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Spectrum and prevalence of BRCA1/2 germline mutations in Pakistani breast cancer patients: results from a large comprehensive study

  • Muhammad Usman Rashid
  • Noor Muhammad
  • Humaira Naeemi
  • Faiz Ali Khan
  • Mariam Hassan
  • Saima Faisal
  • Sidra Gull
  • Asim Amin
  • Asif Loya
  • Ute HamannEmail author
Open Access
Research

Abstract

Background

Pathogenic germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers worldwide. To refine the spectrum of BRCA1/2 mutations and to accurately estimate the prevalence of mutation in the Pakistani population, we studied 539 breast cancer patients selected for family history and age of diagnosis.

Methods

Comprehensive screening for BRCA1/2 germline mutations was performed using state-of-the-art technologies.

Results

A total of 133 deleterious mutations were identified in 539 families (24.7%), comprising 110 in BRCA1 and 23 in BRCA2. The prevalence of BRCA1/2 small-range mutations and large genomic rearrangements was 55.4% (36/65) for families with breast and ovarian cancer, 27.4% (67/244) for families with two or more cases of breast cancer, 18.5% (5/27) for families with male breast cancer, and 12.3% (25/203) for families with a single case of early-onset breast cancer. Nine mutations were specific to the Pakistani population. Eighteen mutations in BRCA1 and three in BRCA2 were recurrent and accounted for 68.2% (75/110) and 34.8% (8/23) of all identified mutations in BRCA1 and BRCA2, respectively. Most of these mutations were exclusive to a specific ethnic group and may result from founder effects.

Conclusions

Our findings show that BRCA1/2 mutations account for one in four cases of hereditary breast/ovarian cancer, one in five cases of male breast cancer, and one in eight cases of early-onset breast cancer in Pakistan. Our study suggests genetic testing of an extended panel of 21 recurrent BRCA1/2 mutations for appropriately selected patients and their families in Pakistan.

Keywords

BRCA1/2 germline mutations breast cancer Pakistan 

Abbreviations

BIC

Breast cancer information core

HGVS

Human genome variation society

LGRs

Large genomic rearrangements

LOVD

The Leiden open variation database

SKMCH&RC

Shaukat Khanum Memorial Cancer Hospital and Research Centre

Background

Individuals harboring BRCA1/2 germline mutations have high lifetime risks of breast and ovarian cancer. The identification of individuals harboring BRCA1/2 mutations is crucial to assess their cancer risk, consider preventive measures and tailor cancer management strategies.

Several studies have investigated the prevalence of BRCA1/2 small-range mutations and/or large genomic rearrangements (LGRs) with frequencies varying from 17.6% to 29.8% in white populations from Europe and Australia [1, 2, 3, 4, 5] and 9.4% to 21.7% in non-whites from Asia [6, 7, 8]. The prevalence and distribution of BRCA1/2 mutations vary across populations, mainly due to population-specific recurrent or founder mutations. Accurate identification of the population-specific mutation spectrum is therefore the first step towards incorporating appropriate genetic BRCA1/2 testing into clinical practice in a particular population. This information is not fully elucidated in Pakistan, a country with one of the highest rates of breast cancer in Asia.

To date, no large comprehensive studies evaluating the BRCA1/2 mutations have been reported in the Pakistani population and mutations in males have not been identified so far. Small-range mutations were previously reported in 341 unselected breast and 120 ovarian cancer patients, in which the analysis was restricted to a few exons only [9]. We conducted two studies in early-onset and familial breast/ovarian cancer patients from Pakistan. In the initial study the complete coding regions and exon-intron boundaries of BRCA1/2 were screened for small-range mutations in 176 patients [10]. In the other study 120 BRCA1/2 small-range mutations negative patients were screened for LGRs [11]. Other Asian studies also had small sample sizes [12, 13], reported small-range mutations only [14, 15], and/or restricted LGR analyses to a small number of study participants [6, 16, 17].

Here, we refined the spectrum of BRCA1/2 mutations and more precisely estimated the mutation frequencies including small-range mutations and LGRs in 539 early-onset and familial breast cancer patients from Pakistan.

Methods

Enrollment of families

Five hundred and ninety-three breast cancer only or breast and ovarian cancer families were enrolled through index breast and/or ovarian cancer patients who presented at the Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH&RC) in Lahore, Pakistan, from September 2004 to August 2015. The recruited families were classified into five risk groups based on family history of breast/ovarian cancer or age at diagnosis (Table 1) as described previously [19]. After enrollment, 54 families were excluded (Fig. 1), leaving 539 families in the study.
Table 1

BRCA1/2 mutation frequencies according to family structure

Risk group

Phenotype of families

No. of families

No. of families with mutations (%) in

BRCA1

BRCA2

BRCA1/2

Small-range

LGRs

All

Small-range

LGRs

All

 

All families

539

101 (18.7)

9 (1.7)

110 (20.4)

23 (4.3)

0 (0)

23 (4.3)

133 (24.7)a

Female breast cancer families

447

67 (15.0)

7 (1.6)

74 (16.6)

18 (4.0)

0 (0)

18 (4.0)

92 (20.6)

A1

1 case ≤ 30 years

203

20 (9.8)

2 (1.0)

22 (10.8)

3 (1.5)

0 (0)

3 (1.5)

25 (12.3)

A2

2 cases, >1 diagnosed ≤50 years

131

20 (15.3)

4 (3.0)

24 (18.3)

6 (4.6)

0 (0)

