Analysis of PALB2 in a cohort of Italian breast cancer patients: identification of a novel PALB2 truncating mutation

Abstract

PALB2 gene is mutated in about 1-2 % of familial breast cancer as well as in 3-4 % of familial pancreatic cancer cases. Few studies have reported mutations in Italian patients with breast or pancreatic cancer. We evaluate the occurrence of PALB2 mutations in Italian patients affected with hereditary breast and ovarian cancers and define the pathological significance of the putative allelic variants. We recruited 98 patients (F = 93, M = 5) affected with breast and/or ovarian cancer, negative for mutations in BRCA1 and BRCA2 (BRCAX). Genomic DNA was isolated from peripheral blood lymphocytes, PALB2 coding regions and adjacent intronic were sequenced; in silico predictions were carried out using prediction programs. Mutational analysis of PALB2 gene revealed the novel mutation c.1919C>A (p.S640X) in a 29 years old woman with breast cancer. The c.1919C>A (p.S640X) mutation causes the lack of C-terminus region inducing alteration of MORF4L1-PALB2 association and the lack of interaction of PALB2 with RAD51 and BRCA2. In addition, we identified two novel PALB2 variants, c.3047T>C (p.F1016S) and c.*146A>G. In silico analysis conducted for c.*146A>G indicates that this variant does not affect the splicing while c.3047T>C (p.F1016S) was predicted as damaging in three classifier algorithms. The proband carrier of c.3047T>C (p.F1016S) showed two breast cancer cases, two ovarian cancer cases and one pancreatic cancer in mother's family. c.3047T>C (p.F1016S) and c.*146A>G should be considered PALB2 UVs even though the genotype-phenotype correlation for these variants remains still unclear. Our findings indicate that the presence of PALB2 mutation should be routinely investigated in hereditary breast and ovarian cancers families since it could be of clinical relevance for clinical management.

Full text

LETTER TO THE EDITOR
Analysis of PALB2 in a cohort of Italian breast cancer patients:
identification of a novel PALB2 truncating mutation
Maria Teresa Vietri •Gemma Caliendo •Concetta Schiano •
Amelia Casamassimi •Anna Maria Molinari •
Claudio Napoli •Michele Cioffi
ÓSpringer Science+Business Media Dordrecht 2015
Abstract PALB2 gene is mutated in about 1–2 % of fa-
milial breast cancer as well as in 3–4 % of familial pan-
creatic cancer cases. Few studies have reported mutations
in Italian patients with breast or pancreatic cancer. We
evaluate the occurrence of PALB2 mutations in Italian
patients affected with hereditary breast and ovarian cancers
and define the pathological significance of the putative
allelic variants. We recruited 98 patients (F =93, M =5)
affected with breast and/or ovarian cancer, negative for
mutations in BRCA1 and BRCA2 (BRCAX). Genomic
DNA was isolated from peripheral blood lymphocytes,
PALB2 coding regions and adjacent intronic were se-
quenced; in silico predictions were carried out using pre-
diction programs. Mutational analysis of PALB2 gene
revealed the novel mutation c.1919C[A (p.S640X) in a
29 years old woman with breast cancer. The c.1919C[A
(p.S640X) mutation causes the lack of C-terminus region
inducing alteration of MORF4L1–PALB2 association and
the lack of interaction of PALB2 with RAD51 and BRCA2.
In addition, we identified two novel PALB2 variants,
c.3047T[C (p.F1016S) and c.*146A[G. In silico analysis
conducted for c.*146A[G indicates that this variant does
not affect the splicing while c.3047T[C (p.F1016S) was
predicted as damaging in three classifier algorithms. The
proband carrier of c.3047T[C (p.F1016S) showed two
breast cancer cases, two ovarian cancer cases and one
pancreatic cancer in mother’s family. c.3047T[C
(p.F1016S) and c.*146A[G should be considered PALB2
UVs even though the genotype–phenotype correlation for
these variants remains still unclear. Our findings indicate
that the presence of PALB2 mutation should be routinely
investigated in hereditary breast and ovarian cancers
families since it could be of clinical relevance for clinical
management.
Keywords PALB2 Hereditary breast cancer Hereditary
ovarian cancer Hereditary pancreatic cancer
Abbreviations
UVs Unclassified variants
HBOC Hereditary breast and ovarian cancer
PALB2 Partner and localizer of BRCA2
MORF4L1 Mortality factor 4 like protein 1
MRG15 MORF4-related gene on chromosome 15
HGVS Human Genome Variation Society
NCBI National Centre for Biotechnology
Information
Hereditary breast cancer genes (BRCA1,BRCA2,CHK2,
TP53,ATM and PALB2) involved in DNA damage re-
sponses suggested that hereditary breast and ovarian cancer
(HBOC) is a consequence of impaired genome stability
control [1]. PALB2, originally identified as a BRCA2-in-
teracting protein, is an essential component for the formation
of the BRCA complex. Indeed, PALB2 acts as a bridge
between BRCA1 and BRCA2 to form a BRCA complex that
then binds RAD51 to initiate homologous recombination
[2]. Mutations in BRCA genes can be found in about 30 % of
M. T. Vietri (&)G. Caliendo A. Casamassimi 
A. M. Molinari C. Napoli M. Cioffi
Department of Biochemistry, Biophysics and General Pathology,
Second University of Naples, Via Luigi De Crecchio, 7,
80138 Naples, Italy
e-mail: mariateresa.vietri@unina2.it
C. Schiano C. Napoli
Institute of Diagnostic and Nuclear Development (SDN),
IRCCS, Via E. Gianturco 113, 80143 Naples, Italy
123
Familial Cancer
DOI 10.1007/s10689-015-9786-z

