HLA class I and II genotype of the NCI-60 cell lines

1479-5876-3-11 1479-5876 Research HLA class I and II genotype of the NCI-60 cell lines Adams Sharon sadams1@mail.cc.nih.gov Robbins Fu-Meei FRobbins@mail.cc.nih.gov Chen Deborah dschen@mail.cc.nih.gov Wagage Devika DWagage@mail.cc.nih.gov Holbeck L Susan holbecks@mail.nih.gov Morse C Herbert III hmorse@niaid.nih.gov Stroncek David DStroncek@mail.cc.nih.gov Marincola M Francesco fmarincola@mail.cc.nih.gov

Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA

Developmental Therapeutics Program, Information Technology Branch, National Cancer Institute, Bethesda, Maryland, 20892, USA

Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, Rockville, USA

Journal of Translational Medicine 1479-5876 2005 3 1 11 http://www.translational-medicine.com/content/3/1/11 15748285 10.1186/1479-5876-3-11 24 1 2005 04 3 2005 04 3 2005 2005 Adams et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation.

Background

A panel of sixty cancer cell lines of diverse lineage (lung, renal, colorectal, ovarian, breast, prostate, central nervous system, melanoma and hematological malignancies) was developed, characterized and extensively used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as a screening tool for anti-cancer drug development 1. This strategy 23456789. yielded data about drug-related cytotoxicity for about 100,000 compounds. In addition, extensive functional characterization of the NCI-60 response to diverse biological or chemical stimulation has been accumulated 101112131415. Although originally developed for chemo-sensitivity testing, with the development of high-throughput analyses the NCI-60 panel has been broadly characterized for other biological applications 16171819202122232425. Thus, patterns incidentally identified provided platforms for further investigations of mechanisms of tumorigenesis and cancer progression 562627282930. More recently, genomic DNA 24 and proteomics analyses have further characterized the profile of these cell lines 31. The combined database provides the most comprehensive phenotyping of commonly accessible cancer cell lines offering correlative information about genetic, transcriptional and post-translational qualities. With growing interest in the identification of novel tumor antigens recognized by T cells as targets for antigen-specific immunization (32, the NCI-60 could become an ideal tool for in silico discovery 33 (34 and for tumor cell-specific T-cell reactivity testing 35. For this purpose, accurate information about the extended human leukocyte antigen (HLA) phenotype of each cell line is necessary for the definition and validation of specific HLA/epitope combinations. Although antiquated and partial information about the HLA phenotype of some of the NCI-60 cell lines is available through the American Type Culture Collection (ATCC), Rockville, MD, no high-resolution information obtained by definitive sequence-based typing (SBT) has ever been published. Since T cell recognition of HLA-epitope complexes is narrowly restricted to unique combinations 36, this information is critical to select reasonable candidates for antigen-discovery choosing cell lines bearing HLA phenotypes most relevant to the disease population studied 37. Accurate information about the HLA genotype of each cell line may, in addition, help their identification, validation and qualification among different laboratories excluding possible errors related to switching of cell lines or culture contamination. Therefore, we provide high-resolution SBT of the complete NCI-60 panel obtained from their original source: the National Cancer Institute's Developmental Therapeutics Program.

Results and Discussion

Previous knowledge of the HLA phenotype of NCI-60 cell lines

We reviewed and collected available information about the HLA phenotype of the NCI-60 cell lines, performed according to serological testing before submission to the ATCC (Table 1). The information was collected through the ATCC website: http://www.atcc.org. Most cell lines had not been previously typed; the large majority of the cell lines from which such information is available had been developed from Caucasian patients. HLA typing was reported according to the old serologic nomenclature at a very low level of resolution. In addition, several reported typings did not match the present typing as shown in Table 2 and 3. This was the case for the colon carcinoma cell line HT29 that maintained a correct haplotype (with the exclusion of the HLA-Cw locus) but had a completely different second haplotype. The melanoma cell line SK-MEL-5 had an almost identical haplotype with the exception of one HLA-B allele originally typed as Bw16 (inclusive of the molecularly-defined alleles: B*38 and B*39), while the present typing was HLA-B*07. Another melanoma cell line SK-MEL-28 maintained a haplotype similar to the previously reported HLA-A11, -B40 but appeared to have lost an HLA-A allele (HLA-A26) compared with the original ATCC description. Finally, the multiple myeloma cell line RPMI 8226 was matched at one haplotype (HLA-A19, -B15 and -Cw2) but was totally discrepant at the second haplotype (HLA-A*6802, -B*1510 and -Cw*0304). The HLA typing of the other two previously typed cell lines was confirmed in the present study. Overall, in spite of the discrepancies in HLA typing observed between the previous and the present analyses, a resemblance was noted in the cell line genotype suggesting that mis-typing related to the low accuracy of serological methods might have been at the basis of the discrepancy rather than contamination or switching of the cell lines.

