGATCTGAATGCTGATC	7→8	38	n.a	<10%
	BRCA2 c.994del	CTAGMAAGACTAGGAAAAAAATTTTCCATGARGCAAACGCT	7→6	219	n.a	<10%
	BRCA1 c.1010del	ACTCCCAGCACAGAAAAAAAGGTAGATCTGAATGCTGATCC	7→6	39	25	23%
	etected variants in homopolymeric regions
	BRCA1 c.3329dup	ATCCTGAAATAAAAAAAGCAAGAATATGAAGAAGTAGTTC	6→7	99	31	47%
	BRCA2 c.5577_5580del	ACATGAAACAATTAAAAAAGTGAAAGACATATTTACAGAC	4→6	105	34	50%
	BRCA1 c.2989_2990dup	AAAACTAAATGTAAGAAAAAAATCTGCTAGAGGAAAACTTT	5→7	167	32	40%
	esults with Nextgene sofware
	xamples of variants with low mutation score			Mutation
					score
	RCA2 c.6351dup	GAAGATCAAAAAAAACACTAGTTTT	7→8	221	10	33%
	RCA1 c.1010del	ACTCCCAGCACAGAAAAAAAGGTAGATCTGAATGCTGATCC	7→6	20	12	50%
	RCA1 c.1961delA	AGAGATAAAGAAAAAAAAGTACAACCAAA	8→7	76	11	52%

