824
`
`Clinical Cancer
`Genetics and Human
`Cancer Genetics
`Programs,
`Comprehensive Cancer
`Center, and Division of
`Human Genetics,
`Department of Internal
`Medicine, The Ohio
`State University,
`Columbus, OH, USA
`and CRC Human
`Cancer Genetics
`Research Group,
`University of
`Cambridge,
`Cambridge, UK
`C Eng
`
`National Institutes of
`Health, Bethesda, MD,
`USA
`L C Brody
`
`Division of Senology,
`Department of
`Obstetrics and
`Gynecology, University
`of Vienna, Austria¶
`T M U Wagner
`
`Departments of
`Human Genetics and
`Pathology, Leiden
`University Medical
`Centre, Leiden, The
`Netherlands‡
`P Devilee
`
`Cancer Therapy and
`Research Center and
`University of Texas
`Health Science Center,
`San Antonio, TX, USA§
`J Vijg
`
`International Agency
`for Research on
`Cancer, Lyon, France
`C Szabo
`
`Myriad Genetic
`Laboratories, Salt Lake
`City, UT, USA**
`S V Tavtigian
`T S Frank
`
`Department of
`Medicine, University of
`Pennsylvania,
`Philadelphia, PA, USA
`K L Nathanson
`
`Clinical Research
`Division, Fred
`Hutchinson Cancer
`Research Center,
`Seattle, WA, USA†
`E Ostrander
`
`Correspondence to:
`Professor Eng, Ohio State
`University Human Cancer
`Genetics Program, 420 W
`12th Avenue, Suite 690
`Tzagournis MRF,
`Columbus, OH 43210, USA,
`eng-1@medctr.osu.edu or
`ceng@hgmp.mrc.ac.uk
`
`Revised version received
`1 October 2001
`Accepted for publication
`2 October 2001
`
`J Med Genet 2001;38:824–833
`
`Interpreting epidemiological research: blinded
`comparison of methods used to estimate the
`prevalence of inherited mutations in BRCA1
`
`Charis Eng, Lawrence C Brody, Teresa M U Wagner, Peter Devilee, Jan Vijg, Csilla Szabo,
`Sean V Tavtigian, Katharine L Nathanson, Elaine Ostrander, Thomas S Frank, on behalf
`of the Steering Committee of the Breast Cancer Information Core (BIC) Consortium*
`
`Abstract
`While sequence analysis is considered by
`many to be the most sensitive method of
`detecting unknown mutations in large
`genes such as BRCA1, most published
`estimates of the prevalence of mutations
`in this gene have been derived from stud-
`ies that have used other methods of gene
`analysis. In order to determine the rela-
`tive sensitivity of
`techniques that are
`widely used in research on BRCA1, a set of
`blinded samples containing 58 distinct
`mutations were analysed by four separate
`laboratories. Each used one of the follow-
`ing methods: single strand conformational
`polymorphism analysis (SSCP), confor-
`mation
`sensitive
`gel
`electrophoresis
`(CSGE), two dimensional gene scanning
`(TDGS), and denaturing high perform-
`ance liquid chromatography (DHPLC).
`Only the laboratory using DHPLC cor-
`rectly identified each of the mutations.
`The laboratory using TDGS correctly
`identified 91% of the mutations but pro-
`duced three apparent false positive re-
`sults. The laboratories using SSCP and
`CSGE detected abnormal migration for
`72% and 76% of the mutations, respec-
`tively, but subsequently confirmed and
`reported only 65% and 60% of mutations,
`respectively. False negatives therefore re-
`sulted not only from failure of the tech-
`niques to distinguish wild type from
`mutant, but also from failure to confirm
`the mutation by sequence analysis as well
`as from human errors leading to misre-
`porting of results. These findings charac-
`terise sources of error in commonly used
`methods of mutation detection that should
`be addressed by laboratories using these
`methods. Based upon sources of error
`identified in this comparison, it is likely
`that mutations in BRCA1 and BRCA2 are
`more prevalent than some studies have
`previously reported. The findings of this
`
`*Names of the BIC Steering Committee appear in the Appen-
`dix.
`
`SSCP team†: Renata Zaucha, Nicola M Suter, Elaine Ostrander.
`
`CSGE Team‡: Marlies Hoogenboom, Ronald van Eijk, Cees J
`Cornelisse, Peter Devilee.
`
`TDGS team§: Nathalie J van Orsouw, Loyda Torres, Esther
`Vrins, Sean McGrath, Jan Vijg.
`
`DHPLC team¶: Regina Moeslinger, Peter J Oefner, Daniela
`Muhr, Teresa M U Wagner.
`
`Myriad team**: Amie M DeVenbaugh, Robin K Zawacki, Tho-
`mas S Frank.
