multiple endocrine neoplasia type 2 ( men2 ) is a rare autosomal dominant inherited disorder with a high lifetime risk of medullary thyroid carcinoma ( mtc ) [ 1 , 2 ] .
men2 consists of three syndromes : familial medullary thyroid carcinoma ( fmtc ) , men2a , and men2b [ 1 , 3 ] .
men2a families have mtc , with at least one individual developing pheochromocytomas , parathyroid hyperplasia , or both .
men2b patients have mtc ( with or without pheochromocytoma ) and other characteristic clinical features : mucosal ganglioneuromas , gi ganglioneuromas , eye abnormalities , and skeletal abnormalities including marfanoid body habitus [ 47 ] .
men2 is caused by pathogenic mutations found exclusively within the ret proto - oncogene ( rearranged during transfection ) .
these are gain - of - function dominant mutations which are commonly heterozygous missense mutations found at specific codons within ret exons 10 , 11 , and 1316 and rarely found within exons 5 and 8 [ 1 , 810 ] .
the medical management for the patient and potentially their family members is based on the familial ret variation , which is usually determined by sanger sequencing .
discovery of a known men2 pathogenic ret mutation within a family leads to screening for mtc , pheochromocytomas , or parathyroid hyperplasia , and potentially prophylactic thyroidectomy to increase survival rate for the intractable , aggressive mtc .
approximately 7580% of mtc patients have the sporadic form of mtc ( i.e. , isolated , nonfamilial mtc ) , not men2 .
patients with apparent sporadic mtc are always tested for an ret germline mutation , in case they actually have men2 and require different medical management .
although there are many well - known pathogenic ret mutations causative of men2 , it may be difficult to know if a rare or novel germline ret variant is a pathogenic mutation ( patient has men2 ) or nonpathogenic polymorphism ( patient has sporadic mtc ) .  
interpretation of rare and novel variants will increase in importance as more people are sequenced at the exome , whole genome , or targeted gene levels .
many new changes will be found with unknown clinical significance and their presence and allele frequency within the general population is of importance to help determine pathogenicity status of a variant .
consortiums like the 1000 genome and other large sequencing projects are making great progress in understanding population sequence variation .
yet more direct studies on single genes or gene panels can yield higher sequencing read coverage and more cost - effective sequencing over a smaller genetic area . also , a particular chosen cohort can be sequenced for a particular locus , such as in the case of this study , where a cohort that was self - reported to have no personal or family history of men2 or mtc was sequenced for a section of the ret protooncogene where most pathogenic men2 causative mutations are located .
ret sequence variation detected in this men2 unaffected population can then be added to the men2 ret database .
this data could be used for several reasons : ( 1 ) to help interpret the pathogenicity of clinically detected ret sequence variation ; ( 2 ) as a reference for any future men2 case studies ( variant was not found in those unaffected by men2 disease ) ; ( 3 ) for improved genetic test design , to avoid or minimize designing probes or primers over known ret sequence variation . to further reduce costs of sequencing large numbers of individuals ,
multiple samples can be pooled ( without indexing ) before next - generation sequencing ( ngs ) .
this was the focus of several studies that analyzed the ability to detect true variants within nonindexed pooled sample sets [ 1116 ] .
thirty samples ( 60 alleles ) were the maximum pooling number indicated by our prior studies and in other reports [ 12 , 14 , 17 , 18 ] , for reproducible and accurate singleton allele detection within the pool ( a singleton is a unique allele within the pool ) .
a pool of this size was expected to a have a singleton allele read frequency of 1.67% , and with consideration of sequencing error rates and potential variance in ngs determined variant read frequencies , singleton variants are expected to be detected above a cutoff of > 1% variant reads [ 17 , 18 ] . in this study
, 136 individuals of an men2 unaffected cohort were sequenced on the illumina genome analyzer utilizing laboratory and bioinformatics protocols from our previous studies for nonindexed , multiple sample pooling .
the pool size was limited to less than 30 , which is the previously determined optimal pooling size for accurate singleton variant detection [ 12 , 14 , 17 , 18 ] . in total
, 61 variants were detected within the men2 unaffected cohort , which included 20 novel variants .
peripheral blood samples from 136 adult volunteers ( 113 caucasian and 23 non - caucasians for ethnic diversity ) were collected and deidentified using university of utah irb protocol no .
