Rapid Mutation Scanning of Genes Associated with Familial Cancer ...

RESEARCH ARTICLE

Neoplasia . Vol. 3, No. 3, 2001, pp. 236 – 244

236

www.nature.com/neo

Rapid Mutation Scanning of Genes Associated with Familial Cancer Syndromes Using Denaturing High-Performance Liquid Chromatography1 Deborah J. Marsh* y, George Theodosopoulos*, Viive Howell z, Anne -Louise Richardson*, Diana E. Benn*, Anne´ L. Proos z, Charis Eng x { and Bruce G. Robinson* y *Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia; yDepartment of Medicine, University of Sydney, NSW 2006, Australia; zLaboratory and Community Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia; xClinical Cancer Genetics and Human Cancer Genetics Program, Comprehensive Cancer Center, and Division of Human Genetics, Department of Internal Medicine, The Ohio State University, 690C Medical Research Facility, 420 West 12th Avenue, Columbus, OH 43201; and {CRC Human Cancer Genetics Research Group, University of Cambridge, UK

Abstract Germline mutations in tumor suppressor genes, or less frequently oncogenes, have been identified in up to 19 familial cancer syndromes including Li - Fraumeni syndrome, familial paraganglioma, familial adenomatous polyposis coli and breast and ovarian cancers. Multiple genes have been associated with some syndromes as approximately 26 genes have been linked to the development of these familial cancers. With this increased knowledge of the molecular determinants of familial cancer comes an equal expectation for efficient genetic screening programs. We have trialled denaturing high performance liquid chromatography ( dHPLC ) as a tool for rapid germline mutation scanning of genes implicated in three familial cancer syndromes — Cowden syndrome ( PTEN mutation ), multiple endocrine neoplasia type 2 ( RET mutation ) and von Hippel - Lindau disease ( VHL mutation ). Thirty - two mutations, including 21 in PTEN, 9 in RET plus a polymorphism, and 2 in VHL, were analyzed using the WAVE DNA fragment analysis system with 100% detection efficiency. In the case of the tumor suppressor gene PTEN, mutations were scattered along most of the gene. However, mutations in the RET proto - oncogene associated with multiple endocrine neoplasia type 2 were limited to specific clusters or ‘‘hot spots.’’ The use of GC - clamped primers to scan for mutations scattered along PTEN exons was shown to greatly enhance the sensitivity of detection of mutant hetero - and homoduplex peaks at a single denaturation temperature compared to fragments generated using non – GC - clamped primers. Thus, when scanning tumor suppressor genes for germline mutation using dHPLC, the incorporation of appropriate GC clamped primers will likely increase the efficiency of mutation detection. Neoplasia ( 2001 ) 3, 236 – 244. Keywords: dHPLC, mutation detection, PTEN, RET, VHL.

Introduction The identification of genes associated with familial cancer syndromes has created a commensurate requirement for the efficient scanning of these genes in at - risk family members. Familial cancers account for up to 10% of all malignancies. Germline mutation in a tumor suppressor or, less frequently an oncogene, has been identified as the primary genetic event linked to an increased risk of developing specific malignancies in approximately 19 familial syndromes. In some cases, such as hereditary nonpolyposis colon cancer ( HNPCC ), mutation of more than one gene has been associated with the clinical phenotype, implicating approximately 26 genes as ‘‘caretakers’’ or ‘‘gatekeepers’’ for the development of human cancer in the familial setting ( Ref. [ 1 ], reviewed in Ref. [ 2 ] ). It has been estimated that as many as 50 different genes may eventually be identified where carriers of heterozygous mutations will have an increased risk of developing cancer [ 3 ]. With this increased clarification of the molecular basis of many inherited cancers comes the opportunity to offer efficient genetic screening programs coupled with the collection of family histories that will allow for noninvasive assessment of asymptomatic at - risk individuals, increase the diagnostic power of clinicians and permit directed cancer surveillance. We, and others, have previously reported

Abbreviations: BRR, Bannayan - Riley - Ruvalcaba; CS, Cowden syndrome; DGGE, denaturing gradient gel electrophoresis; dHPLC, denaturing high - performance liquid chromatography; MTC, medullary thyroid carcinoma; MEN 2, multiple endocrine neoplasia; PTEN, phosphatase and tensin homolog deleted on chromosome ten; RET, Rearranged during transformation; SSCP, single - strand conformation polymorphism; VHL, von Hippel Lindau Address all correspondence to: Dr. Deborah J. Marsh, Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, Sydney, NSW 2065, Australia. E-mail: [email protected] 1 Funding from the American Cancer Society Grant ( RPG - 98 - 211 - 01 - CCE to CE ) and the National Cancer Institute, Bethesda, MD ( P30CA16058 to the Ohio State University Comprehensive Cancer Center ) is gratefully acknowledged. Received 31 January 2001; Accepted 2 March 2001. Copyright # 2001 Nature Publishing Group All rights reserved 1522-8002/01/$17.00

