Breast cancer has a substantial impact on the overall tumor burden in Pakistan comprising 40% of all female malignancies. In Pakistan, monoallelic germline mutations in the high- and moderate-penetrance breast cancer susceptibility genes BRCA1, BRCA2, TP53, CHEK2, and RAD51C account for approximately 25% of early-onset and familial breast cancer suggesting that other susceptibility gene(s) may be involved. Recently identified PALB2 gene, a partner and localizer of BRCA2, acts as a link between BRCA2 and BRCA1 and enables DNA repair [1]. Biallelic mutations in PALB2 (FANCN) cause Fanconi anemia, with clinical features similar to those caused by biallelic mutations in BRCA2 (FANCD1) [2]. Monoallelic mutations in PALB2 confer susceptibility to breast cancer suggesting that PALB2 is another candidate to be a breast cancer susceptibility gene [3]. Deleterious PALB2 mutations are estimated to confer a 35% lifetime risk of breast cancer for the carriers [4].

PALB2 mutation screening in BRCA1/2-negative early-onset or familial breast cancer patients performed previously in different populations have yielded variable results. In a British study, monoallelic mutations in PALB2 were identified in 1.1% of familial breast cancer patients (10/923) and were absent in controls (0/1,084) [3]. In a study conducted in Finland, a recurrent PALB2 mutation, c.1592delT, was identified in 2.7% of familial breast cancer patients (3/113) and in 0.2% of controls (6/2,501) [5]. Mutations were identified in families from Europe, North America, and Australia at frequencies ranging from 0.3% to 5.5% [6-9], while mutations were absent in families from Canada [10], United States [11], and Chile [12].

In Asia, little is known about the contribution of PALB2 mutations to early-onset and familial breast/ovarian cancer. Deleterious PALB2 mutations were reported in families from China, Korea, and Malaysia/Singapore with frequencies varying from 0.8% to 1.6% [13-15] and were absent in Japanese families [16]. Given the limited data on genetic variability of PALB2 in South Asia and the fact that only 25% of hereditary breast/ovarian cancer in Pakistan is attributed to germline mutations in BRCA1/2, TP53, CHEK2, and RAD51C [17-20], we assessed and report the prevalence of PALB2 mutations in 370 early-onset and familial breast/ovarian cancer patients from this population, who had tested negative for mutations in these five breast cancer susceptibility genes. Functional and potentially functional mutations were screened in 372 healthy controls.

The present study included 370 early-onset and familial breast/ovarian cancer patients from Pakistan, who were negative for BRCA1/2, TP53, CHEK2, and RAD51C mutations. Of these, 180 were diagnosed with early-onset breast cancer (≤ 30 years of age), 127 belonged to families with two or more breast cancers, 23 to families with both breast and ovarian cancer, 22 to families with at least one ovarian cancer (diagnosed ≤ 45 years of age), and 18 to families with male breast cancer (Table 1). The median age of the disease onset was 30 years (range, 19 to 73 years) for female breast cancer (n=324), 47.5 years (range, 30 to 73 years) for male breast cancer (n=18), and 33 years (range, 22 to 60 years) for ovarian cancer (n=35). One-hundred and one index patients presented with triple-negative breast cancer (TNBC) and 217 with non-TNBC. The median age of the disease onset was 28 years (range, 19 to 67 years) for patients with TNBC and 30 years (range, 19 to 73 years) for those with non-TNBC.

Overall, 31 different heterozygous PALB2 variants were detected. Of these, 13 were novel: one nonsense mutation, four missense variants, one silent variant, six intronic variants, and one 5′ UTR variant (Table 2). The remaining 18 variants have been previously reported in other populations: eight missense variants, four silent variants, five intronic variants, and one 5′ UTR variant.