6 (4.6)

30 (22.9)

A3

>3 cases, >1 diagnosed ≤50 years

113

27 (23.9)

1 (0.9)

28 (24.8)

9 (8.0)

0 (0)

9 (8.0)

37 (32.7)

A4

Male breast cancer families

        

>1 case of male breast cancer

27

1 (3.7)

0 (0)

1 (3.7)

4 (14.8)

0 (0)

4 (14.8)

5 (18.5)

B

Breast-ovarian cancer families

        

>1 breast cancer and >1 ovarian cancer

65

33 (50.8)

2 (3.0)

35 (53.8)

1 (1.5)

0 (0)

1 (1.5)

36 (55.4)

LGRs large genomic rearrangements

aIncluding 57 previously reported families [11, 18]

Fig. 1

Flow diagram for the study participant’s enrollment, screening methods used and BRCA1/2 mutations detected

Clinical and histopathological data and comprehensive information on personal and family history of cancer(s), and ethnicity were obtained from all study participants. The Institutional Review Board of the SKMCH&RC approved the study. All study participants signed an informed written consent before providing a blood sample.

BRCA1/2 mutation screening

Genomic DNA was extracted from 9 to 18 ml of whole blood samples, as described previously [20]. The entire coding regions of the BRCA1 (Genbank accession number U14680) and BRCA2 (Genbank accession number U43746) genes including exon-intron boundaries were screened in 139 patient DNA samples for small-range mutations using denaturing high-performance liquid chromatography (DHPLC) analysis as previously described [21, 22]. Each sample revealing variants was sequenced as described elsewhere [18]. Four-hundred female patients selected based on triple-negative vs. non triple-negative breast cancer phenotype had been screened for small-range BRCA1/2 mutations and 33 mutations were described [18]. All small-range mutation-negative patients had been screened for LGRs using multiplex ligation-dependent probe amplification and three LGRs were described [11]. For the current study, families were selected on the basis of family history of breast/ovarian cancer, male breast cancer or age at diagnosis.

Mutation classification

All BRCA1/2 alterations identified in the current study were classified into pathogenic mutations, variants of unknown significance, or polymorphisms. Pathogenic mutations were defined as (i) small-range mutations which affect one or a few nucleotides including frameshift, nonsense, or splice-site mutations and generate a premature termination codon, except BRCA2 exon 27 variants generating a premature termination codon after codon 3010 [23] and (ii) LGRs that span one or more exons. Mutations were designated using the Human Genome Variation Society (HGVS) and the Breast Cancer Information Core (BIC) committee nomenclature.

All identified mutations were searched in various mutation databases including BIC (https://research.nhgri.nih.gov/bic/), ClinVar (http://www.ncbi.nim.nih.gov/clinvar/), LOVD (http://databases.lovd.nl/shared/genes/BRCA2), ARUP (http://arup.utah.edu/database/BRCA/), and BRCA Exchange (http://brcaexchange.org/). Mutations not reported in these databases were considered as novel and specific to Pakistani population.

Statistical analyses

Distribution of clinical and histopathological characteristics between BRCA1/2 carriers and non-carriers were estimated using Fisher’s exact test for categorical variables and the Wilcoxon rank-sum test for quantitative variables. All statistical tests were two-sided. Results were considered significant at a p value of <0.05. All statistical analyses were done using StatXact 4 for Windows (Cytel, Cambridge, USA) and R, version 2.1.

Results

A total of 539 index patients from unrelated families were enrolled and stratified into five risk groups (Table 1). The mean age of disease onset was 35.4 years (range 18-78) for female breast cancer (n=502), 45.4 years (range 23-66) for ovarian cancer (n=30) and 54.5 years (range 27-76) for male breast cancer (n=27) patients.

Spectrum of BRCA1/2 mutations

Evaluation of pooled data from 539 patients yielded 71 distinct pathogenic mutations in 133 families (24.7%) (Table 1). Fifty-three BRCA1 mutations were detected in 110 families (20.4%) and 18 BRCA2 mutations in 23 families (4.3%). Five mutations in BRCA1 (9.4%) and four mutations in BRCA2 (22.2%) were novel (Table 2). The phenotypes of all families carrying BRCA1/2 mutations are presented in Table 3.
Table 2

Deleterious BRCA1/2 germline mutations in Pakistani breast/ovarian cancer families

Family

Exon

BIC designation

HGVS designation

Mutation typeb

Reported in databases (No. of entries)c

Nucleotide

Codon

Designation

Nucleotide changea

Effect on protein

BRCA1-small-range mutations

 432

2

185

23

185insA

c.66dup

p.(Glu23Argfs*18)

FS

BIC (32)d

 723

2

185

23

185delAG

c.68_69del

p.(Glu23Valfs*17)

FS

BIC (2036)d

 372

Intron 4

IVS4-2

-

IVS4-2A>G

c.135-2A>G

Splice site

SP

BIC (1)

 254e

7

454

112

454delA

c.335del

p.(Asn112Ilefs*7)

FS

ClinVar (2)

 449

7

509

130

Y130X

c.390C>G

p.(Tyr130*)

NS

LOVD (3)

 296, 317, 340e, 511, 521, 626e, 747

11

804

229

804delT

c.685del

p.(Ser229Leufs*5)

FS

BIC (2)

 470e

11

903

262

Q262X

c.784C>T

p.(Gln262*)

NS

ClinVar (3)

 711

11

1014

299

1014delGT

c.895_896del

p.(Val299Argfs*4)