dHPLC for Mutation Detection in Familial Cancer

genetic screening of patients and their relatives with three distinct inherited cancer syndromes — multiple endocrine neoplasia type 2 ( MEN 2 ) [ OMIM#171400 ] and germline mutation of the proto - oncogene RET; von Hippel - Lindau disease ( VHL ) [ OMIM#193300 ] and germline mutation of the tumor suppressor VHL; and Cowden syndrome ( CS ) [ OMIM#158350 ] with germline mutation of the tumor suppressor PTEN [ 4 – 7 ]. In addition, Bannayan - Riley Ruvalcaba syndrome ( BRR ) [ OMIM#153480 ], has been shown to be part of the clinical spectrum of CS, and approximately half carry a germline mutation of PTEN [ 8 ]. The most common clinical features of MEN 2, VHL, CS, and BRR are summarized in Table 1. In the case of the tumor suppressor genes PTEN and VHL, mutations are scattered along the majority of the gene. However, in contrast, mutations in the RET proto - oncogene associated with MEN 2 are targeted to specific codons. The mutation type, location and the frequency with which these genes are mutated in the germline in association with a familial cancer syndrome are summarized in Table 2. Comprehensive mutation summaries for these and additional genes can be located at the Human Gene Mutation Database ( HGMD ) website, http: / / www.uwcm.ac.uk / uwcm / mg / hgmd0.html. In this study, we report the efficient use of denaturing high - performance liquid chromatography ( dHPLC ) for mutation scanning in three familial cancer syndromes, and

Marsh et al.

237

their variants, where mutations are either clustered in ‘‘hot spots’’ or scattered along the entire gene.

Materials and Methods Patients and Samples Blood samples from the probands of 21 families with CS and / or BRR, 9 families with MEN 2, and 2 families with VHL were collected for analysis. Constitutional DNA was extracted from blood leucocytes using standard techniques [ 18 ]. Germline mutation of PTEN had previously been reported in 20 of the CS or BRR families identified using denaturing gradient gel electrophoresis ( DGGE ), temporal temperature gel electrophoresis ( TTGE ), or sequencing [ 4,5 ] ( Table 3 ). All RET mutants had previously been reported by us or others [ 6,13 ]. The two VHL mutants described in this study had previously been reported by others [ 14,19 ] ( http: / / www.uwcm.ac.uk / uwcm / mg / hgmd0.html ) ( Table 3 ). In addition, blood was collected from four at - risk members of one of the VHL families for analysis. Polymerase Chain Reaction and Sequencing Primer sequences and PCR conditions for the amplification of GC - clamped PTEN fragments have been previously reported [ 20 – 23 ]. In addition, four PTEN exons, or amplicons, and flanking intronic sequences were amplified

Table 1. Common Clinical Features of CS, the Related Syndrome BRR, MEN 2, and VHL. Syndrome

Tumor Types

Other Features

Cowden Syndrome ( CS )

o breast cancer

o trichilemmomas

o thyroid cancer

o papillomatous papules

o endometrial cancer

o Lhermitte - Duclos disease o gastrointestinal polyps o lipomatosis o macrocephaly o macrocephaly

Bannayan - Riley - Ruvalcaba Syndrome ( BRR )

o lipomatosis o pigmented macules of the glans penis o hemangioma o gastrointestinal polyps o mild mental retardation Multiple endocrine neoplasia type 2 ( MEN 2 ) FMTC*

o medullary thyroid carcinoma

MEN 2A


o parathyroid hyperplasia

o pheochromocytoma MEN 2B

von Hippel - Lindau disease ( VHL )


o Marfanoid habitus

o pheochromocytoma

o mucosal neuromas

o pheochromocytoma

o angiomata of the retina

o cerebellar hemangioblastoma o renal cell carcinoma o spinal hemangioblastoma o endolymphatic sac tumors ( rare ) *FMTC — familial medullary thyroid carcinoma.

Neoplasia . Vol. 3, No. 3, 2001

238

dHPLC for Mutation Detection in Familial Cancer Marsh et al.

Table 2. Causative Genes, Their Mutation Type, Location and Mutation Frequency, in CS, BRR, MEN 2, and VHL. Syndrome

Gene

CS

PTEN

Exons 9

Mutation Frequency

( Reference )

Types of Germline Mutations Observed

13 – 81%

( 4, 9 – 12 )

o point nonsense

o exons 2 – 9

Mutation Location

o point missense

o clusters in exons 5,* 7,y and 8.z

o microdeletions o microinsertions o microdeletion – insertions o splice site BRR

PTEN

9

50 – 60%

( 5, 8 )

o ‘‘as above’’

o ‘‘as above’’

o gross hemizygous deletion o balanced translocation MEN 2

RET

20

> 92%

( 6, 13 )

o point missense

o exon 10 ( codons 609, 611, 618, 620 ) o exon 11 ( codons 630, 634 ) o exon 13 ( codons 768, 790, 791 ) o exon 14 ( codon 804 ) o exon 15 ( codon 883 [ MEN 2B only ], 891 ) o exon 16 ( codon 918 [ 95% of MEN 2B ] )

VHL

VHL

3

75 – 100%

( 14 – 16 )

o point nonsense

o all exons

o point missense o microdeletions o microinsertions o microdeletion – insertions o splice site o gross hemizygous deletion o complex rearrangements *Exon 5 encodes the protein tyrosine phosphatase core motif of PTEN. Exons 7 and 8 are believed to encode the potential tyrosine and serine phosphorylation sites ( reviewed in Ref. [ 17 ] ).