7740 . the donors for this unaffected cohort were self - described as not having a personal or family history of neither medullary thyroid carcinoma nor multiple endocrine neoplasia type 2 ( men2 ) .
the 51 samples used as controls were deidentified according to irb no.7275 and were sanger sequenced for ret exons 10 , 11 , and 1316 , including exon / intron boundaries .
the  single - sample control  did not have ret mutations causative of men2 , while the  50 pool 
the 50 pool control was sequenced on the illumina genome analyzer several times previously [ 17 , 18 ] .
dna samples were amplified from ret intron 9 to intron 16 using long - range pcr technology .
amplicons were normalized by sequalprep ( invitrogen corp , carlsbad , ca ) , quantified using quant - it picogreen dsdna kit ( invitrogen corp ) , and equimolar pooled before illumina library prep , utilizing previously described protocols . between 27 and 29 caucasian samples ' amplicons
were combined into four separate pools ( p1 , p2 , p3 , and p4 ) before illumina library prep and ngs .
the non - caucasian cohort 's 23 samples were sequenced in a separate pool ( ethnic pool ) .
the pcr - amplified ret positions 19180 are positions 4360869143617870 in reference sequence nc_000010.10 ( table 1 ) .
two controls were also included in this study , a single sample and also a pool of 50 samples .
each pool and each control were sequenced in a separate flow cell lane on the illumina genome analyzer , using single - end read chemistry .
sequencing image files were processed and reads aligned to the ret reference sequence with seqman ngen version 2.1 software ( dnastar , madison , wi ) , as described previously [ 17 , 18 ] .
reads used were of 67 base lengths since the 3 end read positions of longer reads can have an increase in sequencing background errors , as shown in previous studies [ 17 , 19 , 20 ] .
as previously described , several base quality score screening thresholds ( q - threshold ) evaluated for read coverage , errors ( especially for outlier errors , which could be mistaken for false positives in a pool ) , variant read percentage , and base quality score statistics to determine the 30 q - threshold should be used for analysis of all data sets , which minimized errors while maintaining adequate target read coverage ( data not shown ) [ 17 , 18 ] .
excluded from analysis was a region of repeats and homopolymers that caused misalignment errors in all data sets ( designated  repeat region ,  amplicon positions 7686 to 7720 ) .
changes from the reference sequence were designated variants , and the variant read percentage is the ngs - determined allele frequency .
the previously developed subtractive correction method of variant detection was applied wherein the control 's variant read percentages ( at every position and possible variant change ) are subtracted from the pooled data 's variant read percentages , to yield a pooled data set without background sequencing error [ 17 , 18 ] .
a subset of the ngs - detected ret sequence variants were validated by either high - resolution melting analysis ( hrm ) and/or sanger sequencing .
ret intron 9 used hrm analysis primers ( 5 to 3 ) : forward aca ctg caa tgt gcg ggt ca and reverse gtc ccc caa caa tgc tgc cc . sample dna ( ~5 to 15  ng / ul final concentration ) was amplified and analyzed as described previously , except the lightscanner 32 instrument ( idaho technology , inc . ,
salt lake city , ut ) which was used for both pcr and hrm analysis .
the lightscanner parameters included uracil - dna glycosylase step ( 50c for 10  min ) ; polymerase activation ( 95c for 10  min ) ; 40 pcr cycles ( denaturation at 95c for 1  s , annealing at 62c for 1  s , extension at 72c for 4  s ) ; formation of amplicon heteroduplexes ( 95c for 1  s , then cool rapidly to 40c for 10  s with ramp rate of 20c / s ) ; high - resolution melting protocol ( 70 to 96c with ramp rate of 0.3c / s ) . in order to detect samples with a homozygous ret variant that could not be distinguished from homozygous wild - type samples during hrm analysis ,
the same procedure was performed , except wild - type dna ( ~5  ng / ul final concentration ) which was spiked into the pcr reaction .
the sequence of the single - sample control used in this study exactly matched the reference sequence and therefore had no true variant changes from the reference sequence , only background sequencing error ( table 1 ) .
this sample was an ideal control for error rates since any variant reads from the ret reference at each sequence position reflects the background ngs error rates , and also illumina genome analyzer sequencing has demonstrated reproducible , nonuniform , sequence - specific background error rates , read coverage , and base quality scores between lanes and runs using the same version chemistry [ 11 , 12 , 1720 , 23 , 24 ] .
this single sample controls for the sequence - specific error rates within the pooled data sets by using the subtractive correction method , as described in our previous studies [ 17 , 18 ] . for subtractive correction
, the single - sample control 's variant reads at every possible sequence position , and change from the reference sequence is subtracted from the pool 's variant read percentages .