FS

BIC (2)

 669e

11

1127

336

1127delA

c.1008del

p.(Glu337Lysfs*4)

FS

Nod

 748

11

1307

396

1307delT

c.1188del

p.(Asp396Glufs*14)

FS

LOVD (1)

 241e

11

1309

397

1309delA

c.1190del

p.(Asp397Alafs*13)

FS

ClinVar (3)

 722

11

1518

485

1518_1572dup55

c.1399_1453dup

p.(Ala485Glufs*13)

FS

No

 336e

11

1590

491

Q491X

c.1471C>T

p.(Gln491*)

NS

BIC (4)d

 N12

11

1898

593

1898delTATGGAA

c.1779_1785del

p.(Met594Serfs*3)

FS

LOVD (2)

 N28, 328e, 557e

11

1912

598

L598X

c.1793T>G

p.(Leu598*)

NS

BIC (1)d

 574e

11

2080

655

2080insA

c.1961dup

p.(Tyr655Valfs*18)

FS

BIC (13)d

 488e

11

2268

717

E717X

c.2149G>T

p.(Glu717*)

NS

ClinVar (2)

 236e, 283e, 489e, 493e, 593

11

2388

757

2388delG

c.2269del

p.(Val757Phefs*8)

FS

BIC (10)d

 363

11

2433

772

2433delGT

c.2314_2315del

p.(Val772Thrfs*4)

FS

LOVD (2)

 550h

11

2457

780

Q780X

c.2338C>T

p.(Gln780*)

NS

BIC (36)

 362, 469

11

2459

780

2459delGGAA

c.2340_2343del

p.(Glu781Valfs*10)

FS

LOVD (2)

 421e, 442, 510e, 542, 619e

11

2524

802

2524delTG

c.2405_2406del

p.(Val802Glufs*7)

FS

BIC (5)d

 N34

11

2657

846

2657delAAT-insG

c.2538_2540delinsG

p.(Met847Glyfs*4)

FS

LOVD (2)

 415e, 660e

11

2722

868

S868X

c.2603C>G

p.(Ser868*)

NS

BIC (11)d

 411e

11

3090

991

K991X

c.2971A>T

p.(Lys991*)

NS

ClinVar (2)

 247e

11

3100

994

3100delGT

c.2981_2982del

p.(Cys994*)

FS

ClinVar (5)

 299

11

3248

1043

3248delTATTAATGAA

c.3129_3138del

p.(Asn1043Lysfs*2)

FS

ClinVar (3)

 N13e, 399e

11

3458

1113

3458delTGA

c.3339_3341del

p.(Tyr1113*)

FS

ClinVar (2)

 610

11

3531

1138

G1138X

c.3412G>T

p.(Gly1138*)

NS

ClinVar (3)

 N25

11

3596

1159

3596delAAAG

c.3477_3480del

p.(Ile1159Metfs*50)

FS

BIC (3)

 279e, 445e

11

3717

1200

Q1200X

c.3598C>T

p.(Gln1200*)

NS

BIC (21)d

 382

11

3726

1203

R1203X

c.3607C>T

p.(Arg1203*)

NS

BIC (36)

 646

11

3819

1234

3819delGTAAA

c.3700_3704del

p.(Val1234Glnfs*8)

FS

BIC (61)

 210e, 211e, 313e, 316, 332e, 463, 520, 624, 652e, 653

11

3889

1257

3889delAG

c.3770_3771del

p.(Glu1257Glyfs*9)

FS

BIC (23)d

 N4e, 687, 724, 743

11

4184

1355

4184delTCAA

c.4065_4068del

p.(Asn1355Lysfs*10)

FS

BIC (144)d

 318e

12

4302

1395

Q1395X

c.4183C>T

p.(Gln1395*)

NS

BIC (28)d

 408e

13

4446

1443

R1443X

c.4327C>T

p.(Arg1443*)

NS

BIC (128)

 523e, 555, N18, 598e, 612, 621

Intron 14

IVS14-1

-

IVS14-1G>A

c.4485-1G>A

Splice site

SP

BIC (2)d

 220e, 275e, 512e

15

4627

1503

S1503X

c.4508C>A

p.(Ser1503*)

NS

BIC (1)d

 609e

15

4784

1558

4784delG

c.4665del

p.(Arg1555Serfs*4)

FS

No

 611e

16

4981

1621

4981delA

c.4862del

p.(Asp1621Valfs*12)

FS

No

 249e, 658

17

5154

1679

5154delC

c.5035del

p.(Leu1679*)

FS

BIC (2)

 276e, 679

Intron 17

IVS17+1

-

IVS17+1G>A

c.5074+1G>A

Splice site

SP

BIC (3)

 685

20

5358

1747

5358delC

c.5239del

p.(Gln1747Lysfs*18)

FS

LOVD (2)

 734

20

5385

1756

5385dupC

c.5266dup

p.(Gln1756Profs*74)

FS

LOVD (376)

 706

Intron 20

IVS20-1

-

IVS20-1G>C

c.5278-1G>C

Splice site

SP

LOVD (5)d

 678

21

5429

1771

5429dupG

c.5310dup

p.(Pro1771Alafs*59)

FS

LOVD (1)

 278, 338e

22

5480

1787

5480delTG

c.5361_5362del

p.(Cys1787Trpfs*42)

FS

ClinVar (3)

 682

22

5496

1793

K1793X

c.5377A>T

p.(Lys1793*)

NS

ClinVar (1)