Table 1

Available information from the ATCC about the NCI-60 panel

Name

ATCC no.

Sex

Race

Tumor Type

ATCC HLA typing

Discrepant

BT-549

HTB-122

Breast CA

HS 578T

HTB-126

Breast CA

MCF7

HTB-22

Breast CA

MDA-MB-231

HTB-26

Breast CA

MDA-MB-435

HTB-129

Breast CA

T-47D

HTB-133

Breast CA

SF-268

CNS CA

SF-295

CNS CA

SF-539

CNS CA

SNB-19

CNS CA

SNB-75

CNS CA

U251

CNS CA

COLO 205

CCL-222

Colon CA

HCC-2998

Colon CA

HCT-116

CCL-247

Colon CA

HCT-15

CCL-225

Colon CA

HT29

HTB-38

Colon CA

A1,3,B12,17 Cw5

Yes

KM12

Colon CA

SW-620

CCL-227

Colon CA

MOLT-4

CRL-1582

Leukemia, ALL

CCRF-CEM

CCL-119

Leukemia, ALL

HL-60

CCL-240

Leukemia, APL

K-562

CCL-243

Leukemia, CML

CRL-2262

Leukemia, LCIL

LOX IMVI

Melanoma

M 14

Melanoma

SK-MEL-2

HTB-68

Melanoma

SK-MEL-5

HTB-70

Melanoma

A2,11, B40,Bw16

Yes

SK-MEL-28

HTB-72

Melanoma

A11,26, B40,DRw4

Yes

UACC-62

Melanoma

UACC-257

Melanoma

RPMI 8226

CCL-155

Aw19, B15,37, Cw2

Yes

A549/ATCC

CCL-185

NSCLC

EKVX

NSCLC

HOP-62

NSCLC

HOP-92

NSCLC

NCI-H23

CRL-5800

NSCLC

NCI-H226

CRL-5826

NSCLC

NCI-H322M

NSCLC

NCI-H460

HTB-177

NSCLC

NCI-H522

CRL-5810

NSCLC

IGROV1

Ovarian CA

OVCAR-3

HTB-161

Ovarian CA

OVCAR-4

Ovarian CA

OVCAR-5

Ovarian CA

OVCAR-8

Ovarian CA

NCI/ADR-RES

Ovarian CA

SK-OV-3

HTB-77

Ovarian CA

DU-145

HTB-81

Prostate CA

PC-3

CRL-1435

Prostate CA

A1,9

786-O

Renal CA

A498

HTB-44

Renal CA

ACHN

CRL-1611

Renal CA

CAK1-1

HTB-46

Renal CA

A9,B12,35

SN12C

Renal CA

TK-10

Renal CA

UO-31

Renal CA

RXF-393

Renal CA

AA = African American; ALL = Acute Lymphoblastic Leukemia; APL = Acute promyelocytic leukemia; C = Caucasian; CA = Carcinoma; CML = Chronic Myelogenous Leukemia; CNS = Central Nervous System; F = Female; LCIL = Large Cell Immunoblastic Lymphoma; M = Male; MM = Multiple Myeloma; NA = Not Available; NSCLC = Non Small Cell Lung Cancer.

The information about the ATCC cell lines (Cell Lines with ATCC no.) was obtained accessing the following URL: http://www.atcc.org. Additional information was obtained through the National Cancer Institute's Developmental Therapeutics Program URL: http://dtp.nci.nih.gov/branches/btb/tumor-catalog.pdf.

