Nelly Sabbaghian

Analysis of PALB2/FANCN-associated breast cancer families

No more than ≈30% of hereditary breast cancer has been accounted for by mutations in known genes. Most of these genes, such as BRCA1, BRCA2, TP53, CHEK2, ATM, and FANCJ/BRIP1, function in DNA repair, raising the possibility that germ line mutations in other genes that contribute to this process also predispose to breast cancer. Given its close relationship with BRCA2, PALB2 was sequenced in affected probands from 68 BRCA1/BRCA2-negative breast cancer families of Ashkenazi Jewish, French Canadian, or mixed ethnic descent. The average BRCAPRO score was 0.58. A truncating mutation (229delT) was identified in one family with a strong history of breast cancer (seven breast cancers in three female mutation carriers). This mutation and its associated breast cancers were characterized with another recently reported but unstudied mutation (2521delA) that is also associated with a strong family history of breast cancer. There was no loss of heterozygosity in tumors with either mutation. Moreover, comparative genomic hybridization analysis showed major similarities to that of BRCA2 tumors but with some notable differences, especially loss of 18q, a change that was previously unknown in BRCA2 tumors and less common in sporadic breast cancer. This study supports recent observations that PALB2 mutations are present, albeit not frequently, in breast cancer families. The apparently high penetrance noted in this study suggests that at least some PALB2 mutations are associated with a substantially increased risk for the disease.

DICER1 Mutations in Familial Multinodular Goiter With and Without Ovarian Sertoli-Leydig Cell Tumors

CONTEXT Nontoxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported, and published reports describe 5 families that also contain at least 1 individual with a Sertoli-Leydig cell tumor of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have been identified in families affected by pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCTs. OBJECTIVE To determine whether familial MNG with or without SLCT in the absence of PPB was associated with mutations in DICER1. DESIGN, SETTING, AND PATIENTS From September 2009 to September 2010, we screened 53 individuals from 2 MNG and 3 MNG/SLCT families at McGill University for mutations in DICER1. We investigated blood lymphocytes and MNG and SLCT tissue from family members for loss of the wild-type DICER1 allele (loss of heterozygosity), DICER1 expression, and microRNA (miRNA) dysregulation. MAIN OUTCOME MEASURE Detection of germline DICER1 gene mutations in familial MNG with and without SLCT. RESULTS We identified and characterized germline DICER1 mutations in 37 individuals from 5 families. Two mutations were predicted to be protein truncating, 2 resulted in in-frame deletions, and 1 was a missense mutation. Molecular analysis of the 3 SLCTs showed no loss of heterozygosity of DICER1, and immunohistochemical analysis in 2 samples showed strong expression of DICER1 in Sertoli cells but weak staining of Leydig cells. miRNA profiling of RNA from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers. CONCLUSIONS DICER1 mutations are associated with both familial MNG and MNG with SLCT, independent of PPB. These germline DICER1 mutations are associated with dysregulation of miRNA expression patterns.

Extending the phenotypes associated with DICER1 mutations

DICER1 is crucial for embryogenesis and early development. Forty different heterozygous germline DICER1 mutations have been reported worldwide in 42 probands that developed as children or young adults, pleuropulmonary blastoma (PPB), cystic nephroma (CN), ovarian sex cord‐stromal tumors (especially Sertoli‐Leydig cell tumor [SLCT]), and/or multinodular goiter (MNG). We report DICER1 mutations in seven additional families that manifested uterine cervix embryonal rhabdomyosarcoma (cERMS, four cases) and primitive neuroectodermal tumor (cPNET, one case), Wilms tumor (WT, three cases), pulmonary sequestration (PS, one case), and juvenile intestinal polyp (one case). One carrier developed (age 25 years) a pleomorphic sarcoma of the thigh; another carrier had transposition of great arteries (TGA). These observations show that cERMS, cPNET, WT, PS, and juvenile polyps fall within the spectrum of DICER1‐related diseases. DICER1 appears to be the first gene implicated in the etiology of cERMS, cPNET, and PS. Young adulthood sarcomas and perhaps congenital malformations such as TGA may also be associated. 32:1381–1384, 2011. ©2011 Wiley Periodicals, Inc.