this yields an estimation of the pooled data without background sequencing error rates contributing to the variant read percentages ( examples in figure 1 ) . the single - sample control and
50-pool data were also used for selection of the 30 q - threshold used for quality screening of the data before analysis ( data not shown ) [ 17 , 18 ] .  
the 50-pool control demonstrated sensitivity to detect known variants with low read percentages for this ngs run and for using the subtractive correction method ( as shown in figure 1 ) .
the 50-pool contained 100 alleles and had an expected 1% singleton variant read frequency ( singleton is unique within the pool ) .
all 59 variants previously detected in the 50-pool were present at > 0.5% variant reads and at similar percentage variant read values as determined in a previous ngs run with the same library ( r = 0.9991 , figure 1(a ) ) .
the 50-pool data had some potential false positives around the cutoff of 0.5% variant reads but after subtractive correction with the single - sample control data , and all true variants were readily detected from the background error ( figure 1(b ) ) .
based on our previous work and other studies , sample pools were restricted to 30 or less samples within each pool to result in a variant read percentage above 1% , the chosen cutoff for the most accurate singleton variant detection [ 12 , 14 , 17 ] .
caucasian pool p1 had 27 samples , p2 had 29 , p3 had 28 , and p4 had 29 samples , with an expected 1.85% , 1.72% , 1.77% , and 1.72% singleton variant read frequency , respectively .
all pool data sets were evaluated with and without subtractive correction of sequencing background error rates using the single - sample control ( figure 1(c ) and data not shown ) .
a total of 51 variants were detected in the caucasian men2 unaffected cohort with > 1% variant read values , of which 23 were not found in the non - caucasian men2 unaffected cohort ( ethnic pool ) ( table 1 ) .
the lowest singleton variant in the caucasian data sets was in p2 with 1.12% variant read frequency .
the 23 non - caucasian samples were in one pool ( ethnic pool ) , with an expected 2.17% singleton allele read frequency .
a total of 38 variants were detected in this ethnically diverse men2 unaffected cohort with > 1% variant read values , of which 10 were not found in the caucasian data sets .
the variant read percentages for each detected variant is shown in table 1 per pool and also summarized for the four caucasian pools . for comparison , the ncbi dbsnp allele frequency values for detected variants
all variants detected were intronic changes , except the expected common polymorphisms found in exons 11 , 13 , 14 , and 15 .
of the total 61 variants found in the men2 unaffected cohort , 20 variants were novel changes , not seen in the 50-pool control or in ncbi dbsnp132 .
since the 136 unaffected cohort samples had not been sequenced previously by either sanger or ngs methods , several variant locations within three pools ( total of 79 samples ) were chosen for validation .
the high - resolution melting ( hrm ) analysis method , which is a rapid , closed - tube mutation scanning assay , was chosen to genotype each individual sample for validation of ngs variant detection and the ngs determined variant allele frequency ( table 2 ) .
high - resolution melting analysis detects sequence variation within the pcr amplicon using a saturating dsdna dye and in many cases can uniquely identify each variant based on differential melting profiles ( figure 2(a ) ) [ 2528 ] .
hrm assay states 100% specificity and sensitivity for detection of heterozygous variants within small amplicons ( < 300  bp ) .
hrm analysis was used to detect sequence variations within a section of ret intron 9 and exons 13 and 15 ( figure 2 and data not shown ) .
ret exons 13 and 15 were chosen since they each contain a common polymorphism present in all pools that could be detected using the previously developed hrm assay .
exon 13 contains c.2307g > t variant and exon 15 contains c.2712c > g variant ( ret amplicon positions 5153 and 6943 , respectively , tables 1 and 2 ) .
intron 9 was chosen since it contains three ngs - detected variants in close proximity at ret amplicon positions 117 , 156 , and 174 ( c.1760197g > t , c.1760158c > g , and c.1760140c > g , resp . ) , and also to verify the novel c.1760158c > g variant detected in caucasian pool p2 which had the lowest variant read percentage of 1.12% ( expected 1.72% singleton read frequency within that pool of 29 samples ) .
since some homozygous variants can have similar melting profiles as the wild - type sample , a technique that spikes wild - type dna into the pcr reaction to allow distinction of homozygous variants was performed on any sample that appeared wild - type after testing in the first hrm assay .
this technique identified four homozygous variants in ret exon 15 and one homozygous variant in ret intron 9 ( figure 2(b ) and data not shown ) .
the hrm determined allele frequency correlated well with the ngs variant read percentage for each variant in each pool ( table 2 ) .