`
`www.jmedgenet.com
`
`comparison provide a basis for interpret-
`ing studies of mutations in susceptibility
`genes across many inherited cancer syn-
`dromes.
`(J Med Genet 2001;38:824–833)
`
`Keywords: BRCA1; mutation detection; cancer genetics
`
`The first inherited cancer syndrome for which
`clinical molecular genetic testing became con-
`sidered to be the “standard of care” was mul-
`tiple endocrine neoplasia type 2 (MEN 2).1 2
`The germline mutations in the RET gene that
`are responsible for MEN 2 are limited in
`number; consequently, a variety of techniques
`that are of equivalent sensitivity and specificity
`could be used for detecting mutations.3 In the
`last decade, additional autosomal dominant
`inherited cancer
`syndromes have become
`genetically characterised and clinical testing
`made available. One of the most common
`inherited cancer syndromes is the hereditary
`breast-ovarian cancer
`syndrome (HBOC),
`which is primarily attributable to two genes,
`BRCA1 and BRCA2,4 which together com-
`prise approximately 15 700 nucleotides of
`open reading frame. To date, more than 1000
`mutations of deduced or established clinical
`significance have been identified; these are
`distributed throughout the 48 coding exons
`and respective splice junctions of the two
`genes. Therefore, molecular diagnostic testing
`for HBOC as well as molecular epidemiologi-
`cal studies in most populations require analyti-
`cal methods that are capable of identifying
`hundreds of distinct mutations distributed
`along the lengths of
`these relatively large
`genes
`(http://www.nhgri.nih.gov/Intramural_
`research/Lab_transfer/Bic).
`Direct nucleotide sequence analysis is con-
`sidered the gold standard for mutation detec-
`tion for genes such as BRCA1 and BRCA2.
`However, this is one of the most expensive
`methods for analysing genes, not only because
`of reagent costs but also because of the labour
`required to analyse the more than 15 000 data
`points that it generates. Thus, many laborato-
`ries that analyse BRCA1 or BRCA2, particu-
`larly in the context of performing epidemio-
`logical studies requiring analysis of numerous
`samples, use gene “scanning” technologies to
`identify sequence variants in PCR amplicons in
`order
`to avoid labour and cost
`intensive
`sequencing of wild type exons.5 Although clini-
`cal cancer geneticists around the world counsel
`and manage patients based on the likelihood of
`
`GeneDX 1016, pg. 1
`
`

`
`Methods used to estimate prevalence of BRCA1 mutations
`
`825
`
`mutations derived from such studies, the sensi-
`tivity and specificity of these methods, and thus
`the accuracy of these data, have not been
`systematically evaluated. Since these estimates
`are used for patient management, the accuracy
`of data derived by these methods has substan-
`tial implications, and some4 but not all such
`studies take into account the potential for error
`with such methods. Further, research laborato-
`ries engaged in large scale molecular epidemio-
`logical studies need to understand the potential
`sources of error in such methods in order to
`maximise their sensitivity and specificity.
`In an eVort to assist the clinical cancer
`genetics community to evaluate results from
`diVerent methods used for diagnosing HBOC
`through mutation detection, we sought
`to
`compare the sensitivity, specificity, and cost
`eYciency of four common mutation scanning
`technologies for detecting 58 distinct muta-
`tions in the BRCA1 gene. Two of the methods,
`single strand conformational polymorphism
`analysis (SSCP) and conformation sensitive
`gradient gel electrophoresis (CSGE), screen
`for mutations on the basis of conformational
`changes in PCR products induced by muta-
`tions when compared to the wild type. The
`other two methods, two dimensional gene
`scanning (TDGS) and denaturing high per-
`formance liquid chromatography (DHPLC),
`separate mutational variants on the basis of
`their melting temperatures (TDGS also in-
`cludes a size separation). It is believed that the
`value of the information derived from this
`comparison of mutation scanning methods is
`not
`limited to detection of mutations
`in
`BRCA1 but has implications for the analysis of
`other large genes as well.
`
`Materials and methods
`SAMPLES
`Samples were selected and anonymised for
`blinded analysis by Myriad Genetic Laborato-
`ries. All samples had been analysed following
`the routine procedures used for diagnostic
`testing. DNA was first extracted by Proteinase
`K digestion from peripheral blood mono-
`nuclear cells isolated from each sample and
`then column purified (QIAGEN Inc, Chats-
`worth, CA, USA). Aliquots of DNA were
`amplified by polymerase chain reaction (PCR)
`using 35 M13 forward and reverse tagged
`primer pairs to cover coding exons 2-24 of
`BRCA1 (although exon 4,
`like exon 1,
`is
`non-coding and no variants in either were
`included in the subsequent inter-laboratory
`comparison). The amplified products were
`each directly sequenced in forward and reverse
`directions using fluorescent dye labelled se-
`quencing primers. Chromatographic tracings
`of each amplicon were analysed by a propri-
`etary computer based review followed by visual
`inspection and confirmation. Genetic variants
`were detected by comparison with a consensus
`wild type sequence constructed for BRCA1. As
`part of routine analytic processing, all potential
`genetic variants had been independently con-
`firmed by repeated PCR amplification of the
`indicated gene region(s) and sequence deter-
`mination as above.