HBOC (mutations in both genes have been described in 1 %
of patients) [3]whereasPALB2 mutations have been iden-
tified about in 1–2 % of familial breast cancer and in 3–4 %
of familial pancreatic cancer [4]. To date, only few studies
have been conducted in Italian patients with breast or pan-
creatic cancer [5–10]. We aimed to establish the occurrence
and the possible pathological significance of PALB2 muta-
tions and/or variations in a cohort of Italian patients affected
with hereditary breast/ovarian cancers that were negative for
BRCA1 and BRCA2 mutations.
A total of 98 patients (F =93, M =5) were recruited.
Of these, 86 were affected with breast cancer, 2 with breast
and ovarian cancer, 7 with bilateral breast cancer and 3
with ovarian cancer. All subjects were from Campania, a
region of southern Italy. Their age ranged between 26 and
83 years. Patients were selected according to the selection
criteria for hereditary breast cancer based on the Breast
Cancer Linkage Consortium [11]. Informed consent was
obtained from all patients before genetic analysis and
pedigree was traced. PALB2 mutational analysis was con-
ducted in all patients previously screened for BRCA1 and
BRCA2 mutations. Samples from 103 patients (age
40–80 years) affected with breast cancer, collected without
selection for family history of cancer, were used to eval-
uate the frequency of the identified PALB2 variations. As
control, 102 females (age 25–87 years) from the same
geographical area were anonymously enrolled. They were
all cancer-free at the time of blood donation.
The extraction of genomic DNA from peripheral blood
lymphocytes was performed using Wizard Genomic DNA
purification kit (Promega Corporation, Madison, WI,
USA). The presence of germline mutations in PALB2 was
evaluated by direct sequencing of 13 exons and adjacent
intronic regions. One set of primers was used to amplify
each exon, except for exons 4 and 5, which were amplified
in 4 and 2 PCR products respectively, as previously re-
ported [12].
All PCR products were sequenced on both strands using
the ABI PRISM di-Deoxy Terminator Cycle sequencing kit
on ABI 9700 thermal cycle (Applied Biosystems, Fotser
City, CA, USA) and ABI 3100 Genetic Analyser (Applied
Biosystems, Fotser City, CA, USA). The results were
analysed by using the software Mutation Surveyor version
3.24 (Softgenetics, State Collage PA, USA). Mutations
and/or variants were identified as new by referring to the
Human Gene Mutation Database (http://www.biobase-
international.com/product/hgmd) and Leiden Open Varia-
tion Database (https://grenada.lumc.nl/LOVD2/shared1/
home.php?select_db=PALB2). GenBank reference se-
quences used for naming the novel mutation and variants
were NM_024675.3 and NT_010393.15. The sequence
variants and mutation were named and referred in the text
according to the nomenclature used by Human Genome
Variation Society (HGVS; http://www.hgvs.org) and to the
descriptions suggested by den Dunnen and Antonarakis
[13].
Mutation analysis of PALB2 gene showed the presence
of a mutation in 1/98 (1.02 %) breast and/or ovarian cancer
patients. This mutation c.1919C[A (p.S640X) was identi-
fied in a 29 years old woman who was affected with breast
cancer. In addition, we identified two novel PALB2 vari-
ants in two breast cancer patients; the first one was a
missense substitution c.3047T[C (p.F1016S) and the sec-
ond one, c.*146A
[G, was localized in the 30UTR region.
Noteworthy, these variants have not previously reported in
published literature or in the NCBI or in the Ensembl
genome databases. None of 103 sporadic breast cancer
patients and 102 healthy female were found to carry the
nonsense mutation c.1919C[A (p.S640X) or the novel
variants c.3047T[C (p.F1016S) and c.*146A[G. Patients
that were carriers of the novel nonsense mutation
c.1919C[A (p.S640X) or the novel missense variant
c.3047T[C (p.F1016S) were exposed to a second periph-
eral blood sample to confirm the presence of the mutation
also on mRNA. Total RNA was isolated from peripheral
blood lymphocytes using TriZol reagent (Invitrogen,
Carlsbad, CA, USA) and reverse transcribed using Su-
perScript First-Strand Synthesis System (Invitrogen,
Carlsbad, CA), according to the manufacturer’s protocol.
To amplify the region spanning the mutations, we used a
pair of primers complementary to exons 4 and 5 for
c.1919C[A (p.S640X) and to exons 9–10 for c.3047T[C
(p.F1016S). Then, PCR products were sequenced. Besides,
RNA sample analysis confirmed the presence of the iden-
tified mutation and variant.
The novel PALB2 c.1919C[A (p.S640X) mutation is a
nonsense mutation, localized in exon 5, which results in the
introduction of a stop codon at amino acid position 640
(Fig. 1). The proband carrying the c.1919C[A (p.S640X)
mutation was diagnosed with breast cancer at age 29. At
the time of diagnosis, she underwent a right mastectomy.
She reported a family history of breast cancer and other
malignancies, including prostate, colorectal and brain
cancer (Fig. 2). Thus, the major finding of this study is the
novel PALB2 mutation in a group of patients affected with
hereditary breast and/or ovarian cancer and previously
tested for BRCA1 and BRCA2 mutations. Accordingly to
the published literature, which reported PALB2 mutations
in about 1–2 % of hereditary breast cancer [14], also in
Italian series [5,6,10], we found a frequency of 1.02 %.
The nonsense mutation c.1919C[A (p.S640X) was iden-
tified in a patient who was affected with hereditary breast
cancer. Unfortunately, although breast cancers and other
related tumours were reported in both paternal and mater-
nal branches of her family, not all family members/rela-
tives were available for genetic testing. Thus, the lack of
M. T. Vietri et al.
123