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	Page 47 of 54 Page 49 of 54 Page 51 of 54				Human Mutation Human Mutation Human Mutation Human Mutation Human Mutation Human Mutation Human Mutation							Page 46 of 54 Page 48 of 54 Page 50 of 54 Page 52 of 54
	BRCA2_23_1 0.016 µM BRCA2_11_4 0.004 µM BRCA1_15_2 0.008 µM BRCA1_11_5 0.008 µM BRCA2_21 Total 553	BRCA2_11_22 BRCA2_11_26 BRCA2_10_7 0.016 µM BRCA1_11_16 BRCA2_19 0.007 µM BRCA2_27_3 0.024 µM BRCA2_10_6 0.008 µM BRCA2_11_9 0.008 µM BRCA2_2 144 42 29 3	3	BRCA2_11_19 BRCA2_11_25
	BRCA2_10_5	BRCA2_11_8			BRCA2_14_1			BRCA2_11_5	BRCA1_11_3
	BRCA1_11_9 0.024 µM BRCA2_11_21 0.004 µM BRCA2_23_2 0.008 µM BRCA2_11_26 0.008 µM BRCA2_13	BRCA2_5/6			0.002 µM BRCA1_18 0.016 µM BRCA2_10_7 0.008 µM BRCA1_7			BRCA2_24
	0.004 µM BRCA1_6 SET 9 0.01 µM BRCA2_11_27				0.003 µM BRCA2_18_2 SET 10 0.008 µM BRCA2_14_1 0.008 µM BRCA2_4			BRCA2_11_16
	F o r P e e r R e v i e w Touch60 Touch60 Touch60 Touch60 Touch58 Titanium Taq Titanium Taq Titanium Taq Titanium Taq Titanium Taq Supp T3: Primer concentrations in all multiplex reactions PCR program Touch60 Taq polymerase Titanium Taq SET 1 SET 2 concentration primers/amplicon concentration primers/amplicon 0.005 µM BRCA2_10_4 0.009 µM BRCA1_21 0.002 µM BRCA2 9 0.001 µM BRCA1_22 0.004 µM BRCA1_11_12 0.007 µM BRCA2_14_2 F o r P e e r R e v i e w SET 3 SET 4 concentration primers/amplicon concentration primers/amplicon 0.003 µM BRCA1_23 0.006 µM BRCA1_10 0.004 µM BRCA1_17 0.011 µM BRCA2_14_3 0.008 µM BRCA1_3 0.032 µM BRCA1_5 0.008 µM BRCA2_11_10 0.003 µM BRCA1_24 0.008 µM BRCA1_11_15 0.012 µM BRCA2_11_3 0.008 µM BRCA2_25_2 0.024 µM BRCA2_10_2 0.012 µM BRCA1_13 0.024 µM BRCA2_11_11 0.008 µM BRCA1_11_21 F o r P e e r R e v i e w 0.016 µM BRCA2_7 0.008 µM BRCA2_11_12 0.003 µM BRCA2_25_1 0.008 µM BRCA1_11_18 0.024 µM BRCA1_11_6 SET 7 SET 8 concentration primers/amplicon concentration primers/amplicon 0.016 µM BRCA1_11_1 0.007 µM BRCA1_11_14 0.004 µM BRCA1_19 0.008 µM BRCA2_27_2 0.008 µM BRCA1_15_1 0.008 µM BRCA1_11_8 0.008 µM BRCA2_22_1 0.005 µM BRCA2_15 F o r P e e r R e v i e w concentration primers/amplicon concentration primers/amplicon 0.006 µM BRCA1_11_17 0.006 µM BRCA1_11_20 0.008 µM BRCA2_27_5 0.002 µM BRCA1_14 0.003 µM BRCA1_20 0.008 µM BRCA2_11_13 0.008 µM BRCA2_11_14 0.014 µM BRCA1_11_10 0.008 µM BRCA2_23_1 0.024 µM BRCA2_11_22 0.024 µM BRCA2_10_5 0.024 µM BRCA2_11_8 0.008 µM BRCA1_11_9 0.024 µM BRCA2_5en6 SET 11 SET 12 F o r P e e r R e v i e w SET 13 SET 14 concentration primers/amplicon concentration primers/amplicon 0.002 µM BRCA2_18_1 0.008 µM BRCA2_11_6 0.024 µM BRCA2_17 0.016 µM BRCA2_11_20 0.004 µM BRCA2_3 0.008 µM BRCA1_11_19 0.024 µM BRCA2_16 0.016 µM BRCA2_26 0.008 µM BRCA2_19 0.016 µM BRCA2_11_19 0.008 µM BRCA2_11_5 0.016 µM BRCA2_24 F o r P e e r R e v i e w 0.01 µM BRCA1_11_11 0.024 µM BRCA2_11_23 0.008 µM BRCA2_11_25 0.016 µM BRCA1_11_3 Supp T4A: Summary of sensitivity data Touch60 Touch58 Titanium Taq PlatinumTaq (VIP) Sensitivity Detected variants False negatives (coverage) True False negatives # unique # unique PoC 1 146 30 16 14 0 0 PoC 2 160 40 12 5 1 1 PoC 3 43 29 12 8 0 0 PoC 4 204 115 2 2 2 2 F o r P e e r R e v i e w Supp T4B: Summary of specificity data (VIP)
	Titanium Taq 0.016 µM BRCA2_11_15 0.016 µM BRCA2_10_3 concentration primers/amplicon raw data	Titanium Taq Filter 1 (coverage > 38) PlatinumTaq 0.002 µM BRCA1 11_2 PlatinumTaq Titanium Taq 0.024 µM BRCA2_11_17 0.004 µM BRCA2_11_24 concentration primers/amplicon Specificity (false positives) 0.024 µM BRCA2_11_16 Filter 2 (AF >25%) PlatinumTaq	PlatinumTaq Filter 3 (Q>30) Titanium Taq Filter 4 (Hp>7)
	SET 9 0.016 µM BRCA1_8 0.008 µM BRCA1_11_13 0.006 µM BRCA2_8 # unique PoC 1 1145 205	SET 10	# 1040	SET 11	SET 12 0.024 µM BRCA1_9 0.016 µM BRCA2_11_28 SET 13 unique 0.016 µM BRCA2_12 168	# 382	SET 14	unique 48	SET 15 # 96	unique SET 16 # 27 43	unique 14
	BRCA1_11_17 BRCA1_11_20 BRCA2_8 BRCA2_27_5 BRCA1_14 BRCA2_10_1 BRCA1_20 BRCA2_11_13 BRCA2_11_7 0.016 µM BRCA2_22_2 0.004 µM BRCA1_11_4 BRCA2_12 BRCA2_18_1 BRCA2_11_6 BRCA1_11_7 BRCA2_17 BRCA2_11_20 BRCA2_13 BRCA2_21 BRCA2_27_1 BRCA2_3 BRCA1_11_19 BRCA2_11_27 BRCA2_4 BRCA2_2 BRCA1_7 0.008 µM BRCA1_16_1 0.008 µM BRCA1_16_2 SET 5 SET 6 concentration primers/amplicon concentration primers/amplicon 0.008 µM BRCA1_12 0.024 µM BRCA2_20 0.032 µM BRCA2_27_4 0.016 µM BRCA2_11_1 0.024 µM BRCA2_10_1 0.016 µM BRCA1_11_7 0.008 µM BRCA2_11_7 0.008 µM BRCA2_27_1 SET 15 SET 16 concentration primers/amplicon concentration primers/amplicon PoC 2 2948 693 1955 474 444 112 165 59 PoC 3 764 377 480 248 145 76 74 36 PoC 4 213 177 163 140 39 47 13 19 AMPLICONS BRCA2_11_14 BRCA1_11_10 BRCA1_11_5 BRCA2_11_9 BRCA2_16 BRCA2_26 BRCA1_11_11 BRCA2_11_23 161 61 11 Total 5070 1452 3638 1030 1010 283 348 141 276	55 28 7 104
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John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