y,z

using non – GC - clamped primers, identical to the GC clamped primers previously published, however, missing the GC - clamp as follows: amplicon 5I, 5IFnogc 50 - TTT TTT CTT ATT CTG AGG TTA TC - 30 and reverse primer as previously reported [ 21 ]; amplicon 5II, 5IIRnogc 50 - GAA GAG GAA AGG AAA AAC ATC - 30 and forward primer as previously reported [ 21 ]; exon 7, 7Fnogc 50 - AAT AAT ACT GGT ATG TAT TTA AC - 30 and 7Rnogc 50 - GAT ATT TCT CCC AAT GAA AG - 30; exon 8, 8Fnogc 50 - GTT TCA CTT TTG GGT AAA TA - 30 and 8Rnogc 50 - TGC TTC GAA ATA ATA CAT AA - 30. PCR conditions were identical to those previously published for the amplification of GC - clamped PTEN fragments [ 20,21,23 ]. PCR conditions and primer sequences for amplification of the RET proto - oncogene have, in general, been previously reported. Exon 10 was amplified using 7F and 7R with the addition of 10% dimethyl sulfoxide ( Sigma Chemical Co, Castle Hill, NSW, Australia ) [ 24 ]; exon 11 was amplified using 8AR [ 24 ] and 11F, 50 - AGC CAT GAG GCA GAG CAT AC - 30; exon 15 was amplified using 17B and 17G [ 25 ], and exon 16 was amplified using fRET16 and rRET16 [ 26 ]. Primer sequences for the amplification of VHL exon 3 were modified from those previously published [ 27 ], specifically 40Fmod, 50 - CTG AGA CCC TAG TCT GCC ACT GAG - 30 and 41Rmod, 50 - CAA AAG CTG AGA TGA AAC AGT GTA - 30. PCR reaction conditions consisted of one cycle of denaturation at 948C for 5 minutes, followed by 40

cycles of denaturation at 948C for 1 minute, annealing at 618C for 1 minute and extension at 728C for 1 minute, followed by 728C for 10 minutes. The final concentration of PCR reagents were as follows: 0.4 M of each primer, 0.05 mM of each deoxyribonucleotide triphosphate, 1PCR buffer without MgCl2 ( Perkin - Elmer Corp., Norwalk, CT ), 1.5 mM MgCl2 and 0.5 l of Taq polymerase ( 5 U / l ) ( Perkin - Elmer ). PCR reactions were performed in a final volume of 50 l. All PCR products were generated using a MJ Peltier Thermal Cycler PTC - 100 ( MJ Research, Watertown, MA ). PCR products destined for dHPLC analysis underwent additional cycling after amplification, consisting of a denaturation step of 948C for 5 minutes followed by ramping to 258C over 45 minutes to allow the formation of homo - and heteroduplexes in samples heterozygous for a mutant allele. No additional purification procedures were required. Product sizes ranged from 178 to 396 nucleotides. PCR products destined for sequence analysis were purified using the Wizard PCR Preps DNA Purification System ( Promega, Madison, WI ). Direct sequencing of these products was performed using the ABI Prism BigDye Terminator cycle sequencing ready reaction kit ( Applied Biosystems, Perkin Elmer ). Purified cycle sequencing products were electrophoresed on 4.8% PAGE Plus gels ( Amresco, Cleveland, OH ) and analyzed on a 377XL automated DNA sequencer ( Applied Biosystems, Perkin - Elmer ).



Marsh et al.

239

Table 3. Mutation Summary and Temperatures for Detection Using the WAVE. Gene

PTEN

Family Identifier Ban1

Exon

2

CDdl Ban2

3

Temperature of mutant detection

A34D

56 – 578C

54 – 568C

578C*

55 – 578C

54 – 568C

568C*

59 – 618C

58 – 608C

608C*; 598C

58 – 608C

57 – 598C

598C*; 588C

58 – 608C

58 – 608C

598C*

58 – 608C

57 – 598C

598C*; 588C

56 – 588C

56 – 588C

578C*; 578C

N/A

66 – 688C

668C

N/A

65 – 678C

658C

N/A

63 – 658C

638C

Y68H

5Iy

Q110X

Cdve

c.302 – 304delTCAinsCC

Ban4

C105Y

Ban5

c.347delG

CDtm

c.347 – 351delACAAT 5IIy

Ban7 Ban9

6

S170R 7

B / CS5 B / CS7

c.520 – 544del25 IVS6 + 5G > T

Ban8 Ban11

R130X I135V

Ban10

R233X IVS6 - 1G > C

8

c.987 – 990TAAAdel

Ban15

c.950 – 953delTACT

CD93

R335X

B / CS6

IVS7 - 4insT

Ban14 073

c.886insCT 10

023 029

C618R C620R

11

C634R

126

C634G

068

C634Y

144 041

C634S 15

A883Fz;S904S

180 - 01

S891A

180 - 02

S891A;S904S

181 VHL

Predicted temperature range ( non GC - clamped fragment )

IVS2 - 2A > G

CDst

RET

Predicted temperature range ( GC - clamped fragment )

R47G

CDmy CDcl

Mutation / Polymorphism

S904S

174

16

M918T

N/A

59 – 608C

598C

V01

3

T157I

N/A

60 – 628C

618C

V02

R161X

*Temperatures indicated for GC - clamped fragments only. PTEN exon 5 is amplified in two amplicons. z 508C will detect A883F ( Figure 2C ) but not S904S. y