Table 2

Sequence-based typing of NCI-60 HLA class I Loci

Cell Line

Tissue

A locus

B Locus

Cw Locus

BT-549

41292-D

Breast CA

N.R.

151701, 5501

030301, 07^a

HS 578T

41293-D

Breast CA

03^a, 24^a

35^a, 40^a

030401, 04^a

MCF7

41294-D

Breast CA

020101

18^a, 44^a

05^a

MDA-MB-231

41296-D

Breast CA

0201, 0217

4002, 4101

020202, 17^a

MDA-MB435

41297-D

Breast CA

110101, 240201

15^a, 35^a

030301, 04^a

T47D

41298-D

Breast CA

3301

1402

0802

SF-268

41286-D

CNS CA

010101, 3201

0801, 4002

020202, 07^a

SF-295

41287-D

CNS CA

010101, 2601

070201, 5501

03^a, 07^a

SF-539

41288-D

CNS CA

020101

08^a, 35^a

04^a, 07^a

SNB-19

41289-D

CNS CA

020101

18^a

05^a

SNB-75

41290-D

CNS CA

020101, 110101

35^a, 39^a

04^a, 120301

U251

41291-D

CNS CA

020101

18^a

05^a

COLO 205

41299-D

Colon CA

01^a, 02^a

07^a, 08^a

070201, 07^a

HCC-2998

41300-D

Colon CA

02^a, 24^a

3701, 400601

04^a, 0602

HCT-116

41301-D

Colon CA

01^a, 02^a

18^a, 4501

05^a, 07^a

HCT-15

41302-D

Colon CA

02^a, 24^a

08new, 350101

04^a, 07^a

HT29

41303-D

Colon CA

01^a, 24^a

35^a, 440301

04^a

KM12

41304-D

Colon CA

02new

70201

SW-620

41305-D

Colon CA

02^a, 24^a

07^a, 15^a

070201, 07^a

MOLT 4

41281-D

Leukemia, ALL

010101, 2501

18^a, 570101

0602, 120301

CCRF-CEM

41282-D

Leukemia, ALL

N.R.

08^a, 40^a

030401, 07^a

HL-60

41284-D

Leukemia, APL

10101

570101

0602

K-562

41280-D

Leukemia, CML

110101, 310102

18^a, 40^a

03^a, N.R.