This project was realized with the funding of an Emmanuel van der Schueren scholarship of the Flemish foundation against cancer to Kim De Leeneer. This research was supported by grant 1.5.150.07 from the Fund for Scientific Research Flanders (FWO) to Kathleen Claes and by GOA grant BOF10/GOA/019 (Ghent University). Bruce Poppe is a senior clinical investigator from FWO. This study was supported by a StepStone grant from the Industrial Research Fund from Ghent University; the Roche 454 GS-FLX instrument is part of the NXTGNT infrastructure, funded by a Hercules grant (middle heavy infrastructure). NXTGNT (initiated and supervised by Sofie Bekaert, Jo Vandesompele, Dieter Deforce, Philippe van Nieuwerburgh, Wim Van Criekinge, Jan Hellemans, Paul Coucke) is a genome analysis platform from Ghent University.

John Wiley & Sons, Inc. Human Mutation F o r P e e r R e v i e w c.1292dupT (1411insT) p.Leu431fs Mutation c.1319delT (1438delT) p.Leu440fs Mutation c.1504_1508del5 (1623del5) p.Leu502fs Mutation c.1881_1884del4 (2000del4) p.Val627fs Mutation c.1961delA (2080delA) p.Lys654fs Mutation c.2019delA (2138delA) p.Glu673fs Mutation c.2197del5 (2316del5) p.Glu733fs Mutation c.2210delC (2329delC) p.Thr737fs Mutation c.2212_2215del4 (2331del4) p.Val738fs Mutation c.232delA (351delA) p.Arg78fs Mutation c.2380dupG (2478insG) p.Glu787fs Mutation c.2405_2406delTG (2524delTG) p.Val802fs Mutation c.2646_2648delTGC (2765delTGC) p.Cys882del Mutation c.2685_2686delAA (2804delAA) p.Gln895fs Mutation c.2689insA (2809insA) p.Pro897fs Mutation c.2726delA (2845delA) p.Asn909fs Mutation c.2727_2730del4 (2846del4) p.Asn909fs Mutation c.2728delC (2847delC) p.Gln910fs Mutation c.2764_2767del4 (2883delACAG) p.Thr922fs Mutation c.2934T>G (3053T>G) p.Tyr978X Mutation c.2989_2990dupA (3109insAA) p.Asn997fs Mutation c.3329dupA (3448insA) p.Lys1110fs Mutation c.3481_3491del11 (3600del11) p.Glu1161fs Mutation c.3485delA (3604delA) p.Asp1162fs Mutation c.3494_3495delTT (3613-3614delTT) p.Phe1169fs Mutation c.3549AG>T (3668AG>T) p.Lys1183fs Mutation c.3756_3759del4 (3875del4) p.Leu1252fs Mutation c.3770_3771delAG (3889delAG) p.Glu1257fs Mutation c.3820dupG (3939insG) p.Val1274fs Mutation c.3891_3893delTTC (4010delTTC) p.Ser1297fs Mutation


Kathleen ; Blondeau

Veerle Boecxstaens

Nathalie Rommel

Jan Tack

Kuleuven

pathophysiology, differential diagnosis and management of rumination syndrome

Keywords: Functional GI diseases < Disease-based, Oesophagus < Organbased, Diagnostic tests < Topics, Motility rumination, high resolution manometry, esophageal impedance monitoring, behavioral therapy, baclofen

The review was conducted on the basis of a medline search to identify relevant publications pertaining to the pathophysiology, clinical diagnosis and management of rumination syndrome. Results: The Rome III consensus established diagnostic criteria for rumination syndrome in adults, children and infants. A typical history can be highly suggestive but esophageal (high resolution) manometry/impedance with ingestion of a meal may help to distinguish rumination syndrome from other belching/regurgitation disorders. The pathophysiology is incompletely understood, but involves a rise in intragastric pressure, generated by a voluntary, but often unintentional, contraction of the abdominal wall musculature, at a time of low pressure in the lower esophageal sphincter, causing retrograde movement of gastric contents into the esophagus. To date, controlled trials in the treatment rumination syndrome are lacking. The mainstay of treatment for rumination syndrome is explanation and behavioral treatment which consists of habit reversal techniques that compete with the urge to regurgitate. Chewing gum, prokinetics, baclofen and even anti-reflux surgery have been proposed as adjunctive therapies, but high quality studies are generally lacking. Conclusion: The rumination is an under-recognized condition with incompletely understood pathophysiology. Behavioral therapy seems effective, but controlled treatment trials are lacking.