The novel nonsense mutation at nucleotide position 2229 in exon 5, c.2229T>A (p.Y743*), was detected in a 29-year-old breast cancer patient of Punjabi ethnicity. The patient presented with a grade 3, invasive ductal carcinoma (IDC) of TNBC phenotype. A sister of the index patient was diagnosed with breast cancer at the age of 41 years.

The remaining 12 novel variants were analyzed for their potential functional effect by in silico analysis. Two missense variants p.G644R and p.E744K and the previously reported missense variant p.D498Y [14,16,27] were predicted to be potentially deleterious by three out of five in silico analysis tools (Table 3). The 5′ UTR variant c.-134_-133-del-TCinsGGGT was predicted to create a splice donor site by three out of five splice-site prediction tools implying that it was disease associated (Table 4). This variant was also predicted to create the binding sites for two transcription factors, GR-alpha (T00337) and RXR-alpha (T01345), using PROMO (v3.0.2) on-line program.

The potentially disease-causative missense mutations p.G644R and p.E744K were identified in two women of Punjabi or Pathan ethnic group, who were diagnosed with early-onset breast cancer at the ages of 30 years, respectively. Their tumors were grade 3 IDCs, which were, ER-positive, PR-positive, and HER2-negative. The other missense mutation, p.D498Y, was identified in a patient of Pathan origin, who was diagnosed with synchronous bilateral breast cancer at 39 years of age. Left-sided breast tumor was grade 2 IDC and ER-positive, PR-negative, and HER2-negative. Right-sided breast tumor was grade 3 IDC and ER-negative, PR-negative, and HER2-positive. The index patient reported a family history of breast cancer and other cancers. The mother and one sister of the index patient were diagnosed with breast cancer at the ages of 53 and 44 years, respectively. Her father was diagnosed with lung cancer at age 69. One sister and paternal uncle of the index patient were diagnosed to have brain cancer at the ages of 34 and 48 years, respectively.

The 5′ UTR variant, c.-134_-133delTCinsGGGT, was identified in an early-onset breast cancer patient of Pathan ethnic group, who was diagnosed with disease at 26 years of age. Her tumor was a grade 2 IDC of TNBC phenotype. This patient had also been diagnosed with adenocarcinoma of the rectum at 25 years of age.

The nonsense mutation and the four in silico-predicted potentially functional mutations were not identified in 372 controls implying that they were disease-causative (Table 2). The remaining 17 variants (seven missense variants, four silent variants, five intronic variants, and one 5’ UTR variant) were reported previously and classified as polymorphisms (Table 2).

In this large study conducted in Pakistan, we assessed for the first time the prevalence of PALB2 germline mutations in 370 early-onset and familial breast/ovarian cancer patients negative for BRCA1/2, TP53, CHEK2, and RAD51C mutations. One novel deleterious and four in silico-predicted potentially functional PALB2 mutations (including three novels) were identified. Our study provides additional data on the contribution of PALB2 mutations to hereditary breast/ovarian cancer in an Asian population from Pakistan.

The novel pathogenic PALB2 mutation (p.Y743*) was identified in 0.8% of families with two or more breast cancers (1/127). Similar mutation frequencies were reported in other Asian breast and/or ovarian cancer families from Saudi Arabia (5/725, 0.7%) [28], China (3/360, 0.8%) [13], and Korea (2/235, 0.8%) [14]. Higher frequencies were reported in studies conducted in India (9/752, 1.2%) [29], Malaysia/Singapore (2/122, 1.6%) [15], China (2/108, 1.8%) [30], and Korea (3/120, 2.5%) [31]. This could be due to the Next-Generation Sequencing approach employed in most of these studies. Furthermore, our results are likely to underestimate the PALB2 mutation frequencies as the recently reported large genomic rearrangements were not screened [29,32]. Mutations were not identified in few studies from Japan [16], Chile [12], and United States [11]. In European and North American studies including more than 100 families from Czech Republic, France, Ireland, Germany, Poland, Italy, Finland, Canada, United States, and Australia the mutation frequencies ranged from 0.3% to 5.5% [5-9,33-38]. Altogether these findings suggest that PALB2 mutations account for a small proportion of hereditary breast/ovarian cancer in most populations including the Pakistani population.