Identification of a novel truncating PALB2 mutation and analysis of its contribution to early-onset breast cancer in French-Canadian women.

BackgroundPALB2 has recently been identified as a breast cancer susceptibility gene. PALB2 mutations are rare causes of hereditary breast cancer but may be important in countries such as Finland where a founder mutation is present. We sought to estimate the contribution of PALB2 mutations to the burden of breast cancer in French Canadians from Quebec.MethodsWe screened all coding exons of PALB2 in a sample of 50 French-Canadian women diagnosed with either early-onset breast cancer or familial breast cancer at a single Montreal hospital. The genetic variants identified in this sample were then studied in 356 additional women with breast cancer diagnosed before age 50 and in 6,448 newborn controls.ResultsWe identified a single protein-truncating mutation in PALB2 (c.2323 C>T, resulting in Q775X) in 1 of the 50 high-risk women. This variant was present in 2 of 356 breast cancer cases and in none of 6,440 newborn French-Canadian controls (P = 0.003). We also identified two novel new non-synonymous single nucleotide polymorphisms in exon 4 of PALB2 (c.5038 A>G [I76V] and c.5156 G>T [G115V]). G115V was found in 1 of 356 cases and in 15 of 6,442 controls (P = 0.6). The I76V variant was not identified in either the extended case series or the controls.ConclusionWe have identified a novel truncating mutation in PALB2. The mutation was found in approximately 0.5% of unselected French-Canadian women with early-onset breast cancer and appears to have a single origin. Although mutations are infrequent, PALB2 can be added to the list of breast cancer susceptibility genes for which founder mutations have been identified in the French-Canadian population.

Analysis of the Gene Coding for the BRCA2-Interacting Protein PALB2 in Familial and Sporadic Pancreatic Cancer