`
`www.jmedgenet.com
`
`For the purposes of this study, “mutations”
`were defined as protein truncating and mis-
`sense mutations located within exons 2-3 and
`5-24 of BRCA1 as well as intronic sequence
`alterations occurring no more than 20 bp
`proximal or 10 bp distal to the ends of these
`exons. Non-truncating genetic variants were
`excluded from consideration for the purposes
`of this study if they had been observed at an
`allele frequency of greater than 1% of a suitable
`control population with no evidence for signifi-
`cantly higher frequency in cases than controls,
`or if published data indicated absence of
`substantial clinical significance, or
`if
`they
`neither altered the amino acid sequence nor
`were predicted to aVect exon splicing signifi-
`cantly.
`The sample set consisted of 65 samples,
`including 50 that contained a total of 58 muta-
`tions of established or potential clinical signifi-
`cance and 15 additional samples in which no
`mutation had been identified through se-
`quence analysis as above. The positive samples
`included 20 frameshift mutations (17 dele-
`tions, three insertions), 18 nonsense muta-
`tions, 15 missense mutations, and five muta-
`tions occurring in the non-coding regions
`adjacent to the beginning or end of the exon
`(table 1). All mutations and genetic variants
`were named according to a designated conven-
`tion,6 numbering the nucleotides from the first
`transcribed base of BRCA1 GenBank entry
`U14680.
`Ten µg of genomic DNA that remained after
`the completion of routine analysis by Myriad
`Genetic Laboratories were aliquotted to the
`participating laboratories per their stated re-
`quirements as follows: 4 µg each for SSCP and
`CSGE and 1 µg each for TDGS and DHPLC.
`A letter of agreement was provided to each
`participating laboratory that delineated the
`principles of the exercise, including the criteria
`by which sensitivity and specificity would be
`derived. DiVerences between laboratories lim-
`ited to the names or cDNA locations of the
`mutations were not considered discrepancies
`for the purpose of this comparison. A Myriad
`Genetic Laboratories
`representative (TSF)
`provided the number and identity of the muta-
`tions to a designated representative of the
`Breast Cancer Information Core (BIC) (LCB)
`to whom all laboratories subsequently submit-
`ted their results. Only when each of the labora-
`tories had completed and submitted final
`results to the BIC representative were the
`authors provided with each other’s results.
`
`SSCP
`All coding regions and exon-intron boundaries
`of BRCA1 were amplified from genomic DNA
`by PCR using either previously described sets
`of primers7 8 or primer pairs designed in the
`Ostrander laboratory. PCR was carried out in
`12.5 µl volumes with 25 ng genomic DNA as
`template, 1 · PCR buVer, 1.5 mmol/l magne-
`sium, 0.048 mmol/l each dATP, dTTP, and
`dGTP, 0.0048 mmol/l dCTP, 0.2 U Taq, (Bio-
`line, USA), and 0.004 mCi [♡-P32]) dCTP
`(Amersham, USA). Initial denaturation was
`done at 95(cid:176)C for one minute followed by 35
`
`GeneDX 1016, pg. 2
`
`

`
`826
`
`Eng, Brody, Wagner, et al
`
`Table 1 Mutations subject to blinded analysis by SSCP, CSGE, TDGS, and DHPLC
`
`Mutation name
`
`Exon
`
`Base change
`
`Mutation type Mutation eVect
`
`2
`187delAG
`2
`M1I (122G>T)
`5
`C64Y
`5
`C61G
`6
`IVS5-11T>G
`7
`E143X
`7
`525insA
`8
`Y179C*
`8
`639delC
`8
`IVS8+1G>T
`11
`1629delC
`11
`2576delC
`11
`K679X
`11
`L246V*
`11
`F486L*
`11
`N550H*
`11
`E1222X