 248e

Intron 23

IVS23-2

-

IVS23-2A>T

c.5468-2A>T

Splice site

SP

ClinVar (1)

 260, 264, 329e, 377e, 389, 439, 481, 501, 522

24

5622

1835

R1835X

c.5503C>T

p.(Arg1835*)

NS

BIC (74)d

BRCA1-large genomic rearrangementse

 229, 291, 314, 379, 406, 498, 549

1-2

-

-

del exon 1-2

g.41271967_41308900delf

 

LGR

(42)g

 261, 719

21-24

-

-

del exon 21-24

g.41172653_41205744delf

 

LGR

No

BRCA2-small-range mutations

 497, 700

3

320

31

W31X

c.92G>A

p.(Trp31*)

NS

ClinVar (4)

 N26

Intron 4

IVS4-2

-

IVS4-2A>G

c.426-2A>G

Splice site

SP

ClinVar (4)

 545

9

993

255

993delCACAA

c.765_769del

p.(Asn255Lysfs*19)

FS

No

 330

10

1528

434

1528delAAAA

c.1300_1303del

p.(Lys434Glufs*25)

FS

ClinVar (2)

 602

11

3048

941

3048delA

c.2820del

p.(Val941Cysfs*19)

FS

No

 206

11

3063

945

3063delA

c.2835del

p.(Asp946Ilefs*14)

FS

ClinVar (2)

 505

11

4088

1287

4088delA

c.3860del

p.(Asn1287Ilefs*6)

FS

BIC (2)

 222, 407h, 525, 540h

11

5450

1741

5450delGTAA

c.5222_5225del

p.(Ser1741Thrfs*35)

FS

BIC (1)

 627, 684

11

5910

1894

Y1894X

c.5682C>A

p.(Tyr1894*)

NS

BIC (3)

 295e

11

5950

1908

5950delCT

c.5722_5723del

p.(Leu1908Argfs*2)

FS

BIC (43)d

 447

11

6696

2156

6696delTC

c.6468_6469del

p.(Gln2157Ilefs*18)

FS

BIC (24)d

 548h

11

7044

2274

7044delAAGAG

c.6816_6820del

p.(Gly2274Alafs*17)

FS

ClinVar (6)

 579

15

7803

2526

7803delA

c.7575del

p.(Ala2526Glnfs*2)

FS

LOVD (2)

 492

Intron 17

IVS17+2

-

IVS17+2C>A

c.7976+2C>A

Splice site

SP

ClinVar (1)

 713

20

8773

2849

8773delAA

c.8545_8546del

p.(Lys2849Glyfs*19)

FS

No

 702

20

8779

2860

8779_8798dup20

c.8551_8570dup

p.(Lys2860Asnfs*10)

FS

No

 207h

21

8897

2890

8897insT

c.8669dup

p.(Thr2891Asnfs*16)

FS

ClinVar (1)

 538

Intron 21

IVS21+4

-

IVS21+4A>G

c.8754+4A>G

Splice site

SP

BIC (7)

aNumbering starts at the first A of the first coding ATG (located in exon 2) of NCBI GenBank accession number U14680 (BRCA1) and U43746 (BRCA2)

bFS frameshift mutation, LGR large genomic rearrangement, MS missense mutation, NS nonsense mutation, SP splice-site mutation

cBIC Breast Cancer Information Core database (https://research.nhgri.nih.gov/projects/bic/), LOVD Leiden Open Variation Database (http://databases.lovd.nl/shared/genes/BRCA2); ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), date last accessed June 26, 2018

dPreviously reported in Pakistani breast/ovarian cancer cases [9, 10]

eFamilies and mutation description have been previously reported [11, 18]

fGenomic locale for chromosome 17, from the UCSC genome browser, Feb 2009 assembly

gNot available in databases; reported in various studies [1, 11]

hFamilies with male breast cancer

Table 3

Characteristics of the 133 families with deleterious BRCA1/2 mutations

Family

No. of cancers

Age at onset (years)

Other cancer(s)c

(age at onset in years)

Ethnicity

Female BC (Bilateral)

OC (OC+BC)

BC

OC

Families carrying BRCA1-small-range mutations

 236a

1

-

22b

-

-

Pathan

 316

1

-

25b

-

-

Punjabi

 264

1

-

26b

-

-

Punjabi

 706

1

-

26b

-

Uterus (67)

Punjabi

 N12

1

-

26b

-

-

Punjabi

 624

1

-

27b

-

-

Punjabi

 N25

1

-

28b

-

-

Punjabi

 276a

1

-

28b

-

-

Punjabi

 610

1

-

28b

-

-

Punjabi

 678

1

-

28b

-

-

Punjabi

 411a

1

-

29b

-

Stomach (70)

Punjabi

 724

1

-

29b

-

Renal (48), lung (65), throat (65), unknown

Punjabi

 N28

1

-

30b

-

-

Punjabi

 279a

1(1)

-

27/36b

-

-

Punjabi

 278

2

-

25b,32

-

-

Kashmiri

 332a

2

-

26b,51

-

Leukemia (45)

Punjabi

 682

2

-

28b,40

-

Uterus (<62,65), throat (<72)

Punjabi

 N18

2

-

29b,<50

-

-

Punjabi

 421a

2

-

30b,33

-

-

Punjabi

 432

2

-

30b,53

-

Skin (12), oral (54)

Punjabi

 520

2

-

30b,47

-

Uterus (32)