useful DNA samples has implicated that transmission of
this mutation is uncertain in the patient’s family.
Several studies have identified different monoallelic
PALB2 truncating in individuals with familial breast cancer
from various geographic areas [15–18]. Most of the
pathogenic PALB2 mutations detected in these previous
analyses are truncating, frameshift or stop codon mutations
and they were scattered throughout the entire gene region
with no hot-spot areas [19]. The c.1919C[A (p.S640X)
PALB2 mutation leads to protein truncation and it occurs in
Fig. 1 Partial electropherogram
of PALB2 exon 5 evidencing the
nonsense mutation c.1919C[A
(p.S640X). with forward (a) and
reverse (b) primers. The novel
mutation leads to the
introduction of a stop codon at
amino acid position 640
Fig. 2 Pedigree of a breast cancer family carrying the PALB2 mutation c.1919C[A (pS640X). Age at diagnoses (bold) and age at the present
time or age at exitus (italic) are reported. The proband is marked with an arrow
Novel PALB2 mutation in breast cancer patients
123

a region responsible for binding between PALB2 and
MRG15. The PALB2 protein has a coiled-coil motif at the
N terminus required for interaction with BRCA1 and a
C-terminal domain containing four WD-repeats that me-
diate the interaction with BRCA2 [20]. Moreover, it in-
teracts with RAD51 by two regions, one at the N-terminus
and the other one at the C-terminus. The fourth protein,
which interacts with PALB2, is MORF4L1 that is encoded
by the gene MRG15. MORF4L1 has a binding motif re-
sponsible for the interaction with amino acids 611–764 of
PALB2 [21]. PALB2 links MORF4L1 to the BRCA
complex that is formed in response to DNA damage [22].
The c.1919C[A (p.S640X) mutation causes the absence of
the C-terminus region, thereby inducing an alteration of
MORF4L1–PALB2 association and subsequently the lack
of interaction of PALB2 with RAD51 and BRCA2.
In addition, mutational analysis of our series of patients
showed further two novel variants, c.3047T[C (p.F1016S)
and c.*146A[G. These variants were identified in two
patients affected with hereditary breast cancer but not in
healthy controls or in sporadic breast cancers patients. The
c.3047T[C (p.F1016S) variant was identified in a patient
diagnosed with breast cancer at the age of 28. In mother’s
family there were many relatives with tumour (Fig. 3).
This family showed two breast cancer cases, two ovarian
cancer cases and one pancreatic cancer. The c.*146A[G
variant was identified in a 45 years old woman affected
with breast cancer. The patient showed other malignancies
in her family; particularly, two breast cancer cases, one
hepatic cancer and one Hodgkin lymphoma (Fig. 4).
The novel missense variant, c.3047T[C (p.F1016S),
identified in this study, was analyzed with PolyPhen-2
(http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.
bii.a-star.edu.sg/) and A-GVGD (http://agvgd.iarc.fr/
agvgd_input.php). Concordance between these methods
will be therefore a strong predictor. To evaluate potential