Denaturing HPLC DHPLC was undertaken on a WAVE DNA fragment analysis system ( Transgenomic, Omaha, NE ). Two to five microliters of heteroduplexed PCR fragments was injected onto the DNASep cartridge. Products were eluted at a constant flow rate of 0.9 ml / min with a linear acetonitrile gradient determined by WAVEMaker software ( Transgenomic, Omaha, NE ) based on the size and GC content of the amplicon. The gradient was achieved by combining 0.1 M triethylammonium acetate ( TEAA ) buffer ( pH 7 ) ( Transgenomic ) and Buffer B ( 0.1 M TEAA with 25% acetonitrile ) ( Transgenomic ). Eluted DNA fragments were detected by the system’s UV detector and analyzed as chromatograms. The processing time for each sample was comprised of a 2.2 - minute lag for the detector, 0.5 minutes loading with a 5% decrease in Buffer B, a 4.5 - minute linear gradient with a slope of 2% increase in Buffer B per minute, a 0.5 - minute cleaning stage using 75% acetonitrile, and a 0.9 - minute


equilibration before the next injection. Homo - and heteroduplex peaks were detected between the initial injection peak, produced by residual nucleotides and primers in the reaction, and the wash peak, produced by the acetonitrile flush at the end of each analysis. Melt profiles were constructed using the WAVEMaker software ( Transgenomic ) using PCR fragments in the size range of 178 to 344 base pairs ( non – GC - clamped fragments ) and 218 to 396 base pairs ( GC - clamped fragments ). A range of partial denaturation temperatures was predicted by this software ( Table 3 ). PCR products were initially analyzed under nondenaturing conditions, 508C, to assess the quality of the peak representing both hetero - and homoduplex DNA fragments in a mutant sample. All temperatures within the predicted range for partial denaturation were then assessed for their ability to resolve homo - and heteroduplexes. A single temperature was chosen where all mutants could be resolved. Given that heteroduplexes are

240


less stable, they denatured earlier than the homoduplexes and so were seen first in the elution profile.

Results Thirty - two known mutations and one polymorphism previously determined by sequence analysis and / or restriction endonuclease digestion, 21 in PTEN, 9 in RET plus a polymorphism, and 2 in VHL, were detected by dHPLC at temperatures within the range predicted by the WAVEMaker software ( Transgenomic ) ( Table 3 ). The mutation R47G in exon 2 of PTEN identified in a family with CS has not been previously reported. The clinical symptoms of CS in this family included gastrointestinal polyps, multiple fibroadenomas of the breast, Hashimoto’s thyroiditis and goiter, lipomas, and papules of the oral mucosa. The value of GC - clamped primers for use in dHPLC mutation detection was directly assessed in four PTEN exons or amplicons — 5I, 5II, 7, and 8. Two to five separate mutations, well scattered over the exon, were analyzed in each case for detection of all mutants at a single temperature. The nonsense PTEN point mutation R233X in exon 7 was poorly discernible when analysis was undertaken using non – GC - clamped primers at all temperatures predicted by the WAVEMaker software ( Transgenomic ). In addition, temperatures up to 708C, at which the peak had largely

collapsed, were unable to resolve this mutant. However, resolution of the heteroduplexed fragments was clearly observed using a PCR amplicon generated by identical primers with the addition of a GC - clamp ( Figure 1A ). Of interest, the elution profile of the wild - type DNA of GC clamped PTEN exon 7 shows an apparent peak preceding the homoduplex peak ( Figure 1A ). This peak was not observed in non – GC - clamped PTEN exon 7. The same wild - type DNA was used for both experiments and shown by sequence analysis to be free of sequence variants. It is most likely that this apparent peak represents an artifact introduced by the GC clamp. Clearly, however, the GC - clamped PTEN exon 7 wild type shows a distinctly different elution profile to both the R233X and IVS6 - 1G > C exon 7 mutants. Artifact peaks before the appearance of homo - and heteroduplex peaks in the elution profile likely due to the method of sample preparation were also detected ( Figure 2A ). These artifact peaks could be decreased relative to true homo - and heteroduplex peaks by dilution of the template DNA before PCR amplification, excluding the possibility that they represented true peaks. The presence of artifacts highlights the importance of including wild - type DNA controls in each experiment. An intronic polymorphism present in amplicon 8, IVS8 + 32 T / G [ 28 ] was not detectable at the temperature used for mutation analysis of exon 8 ( Table 3, data not shown ). This

Figure 1. dHPLC analysis of PTEN exons or amplicons. ( A ) PTEN exon 7 analyzed as both a GC - clamped and a non – GC - clamped fragment. The mutation R233X is scarcely detectable in the elution profile of the nonclamped exon 7 fragment compared to the GC - clamped fragment. Increased resolution of the elution profile is also observed for the splice site mutation IVS6 - 1G > C. ( B ) PTEN amplicon 5I analyzed as both a GC - clamped and a non – GC - clamped fragment. Two missense point mutations, C105Y and Q110X, a small deletion – insertion, c.302 – 304delTCAinsCC, and a deletion, c347 – 351delACAAT, were able to be discriminated from the wild - type sequence using non – GC - clamped primers, however, resolution was significantly improved using primers with a GC clamp.



Marsh et al.

241

Figure 2. dHPLC analysis of RET and VHL exons. ( A – E ) Nine clustered mutations and a polymorphism in RET exon 10, 11, 15, and 16 were able to be resolved. C618R and C620R ( exon 10 ), C634R and C634Y ( exon 11 ), A883F, S891A and S904S ( exon 15 ), and M918T ( exon 16 ) showed elution profiles distinctly resolvable from the wild - type amplicon for each exon. C634G and C634S ( exon 11 ) showed elution profiles with more subtle variation from wild - type exon 11. ( F ) The elution profiles of two missense point mutations in VHL exon 3, T157I and R161X, were clearly distinct from wild - type sequence.