Punjabi

 449

2

-

32b,55

-

-

Punjabi

 557a

2

-

32b,45

-

Unknown (<55), renal (70)

Punjabi

 747

2

-

33b,38

-

-

Unknown

 722

2

-

20,34b

-

Unknown (<18,<40)

Punjabi

 687

2

-

37b,45

-

-

Punjabi

 470a

2

-

40b,40

-

Stomach (46), colon (59), lung

Punjabi

 510a

2

-

40b,55

-

-

Punjabi

 N13a

2

-

40b,>50

-

-

Punjabi

 593

2

-

43,44b

-

Leukemia (22)

Pathan

 299

2(1)

-

24/27b,55

-

-

Punjabi

 660a

2(1)

-

25/26b,70

-

Bladder

Punjabi

 260

2(1)

-

25/26b,28

-

-

Punjabi

 511

2(1)

-

30/33b,<32

-

Brain (75)

Punjabi

 N34

3

-

24b,<30,31

-

-

Punjabi

 669a

3

-

25b,<40,<50

-

Brain (<78), oral (<80)

Punjabi

 685

3

-

26b,26,?

-

Blood (2x)

Mohajir

 723

3

-

28b,40,?

-

-

Pathan

 612

3

-

29b,<30,40

-

Throat (45), uterus (48)

Punjabi

 313a

3

-

30b, 48,?

-

-

Punjabi

 336a

3

-

23,30b,38

-

Prostate (29)

Punjabi

 493a

3

-

35b,55,>55

-

-

Pathan

 382

3

-

36b,50,?

-

-

Punjabi

 489a

3

-

25,42b,45

-

-

Punjabi

 743

3

-

40,44b,62

-

Bone (60), leukemia (60)

Pathan

 658

3

-

26,44b,50

-

-

Punjabi

 377a

3

-

31,50b,85

-

Thyroid (59), intestine (70), bladder (75), liver

Punjabi

 550d

3

-

50b,55,>50

-

Lung, unknown

Punjabi

 372

3(1)

-

21/21b,29,45

-

Squamous cell carcinoma scalp (22)b

Pathan

 626a

3(1)

-

35b,36/37, 42

-

-

Balochi

 389

3(1)

-

22/32,42,48b

-

Brain (36), uterus (70)

Punjabi

 247a

4

-

27,28b,40,42

-

Uterus (31, 55)

Siriaki

 652a

4

-

31b,33,42,50

-

-

Punjabi

 362

4

-

31,32,35b,45

-

Liver (>40), abdomen

Pathan

 399a

4

-

43, 44b,50,?

-

Abdomen (45), lung (45), prostate (53)

Punjabi

 338a

4

-

30,40,44,48b

-

Stomach (73)

Punjabi

 408a

4(1)

-

24b,31/31,33,50

-

Abdomen (54), esophagus (74)

Punjabi

 521

4(1)

-

25/38b,27,33,70

-

Stomach (60, 65), lung, unknown

Punjabi

 653

5

-

24,32,35b,37,50

-

Colon (42), throat (66)

Punjabi

 734

5

-

37,38b,55,?,?

-

-

Punjabi

 296

7(1)

-

21b,<30,34,43,44/44,51,52

-

-

Punjabi

 439

8

-

30,<35,40b,40,40,40,>50,>65

-

Uterus (40), prostate, unknown

Punjabi

 249a

8(1)

-

30/31b,37,45,?,?,?,?,?

-

-

Punjabi

 619a

1

1

30b

>50

-

Punjabi

 646

1

1(1)

34b

36b

-

Punjabi

 748

1

1(1)

47b

52b

Skin (45), liver (50)

Punjabi

 542

1

2(1)

40b

46b,70

Leukemia (74)

Punjabi

 210a

1

4

45b

?,?,?,?

Brain (32), abdomen, lung, leukemia

Punjabi

 241a

2

1

29b,64

35

Lymphoma

Punjabi

 598a

2

1

30b,56

>50

Stomach (>50, >50), tongue (>50)

Punjabi

 463

2

1

35b,58

48

-

Punjabi

 481

2

1

<25,47b

36

Lung (55), uterus

Punjabi

 621

2

1

50,>60

50b

Prostate (65)

Punjabi

 211a

2

1(1)

26b,50

50

-

Punjabi

 415a

2

1(1)

34,35b

35b

Leukemia (45), unknown

Punjabi

 679

2

1(1)

28b,<33

49b

-

Punjabi

 N4a

2

1 (1)

41b,45

45

Uterus (38)

Punjabi

 488a

2

2(1)

40b,55

42b,45

-

Punjabi

 555

2

2(1)

29b,36

36b,>50

Leukemia (10), vocal cord (45)

Punjabi

 317

2

3(1)

41,46

47b,52,55

Fallopian tube (47)b

Punjabi

 318a

2

4(2)

40,46b

40,42b,44,58

Bladder (50, 50)

Pathan

 442

2(1)

2

34,50/50b

28,52

Leukemia (15)

Punjabi

 283a

2(1)

3

34/38b,56

54,55,65

-

Pathan

 254a

3

1

27,32b,43

41

Brain

Punjabi

 711

3

1

40b,?,?

?

Gall bladder

Sindhi

 445a

3

1(1)

44b,>60,73

74

Gall bladder

Punjabi

 363

3

2

32,35,70

47b, ?

Lung (65), oral (70), liver

Kashmiri

 329a

3

3

34b,39,?

39,<50,?