41285-D

Leukemia, LCIL

02^a, 03^a

3701, 3901

0602, 120301

LOX IMVI

41315-D

Melanoma

110101, 2902

070201, 440301

070201, 1601

M 14

41316-D

Melanoma

110101, 240201

15^a, 35^a

030301, 04^a

SK-MEL-2

41317-D

Melanoma

03^a, 26^a

35^a, 38^a

04^a, 120301

SK-MEL-5

41319-D

Melanoma

020101, 110101

07^a, 40^a

030401, 070201

SK-MEL-28

41318-D

Melanoma

110101

4001

030401

UACC-62

41321-D

Melanoma

02^a, 32^a

39^a, 44^a

05^a, 12^a

UACC-257

41320-D

Melanoma

020101

18^a, 44^a

05^a, 07^a

RPMI-8226

41283-D

3001, 6802

1503, 1510

020204, 030402

A549/ATCC

41306-D

NSCLC

2501, 3001

18^a, 440301

120301, 1601

EKVX

41307-D

NSCLC

010101

3701

0602

HOP-62

41308-D

NSCLC

030101

07^a, 44^a

05^a, 070201

HOP-92

41309-D

NSCLC

03^a, 24^a

27^a, 470101

01^a, 06^a

NCI-H23

41312-D

NSCLC

8001

5001

0602

NCI-H226

41311-D

NSCLC

010101, 240201

07^a, 39^a

070201, 120301

NCI-H322M

41310-D

NSCLC

2902

440301

1601

NCI-H460

41313-D

NSCLC

24^a, 68^a

35^a, 51^a

03^a, 15^a

NCI-H522

41314-D

NSCLC

020101

44^a, 5501

030301, 05^a

IGROV1

41322-D

Ovarian CA

240201, 3301

4901

07^a

OVCAR-3

41323-D

Ovarian CA

020101, 2902

070201, 5801

070201, 07^a

OVCAR-4

41324-D

Ovarian CA

010101, 3201

0801, 4002

07^a, 15^a

OVCAR-5

41325-D

Ovarian CA

01^a, 02^a

08^a, 44^a

05^a, 07^a

OVCAR-8

41326-D

Ovarian CA

010101, 2501

570101

0602

NCI/ADR-RES

41295-D

Ovarian CA

010101, 2501

570101

0602

SK-OV-3

41327-D

Ovarian CA

03^a, 68^a

18^a, 35^a

04^a, 05^a

DU-145

41328-D

Prostate CA

030101, 3303

5001, 570101

0602

PC-3

41329-D

Prostate CA

010101, 240201

1302, 5501

01^a, 06^a

786-O

41330-D

Renal CA

030101

07^a, 44^a

05^a, 070201

A498

41331-D

Renal CA

020101

0801

07^a

ACHN

41332-D

Renal CA

2601

4901

07^a

CAKI-1

41333-D

Renal CA

2301, 240201

3502, 440301

04^a, 04new

SN12C

41334-D

Renal CA

03, 24new

07^a, 44^a

05^a, 070201

TK-10

41335-D

Renal CA

3301

1402

0802

UO-31

41336-D

Renal CA

010101, 030101

07^a, 14^a

07^a, 08^a

RXF-393

41337-D

Renal CA

02^a, 24^a

1401, 44^a

05^a, 0802

Sequence-based typing for the HLA class I loci are reported with the highest degree of resolution. Non-resolved ambiguities are reported as two digit denominations with a superscript ^aas previously described 43. HLA typings divergent from those originally described in the ATCC database are reported in red. ID# refers to the HLA laboratory reference number. New alleles are indicated by the suffix new following the allele. N.R. – Ambiguity not resolved at the lower level of resolution.

Table 3

Sequence-based typing of NCI-60 HLA class II Loci

Cell Line

Tissue

DRβ1 Locus

DQB1 Locus

DPB1 Locus

BT-549

41292-D

Breast CA

11^a, 13^a

030101, 060401

020102, 0401

HS 578T

41293-D

Breast CA

01^a, 150101

050101, 0602

0401, 7801

MCF7

41294-D

Breast CA

03^a, 15^a

0201, 0602

020102, 0401

MDA-MB-231

41296-D

Breast CA

0701, 1305

0202, 030101

020102, 1701

MDA-MB435

41297-D

Breast CA

040501, 130101

0302, 0603

1301, 1901

T47D

41298-D

Breast CA

010201

050101

020102, 0401

SF-268

41286-D

CNS CA

03^a, 04^a

0201, 0302

0401, 0601

SF-295

41287-D

CNS CA

14^a, 15^a

050301, 0602

0401

SF-539

41288-D

CNS CA

030101, 12^a

0201, 030101

010101, 0401

SNB-19

41289-D

CNS CA

030101

0201

0402

SNB-75

41290-D

CNS CA

0103, 11^a

03^a, 050101

0401, 0402

U251

41291-D

CNS CA

030101

0201

0402

COLO 205

41299-D

Colon CA

040101, 130101

0603

0401

HCC-2998

41300-D

Colon CA

11^a, 16^a

030101, 050201

0401

HCT-116

41301-D

Colon CA

N.R.

02new, 03new

030101, 0402

HCT-15

41302-D

Colon CA

03^a, 14^a

02^a, 050301

010101, 0401

HT29

41303-D

Colon CA

0402, 0701

02^a, 0302

0401

KM12

41304-D

Colon CA

040101

0302

1301

SW-620

41305-D

Colon CA

0103, 130101

050101, 0603

010101, 0401

MOLT 4

41281-D

Leukemia, ALL

07new, 12new

0202, 030101

20102

CCRF-CEM

41282-D

Leukemia, ALL

030101, 0701

0201, 0202

0401, 1301

HL-60

41284-D

Leukemia, APL

N.R.