was likely due to this variant’s location in an almost fully denatured region of the melt profile. In general, the use of GC - clamped primers for PCR amplification of PTEN greatly improved the resolution of hetero - and homoduplex peaks, dramatically improving the efficiency of user interpretation ( Figure 1, A and B ). Given the clustered nature of RET mutations, dHPLC was optimized using non – GC - clamped primers ( Table 3 ). All nine missense mutations were able to be differentiated


from the wild - type DNA homoduplexes in the four exons analyzed ( Figure 2, A – E ). C634G and C634S in RET exon 11 ( Figure 2B ) showed more subtle changes in their elution profiles, yet were still discernible from wild - type DNA. A silent polymorphism in RET exon 15 resulting from a C!G substitution, S904S, was detected at 638C ( Figure 2D ) [ 29 ]. This polymorphism was also present in the individual shown to carry the A883F mutation ( 041, Table 3 ). Of interest, this polymorphism was not detected at

242


508C in the wild - type DNA, subsequently shown by sequence analysis to be positive for S904S. However, DNA from individual 041 containing both the A883F mutation and the S904S polymorphism did form heteroduplexes at this temperature ( Figure 2C ). The mutant S891A was unable to be resolved at 508C. Therefore, even though the polymorphism was unable to be detected at 508C, thus excluding it as a normal variant in the elution profile, the inability to detect all RET exon 15 mutants at this temperature excluded the possibility of conducting mutation scanning at this temperature. Only two VHL mutants, mapping within nine nucleotides of each other in VHL exon 3, were available for analysis. Again, given the close proximity of these mutants, dHPLC was optimized using non – GC - clamped primers ( Table 3, Figure 2F ). Further, dHPLC was used to screen four additional asymptomatic at - risk members of one of the VHL families, V02. The elution profile for all asymptomatic individuals, aged 5 to 11 years, matched that of the proband and was clearly different to wild - type DNA analyzed as a control in the same experiment. Sequence analysis confirmed that these individuals were positive for R161X.

Discussion The underlying primary genetic mutations associated with an increasing number of familial cancer syndromes are rapidly being identified. Greater understanding of the molecular basis of many of these inherited syndromes allows for the development of efficient genetic screening programs to identify family members at risk of developing cancer, thus allowing directed cancer surveillance for mutation carriers. We have employed a dHPLC system, WAVE DNA fragment analysis system ( Transgenomic ), for rapid germline mutation screening in two tumor suppressor genes, PTEN and VHL, and one oncogene, RET, mutations of which are associated with CS, VHL disease, and MEN 2, respectively. The efficiency of mutation detection in PTEN was increased by the use of GC - clamped primers resulting in 100% ( N = 32 ) of mutations plus a polymorphism being identified by dHPLC. These mutations had previously been identified by the more labor - intensive techniques of DGGE and TTGE ( PTEN ) [ 4,5 ]; sequencing and / or restriction endonuclease digestion ( PTEN, VHL, and RET ) [ 4,5,16,30 ]. In addition, dHPLC was used to provide a rapid screen of four asymptomatic individuals, age range 5 to 11 years, of unknown genetic carrier status in the VHL family V02. All individuals were shown to be positive for the R161X mutation and appropriate, directed cancer surveillance has now been recommended for these patients. Although mutation hot spots are seen in many of the tumor suppressor genes linked to familial cancer syndromes, in general, germline mutations are scattered largely along the entirety of the gene [ 31 – 33 ]. Germline mutation of PTEN in CS and BRR also follows this pattern, making efficient dHPLC mutation detection at a single temperature for each specific amplicon impossible due to variation of the fragment’s melt profile. For reliable mutation detection using

non – GC - clamped primers, analyses will need to be performed at multiple temperatures to ensure detection of all mutants as has been previously reported [ 34 ]. In general, the use of GC - clamped primers allows the generation of a more uniform melt profile, thus leading to improved resolution of hetero - and homoduplex peaks in the elution profile. Analyses performed at a single temperature are then more likely to generate elution profiles clearly defining mutant samples, regardless of the position of the mutation in the amplicon. In a direct comparison of GC - clamped and non – GC - clamped PTEN amplicons, mutants that were poorly discernible as nonclamped fragments were able to be clearly resolved when generated as part of a GC clamped amplicon ( Figure 1 ). Perhaps the most dramatic example of this increased resolution was seen in the case of the germline mutant R233X ( Figure 1A ). This mutant was reported in a study of somatic PTEN mutations in small cell lung carcinoma using dHPLC where it was also shown to be poorly resolved [ 35 ]. In general, the use of GC - clamped primers for dHPLC greatly improved the resolution of homo and heteroduplex peaks, clearly improving the efficiency of this methodology. Similar to other more traditional mutation scanning methods such as DGGE and SSCP, the presence of polymorphisms can present a complicating factor when using dHPLC to screen for germline mutations. Although a known intronic polymorphism in PTEN, IVS8 + 32 T / G, was not detected at the temperature chosen to screen for mutants, the RET polymorphism S904S was clearly evident in the elution profile of the fragment containing RET exon 15 ( Figure 2D ). The inability to detect the PTEN intronic polymorphism was likely due to its location in an almost fully denatured region of the melt profile. However, the presence of the RET exon 15 polymorphism likely influenced the elution profile of the A883F mutant, as the single patient with this mutation, a case of de novo MEN 2B, also had this polymorphism ( Figure 2D ). An earlier study using dHPLC to screen for RET mutations only analyzed exon 10 mutants [ 34 ]. To date, polymorphisms have not been reported in this exon. The presence of polymorphisms must be considered when using dHPLC to screen members of hereditary cancer families at risk of inheriting the disease allele. A second genetic aberration that complicates screening with dHPLC is gross germline deletion. To date, gross germline deletion of PTEN has not been reported to be associated with CS; however, three cases of gross germline deletion of the 10q23 region encompassing PTEN have been reported [ 5,36,37 ]. Gross germline deletion of VHL and its flanking intervals is a more common phenomenon and has been reported in up to 25% of VHL families [ 7 ]. It is important to recognize that dHPLC would be an unsuitable mutation detection method in cases of germline whole gene deletion as the presence of only one wild - type allele would preclude the formation of heteroduplexes and thus a definitive elution profile. In the case of VHL screening, dHPLC could be used as an initial screen for the detection of germline intragenic mutations. If no mutations were detected in this first screen, DNA from