`11
`E1134X
`11
`Q1111X
`11
`Q957X
`11
`3600del11
`11
`1294del40
`11
`V772A*
`11
`E1250X
`11
`Q780X
`11
`L668F*
`11
`2322delC
`11
`3347delAG
`11
`E908X
`11
`2072del4
`11
`2080delA
`11
`W321X
`11
`2594delC
`11
`3171ins5
`11
`3829delT
`11
`3875del4
`11
`4154delA
`11
`Q563X
`11
`Q1240X
`11
`2190delA
`12
`Q1395X
`13
`H1402Y*
`14
`Y1463X
`14
`4510del3insTT
`15
`W1508X
`16
`P1637L*
`16
`IVS16+1G>A
`17
`IVS17+1G>T
`18
`A1708E
`18
`Y1703X
`18
`IVS17-1G>A
`5385insC (“5382insC”) 20
`E1754X
`20
`M1775R
`21
`C1787S*
`22
`G1788D*
`22
`5454delC
`22
`R1835X
`24
`
`Frameshift
`Del AG
`Missense
`122 G>T
`Missense
`310 G>A
`Missense
`300 T>G
`Splice
`4795-11T>G
`Nonsense
`546 G>T
`Frameshift
`Ins A
`Missense
`655 A>G
`Frameshift
`Del C
`Splice
`666+1 G>T
`Frameshift
`Del C
`Frameshift
`Del C
`Nonsense
`2154 A>T
`Missense
`855 T>G
`Missense
`1575 T>C
`Missense
`1767 A>C
`Nonsense
`3783 G>T
`Nonsense
`3519 G>T
`Nonsense
`3450 C>T
`Nonsense
`2988 C>T
`Del GAAGATACTAG Frameshift
`Del 40
`Frameshift
`2434 T>C
`Missense
`3867 G>T
`Nonsense
`2457 C>T
`Nonsense
`2121 C>T
`Missense
`Del C
`Frameshift
`Del AG
`Frameshift
`2841 G>T
`Nonsense
`Del GAAA
`Frameshift
`Del A
`Frameshift
`1081 G>A
`Nonsense
`Del C
`Frameshift
`Ins TGAGA
`Frameshift
`Del T
`Frameshift
`Del GCTC
`Frameshift
`Del A
`Frameshift
`1806 C>T
`Nonsense
`3837 C>T
`Nonsense
`Del A
`Frameshift
`4302 C>T
`Nonsense
`4323 C>T
`Missense
`4508 C>A
`Nonsense
`Del CTA Ins TT
`Frameshift
`4643 G>A
`Nonsense
`5029 C>T
`Missense
`5105+1G>A
`Splice
`5193+1 G>T
`Splice
`5242 C>A
`Missense
`5228 T>G
`Nonsense
`5194-1 G>A
`Splice
`Ins C
`Frameshift
`5379 G>T
`Nonsense
`5443 T>G
`Missense
`5478 T>A
`Missense
`5482 G>A
`Missense
`Del C
`Frameshift
`5622 C>T
`Nonsense
`
`Premature stop
`Missense
`Missense
`Missense
`Splice
`Nonsense
`Premature stop
`Indeterminate
`Premature stop
`Splice
`Premature stop
`Premature stop
`Nonsense
`Indeterminate
`Indeterminate
`Indeterminate
`Nonsense
`Nonsense
`Nonsense
`Nonsense
`Premature stop
`Premature stop
`Indeterminate
`Nonsense
`Nonsense
`Indeterminate
`Premature stop
`Premature stop
`Nonsense
`Premature stop
`Premature stop
`Nonsense
`Premature stop
`Premature stop
`Premature stop
`Premature stop
`Premature stop
`Nonsense
`Nonsense
`Premature stop
`Nonsense
`Indeterminate
`Nonsense
`Premature stop
`Nonsense
`Indeterminate
`Splice
`Splice
`Missense
`Nonsense
`Splice
`Premature stop
`Nonsense
`Missense
`Indeterminate
`Missense
`Premature stop
`Nonsense
`
`*The following groups of mutations were concurrently present in their respective samples:
`C1787S and G1788D; 1294del40 and V772A; 5385insC and H1402Y; Y1703X and L668F;
`2576delC and P1637L; K679X and L246V; Y179C, F486L, and N550H.
`cycles of 30 seconds at 94(cid:176)C, 15 seconds at an
`appropriate annealing temperature, 15 seconds
`at 72(cid:176) C, followed by final elongation at 74(cid:176)C
`for three minutes. Samples were then diluted
`1:3 in formamide buVer (98% formamide, 10
`mmol/l EDTA, pH 8, 0.05% bromophenol
`blue, and 0.05% xylene cyanol), denatured at
`99(cid:176)C for five minutes, immediately placed on
`ice, and loaded on two types of gels, multiplex
`0.5 MDE and non-multiplex 3% glycerol.
`Selected amplicons were then pooled for
`electrophoresis;
`this allowed simultaneous
`analysis of several fragments chosen according
`to band size and migration patterns. Electro-
`phoresis was performed at room temperature
`for 16 to 20 hours at 6 W and eight hours at 8
`W for MDE and glycerol gels, respectively.