030302

0401, 1301

K-562

41280-D

Leukemia, CML

03^a, 04^a

0201, 0302

0401, 0402

41285-D

Leukemia, LCIL

01^a, 160101

050101, 050201

0401

LOX IMVI

41315-D

Melanoma

0701, 150101

0202, 0602

0401, 110101

M 14

41316-D

Melanoma

040501, 130101

0302, 0603

1301, 1901

SK-MEL-2

41317-D

Melanoma

0402, 130101

030101, 0603

020102, 0401

SK-MEL-5

41319-D

Melanoma

040101, 130101

0302, 0603

030101, 1601

SK-MEL-28

41318-D

Melanoma

0404

0302

030101

UACC-62

41321-D

Melanoma

12^a, 130101

030101, 0603

0401, 1401

UACC-257

41320-D

Melanoma

040101

030101, 0302

0401

RPMI-8226

41283-D

030101, 0701

0201, 0202

010102, 1301

A549/ATCC

41306-D

NSCLC

0701, 110401

0202, 030101

N.R.

EKVX

41307-D

NSCLC

150101

0602

0401

HOP-62

41308-D

NSCLC

13^a, 15^a

06^a, 06^a

0402

HOP-92

41309-D

NSCLC

01^a, 150101

050101, 0602

0401, 0402

NCI-H23

41312-D

NSCLC

130101

0603

1901

NCI-H226

41311-D

NSCLC

150101, 160101

050201, 0602

020102, 0401

NCI-H322M

41310-D

NSCLC

0701

0202

0401

NCI-H460

41313-D

NSCLC

01^a, 04^a

030101, 050101

N.R.

NCI-H522

41314-D

NSCLC

040101, 150101

03^a, 0602

0401

IGROV1

41322-D

Ovarian CA

11^a, 11^a

03new

new, 0501

OVCAR-3

41323-D

Ovarian CA

080101, 080401

0402

020102, 0401

OVCAR-4

41324-D

Ovarian CA

030101, 040101

0201, 030101

0401, 1301

OVCAR-5

41325-D

Ovarian CA

030101, 040101

0201, 030101

0401

OVCAR-8

41326-D

Ovarian CA

0701, 150101

030302, 0602

020102, 1301

NCI/ADR-RES

41295-D

Ovarian CA

0701, 150101

030302, 0602

020102, 1301

SK-OV-3

41327-D

Ovarian CA

01^a, 030101

0201, 050101

020102, 0401

DU-145

41328-D

Prostate CA

N.R.

030302, 050101

0401

PC-3

41329-D

Prostate CA

0701, 130101

0202, 0603

0401

786-O

41330-D

Renal CA

13^a, 15^a

06^a, 06^a

0402

A498

41331-D

Renal CA

030101

0201

010101

ACHN

41332-D

Renal CA

160101

050201

020102

CAKI-1

41333-D

Renal CA

0701, 110401

0202, 03^a

020102, 1001

SN12C

41334-D

Renal CA

040101, 150101

03^a, 0602

N.R.

TK-10

41335-D

Renal CA

010201

050101

0402

UO-31

41336-D

Renal CA

130201, 150101

0602, 0609

0402, 0501

RXF-393

41337-D

Renal CA

110101, 150101

030101, 0602

010101, 0401

Sequence-based typing for the HLA class II loci are reported with the highest degree of resolution. Non-resolved ambiguities are reported as two digit denominations with a superscript ^aas previously described [43]. HLA typings divergent from those originally described in the ATCC database are reported in red. ID# refers to the HLA laboratory reference number. New alleles are indicated by the suffix new following the allele. N.R. = Ambiguity not resolved at the lower level of resolution.