probands of these families could undergo alternative screening to detect putative deletions of the entire VHL gene using methods such as quantitative Southern blotting or fluorescence in situ hybridization as have been previously recommended [ 16 ]. The arising complication from the presence of a single allele when screening with dHPLC is also relevant when studying somatic mutations of tumor suppressor genes in tissues where the wild - type allele is lost. A study of somatic PTEN mutation by dHPLC in 63 malignant gliomas showed that low - level amounts of normal contaminant DNA, 10% to 20%, enabled the formation of detectable heteroduplexes in 13 samples where the wild - type PTEN allele had been lost and a mutation was present in the retained allele [ 34 ]. However, perhaps more reliably for the analysis of DNA from tumor tissue, wild - type PCR product could be admixed with the tumor or cell line PCR product known to show allelic loss to mimic the presence of a wild - type allele, then denatured, reannealed and screened for heteroduplex peaks by dHPLC [ 35 ]. Given the examples of VHL and PTEN where whole gene deletion has been identified in the germline, a wild - type allele is also retained. Therefore the method of admixing wild - type DNA with a test sample would not be suitable for germline DNA analyses. A number of studies have been performed to directly compare dHPLC with the more established mutation detection methodologies of single - strand conformation polymorphism and heteroduplex analysis. In studies of known mutations of BRCA1, the cystic fibrosis transmembrane conductance regulator gene, CFTR, and the tuberous sclerosis complex genes, TSC1 and TSC2, dHPLC detected 96% to 100% of mutations, SSCP 85% to 94%, and heteroduplex analysis only 82% [ 31,38 ]. In a further study of 150 unrelated cases with tuberous sclerosis, dHPLC was shown to detect mutations in 68% of cases, SSCP in 61% and heteroduplex analysis in only 58% [ 39 ]. In addition, a blinded comparison of DGGE and dHPLC used to screen BRCA1 in 41 probands from hereditary breast and ovarian cancer families showed that dHPLC was able to detect a mutant that was not identified by DGGE [ 40 ]. To date, no false - positives have been identified using dHPLC [ 31,33,34,41 ]. In summary, we have described a novel application for GC - clamped amplicons to increase the sensitivity of dHPLC allowing accurate screening at a single temperature of the tumor suppressor gene PTEN associated with CS and the related syndrome BRR. This method has applicability to other tumor suppressor genes associated with familial cancer syndromes including BRCA1, BRCA2, and MEN1 where mutations are scattered largely along the entirety of the gene. When planning the implementation of a germline genetic screening program for inherited cancer syndromes incorporating dHPLC, it is essential to take into consideration the spectrum of mutations, including gross germline deletion and polymorphisms, that have been previously described to permit accurate interpretation of dHPLC elution profiles.


Marsh et al.

243

Acknowledgements We are grateful to the patients who participated in this study. Roberto Zori is thanked for his critical reading of this manuscript. The Australian Genome Research Facility ( AGRF ) is acknowledged for commercial sequencing. John Morris Scientific ( Sydney, Australia ) is acknowledged for allowing use of the WAVE DNA Fragment Analysis System ( Transgenomic ).