Four in silico-predicted potentially functional PALB2 mutations (p.G644R, p.E744K, p.D498Y, and c.-134_-133del-TCinsGGGT) were also identified in this study. The novel missense mutations p.G644R and p.E744K, each identified in an early-onset breast cancer patient, were located in the highly conserved MRG15-binding domain of PALB2 spanning amino acid residues 611 to 764. They may ablate PALB2 interacting with MRG15, which may result in impaired DNA repair [39] leading to genomic instability and cancer. The p.G644R variant was predicted to be deleterious by SIFT, Align-GVGD and SNAP-2 algorithms, while it was predicted as benign by PolyPhen-2 and MutationTaster. The differential prediction may be due to the underlying algorithm’s differences as SIFT uses evolutionary sequence conservation whereas PolyPhen-2 utilizes protein structure information [40]. Since none of these algorithms is 100% predictive, a consensus prediction is reported to improve the prediction performance [41]. Overall, this variant was considered as deleterious by three of the five protein function algorithms. The missense mutation p.D498Y was identified in a familial breast cancer patient. Due to lack of DNA samples, co-segregation of the mutation with breast cancer could not be studied. Previously, this was also reported as an in silico-predicted potentially disease-causative mutation in families from Korea, Japan, and Australia [14,16,27]. The 5′ UTR variant, c.-134_-133delTCinsGGGT, was detected in a patient with personal history of breast and rectal cancer. It was predicted to result in activation of a cryptic splice site. This variant was also predicted to affect two transcription factor binding sites and may alter the promoter activity of PALB2. None of these mutations were detected in 372 controls further suggesting that they are disease-causative; however, functional analyses of the potentially disease-causative mutations will be required for final mutation classification.

No deleterious PALB2 mutations were identified in early-onset breast cancer patients, families with breast and ovarian cancer, ovarian cancer, or male breast cancer. These findings are in keeping with those previously reported in families from Turkey [42], Finland [43], Japan [16], the Netherlands [44], Spain [32], Italy [45], and Australia [46]. In contrast, PALB2 mutations have also been reported in families from Poland [37], Italy [47], Czech Republic [8], and China [13]. These findings suggest that PALB2 mutations may not substantially confer susceptibility to early-onset breast cancer patients and patients from breast and ovarian cancer, male breast cancer or ovarian cancer families.

In our study, the breast tumors associated with the PALB2 truncating (p.Y743*) and in silico-predicted potentially functional (p.D498Y, p.G644R, and p.E744K) mutations presented with high-grade tumors of IDC histology, which is consistent with previous studies conducted among Asian [13-15], European [24,25,48], and North-American patients [49]. In the Pakistani study, the breast tumor linked with the truncating mutation displayed the TNBC phenotype, in agreement with other studies from Europe, North-America and Australia [50-52]. Breast tumors associated with missense mutations were ER-positive, PR-positive, and HER2-negative. Similar hormone receptor expression patterns were also reported in breast tumors of Asian [14], European [48], and North-American patients [53], who harbored PALB2 truncating mutations. The differential expression of hormone receptors could be due to mutation-specific tumor phenotypes in the studied population.

In summary, we have identified one novel pathogenic and four potentially pathogenic PALB2 mutations, three being novel in 370 Pakistani early-onset and familial breast/ovarian cancer patients, negative for mutations in BRCA1/2, TP53, CHEK2, and RAD51C. The frequency of PALB2 mutations in patients with familial breast cancer was 0.8% (1/127), while no mutations were identified in early-onset breast cancer patients and patients from breast and ovarian cancer, male breast cancer or ovarian cancer families. Our findings suggest a marginal contribution of PALB2 mutations to breast cancer susceptibility in Pakistan.