Introduction

Rumination is a phenomenon that occurs in animals such as sheep, cattle, and goats, with compartmentalized stomachs consisting of multiple chambers. [START_REF] Winship | Esophagus in rumination[END_REF] In these animals, food moves back into the mouth from the proximal two chambers of the stomach by retrograde peristalsis which is coordinated with a relaxation of the lower esophageal sphincter. The animals then re-chew and re-swallow the regurgitated food, and this aids in the digestion and absorption of food by reducing particle size and enhancing mechanical degradation [START_REF] Winship | Esophagus in rumination[END_REF].

Rumination syndrome in humans refers to a clinical entity which is characterized by the effortless, often repetitive, regurgitation of recently ingested food into the mouth [START_REF] Levine | Habitual rumination: a benign disorder[END_REF][START_REF] Malcolm | Rumination syndrome[END_REF].

The regurgitated material can be chewed and swallowed again, or is spat out. Typically, the regurgitation in rumination is not preceded by nausea or retching. Rumination syndrome was originally described in children and in developmentally disabled (4-7), but it is now well-recognized that rumination syndrome occurs in patients of all ages and cognitive abilities, the majority of them female [START_REF] Levine | Habitual rumination: a benign disorder[END_REF][START_REF] Malcolm | Rumination syndrome[END_REF][START_REF] Ball | A special feeding technique for chronic regurgitation[END_REF][START_REF] Rogers | Chronic regurgitation among persons with mental retardation: A need for combined medical and interdisciplinary strategies[END_REF][START_REF] Fredericks | Overview of the treatment of rumination disorder for adults in a residential setting[END_REF][START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] O'brien | The rumination syndrome: clinical features rather than manometric diagnosis[END_REF][START_REF] Khan | Rumination syndrome in adolescents[END_REF].

Clinically, rumination syndrome is probably under-diagnosed, and adults with rumination may be erroneously considered to have (refractory) vomiting secondary to gastroparesis or regurgitation due to gastroesophageal reflux disease. Lack of awareness amongst physicians probably contribute to missed diagnoses of rumination syndrome, which should be considered in the differential diagnosis of all patients presenting with "regurgitation" and "vomiting", especially when they occur postprandially and are associated with weight loss [START_REF] Quigley | AGA technical review on nausea and vomiting[END_REF][START_REF] Tack | Functional Gastroduodenal Disorders[END_REF]. Poor understanding of the syndrome by physicians may also reduce the effectiveness of therapeutic interventions in patients with this complex disorder.

Methods

To identify relevant studies, both computerized (Medline) and manual searches were performed, using the cited references of the retrieved articles. MeSH and free-text terms for rumination syndrome were used alone or combination with the terms pathogenesis, pathophysiology, pathogenesis, epidemiology, treatment and management for searches conducted for the period up to November 2010.

Epidemiology

The epidemiology of rumination syndrome in the adult general population remains to be carefully defined. Reliable data on the prevalence of the disorder are lacking, and data from clinical practice are probably inadequate for this underdiagnosed condition which is often not considered in the differential diagnosis of refractory vomiting or regurgitation.

Based on the case numbers seen at referral centers, rumination syndrome is probably uncommon [START_REF] Fredericks | Overview of the treatment of rumination disorder for adults in a residential setting[END_REF][START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] O'brien | The rumination syndrome: clinical features rather than manometric diagnosis[END_REF][START_REF] Khan | Rumination syndrome in adolescents[END_REF][START_REF] Quigley | AGA technical review on nausea and vomiting[END_REF][START_REF] Tack | Functional Gastroduodenal Disorders[END_REF].

Diagnostic Criteria

According to the Rome I and II consensus, rumination syndrome in adults was defined as effortless regurgitation of recently ingested food into the mouth with subsequent remastication and reswallowing or spitting out, in the absence of structural disease.

Furthermore, the Rome II definition stated that the regurgitation is effortless, not associated with abdominal discomfort, heartburn, or nausea, and can occurs in the postprandial period to cease when the food taste becomes acidic [START_REF] Richter | Functional esophageal disorders[END_REF][START_REF] Clouse | Functional esophageal disorders[END_REF].