`Results were visualised by autoradiography.
`Amplification and electrophoresis were re-
`peated for confirmation of altered migration
`
`patterns. Variant bands were subsequently cut
`from gels, resuspended in distilled water,
`resubjected to PCR, and then sequenced with
`Big Dye Terminator Cycle Sequencing kits (PE
`Applied Biosystems) in both forward and
`reverse strand directions.
`Following the routine practice of the labora-
`tory, each abnormally migrating fragment was
`subject
`to sequence analysis regardless of
`whether it was also the location of a common
`BRCA1 polymorphism, since it has been
`shown that
`the abnormal SSCP migration
`associated with common polymorphisms may
`mask a coexistent deleterious mutation.9
`
`CSGE
`The entire coding region of BRCA1 including
`at least 15-50 bp of each flanking intron was
`subdivided into 33 segments. To facilitate PCR
`multiplexing and direct sequencing of selected
`fragments afterwards, all forward primers were
`tagged with M13-Forward tails and labelled
`with fluorescent FAM, HEX, or TET. Reverse
`primers contained M13-Reverse tails. Oligonu-
`cleotides were purchased from Eurogentec,
`Belgium; their sequences are available from the
`Devilee
`lab website
`(http://www.medfac.
`leidenuniv.nl/lab-devilee/Lab/csgeolig.htm).
`The 33 fragments were amplified in one
`mono and 16 duplex PCRs as detailed on the
`website provided above. A 14 µl reaction
`mixture prepared in each well of a 96 well
`microtitre plate contained 10 pmol primers, 1 ·
`PCR buVer (50 mmol/l KCl, 10 mmol/l TRIS-
`HCl, pH 8.4, 2.5 mmol/l MgCl2, 0.2 mg/ml
`BSA, 0.2 mmol/l dNTPs), and 0.1 U Goldstar
`Taq polymerase (EuroGentech, Seraing, Bel-
`gium). Subsequently, 1 µl of each DNA sample
`(50 ng/µl) was added to the reaction mixtures.
`PCR was performed for 40 cycles consisting of
`30 seconds at 94(cid:176)C, 30 seconds at 58 (cid:176)C, and
`30 seconds at 72(cid:176)C.
`After PCR, reaction mixtures corresponding
`to a given DNA sample were pooled into a 96
`well microtitre plate in a HEX:FAM:TET ratio
`of 3:2:2 for a final volume of 24 µl, in a total of
`six pools per DNA sample (see above website
`for details). Seven µl of this mixture were ali-
`quotted into a fresh plate and heat/air dried by
`exposing to 45(cid:176)C for one hour. The mixture
`was dissolved in 2.5 µl of Pink Loading Dye
`(Amersham Pharmacia, Benelux, Roosendaal,
`The Netherlands), to which 0.25 µl GeneScan-
`500 TAMRA size standard and 0.25 µl loading
`buVer were added (Applied Biosystems). Using
`an eight channel
`loading device (Hamilton,
`Bonaduz, Switzerland), 1.5 µl of this mixture
`was loaded onto an f-CSGE gel, which had
`been pre-run for 15 minutes. The samples were
`subjected to electrophoresis through these gels
`for 4.5 hours at 1680 V at 30(cid:176)C. Gels were
`analysed with GeneScan® and Genotyper®
`software (Applied Biosystems). Each abnor-
`mally migrating fragment was reamplified from
`the DNA sample using the same primers as
`above and sequenced in the forward direction
`using Big Dye Terminator Cycle Sequencing
`kits (PE Applied Biosystems).
`
`www.jmedgenet.com
`
`GeneDX 1016, pg. 3
`
`

`
`Methods used to estimate prevalence of BRCA1 mutations
`
`827
`
`TDGS
`All BRCA1 coding exons were amplified from
`genomic DNA in a 7-plex long distance PCR.
`Individual exons or parts of exons were ampli-
`fied in four multiplex groups of nine or 10
`fragments each, using the long distance 7-plex
`PCR products as template, so that the entire
`BRCA1 coding region was resolved in a total of
`37 fragments. Primers for the multiplex short
`PCR were designed as described.10 11 Products
`of the four multiplex groups were combined,
`mixed with sample buVer, and loaded directly
`into the slot of a 2D gel. Electrophoresis was
`performed in an automated 2D DNA electro-
`phoresis system12 and gels were stained with
`ethidium bromide. Spot patterns were inter-
`preted by eye for the appearance of four spots
`rather than one, indicating the presence of a
`heterozygous mutation or polymorphism. The
`complete protocol for BRCA1-TDGS has been
`described previously.11 Each sample was ana-
`lysed only once, under the same conditions,
`and fragments that were absent or faint were
`repeated by one dimensional DGGE (an aver-
`age of five fragments per BRCA1 gene sample).