-

Punjabi

 609a

4(1)

1

29b,31,48/55,65

30

-

Mohajir

 328a

4

1

29,30b,31,39

55

-

Punjabi

 501

4

1

34b, 35, >50,?

?

Brain (42)

Punjabi

 522

4

2(1)

30b,<40,45,45

35,60

Uterus (41)

Punjabi

 611a

4

2(1)

31b,36,37,42

50,55

Blood (30)

Punjabi

 523a

3(1)

4

39/46,40,53b

30,45,51,60

-

Punjabi

 275a

4(1)

1(1)

34/40b,40,42,50

40

-

Punjabi

 469

5

1(1)

29b,29,<35,>35,>55

32b

-

Mohajir

 340a

6

1

34b,42,<50,<50,<50,<50

54

-

Balochi

 574a

6

2

32,32,35,45b,48,>50

48,55

-

Mohajir

 512a

6(1)

1

<25,<30,<40,46/55b,<50,>50

<50

Uterus (<50)

Kashmiri

 248a

7

2(2)

23b,<25,34,<40,<46,<60,?

23b,<60

-

Punjabi

 220a

8

1(1)

25,27b,<30,53,58,63,77

<30

-

Punjabi

Families carrying BRCA1-LGRsa

 229

1

-

28b

-

-

Punjabi

 379

2

-

29,31b

-

Liver (38)

Punjabi

 261

2

-

33,34b

-

-

Punjabi

 406

2

-

39b,40

-

Abdomen (65)

Punjabi

 498

2

-

40,41b

-

-

Siriaki

 549

2

-

38,72b

-

Unknown

Punjabi

 314

6

-

32b,42,56,70,?,?

-

Uterus (54), pharynx (59), brain (63), abdomen

Punjabi

 291

3

1

39,42,48

48b

Stomach, brain

Punjabi

 719

3(1)

1

>40,42b,?

?

-

Punjabi

Families carrying BRCA2-small-range mutations

 330

1

-

29b

-

Lung (48, 58, 66), tongue (55)

Punjabi

 206

1

-

30b

-

-

Pathan

 540d

1

-

67b

-

-

Mohajir

 207d

1

-

76b

-

Intestine (60)

Punjabi

 295a

1(1)

-

23/23b

-

Leukemia (49), esophagus (50)

Punjabi

 N26

2

-

26b,35

-

-

Pathan

 602

2

-

31b,43

-

-

Punjabi

 492

2

-

38b,39

-

-

Mohajir

 505

2

-

43,46b

-

-

Pathan

 713

2

-

35,56b

-

-

Kashmiri

 700

2(1)

-

35/43b,46

-

Throat (72)

Punjabi

 627

2(1)

-

42b,51/51

-

-

Pathan

 545

3

-

35,36b,47

-

Brain (50), uterus (50), bone (54)

Punjabi

 497

3

-

51b,55,50

-

Brain

Siriaki

 548d

3

-

45,50,69b

-

-

Pathan

 702

4

-

26,30b,<33,70

-

Throat (72)

Punjabi

 579

4

-

35,49,<50,51b

-

Oral (35), gall bladder (42)

Kashmiri

 407d

4

-

31,45(male),45,54b

-

Esophagus (39,59), leukemia (64)

Mohajir

 538

5

-

34b,38,45,50,58

-

Retinoblastoma (3), pancreas (75), liver (83)

Pathan

 684

5

-

40b,45,48,50,<57

-

Throat (<48, <82), stomach (53), intestine (60)

Pathan

 447

3(1)

2

31b,<50,55/65

50,>50

Abdomen (>50), colon (62), brain (65)

Punjabi

 525

4(1)

1

32,33/35b,<50,60

<45

Bladder (>60)

Mohajir

 222

7

2(1)

35,42,43,50,54b,60,?

47,53b

Lung (60), prostate (70), lung

Kashmiri

BC breast cancer, OC ovarian cancer, Unknown cancer phenotype is not known

aMutations previously described [11, 18]; bProband; cAge at cancer diagnosis is mentioned along with cancer phenotype. For relatives with unknown age at cancer onset, only cancer phenotype is mentioned; dFamilies with male breast cancer

Twenty-one (21/71; 29.6%) mutations including 18 in BRCA1 and three in BRCA2 occurred more than once (Fig. 2a, b). These mutations were identified in 83 unrelated families and accounted for 62.4% (83/133) of all families with mutations. The most common BRCA1 mutation was c.3770_3771del (ten Punjabi families), followed by c.5503C>T (nine Punjabi families), exon 1-2 deletion (seven Punjabi families), c.685del (five Punjabi and two Balochi families), c.4485-1G>A (six Punjabi families), c.2269del (one Punjabi and four Pathan families), c.2405_2406del (five Punjabi families), c.4065_4068del (three Punjabi and one Pathan families), c.1793T>G and c.4508C>A (three Punjabi families each), exon 21-24 deletion, c.2603C>G, c.3339_3341del, c.3598C>T, c.5035del, c.5074+1G>A, and c.5361_5362del (two Punjabi families each) and c.2340_2343del (one Pathan and one Mohajir families). The most common BRCA2 mutation was c.5222_5225del (one Punjabi and three Mohajir families), followed by c.92G>A (two Punjabi families) and c.5682C>A (two Pathan families).
Fig. 2

Distribution of deleterious germline mutations identified in Pakistani breast/ovarian cancer families across the BRCA1 and BRCA2 genes. Recurrent mutations are marked with (RM). The distribution of breast cancer (BC) and ovarian cancer (OC) in families according to the position of the mutations in BRCA1 (a) and BRCA2 (b) is shown. Regions inferred to be breast cancer cluster regions (BCCRs) and ovarian cancer cluster regions (OCCRs) according to Rebbeck and colleagues [24] are shown at the bottom

In addition to the deleterious mutations, 153 (28.4%) distinct BRCA1/2 sequence variants were detected: 79 missense variants, 48 non-coding variants, 24 synonymous variants, one in-frame deletion, and one polymorphic nonsense variant in exon 27 of BRCA2 (data not shown).