Rumination is well described in infants with the typical age of onset between three and six months. The Rome III committee based adult diagnostic criteria on the pediatric criteria and on the Rome II definitions. The definition was adapted to stress the absence of retching and nausea in most cases, as well as the time frame of 3 months for all functional disorders [START_REF] Quigley | AGA technical review on nausea and vomiting[END_REF]. The Rome III definition for rumination syndrome in adults is summarized in Table 1 [START_REF] Tack | Functional gastroduodenal disorders[END_REF]. The definition for rumination in childhood and adolescence is closely related [START_REF] Rasquin | Childhood functional gastrointestinal disorders: child/adolescent[END_REF], but includes failure to respond to anti-reflux therapy. In infants an adapted definition has been proposed [START_REF] Hyman | Childhood functional gastrointestinal disorders: neonate/toddler[END_REF]. It is often stated that rumination syndrome can easily be diagnosed by history alone [START_REF] Tack | Functional Gastroduodenal Disorders[END_REF][START_REF] Clouse | Functional esophageal disorders[END_REF]. Typical elements are the start of regurgitation during or immediately after the meal, the effortless nature, the absence of prodromal nausea and especially the ability to swallow the regurgitated material. In typical cases, repetitive regurgitation of gastric contents starts within minutes, often already during the meal, and usually persists for 1 up to 2 hours. To the subject, the regurgitated material is recognized as food, and often has a pleasant taste [START_REF] O'brien | The rumination syndrome: clinical features rather than manometric diagnosis[END_REF]. The regurgitation is effortless or may be preceded by an immediately preceding sensation of belching. These features may help to distinguish rumination from vomiting, although history alone is not always accurate [START_REF] Rommel | Rumination or belching-regurgitation? Differential diagnosis using oesophageal impedancemanometry[END_REF]. In case of a sufficiently convincing history, a firm diagnosis of rumination can be made, the nature of the disorder can be explained to the patient [START_REF] Malcolm | Rumination syndrome[END_REF][START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] O'brien | The rumination syndrome: clinical features rather than manometric diagnosis[END_REF][START_REF] Tack | Functional Gastroduodenal Disorders[END_REF][START_REF] Tack | Functional gastroduodenal disorders[END_REF], and treatment can promptly be initiated (see below). In some cases, there may still be diagnostic uncertainty, or the patient may be reluctant to accept this simple diagnosis [START_REF] Rommel | Rumination or belching-regurgitation? Differential diagnosis using oesophageal impedancemanometry[END_REF].

Clinical Evaluation and diagnostic approach

In spite of the often typical presentation, a diagnosis of rumination is often delayed in clinical practice, and patients may initially be misdiagnosed as having gastroesophageal reflux disease (GERD) or dyspepsia/gastroparesis with vomiting [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] Chial | A twenty-one-year-old college student with postprandial regurgitation and weight loss[END_REF].

Distinguishing rumination from regurgitation as a part of the typical GERD spectrum is sometimes difficult, especially if some heartburn is or was present and when the patient has already been treated with a proton pump inhibitor. Regurgitation, like rumination, occurs mainly postprandially, is effortless, may be swallowed again and is not preceded by nausea. Furthermore, endoscopy may reveal low-grade esophagitis, a consequence of caustic gastric contents that are brought up during rumination events, which may further complicate the diagnostic picture [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF]. Ambulatory esophageal pH/impedance monitoring may actually be suggestive of reflux disease in these patients, by showing a high number of reflux events and an elevated percentage of time with intra-esophageal pH below 4 [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] Oelschlager | Effective treatment of rumination with Nissen fundoplication[END_REF]. Upon detailed examination, the traces show a high number of repetitive events in the first postprandial hour, a busy erratic pattern of back-and forthmovement by regurgitation and re-swallowing, and absence of events at night (Figure 1). Furthermore, when studied off acid suppression, there is often a discrepancy between the high number of reflux events and the relatively modest percentage of time that the esophageal pH is below 4, due to food buffering of gastric acidity during the postprandial period when repetitive regurgitation occurs [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF].