the variant with the lowest read percentage ( position 156 in p2 , 1.12% ) was verified as present and heterozygous in one sample within caucasian pool p2 .
this paper describes ret proto - oncogene sequence variation detected in an men2 unaffected cohort of 136 individuals .
the previous genome analyzer sequenced 50-pool library [ 17 , 18 ] was used to control for the detection of variations with low read frequency , and all known variants were detected > 0.5% variant reads . with similar error rates between genome analyzer lanes of the same run [ 11 , 12 , 17 , 19 , 20 , 23 , 24 ] , the singleton variants in a less than or equal to 30-sample pool should be accurately detected above background error using our previously determined cutoff value of > 1% variant read frequency . the single sample controlled for background sequencing error rates across each ret sequence position and was used for the subtractive correction method of variant detection for pools [ 17 , 18 ] .
the majority of the men2 unaffected cohort were of caucasian ethnicity , while 23 samples were non - caucasian ( ethnic pool ) and were used to identify ret variants within a more ethnically diverse sample set .
the 136 samples were distributed into five nonindexed pools and were sequenced in five separate flow cell lanes .
using previously described protocols for bioinformatics , subtractive correction , and variant read cutoff value of > 1% [ 17 , 18 ] , a total of 61 ret variants were detected within the men2 unaffected cohort .
twenty of these variants were novel , not in ncbi dbsnp 132 ( which includes 1000 genome data ) or found in our previous sample pooling studies on the ret proto oncogene [ 17 , 18 ] .
many of these novel changes were specific to either the caucasian or ethnic samples and were of low variant read frequency , so they were likely to be singleton or doubleton variants within the pools .
several variants were verified by hrm and sanger sequencing , including the novel change ( c.1760158c > g ) with the lowest variant read percentage ( 1.12% ) .
the ret men2 database developed by the author so far has 147 entries , of which 74 are known pathogenic mutations and 62 are variants of uncertain significance .
this database has been used as a model for predictions of phenotypic severity of variants of unknown clinical significance within the ret proto oncogene .
ret sequence variation data for these men2 unaffected cohorts will be added to the men2 ret database .
this variant data will help in medical interpretation of variations found in this ret proto - oncogene region using methods such as sanger sequencing or ngs ( targeted to the ret gene , the whole exome , or whole genome sequencing ) .
any ret sequence change detected in individuals with a family history of men2 symptoms or where men2 is suspected ( patient with apparent sporadic mtc or pheochromocytoma ) can be compared to the available ret men2 database , and also to the benign ret sequence variation present in the large cohort of unaffected individuals that was generated in this paper .
this highlights the importance of clinically relevant databases with not only known pathogenic changes , but also the inclusion of known benign changes for clinical test interpretation .
the men2 unaffected cohort 's variant results can also be used in comparison to variants detected in suspected men2 patients for case reports .
a potential problem for genetic test design is unknown variants present in the location of the pcr primers , sanger sequencing primers , or melting analysis probes .
results from this unaffected population will help with genetic test design , so that primers , and probes will not be designed over the known ret sequence changes .
this paper presents sequence variation detection methods that could be used for other genes and analysis for specific cohorts ( unaffected versus affected , by different ethnicities , or those with specific symptoms of disease ) .
the resulting data can be added to locus - specific databases to help interpret the pathogenicity of clinically detected sequence variation .
these validated methods can also apply to other pooled samples ( such as genetic locations for gwas followup or for testing - specific populations ) and natural pools ( such as mitochondrial heteroplasmy or mixed tumor populations ) .
 multisample , nonindexed pooling combined with next - generation sequencing ( ngs ) was used to discover ret proto - oncogene sequence variation within a cohort known to be unaffected by multiple endocrine neoplasia type 2 ( men2 ) . 
 dna samples ( 113 caucasians , 23 persons of other ethnicities ) were amplified for ret intron 9 to intron 16 and then divided into 5 pools of < 30 samples each before library prep and ngs . 
 two controls were included in this study , a single sample and a pool of 50 samples that had been previously sequenced by the same ngs methods . 
 all 59 variants previously detected in the 50-pool control were present . 
 of the 61 variants detected in the unaffected cohort , 20 variants were novel changes . 
 several variants were validated by high - resolution melting analysis and sanger sequencing , and their allelic frequencies correlated well with those determined by ngs . 
 the results from this unaffected cohort will be added to the ret men2 database . 
1. Introduction
2. Materials and Method
3. Results
4. Discussion