splicing effects of the novel c.*146A[G variant in silico
sequence analysis tool NNSPLICE (http://www.frutfly.org/
seq_tools/splice.html) was used. When the splice site is
mutated the program assigns a score considerably lower
Fig. 3 Pedigree of a breast cancer family carrying the novel PALB2 variant c.3047T\C (p.F1016S). Age at diagnoses (bold) and age at the
present time or age at exitus (italic) are reported. The proband is marked with an arrow
M. T. Vietri et al.
123

than the wild-type sequence (\0.40). Overall, in silico
analysis predicted c.3047T[C (p.F1016S) as deleterious in
three classifier algorithms. Moreover, no evidence of a
possible splicing defect for the novel non-coding variant
c.*146A[G was showed by NNSPLICE (Table 1).
The pathogenicity of a variant of unknown clinical
significance is assessed by a number of approaches that
include evaluation of the frequency of the variant in cases
and unaffected controls, mutation segregation analysis with
disease in families, the usage of in silico prediction pro-
grams and in vitro studies. Prediction programs for detec-
tion of splicing defects or protein alterations should not be
considered a replacement for in vitro studies. However, co-
segregation analysis may support the clinical importance of
variants of unknown pathogenetic significance. Unfortu-
nately, in both families of patients with the c.3047T[C
(p.F1016S) missense variant and the untranslated-30UTR
c.*146A[G variant there were not relatives that agreed to
the genetic test. Therefore, the genotype–phenotype cor-
relation for these variants remains unknown and
c.3047T[C (p.F1016S) and c.*146A[G should be con-
sidered UVs of PALB2. Interestingly, in the lineage of
patient with c.3047T[C (p.F1016S), a maternal uncle with
pancreatic cancer was described (Fig. 3). Mutations in
PALB2 occur with a prevalence of 2.1 % in breast cancer
patients when they are selected for a personal and/or family
history of pancreatic cancer [4]. Further studies could
corroborate the pathogenicity of the c.3047T[C
(p.F1016S) variant and confirm the contribution of PALB2
gene mutation in pancreatic cancer.
The analyzed patients also showed ten previously reported
variants, c.-109delG, c.-47G[A, c.212-58A[C(IVS3-58A[C),
c.1676A[G (p.Q559R), c.2014G[C (p.E672Q), c.2590C[T
(p.P864S), c.2794G[A (p.V932M), c.2816T[G (p.L939 W),
c.2993G[A (p.G998E) and c.3300T[G (p.T1100T). The fre-
quencies and their effects are reported in Table 2.Amongthese
variants, -109delG had not yet been previously tested with pre-
diction programs. In silico analysis conducted with NNSPLICE
indicates that also this variant does not affect the splicing
(Table 2). In a previous study, computational analysis for non-
Fig. 4 Pedigree of a breast cancer family carrying the novel PALB2 variant c.*146A[G. Age at diagnoses (bold) and age at the present time or
age at exitus (italic) are reported. The proband is marked with an arrow
Novel PALB2 mutation in breast cancer patients
123