BRCA1/2 mutation frequencies

The frequencies of BRCA1/2 mutations by risk group are provided in Table 1. For BRCA1, the highest mutation frequency was noted in families with breast and ovarian cancer (53.8%), followed by families with at least three breast cancer cases (24.8%), families with two breast cancer cases (18.3%), or families with one early-onset breast cancer case (<30 years) (10.8%). For BRCA2, the highest frequency was observed in families with male breast cancer (14.8%).

Patient and tumors characteristics by BRCA1/2 status

BRCA1 carriers (n=110) were more often identified among female patients (99.1% vs. 94.6%, p=0.039) and belonged to the Punjabi ethnic group (81.8% vs. 68.7%, p=0.030) compared to non-carriers (n=406). In contrast, BRCA2 carriers (n=23) were more common among male patients (17.4% vs. 5.4%, p=0.043) and more often belonged to Pathan ethnic group (34.8% vs. 15.5%, p=0.009).

Female breast cancer patients with mutations in BRCA1 (n=106) or BRCA2 (n=19) had a similar mean age of diagnosis (34.0 years (range 21–72) and 37.7 years (range 23-56), respectively, p=0.073, Wilcoxon rank-sum test), which did not differ to that of non-carriers (n=377) (35.7 years (range 18-78). In contrast, male breast cancer patients harboring BRCA2 mutations (n=4) had an older mean age of diagnosis than non-carriers (n=22) (66.5 years (range 54-76) and 52.5 years (range 27-69) years, respectively, p=0.039, Wilcoxon rank-sum test).

BRCA1-associated breast tumors more often were invasive ductal carcinomas (99.0% vs. 91.4%, p=0.004), triple-negative (60.8% vs. 22.6%, p=<0.0001), and of higher tumor grade (grade 3: 94.9% vs. 63.2%, p=<0.0001) compared to tumors of non-carriers. BRCA2-associated breast tumors more often were PR positive compared to tumors of non-carriers (81.8% vs. 57.2%, p=0.025) (data not shown).

Discussion

To our knowledge, this is the largest Pakistani study that comprehensively investigated the spectrum of BRCA1/2 small-range mutations and LGRs and prevalence of mutations in 539 high-risk families. Mutations were identified in 24.7% (133/539) of families. Eighteen BRCA1 and three BRCA2 mutations were recurrent and accounted for 68.2% and 34.8% of all mutations in BRCA1 and BRCA2, respectively. Nine mutations were specific to the Pakistani population, whereas other mutations had been reported elsewhere.

The most common type of identified mutations were frameshift mutations (60.6%) followed by nonsense mutations (25.4%). These data are consistent with our previous report [10] and a recent worldwide study [25]. In Pakistani patients, BRCA1 mutations were about 5-times more frequent than BRCA2 mutations. A similar distribution was observed in two Asian studies from South India [26] and Saudi Arabia [27] and most studies among white populations [3, 4, 5, 28]. This is likely due to the predominance of recurrent BRCA1 mutations in these populations. Contradictory results were reported in other Asian studies from China, Hong Kong, Korea, and Indonesia, where BRCA2 mutations were observed at an equal or a higher frequency than BRCA1 mutations [6, 12, 15, 16, 17].

Among the 133 mutations identified in our study, 18 BRCA1 and three BRCA2 mutations were recurrent, accounting for 68.2% and 34.8% of all mutations in BRCA1 and BRCA2, respectively. The proportion of recurrent BRCA1 mutations to the total number of identified BRCA1 mutations is higher than our previous report [10], which is likely due to the larger size of the present study. Of the identified recurrent mutations, the majority was also reported as recurrent mutations in other populations [1, 4, 25], while few were exclusively identified in a specific ethnic group of Pakistan. Fourteen BRCA1 mutations (c.3770_3771del, c.5503C>T, c.4485-1G>A, c.2405_2406del, c.1793T>G, c.4508C>A, c.2603C>G, c.3339_3341del, c.3598C>T, c.5035del, c.5074+1G>A, c.5361_5362del, exon 1-2 deletion, and exon 21-24 deletion) and one BRCA2 mutation (c.92G>A) were identified only in the Punjabi ethnic group. One BRCA2 mutation (c.5682C>A) was found only in the Pathan ethnic group. Five other recurrent mutations were identified in more than one ethnic group. Our findings imply that a panel of ethnic specific recurrent mutations may be useful for initial screening of high-risk patients from these ethnic groups. Founder effects were previously suggested for six of the 18 recurrent BRCA1 mutations (c.3770_3771del, c.4065_4068del, c.4485-1G>A, c.4508C>A, c.5503C>T, exon 1-2 deletion) [9, 10, 11], while haplotype analyses of the remaining recurrent mutations have not been performed so far. The high percentage of recurrent BRCA1 mutations facilitates the development of a local, economical, and efficient ethnic-specific genetic testing strategy in Pakistan.