In the majority of patients, additional symptoms such as nausea, heartburn, abdominal discomfort, diarrhea and/or constipation are also present [START_REF] Winship | Esophagus in rumination[END_REF][START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] Tack | Functional gastroduodenal disorders[END_REF][START_REF] Chial | A twenty-one-year-old college student with postprandial regurgitation and weight loss[END_REF]. Weight loss can also be a prominent feature of rumination syndrome, particularly in the adolescent population. [START_REF] Malcolm | Rumination syndrome[END_REF][START_REF] Whitehead | Rumination syndrome in children treated by increased holding[END_REF][START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] Tack | Functional gastroduodenal disorders[END_REF][START_REF] Chial | A twenty-one-year-old college student with postprandial regurgitation and weight loss[END_REF]. These features make distinguishing rumination from gastroparesis less obvious in certain cases, but in gastroparesis there is preceding nausea and often retching, and vomiting occurs typically late postprandially, and the food is no longer recognizable as such by taste. While vomiting may be intermittent, or dependent on the meal, rumination seems to occur invariably with every meal, and often also occurs after ingestion of only liquids. Occurrence of weight loss in young female adults may also lead to misdiagnosing as bulimia and/or anorexia nervosa. [START_REF] Eckern | The relationship between rumination and eating disorders[END_REF][START_REF] Larocca | Rumination: its significance in adults with bulimia nervosa[END_REF][START_REF] Fairburn | Rumination in bulimia[END_REF]. The manometric pattern associated with rumination has been described already a long time ago in the literature [START_REF] O'brien | The rumination syndrome: clinical features rather than manometric diagnosis[END_REF][START_REF] Tutuian | Rumination documented by using combined multichannel intraluminal impedance and manometry[END_REF]. More recently, the yield of adding stationary intraluminal impedance was evaluated. Performing stationary esophageal manometry and impedance with administration of a meal and monitoring of postprandial regurgitation events refined the diagnosis and was able to more accurately distinguish between rumination, regurgitation and belching [START_REF] Rommel | Rumination or belching-regurgitation? Differential diagnosis using oesophageal impedancemanometry[END_REF]. In the rumination events, a rise in intra-gastric pressure as assessed by manometry preceded or occurred simultaneously with retrograde intra-esophageal flow as detected by impedance (21) (Figure 2). More recently, the combined use of manometry, high resolution manometry and stationary esophageal impedance in the evaluation of rumination and regurgitation disorders was evaluated, and the combination of high resolution manometry with stationary impedance monitoring had the highest diagnostic accuracy and was able to detect some unusual patterns (Figure 3), including combinations of esophageal belching and rumination [START_REF] Blondeau | Added value of combined high resolution manometry-impedance recordings in patients with belching and/or postprandial regurgitation[END_REF].

Pathophysiology

Rumination is characterized by a rise in intra-gastric pressure which precedes or coincides with retrograde movement of gastric contents into the esophagus [START_REF] Rommel | Rumination or belching-regurgitation? Differential diagnosis using oesophageal impedancemanometry[END_REF][START_REF] Tutuian | Rumination documented by using combined multichannel intraluminal impedance and manometry[END_REF]. The rise in intra-gastric pressure is generated by a voluntary, but often not intentional, contraction of the abdominal wall musculature. In addition, it has been suggested that a [START_REF] Khan | Rumination syndrome in adolescents[END_REF][START_REF] Chitkara | Teaching diaphragmatic breathing for rumination syndrome[END_REF]. The prerequisite for upward movement of gastric contents during straining is a lower esophageal sphincter (LES) pressure which is lower than the generated intragastric pressure. The reasons for this low LES pressure could be a prolonged low LES pressure postprandially, or a temporary lowering during transient LES relaxations (TLESRs) which are sensed by the subject. Indeed, it has been suggested that TLESRs at the time of belching are sensed by the patients and used to strain and bring up gastric content [START_REF] Shay | Rumination, heartburn, and daytime gastroesophageal reflux. A case study with mechanisms defined and successfully treated with biofeedback therapy[END_REF]. Alternatively, an exaggerated response to gastric filling has been proposed to underlie transient drops in LES pressure in rumination. Thumshirn et al demonstrated that patients of normal intelligence with rumination syndrome required significantly lower fundic pressures to induce LES relaxation and had increased sensitivity to gastric balloon distention compared to healthy controls [START_REF] Thumshirn | Gastric mechanosensory and lower esophageal sphincter function in rumination syndrome[END_REF]. The authors suggest that in these patients, the pressure of food within the fundus results in reflex inhibition of lower esophageal pressure leading to the induction of a modified belch reflex. A third hypothesis is a learned, voluntary relaxation of the diaphragmatic crura that allows the normal postprandial increase in intragastric pressure to overcome the resistance to retrograde flow provided by the lower esophageal sphincter [START_REF] Smout | Voluntary induction of transient lower esophageal sphincter relaxations in an adult patient with the rumination syndrome[END_REF].

The reason for the straining is unclear, and both psychological factors (see below) as well as attempts to relieve dyspeptic symptoms with belching leading to a rumination behavior have been suggested [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF].