Overall, there was no evidence of contamination among the cell lines tested with clean homozygous or heterozygous combinations observed in all loci analyzed. SBT of HLA class I and HLA class II loci are reported in Table 2 and 3 respectively. Information about the HLA typing of the cell lines is also available through the Molecular Targets URL: http://dtp.nci.nih.gov/mtargets/mt_index.html. Approximately 17% of the cell lines (10 out of 58 including: T47D, SNB-19, U251, KM12, RPMI-8226, EKVX, NCI-H23, NCI-H322M, A498, ACHN and TK-10) exhibited a pseudo-homozygous pattern suggestive of complete loss of heterozygosity encompassing the HLA class I and HLA class II regions. This frequency is close to the loss of haplotype that we originally described for melanoma cell lines generated at the National Cancer Institute (Bethesda, MD) 3839 and subsequently observed in other cancers 4041. We conclude that this is an unlikely representative of patients' homozygosity because complete HLA class I and II homozygosity is exceedingly rare in the population at large. To corroborate this statement, we analyzed 554 genomic DNA specimens from normal donors recently typed with the same technology in our laboratory. Genomic DNA for the normal donors was obtained from whole blood samples. Only 5 individuals were found to be truly homozygous for all HLA class I and class II loci for a frequency of 0.9%.

Overall, discrepancies between ATCC typings and the present typing or the unbalanced frequency of homozygosity could be related to accumulated genetic alterations between the cell lines since the time of their original expansion from the patient and should not be surprising.

A particular case was represented by the NCI/ADR-RES cell line which was previously believed to be an adriamycin derivative of the breast cancer cell line MCF-7. Subsequently, it was discovered not to be related to MCF-7, but it's derivation was unclear 42. Karyotyping analysis suggested it was related to the ovarian cell line OVCAR-8. Subsequent DNA fingerprinting confirmed that both cell lines were generated from the same individual. HLA genotyping confirms this since the cell lines are indeed identical.

To avoid possible misinterpretations, a large number of alleles are not presented here with their definitive nomenclature but rather at a two digits level of resolution because some of the ambiguities could not be completely resolved by SBT as previously described 43. However, more detailed information about individual cell lines can be obtained by contacting Sharon Adams directly at the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD. As previously described 43, it is possible to resolve most of these ambiguities using various methods including sequence-specific primer PCR or pyro-sequencing 44. If necessary in the future, the NIH HLA laboratory may assist in further characterization of individual HLA alleles. Another caveat is that the identification of HLA alleles at the genomic level does not necessarily correspond to surface expression of their protein products since various abnormalities in transcription, translation and assembling could influence the surface expression of HLA molecules 394546.

Finally, several new alleles were identified (referred to in the tables as new, for which a nomenclature is pending; in detail KM12 HLA-A*02new = Genebank Accession # AY918166; SN12C HLA-A*24new = # AY918167; CAKI-1 HLA-Cw04new = # AY918170). Information regarding the sequence of these alleles could be obtained by directly contacting the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD.

Materials and Methods

Cell Lines

Genomic DNA from the NCI-60 cell line anticancer drug discovery panel was obtained from SH of the National Cancer Institute Developmental Therapeutics Program (Bethesda, MD). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 5 mM L-glutamine.

DNA Isolation

Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE isolation kit (Gentra Systems, Minneapolis, MN, USA). The DNA was re-suspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing.

Sequence-Based Typing (SBT)

HLA class I loci sequence-based typing (SBT) was performed as previously described (43. The primary PCR amplification reaction produced a 1.5 kb amplicon encompassing exon 1 through intron 3 of the HLA class I locus. All reagents necessary for primary amplification and sequencing were included in the HLA-A, HLA-B and HLA-C alleleSEQR Sequenced Based Typing Kits (Atria Genetics, Hayward, CA, U.S.A.). The primary amplification PCR products were purified from excess primers, dNTPs and genomic DNA using ExoSAP-IT (American Life Science, Cleveland, OH, U.S.A.). Each template was sequenced in the forward and reverse sequence orientation for exon 2 and exon 3 according to protocols supplied with the SBT kits. Excess dye terminators were removed from the sequencing products utilizing an ethanol precipitation method with absolute ethanol. The reaction products were reconstituted with 15 μl of Hi-Di™ Formamide (PE Applied Biosystems / Perkin-Elmer, Foster City, CA, U.S.A.) and analyzed on the ABI Prism* 3700 DNA Analyzer with Dye Set file: Z and mobility file: DT3700POP6 [ET].

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