BRCA1/2 mutations were identified in 24.7% of Pakistani breast cancer families. This frequency is higher than that from our initial report (17%) [10], probably due to the larger study size and comprehensive mutation analyses of both genes. This frequency is also higher than those from other Asian reports from Hong Kong, Malaysia, and Korea, ranging from 9.4% to 21.7% [6, 7, 8, 16, 17]. These findings further support the notion that the BRCA1/2 mutation frequencies vary among different populations. Our data are similar to those reported in white populations, ranging from 17.6% to 29.8% [1, 2, 4]. We found the highest mutation frequency in breast and ovarian cancer families (55.4%), in agreement with previous studies from Pakistan [10], Korea [16], and studies in white populations [4, 28]. We observed a 2.52 fold (53.8% vs. 21.3%) increased occurrence of BRCA1 mutations in breast and ovarian cancer families compared to breast cancer only families, in line with previous reports [1, 4, 6, 28]. Our findings support the notion that the presence of ovarian cancer in Pakistani breast cancer families increases the likelihood for the occurrence of BRCA1 mutation.

In the present study on 27 families with male breast cancer, a BRCA1/2 mutation frequency of approximately 19% was observed, with BRCA2 mutations being about 4-times more common than BRCA1 mutations. Our data are in line with previous studies [4, 14]. This observed frequency is higher than that reported in our initial much smaller study, in which no mutations were identified [10]. In agreement with the National Comprehensive Cancer Network (NCCN) guidelines, our data also warrant BRCA1/2 testing in families with male breast cancer (NCCN Guidelines Version 2.2019).

The main strength of this study is its large size of 539 high-risk families, the comprehensive screening of both genes for small-range mutations and LGRs using highly sensitive methods (allowing the identification of recurrent BRCA1/2 mutations in the Pakistani population and the more accurate estimation of BRCA1/2 mutation frequencies among high-risk families), and the confirmations of mutations in an independent patient’s sample. However, our study also has some limitations. Participants were recruited at one tertiary care cancer center in Lahore, which may have introduced selection bias. Families belonging to Punjabi and Pathan ethnic groups are over-represented and, therefore, mutations in these groups may be over-represented. Nevertheless, Punjabi (44.7%) and Pathan (15.4%) are the most common ethnic groups reported in Pakistan (The World Factbook). Further, our data are based on self-reported ethnicity of study participants, which may lead to a misclassification of the ethnic origin of some of them.

Conclusions

In summary, our study showed that BRCA1/2 mutations account for 24.7% of high-risk breast cancer patients in Pakistan. Our results have important clinical implications, such as personalized treatment with platinum-based or PARP-inhibitor therapy for breast/ovarian cancer patients carrying a pathogenic BRCA1/2 mutation and early detection, surgical prevention, and chemoprevention strategies for their unaffected BRCA1/2 mutation positive relatives. Overall, BRCA1/2 mutations account for one in four patients with a family history of breast cancer/breast and ovarian cancer, one in five patients with male breast cancer, and one in eight patients with early-onset breast cancer. Eighteen mutations in BRCA1 and three in BRCA2 were recurrent and accounted for 68.2% and 34.8% of all identified mutations in BRCA1 and BRCA2, respectively. Our data suggest that BRCA1 testing may be justified for families with multiple female breast cancers, breast and ovarian cancer or early-onset breast cancer and BRCA2 testing for families with male breast cancer from Pakistan. Our findings will help in tailoring cost-effective genetic testing approach for the high-risk Pakistani population or for individuals of Pakistani origin residing in other countries.

Notes

Acknowledgements

We are grateful to all study subjects for their participation in this study. We thank the clinicians (Neelam Siddiqui, Mazhar Ali Shah, Narjis Muzaffar, Usman Ahmad, Umm E Kalsoom, Amir Ali Syed, Huma Majeed, Zulqarnain Chaudhry, Muhammad Asad Parvaiz, and Amina Khan) for their help in recruitment of study participants. We thank Jörg Hoheisel for critical reading of the manuscript.

Authors’ contributions

MUR contributed to conception and design of the study, patient recruitment and data acquisition. In addition, he was involved in data analysis, interpretation and in drafting and revising the manuscript. NM performed the molecular analyses and contributed to data analysis and interpretation. He was also involved in writing the first draft of the manuscript. HN, FAK and SG performed the molecular analysis. MH and SF were involved in patient recruitment and clinical data acquisition. AA was involved in the recruitment of study subjects, clinical data collection and revising the manuscript. AL was involved in the pathological data acquisition and interpretation. UH contributed to conception and design of the study, data analysis and interpretation and led the writing of the manuscript. All authors read and approved the final manuscript.

Funding

The study was supported by the Shaukat Khanum Memorial Cancer Hospital and Research Centre (grant number ONC-BRCA-002) and the German Cancer Research Center.

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. It was approved by the ethics committee of Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH&RC), Lahore Pakistan. The ethics committee name is the “Institutional Review Board”. The approval number is ONC-BRCA-002. Written informed consent was obtained from all study participants.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing of interests.

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© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Basic Sciences Research, Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH&RC)LahorePakistan
  2. 2.Molecular Genetics of Breast Cancer, German Cancer Research Center (DKFZ)HeidelbergGermany
  3. 3.Clinical Research Office, SKMCH&RCLahorePakistan
  4. 4.Leibniz Institute on Aging - Fritz Lipmann InstituteJenaGermany
  5. 5.Levine Cancer InstituteCharlotteUSA
  6. 6.Pathology DepartmentSKMCH&RCLahorePakistan

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