References [1] Kinzler KW, and Vogelstein B ( 1998 ). Landscaping the cancer terrain. Science 280, 1036 – 1037. [2] Petersen GM, and Codori A - M ( 1998 ). Genetic testing for familial cancer. In The Genetic Basis of Human Cancer. B Vogelstein and KW Kinzler ( Eds ). The McGraw - Hill Companies, Inc., USA. pp. 591 – 599. [3] Knudson AG ( 1996 ). Hereditary cancer: two hits revisited. J Res Clin Oncol 122, 135 – 140. [4] Marsh DJ, Coulon V, Lunetta KL, Rocca - Sera P, Dahia PLM, Zheng Z, Liaw D, Caron S, Duboue B, Lin AY, Richardson A - L, Bonnetblanc J M, Bressieux JM, Cabbot - Moreau A, Chompret A, Demange L, Eeles RA, ELES, Yahanda AM, Fearon ER, Fricker J - P, Gorlin RJ, Hodgson SV, Huson S, Lacombe D, Leprat F, Odent S, Toulouse C, Olopade OI, Sobol H, Tishler S, Woods CG, Robinson BG, Weber HC, Parsons R, Peacocke M, Longy M, and Eng C ( 1998 ). Mutation spectrum and genotype – phenotype analyses in Cowden disease and Bannayan Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum Mol Genet 7, 507 – 515. [5] Marsh DJ, Kum JB, Lunetta KL, Bennet MJ, Gorlin RJ, Ahmed F, Bodurtha J, Crowe C, Curtis MA, Dasouki M, Dunn T, Feit H, Geraghty MT, Graham JM Jr, Hodgson SV, Hunter A, Korf BR, Manchester D, Miesfeldt S, Murday VA, Nathanson KL, Parisi M, Pober B, Romano C, Tolmie JL, Trembath R, Winter RM, Zackai EH, Zori RT, Weng L - P, Dahia PLM, and Eng C ( 1999 ). PTEN mutation spectrum and genotype – phenotype correlations in Bannayan - Riley - Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum Mol Genet 8, 1461 – 1472. [6] Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, Ploos van Amstel H.K, Lips CJM, Nishisho I, Takai S - I, Marsh DJ, Robinson BG, Frank - Raue K, Raue F, Xue F, Noll WW, Romei C, Pacini F, Fink M, Niederle B, Zedenius J, Nordenskjold M, Komminoth P, Hendy GN, Gharib H, Thibodeau SN, Lacroix A, Frilling A, Ponder BAJ, and Mulligan LM ( 1996 ). The relationship between specific RET proto oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2: International RET mutation consortium analysis. J Am Med Assoc 276, 1575 – 1579. [7] Richards F, Webster A, McMahon R, Woodward E, Rose S, and Maher E ( 1998 ). Moleculargenetic analysis of von Hippel - Lindau disease. J Intern Med 243, 527 – 533. [8] Marsh DJ, Dahia PLM, Zheng Z, Liaw D, Parsons R, Gorlin RJ, and Eng C ( 1997 ). Germline mutations in PTEN are present in Bannayan Zonana syndrome. Nat Genet 16, 333 – 334. [9] Liaw D, Marsh DJ, Li J, Dahia PLM, Wang SI, Zheng Z, Bose S, Call KM, Tsou HC, Peacocke M, Eng C, and Parsons R ( 1997 ). Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 16, 64 – 67. [10] Nelen MR, van Staveren WCG, Peeters EAJ, Hassel MB, Gorlin RJ, Hamm H, Lindboe CF, Fryns J - P, Sijmons RH, Woods DG, Mariman ECM, Padberg GW, and Kremer H ( 1997 ). Germline mutations in the PTEN / MMAC1 gene in patients with Cowden disease. Hum Mol Genet 6, 1383 – 1387. [11] Tsou HC, Teng D, Ping XI, Broncolini V, Davis T, Hu R, Xie X - X, Gruener AC, Schrager CA, Christiano AM, Eng C, Steck P, Ott J, Tavitigian SV, and Peacocke M ( 1997 ). Mutations of MMAC1 in early - onset breast cancer: causative in association with Cowden syndrome and excluded in BRCA1 - negative cases. Am J Hum Genet 61, 1036 – 1043. [12] Kohno T, Takahashi M, Fukutomi T, Ushio K, and Yokota J ( 1998 ). Germline mutations of the PTEN / MMAC1 gene in Japanese patients with Cowden disease. Jpn J Cancer 89, 471 – 474. [13] Smith DP, Houghton C, and Ponder BAJ ( 1997 ). Germline mutation of RET codon 883 in two cases of de novo MEN 2B. Oncogene 15, 1213 – 1217.

244


[14] Chen F, Kishida T, Yao M, Hustad T, Glavac D, Dean M, Gnarra JR, Orcutt ML, Duh F - M, Glenn G, Green J, Hsia YE, Lamiell J, Li H, Wei MH, Schmidt L, Tory K, Kuzman I, Stackhouse T, Latif F, Linehan WM, Lerman M, and Zbar B ( 1995 ). Germline mutations in the von Hippel Lindau disease tumor suppressor gene: correlations with phenotype. Hum Mutat 5, 66 – 75. [15] Maher ER, Webster AR, Richards FM, Green JS, Crossey PA, Payne SJ, and Moore AT ( 1996 ). Phenotypic expression in von Hippel Lindau disease: correlations with germline VHL gene mutations. J Med Genet 33, 328 – 332. [16] Stolle C, Glenn G, Zbar B, Humphrey JS, Choyke P, Walther M, Pack S, Hurley K, Andrey C, Klausner R, and Linehan WM ( 1998 ). Improved detection of germline mutations in the von Hippel - Lindau disease tumor suppressor gene. Hum Mutat 12, 417 – 423. [17] Marsh DJ, and Stratakis C ( in press ). Hamartoma syndromes: clinical and molecular aspects. In: Genetic Disorders of Endocrine Neoplasia. PLM Dahia and C Eng ( Eds ). Karger, Basel, Switzerland ( in press ). [18] Mathew CG, Smith BA, Thorpe K, Wong Z, Royle NJ, Jeffreys AJ, and Ponder BAJ ( 1987 ). Deletion of genes on chromosome 1 in endocrine neoplasia. Nature 328, 524 – 526. [19] Crossey PA, Richards FM, Foster K, Green JS, Prowse A, Latif F, Lerman MI, Zbar B, Affara NA, Ferguson - Smith MA, and Maher ER ( 1994 ). Identification of intragenic mutations in the Von Hippel - Lindau disease tumour suppressor gene and correlation with disease phenotype. Hum Mol Genet 3, 1303 – 1308. [20] Marsh DJ, Roth S, Lunetta KL, Hemminki A, Dahia PLM, Sistonen P, Zheng Z, Caron S, van Orsouw NJ, Bodmer WF, Cottrell SE, Dunlop MG, Eccles D, Hodgson SV, Jarvinen H, Kellokumpu I, Markie D, Neale K, Phillips R, Rozen P, Syngal S, Vijg J, Tomlinson IPM, Aaltonen LA, and Eng C ( 1997 ). Exclusion of PTEN and 10q22 – 24 as the susceptibility locus for juvenile polyposis syndrome. Can Res 57, 5017 – 5021. [21] Guldberg P, Straten PT, Birck A, Ahrenkiel V, Kirkin AF, and Zeuthen J ( 1997 ). Disruption of the MMAC1 / PTEN gene by deletion or mutation is a frequent event in malignant melanoma. Can Res 57, 3660 – 3663. [22] Mutter GL, Lin M - C, Fitzgerald JT, Kum JB, Baak JPA, Lees JA, Weng L - P, and Eng C ( 2000 ). Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst 92, 924 – 931. [23] Marsh DJ, Dahia PLM, Caron S, Kum JB, Frayling IM, Tomlinson IPM, Hughes KS, Eeles RA, Hodgson SV, Murday VA, Houlston R, and Eng C ( 1998 ). Germline PTEN mutations in Cowden syndrome - like families. J Med Genet 35, 881 – 885. [24] Donis - Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lairmore TC, Howe JR, Moley JF, Goodfellow P, and Wells SAJ ( 1993 ). Mutations in the RET proto - oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 2, 851 – 856. [25] Mulligan LM, Eng C, Attie T, Lyonnet S, Marsh DJ, Hyland VJ, Robinson BG, Frilling A, Verellen - Dumoulin C, Safar A, Venter DJ, Munnich A, and Ponder BAJ ( 1994 ). Diverse phenotypes associated with exon 10 mutations of the RET proto - oncogene. Hum Mol Genet 3, 2163 – 2167. [26] Hofstra RM, Landsvater RM, Ceccherini I, Stulp RP, Stelwagen T, Luo Y, Pasini B, Hoppenner JW, van Amstel HK, Romeo G, Lips CJM, and Buys CHCM ( 1994 ). A mutation in the RET proto - oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 367, 375 – 376. [27] Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, Latif F, Liu S, Chen F, Duh F - M, Lubensky I, Duan DR, Florence C, Pozzatti R, Walther MM, Bander NH, Grossman HB, Brauch H, Pomer S, Brooks JD, Isaacs WB, Lerman MI, Zbar B, and Linehan, WM ( 1994 ).