`Fragments that showed a four spot pattern that
`could be recognised as a previously detected
`polymorphism on the basis of their characteris-
`tic configurations were assigned as such. New
`variants were subjected to sequence analysis.
`Sequence analysis was either carried out on a
`Beckman CEQ2000 sequencer (75% of frag-
`ments) or contracted out to DavisSequencing
`(Davis, CA, USA) (25% of fragments). All 2D
`patterns are published on the web (http://
`www.tdgs.saci.org/myriad.html).
`
`DHPLC
`For purposes of PCR, BRCA1 was divided into
`35 amplicons comprising the coding sequence
`and adjacent non-coding sequence in the
`regions of the splice junctions. Primers were
`designed to minimise overlap between frag-
`ments, to improve the robustness of PCR, or to
`increase the length of fragment screened. The
`primers used had originally been described for
`SSCP13 with the exception of the primers for
`exon 5.8 PCR was performed in a 50 µl volume
`containing 15 mmol/l Tris-HCl, pH 8.0, 50
`mmol/l KCl, 1.5-4.5 mmol/l MgCl2, 10 mmol/l
`of dNTPs, 0.25 µmol/l of each primer, and 10
`ng of genomic DNA. For all PCR reactions,
`AmpliTaq Gold (Perkin Elmer, Foster City,
`USA) was used. The PCR cycling conditions
`comprised an initial denaturation step at 95(cid:176)C
`for seven minutes to activate AmpliTaq Gold.
`Subsequent denaturing steps were 94(cid:176)C for 45
`seconds and extension steps of 72(cid:176)C for 30
`seconds. In some instances, annealing tem-
`peratures were decreased from 63(cid:176)C by 0.5 (cid:176) C
`decrements to 56(cid:176)C in 14 cycles, followed by
`21 cycles at 56(cid:176)C for 20 seconds. In one case,
`namely exon 23, the annealing temperature
`was decreased from 67(cid:176)C to 60 (cid:176) C, while in the
`case of exon 11EF, it was decreased from 65(cid:176)C
`to 58(cid:176)C. In all other cases, 35 cycles were per-
`formed at constant annealing temperatures.
`Denaturing high performance liquid chro-
`matography was carried out on an automated
`HPLC instrument (Transgenomics Inc, San
`
`www.jmedgenet.com
`
`Jose, CA, USA). The DNA separation column
`was packed with proprietary 2 µ non-porous
`alkylated poly(styrene-divinylbenzene) parti-
`cles.14 The mobile phase was 0.1 mol/l triethyl-
`ammonium acetate buVer, pH 7.0 (TEAA, PE
`Biosystems, Foster City, CA, USA). Crude
`PCR products were subjected to an additional
`three minute, 95(cid:176)C denaturing step followed
`by gradual reannealing from 95-65(cid:176)C over a
`period of 30 minutes before analysis. Homo-
`and heteroduplex species were eluted with a
`linear acetonitrile (Merck, Vienna, Austria)
`gradient at a flow rate of 0.9 ml/minute. The
`start and end points of the gradient were
`adjusted according to the size of the PCR
`products using an algorithm provided by the
`WAVE Maker™ system control
`software
`(Transgenomics Inc, San Jose, CA, USA).
`Generally, analysis took eight minutes, includ-
`ing column regeneration and re-equilibration
`to the starting conditions. The temperature
`required for successful resolution of heterodu-
`plex molecules was determined by use of the
`DHPLC melting algorithm available at http://
`insertion.stanford.edu/melt.html,15
`respectiv-
`ely, the WAVE Maker™ software. Appropriate
`temperature(s) of analysis were determined for
`each amplicon, with 19 of the 35 amplicons
`requiring analysis at two temperatures and the
`rest at one. Known sequence variants, on aver-
`age four per amplicon, are analysed along with
`the new samples
`to establish the proper
`performance of the DHPLC instrument. The
`appearance of additional peaks or shoulders
`was interpreted as indicative of the presence of
`a mismatch, which was subsequently analysed
`by sequencing. Nine amplicons known to con-
`tain BRCA1 polymorphisms with a heterozy-
`gosity >5% were sequenced routinely when
`observed to be heterozygous.8
`
`COST CALCULATION
`The costs of mutation analysis were calculated
`in two ways. The first only took into account
`the cost of consumable supplies on a per sam-
`ple basis. The second calculation derived a
`“universal cost equivalent” that attempts to
`analyse each method in terms of labour, quan-
`tities of supplies (for example, numbers of ABI
`gels, numbers of oligonucleotide primers,
`number of PCR reactions) and run times nec-
`essary to perform an analysis.