Psychological Features

In some patients, stressful life events can be identified around the period of symptom onset [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF]. However, in most cases, rumination occurs in the absence of such identifiable eliciting factors. Associations between rumination and bulimia nervosa have also been described. Studies reported rumination in 20% of bulimics, and a history of bulimia in 17% of females with rumination [START_REF] O'brien | The rumination syndrome: clinical features rather than manometric diagnosis[END_REF][START_REF] Fairburn | Rumination in bulimia[END_REF]. However, ruminators with bulimia tend to expel rather than to re-swallow their food, and they may also exhibit self-induced vomiting by digital stimulation of the hypopharynx. It has been suggested that rumination in this group is a learned behavior, aimed at controlling body weight without induction of frank vomiting, and therefore may be considered more a variant of an eating disorder. Because of the underlying aim of weight loss, treatment of rumination in bulimics has been reported to be less successful.

TREATMENT

To date, controlled trials in the treatment rumination syndrome are lacking, and the available evidence consists mainly of case series. Rumination is often misdiagnosed as GERD with regurgitation, and proton pump inhibitors (PPIs) are often started. In rumination syndrome, PPIs may provide some benefit by suppressing the symptom of heartburn and by better protect the esophageal mucosa. On the other hand, acid suppression may paradoxically prolong rumination behavior after a meal, which usually ceases when the food in the stomach becomes acidic. Hence, the therapeutic benefit of PPIs is limited and they are at best and adjuvant while more definitive therapy is instituted.

The mainstay of treatment for rumination syndrome is explanation of the condition, the mechanism underlying the rumination events, and behavioral modification. Behavioral treatment consists of learning and using habit reversal techniques, usually diaphragmatic breathing, which compete with the urge to regurgitate [START_REF] Chitkara | Teaching diaphragmatic breathing for rumination syndrome[END_REF][START_REF] Shay | Rumination, heartburn, and daytime gastroesophageal reflux. A case study with mechanisms defined and successfully treated with biofeedback therapy[END_REF][START_REF] Johnson | Behavioral assessment and treatment of postprandial regurgitation[END_REF][START_REF] Prather | An open trial of cognitive behavioral intervention in the treatment of rumination syndrome[END_REF][START_REF] Wagaman | Behavioral intervention for the treatment of rumination[END_REF]. The application of diaphragmatic breathing during the postprandial period effectively eliminates rumination activity [START_REF] Chitkara | Teaching diaphragmatic breathing for rumination syndrome[END_REF]. Diaphragmatic breathing can easily be learned by putting a hand on the chest and on the abdomen during respiration, and instructing that only the hand on the abdomen may move with breathing [START_REF] Chitkara | Teaching diaphragmatic breathing for rumination syndrome[END_REF]. Case series report disappearance of rumination behavior in 30-66% and improvement in another 20-55% [START_REF] Chial | Rumination syndrome in children and adolescents: diagnosis, treatment, and prognosis[END_REF][START_REF] Prather | An open trial of cognitive behavioral intervention in the treatment of rumination syndrome[END_REF][START_REF] Soykan | The rumination syndrome: clinical and manometric profile, therapy, and long-term outcome[END_REF]. Alternatively, chewing gum has been proposed as a treatment of rumination in the pediatric population. A number of case studies reported a positive effect of chewing gum, which led to a reduction of the number of rumination events in young children and adolescents (37,38).

In theory, treatment aimed at restoring the barrier function of the LES should also be effective. In a small group of patients who did not respond to behavior therapy, a Nissen fundoplication was reported to be effective (39). However, clinical experience suggests that the outcome of surgery may be less favorable, potentially leading to persistent symptoms which may include retching, gas-bloat syndrome, and gastroparesis. Medical therapy aimed at improving LES pressure is another potential approach. One study used the prokinetic levosulpiride as adjuvant, but the focus of treatment was also on psychotherapy and education (40). The same authors also found that a higher LES pressure was associated with a better treatment outcome (40). However, prokinetic drugs are generally not very effective at improving LES pressure (41). On the other hand, baclofen was found to significantly enhance postprandial LES pressure and to suppress TLESRs, and this could potentially beneficial in the treatment of rumination syndrome (42). In a preliminary series of 10 patients who underwent combined high resolution manometry with stationary impedance monitoring and administration of a 1000Kcal solid meal, we observed that baclofen 10 mg preprandially was able to decrease symptoms of regurgitation by approximately 50%. In addition we observed a 65% reduction in rumination episodes identified on th HRM-impedance recording (Blondeau et al., unpublished observations).

Future research

The exact epidemiology and the natural history of the rumination syndrome await detailed studies. The causes and onset of rumination syndrome also remain unclear.

Future studies should establish whether this is indeed occurring as a consequence of dyspeptic symptoms, and whether having a low LES pressure or a certain psychological profile is a predisposing factor. Controlled trials are needed to assess the efficacy of

Rumination syndrome in adults:

Must include both of the following:

1. Persistent or recurrent regurgitation of recently ingested food into the mouth with subsequent spitting or re-mastication and swallowing.

