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

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.

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 [1-5] and 9.4% to 21.7% in non-whites from Asia [6-8]. 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 [3–5, 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, 15–17].

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) [9-11], 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% [6–8, 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.