`
`Results
`For the set of samples known to contain
`BRCA1 mutations or not
`(table 1),
`the
`reported overall sensitivity of the methods, as
`summarised in table 2, required not only the
`initial detection of an abnormality in an ampli-
`con, but also the ability to confirm the
`mutation by sequence analysis and to report
`the result correctly to a central source (LCB)
`who compiled the results. Samples for which
`PCR amplification could not be attained or for
`which there was insuYcient DNA for sequence
`confirmation were not counted as “negative”
`results, but were omitted from the total. Only
`DHPLC was able to correctly identify each of
`the 58 mutations in the sample set. Eleven
`mutations were each missed by at least two
`
`GeneDX 1016, pg. 4
`
`

`
`828
`
`Eng, Brody, Wagner, et al
`
`Table 2 Comparison of methods for detecting mutations in BRCA1*
`
`Mutation type (number in
`set)*
`
`Abnormal migration
`(%)
`
`Confirmation of
`mutation in
`abnormally migrating
`fragment (%)
`
`Total mutations
`reported correctly (%)
`
`(A) SSCP
`Frameshift (20)
`Base substitutions
`Nonsense (18)
`Missense (15)
`Splice (5)
`Total
`
`(B) CSGE
`Frameshift (20)
`Base substitutions
`Nonsense (18)
`Missense (15)
`Splice (5)
`Total
`
`(C) TDGS
`Frameshift (20)
`Base substitutions
`Nonsense (18)
`Missense (15)
`Splice (5)
`Total
`
`(D) DHPLC
`Frameshift (20)
`Base substitutions
`Nonsense (18)
`Missense (15)
`Splice (5)
`Total
`
`19/20 (95)
`
`7/18 (39)
`12/15 (80)
`4/5 (80)
`42/58 (72)
`
`14/15 (93)
`
`8/12 (67)
`10/15 (67)
`2/3 (67)
`34/45 (76)
`
`18/20 (90)
`
`17/18 (94)
`14/15 (93)
`4/5 (80)
`53/58 (91)
`
`17/18 (94)
`
`7/7 (100)
`7/7 (100)
`3/3 (100)
`34/35 (97)
`
`11/14 (79)
`
`8/8 (100)
`9/10 (90)
`2/2 (100)
`30/34 (88)
`
`18/18 (100)
`
`16/16 (100)
`14/14 (100)
`3/3 (100)
`51/51 (100)
`
`16/19 (84)
`
`7/18 (39)
`7/10 (70)
`3/4 (75)
`33/51 (65)
`
`10/15 (67)
`
`6/12 (50)
`9/15 (60)
`2/3 (67)
`27/45 (60)
`
`18/20 (90)
`
`16/17 (94)
`14/15 (93)
`3/4 (75)
`51/56 (91)
`
`20/20 (100)
`
`20/20 (100)
`
`20/20 (100)
`
`18/18 (100)
`15/15 (100)
`5/5 (100)
`58/58 (100)
`
`18/18 (100)
`15/15 (100)
`5/5 (100)
`58/58 (100)
`
`18/18 (100)
`15/15 (100)
`5/5 (100)
`58/58 (100)
`
`*Discrepant values between the number of samples in the set and the number of samples analysed
`(denominator) reflect samples for which either PCR amplification failed or for which there was
`insuYcient DNA for sequence analysis following initial screening.
`
`laboratories, of which two (both single nucle-
`otide substitutions
`resulting in premature
`truncation) were each missed by three of the
`four laboratories. The results of the individual
`laboratories are presented below.
`
`SSCP
`The laboratory using SSCP correctly identified
`33 of 51 mutations (65%) (table 2A), with
`seven additional mutations occurring in sam-
`ples that could not be analysed because of
`insuYcient DNA for sequence analysis follow-
`ing SSCP, as discussed below. A false positive
`result that was reported in one of the 15 nega-
`tive samples resulted not from a technical
`error, but instead from a laboratory sample
`switch that also accounted for one of the false
`negative results.