Regurgitation is not preceded by retching

Supportive criteria:

1. Regurgitation events are usually not preceded by nausea 2. Cessation of the process when the regurgitated material becomes acidic 3. Regurgitant contains recognizable food with a pleasant taste

Rumination syndrome in adolescents :

Must include all of the following:

  Rumination syndrome, characterized by the effortless, often repetitive, regurgitation of recently ingested food into the mouth, was originally described in children and in the developmentally disabled. It is now well-recognized that rumination syndrome occurs in patients of all ages and cognitive abilities. Aim: To provide a scholarly review on our current understanding of the rumination syndrome. Methods:

  In case of diagnostic uncertainty, and especially in case of poor patient's acceptance of a possible diagnosis of rumination, manometric evaluation may confirm the diagnosis.

  the head is used to open the upper esophageal sphincter at the same moment

  that enhance LES pressure, and whether these are equivalent or superior to behavioral therapy, or whether they are more suitable as an adjunctive measure. MM, Petti TA, Karwisch G. Case study: chewing gum treatment of rumination in an adolescent with an eating disorder. J Am Acad Child Adolesc Psychiatry. 1997 Aug;36[START_REF] Ball | A special feeding technique for chronic regurgitation[END_REF]:1124-7. 38. Rhine D, Tarbox J. Chewing gum as a treatment for rumination in a child with autism. J Appl Behav Anal. 2009 Summer;42(2):381-5. 39. Oelschlager BK, Chan MM, Eubanks TR, Pope CE 2nd, Pellegrini CA. Effective treatment of rumination with Nissen fundoplication. J Gastrointest Surg. 2002 Jul-Aug;6(4):638-44. 40. Lee H, Rhee PL, Park EH, Kim JH, Son HJ, Kim JJ, Rhee JC. Clinical outcome of rumination syndrome in adults without psychiatric illness: a prospective study. J Gastroenterol Hepatol. 2007 Nov;22(11):1741-7. 41. Tack J, Sifrim D. Anti-relaxation therapy in GORD. Gut. 2002 Jan;50(1):6-7 42. Zhang Q, Lehmann A, Rigda R, Dent J, Holloway RH. Control of transient lower oesophageal sphincter relaxations and reflux by the GABA(B) agonist baclofen in patients with gastro-oesophageal reflux disease. Gut. 2002 Jan;50(1):19-24. 43. Blondeau K, Boecxstaens V, Rommel N, Depeyper S, Holvoet L, Boeckxstaens G and Tack J. Baclofen improves symptoms and postprandial flow events in patients with rumination and supragastric belching/aerophagia. Gastroenterology 2011, in press (abstract).

Figure 1 .

 1 Figure 1. Rumination as observed during ambulatory pH/impedance monitoring.The trace is characterized by a busy erratic pattern of back-and forth-movement by rumination (left arrow) and re-swallowing (right arrow) on the impedance tracings (6 upper channels). There is no acid reflux (lower channel).

Figure 2 .Figure 3 .

 23 Figure 2. Rumination is observed as a rise in intra-gastric pressure on manometry (2 lower channels, block arrow), which precedes retrograde intra-esophageal flow on impedance (7 upper channels, line arrow). LES: Lower Esophageal Sphincter, UES: Upper Esophageal Sphincter

1 .

 1 Repeated painless regurgitation and re-chewing or expulsion of food that a. begin soon after ingestion of a meal b. do not occur during sleep c. do not respond to standard treatment for gastroesophageal reflux 2. No retching 3. No evidence of an inflammatory, anatomic, metabolic, or neoplastic process that explains the subject's symptoms Rumination syndrome in infants : Must include all of the following for at least 3 months: 1. Repetitive contractions of the abdominal muscles, diaphragm, and tongue 2. Regurgitation of gastric content into the mouth, which is either expectorated or rechewed and re-swallowed 3. Three or more of the following: a. Onset between 3 and 8 months b. Does not respond to management for gastroesophageal reflux disease or to anticholinergic drugs, hand restraints, formula changes, and gavage or gastrostomy feedings c. Unaccompanied by signs of nausea or distress d. Does not occur during sleep and when the infant is interacting with

TABLE Rome III definitions of rumination syndrome in adults, adolescents and infants

 Rome

Grant support: Kathleen Blondeau is a postdoctoral research fellow of the FWO Flanders. This work was supported by a Methusalem grant to Jan Tack, M.D., Ph.D.