Table 1 In silico study of PALB2 novel variants identified in two patients affected with hereditary breast cancer
Designation Localization Nucleotide
change
Protein
change
HBOC patients
(n =98)
Number
(frequency)
Sporadic breast
cancer (n =103)
Number (frequency)
Healthy female
control (n =102)
Number (frequency)
PolyPhen-2 SIFT A-
GVGD
NNSplice Neoplasia
(age)
a
c.3047T[C
(p.F1016S)
Exon 10 c.3047T[C p.Phe1016Ser 1 (1.02 %) – – Probably
damaging
Affecting
protein
function
C65 – BC (28)
c.*146A[G Non coding
30-UTR
c.*146A[G – 1 (1.02 %) – – – – – No effect BC (45)
a
Age at diagnosis
Table 2 PALB2 genetic variants in hereditary breast and/or ovarian cancer (HBOC), sporadic breast cancer patients and healthy controls
Designation Localization Nucleotide
change
Protein
change
HBOC patients
(n =98)
Number
(frequency)
Sporadic breast
cancer (n =103)
Number (frequency
Healthy female
control (n =102)
Number (frequency
Bioinformatic
prediction
References
Missense
c.1676A[G (p.Q559R) Exon 4 c.1676A[G p.Gln559Arg 21 (21.42 %) 21 (20.38 %) 20 (19.60 %) Benign Zheng et al. [23]
c.2014G[C (p.E672Q) Exon 5 c.2014G[C p.Glu672Gln 14 (14.28 %) 13 (12.62 %) 11 (10.78 %) Benign Zheng et al. [23]
c.2590C[T (p.P864S) Exon 7 c.2590C[T p.Pro864Ser 2 (2.04 %) – – Possibly benign Zheng et al. [23]
c.2794G[A (p.V932M) Exon 8 c.2794G[A p.Val932Met 1 (1.02 %) – – Potentially deleterious Wong-Brown et al. [24]
Blanco et al. [25]
c.2816T[G (p.L939W) Exon 8 c.2816T[G p.Leu939Tyr 1 (1.02 %) – – Deleterious Blanco et al. [25]
c.2993G[A (p.G998E) Exon 9 c.2993G[A p.Gly998Met 9 (9.18 %) 8 (7.76 %) 9 (8.82 %) Deleterious Zheng et al. [23]
Synonymous
c.3300T[G (p.T1100T) Exon 12 c.3300T[G p.Thr1100Thr 13 (13.26 %) 12 (11.65 %) 12 (11.76 %) No effect Blanco et al. [26]
Intronic
c.-109delG Non coding 50UTR c.-109delG – 1 (1.02 %) – – No effect *
c.-47G[A Non coding 50UTR c.-47G[A – 6 (6.10 %) 4 (3.88 %) 2 (1.96 %) No effect Blanco et al. [26]
c.212-58A[C IVS3-58 Intron 3 c.212-58A[C – 10 (13.20 %) 10 (9.70 %) 12 (11.76 %) No effect Blanco et al. [26]
* Tested in our laboratory using NNSPLICE software
M. T. Vietri et al.
123

synonymous variants predicted c.1676A[G (p.Q559R),
c.2014G[C (p.E672Q) as benign, c.2590C[T (p.P864S) as
possibly benign, whereas, according to all used predictive algo-
rithms, c.2816T[G (p.L939 W) and c.2993G[A (p.G998E)
were reported as deleterious [23]. Moreover, c.2794G[A
(p.V932M) was reported as a variant potentially affecting protein
functionbyinsilicostudies[24,25]. In our series, c.2993G[A
(p.G998E) was reported in 9.18 % of patients, in 8.82 % of
controls and 7.76 % of sporadic breast cancers, in agreement
with the frequencies obtained in the study of Silvestri et al. [7],
suggesting that this variant does not have a pathogenic role. On
the contrary, c.2794G[A (p.V932M) and c.2816T[G
(p.L939 W) were observed only in the group of patients, thus
confirming its possible damaging effect previously reported [23,
24]. Also c.2590C[T (p.P864S) was reported only in HBOC
patients; however, it can be considered benign based on in silico
analysis [24]. Furthermore, no evidence for a possible splicing
defect was provided by analysis with NNSPLICE for c.-47G[A,
c.212-58A[C (IVS3-58A[C) and c.3300T[G (p.T1010T) [26].
Our results show that also c.-109delG does not affect splicing.
Noteworthy, c.-109delG was found in 1.02 % of patients but not
in controls.
Further studies, such as in vitro studies and co-segrega-
tion analyses, are warranted to understand the involvement
of the above-described PALB2 alterations in HBOC and to
elucidate the role of the identified UVs; thus, they have been
planned in our laboratory. In conclusions, PALB2 mutation
testing should be performed routinely to identify mutations
in HBOC families since it may become of clinical relevance
for clinical management. Currently, to optimize molecular
diagnosis of HBOC, which is characterized by genetic
heterogeneity, it is possible to use next generation se-
quencing (NGS). Indeed, this procedure allows evaluating
mutations in several genes including PALB2 [27] and should
be considered as a proper option in the next future.
Conflict of interest The authors declare that they have no conflict
of interest.
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