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet 7, 85 – 90. Dahia PLM, Marsh DJ, Zheng Z, Zedenius J, Komminoth P, Frisk T, Wallin G, Parsons R, Longy M, Larsson C, and Eng C ( 1997 ). Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumours. Can Res 57, 4710 – 4713. Ceccherini I, Hofstra RMW, Luo Y, Stulp RP, Barone V, Stelwagen T, Bocciardi R, Nijveen H, Bolino A, Seri M, Ronchetto P, Pasini B, Boszzano M, Buys CHCM, and Romeo G ( 1994 ). DNA polymorphisms and conditions for SSCP analysis of 20 exons of the ret proto oncogene. Oncogene 9, 3025 – 3029. Marsh DJ, Robinson BG, Andrew S, Richardson A - L, Pojer R, Schnitzler M, Mulligan LM, and Hyland VJ ( 1994 ). A rapid screening method for the detection of mutations in the RET proto - oncogene in multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma families. Genomics 23, 477 – 479. Gross E, Arnold N, Goette J, Schwarz - Boeger U, and Kiechle M ( 1999 ). A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC. Hum Genet 105, 72 – 78. Marx SJ, Agarwal SK, Heppner C, Kim YS, Kester MB, Goldsmith PK, Skarulis MC, Spiegel AM, Burns AL, Debelenko LV, Zhuang Z, Lubensky IA, Liotta LA, Emmert - Buck MR, Guru SC, Manickam P, Crabtree JS, Collins FS, and Chandrasekharappa SC ( 1999 ). The gene for multiple endocrine neoplasia type 1: recent findings. Bone 25, 119 – 122. Arnold N, Gross E, Schwarz - Boeger U, Pfisterer J, Jonat W, and Kiechle M ( 1999 ). A highly sensitive, fast, and economical technique for mutation analysis in hereditary breast and ovarian cancers. Hum Mutat 14, 333 – 339. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, and James CD ( 1998 ). Denaturing high performance liquid chromatography ( DHPLC ) used in the detection of germline and somatic mutations. Nucleic Acids Res 26, 1396 – 1400. Yokomizo A, Tindall DJ, Drabkin H, Gemmill R, Franklin W, Yang P, Sugio K, Smith DI, and Liu W ( 1998 ). PTEN / MMAC1 mutations identified in small cell, but not in non – small cell lung cancers. Oncogene 17, 475 – 479. Arch EM, Goodman BK, Wesep RAV, Liaw D, Clarke K, Parsons R, McKusick VA, and Geraghty MT ( 1997 ). Deletion of PTEN in a patient with Bannayan - Riley - Ruvalcaba syndrome suggests allelism with Cowden’s disease. Am J Med Genet 71, 489 – 493. Tsuchiya KD, Wiesner G, Casidy SB, Limwongse C, Boyle JT, and Schwartz S ( 1998 ). Deletion 10q23.2 – 23.3 in a patient with gastrointestinal juvenile polyposis and other features of a Cowden - like syndrome. Genes Chromosomes Cancer 21, 113 – 118. Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, and O’Donovan MC ( 1999 ). Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single stranded conformation polymorphism and heteroduplex analysis. Clin Chem 45, 1133 – 1140. Jones AC, Sampson JR, Hoogendoorn B, Cohen D, and Cheadle JP ( 2000 ). Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis. Hum Genet 106, 663 – 668. Wagner T, Stoppa - Lyonnet D, Fleischmann E, Muhr D, Pages S, Sandberg T, Caux V, Moeslinger R, Langbauer G, Borg A, and Oefner P ( 1999 ). Denaturing high - performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations. Genomics 62, 369 – 376. Ellis LA, Taylor CF, and Taylor GR ( 2000 ). A comparison of fluorescent SSCP and denaturing HPLC for high throughout mutation scanning. Hum Mutat 15, 556 – 564.