`After the initial SSCP scan, 58 aberrant
`bands were detected on MDE gels and an
`additional five bands were observed on glycerol
`gels. Initially, two of five variants seen on glyc-
`erol gels were not detected on MDE gels owing
`to the presence of overlapping multiplex bands
`representing other exons. Reamplification of all
`possible variants from the initial screen con-
`firmed the presence of 42 abnormal bands out
`of
`the 58 mutations (72%) distributed in
`diVerent exons of BRCA1. Aberrant electro-
`phoresis of bands was identified for 19 of 20
`(95%) frameshift mutations, including 17 of 17
`deletions and two of three insertions. The
`mutation 5385insC (“5382insC”) was missed,
`although it has been previously detected by
`SSCP by this laboratory using the same
`techniques.16–18 This suggests that the eYciency
`of SSCP in detecting very subtle changes is
`variable. Abnormal migration was seen for 23
`
`www.jmedgenet.com
`
`of 38 nucleotide substitutions (61%), including
`five localised to introns (table 2A). SSCP failed
`to detect seven of the nine G to T substitutions
`and four of the 10 C to T substitutions, but
`abnormal migration occurred with five of six G
`to A substitutions. Several of
`the single
`nucleotide substitutions that did not alter elec-
`trophoresis mobility occurred near either end
`of a PCR amplicon. For example, the missed G
`to T substitutions that resulted in M1I and
`E143X each occurred near the ends of exon 2
`and exon 7, respectively.
`Sequence analysis was performed for all
`samples for which abnormal migration was
`identified. In seven instances, the first obtained
`sequence was not diagnostic and there was
`insuYcient DNA to repeat
`the procedure;
`these seven variants were excluded from
`further calculations of sensitivity. Sequence
`analysis identified 34 of the remaining 35
`mutations for which abnormal migration had
`been observed, but
`failed to identify the
`frameshift mutation 2576delC. Finally, as
`mentioned above, mislabelled samples resulted
`in incorrect reporting of two samples, resulting
`in one false positive and one false negative
`interpretation in the final report of results.
`
`CSGE
`The laboratory using CSGE correctly identi-
`fied 27 of 45 mutations (60%), with 13 muta-
`tions that could not be analysed owing to fail-
`ure to amplify by PCR as discussed below
`(table 2B). No mutations were identified in the
`15 samples documented not to harbour a
`sequence alteration.
`Abnormal
`electrophoretic migration by
`CSGE was present in 34 of 45 (76%) samples
`for which PCR amplification was successfully
`performed. Nucleotide
`substitutions
`ac-
`counted for 10 of the 11 mutations that were
`missed at this stage of analysis (table 2B). Ret-
`rospective evaluation showed subtle diVerences
`relative to wild type fragments in three of these
`11 false negatives, and two additional peak
`shifts were suYciently clear that they repre-
`sented erroneous interpretation by the ob-
`server. The
`remaining six (all missense
`changes: G>T, G>T, C>T, T>C, C>A, A>T)
`did not show migration patterns that were dis-
`tinguishable from the wild type.
`Four additional mutations were missed
`because of failure of sequence analysis to con-
`firm a mutation following observation of
`abnormal gel mobility. One of these was a base
`substitution, a T to G at cDNA nt 855, result-
`ing in the substitution of valine for leucine at
`amino acid position 246. This variant pro-
`duced only a very subtle change in the
`sequence trace at the heterozygote position and
`was erroneously called negative. The other
`three were
`small
`frameshifting deletions
`(2072del4, 2080delA, and 2594delC) that are
`ordinarily considered to be easily detectable by
`sequencing. Sequence data were analysed
`using the Staden software, which subtracts the
`sample sequence trace from a wild type control
`trace to highlight sequence diVerences, exclud-
`ing those parts of the trace that do not meet
`
`GeneDX 1016, pg. 5
`
`

`
`Methods used to estimate prevalence of BRCA1 mutations
`
`829
`
`minimum quality. Sequence analysis was per-
`formed only in the forward direction, and since
`these three mutations were each at the very
`ends of sequence traces, the quality of data in
`the region of the mutation was suboptimal.
`This frameshift was therefore not identified by
`the Staden program, which went unnoticed by
`the operator.
`Administrative errors led to the remaining
`three false negative results. In one instance, a
`mutation detected in one sample was incor-
`rectly assigned to another
`that was also
`mutation positive, but both of these mutations
`were subsequently lost in the final report. An
`additional mutation, 3875del4, was identified
`correctly in CSGE and sequence analysis, but
`reported as negative owing to a clerical error.
`
`TDGS
`The laboratory using TDGS correctly identi-
`fied 51 of 56 mutations (91%);
`the two
`remaining mutations could not be identified
`owing to repeated failure of sequence analysis
`(table 2C). In addition, three apparently false
`positive results were reported following se-
`quence analysis as described below.
`Each of the five mutations missed by TDGS
`(L246V, IVS17+1G>T, Y1463X, 3171ins5,
`and 4510del3insTT) appear to be result of
`misinterpretation of the 2D gel. In each of
`these cases, the mutant allele amplified much
`less eYciently than the wild type, resulting in
`one intense wild type homoduplex, one very
`light or absent mutant homoduplex, and two
`very light heteroduplexes.
`The TDGS laboratory reported three muta-
`tions that were otherwise not identified by any
`other techniques, including sequence analysis,
`a