Dear Sir: Current evidence indicates that about 5%–10% of pancreatic cancer has a familial component, although the vast majority of pancreatic cancer families remain unexplained. 1 PALB2 is a recently identified breast cancer susceptibility gene whose protein is closely associated with BRCA2, and is essential for BRCA2 anchorage to nuclear structures. This functional relationship made PALB2 a candidate gene for susceptibility to BRCA2-related cancers such as pancreas cancer. Recently, Jones et al2 screened 96 familial pancreatic cancer patients, 16 of whom had 1 first-degree relative with pancreatic cancer and 80 had ≥2 additional relatives with pancreatic cancer, ≥1 of which was first degree.2 Truncating PALB2 mutations were identified in 3 patients (3.1%) and there was no difference in average age of cancer onset between mutation-positive and -negative families. We sought to screen a larger cohort of pancreatic cancer cases, including both familial and sporadic types, to determine the wider contribution of PALB2 gene mutations in pancreatic cancer. We selected a total of 254 individuals with pancreas cancer (148 male, 106 female) at a median age of diagnosis of 61 years. Patients were identified between 1997 and 2007 via clinic-based recruitment in Toronto and Montreal and through either the Familial Gastrointestinal Cancer Registry at Mount Sinai Hospital in Toronto or the population-based Ontario Pancreas Cancer Study. In total, 203 patients were recruited in Toronto and 51 in Montreal. All probands were confirmed to have pancreatic adenocarcinoma by pathology report; 101 pro-bands had a family history of pancreatic cancer, of which 32 had 2 affected first-degree relatives. In these 101 cases, 74 had 1 affected relative, 18 had 2 affected relatives, 7 had 3 affected relatives, and 2 cases had >3 affected relatives (Table 1). Sixty probands had a family history of breast/ovarian cancer, including 21 cases with a family history of pancreatic cancer (included above). Because the cases were ascertained through pancreas cancer studies, the family history of breast cancer or ovarian cancer was not strong, with most families having a single relative affected, and no family having a BRCAPRO score >0.12 (ie, these were not primarily familial breast/ovarian cancer families with 1 or 2 additional cases of pancreas cancer). The median age at diagnosis of pancreatic cancer cases with no family history was 49 years (range, 31–85); 55% of these were <50 years old and 66% were <60 years old at diagnosis. Genomic DNA was obtained from blood, saliva, or buccal cells using standard extraction methods. Before analysis, 20 ng of total genomic DNA was used for whole genome amplification according to the manufacturer’s instructions using the Repli-g Mini Kit (Qiagen, Mississauga, Ontario, Canada). We screened the 13 coding exons of PALB2 by sequencing (n = 83) or high-resolution melt analysis (n = 171), which has similar sensitivity to sequencing.3 Samples with variants were reamplified by polymerase chain reaction (PCR) using the original, non–whole-genome-amplified DNA as template and the PCR product was sequenced in forward and reverse directions for confirmation. We performed multiplex ligation-dependent probe amplification assay (MLPA) for PALB2 on 228 samples where we had sufficient DNA of adequate quality, as previously described by Foulkes et al.4 Table 1 Pancreas Cancer Cases We identified a heterozygous, 6.7-kb germline deletion including exons 12 and 13 of PALB2 in a patient who was affected by breast and then pancreas cancer (ages 47 and 61, respectively) and whose mother died of pancreas cancer at age 83 (Figure 1). This result was confirmed by long range PCR (Takara Bio Inc., Madison, WI) using 2 different primer pairs, which determined the deleted region to span a region from the middle of intron 11 (2.7 kb from the beginning of exon 12), up to 1.8kb after exon 13. This deletion would disrupt the PALB2 WD40 motif, which is required for interaction with the BRCA2 protein. 5 Aside from the exonic deletion, 2 previously unreported missense variants (S285L and T911I) were identified, but neither were predicted to be pathogenic. Both these variants were seen in young-onset pancreas cancer cases (41 years and 48 years) with no family history. A number of previously reported variants were also identified (Table 2). Figure 1 (A) Pedigree of the proband (shown by arrow). PA, pancreatic cancer; BR, breast cancer. (B) MLPA showing deletion of exon 12 (161-bp fragment) and exon 3 (338-bp fragment)

Biallelic DICER1 mutations occur in Wilms tumours

DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two‐hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single‐base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two ‘hits’ in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours. Copyright

A PALB2 mutation associated with high risk of breast cancer

IntroductionAs a group, women who carry germline mutations in partner and localizer of breast cancer 2 susceptibility protein (PALB2) are at increased risk of breast cancer. Little is known about by how much or whether risk differs by mutation or family history, owing to the paucity of studies of cases unselected for family history.MethodsWe screened 1,403 case probands for PALB2 mutations in a population-based study of Australian women with invasive breast cancer stratified by age at onset. The age-specific risk of breast cancer was estimated from the cancer histories of first- and second-degree relatives of mutation-carrying probands using a modified segregation analysis that included a polygenic modifier and was conditioned on the carrier case proband. Further screening for PALB2 c.3113G > A (W1038X) was conducted for 779 families with multiple cases of breast cancer ascertained through family cancer clinics in Australia and New Zealand and 764 population-based controls.ResultsWe found five independent case probands in the population-based sample with the protein-truncating mutation PALB2 c.3113G > A (W1038X); 2 of 695 were diagnosed before age 40 years and 3 of 708 were diagnosed when between ages 40 and 59 years. Both of the two early-onset carrier case probands had very strong family histories of breast cancer. Further testing found that the mutation segregated with breast cancer in these families. No c.3113G > A (W1038X) carriers were found in 764 population-based unaffected controls. The hazard ratio was estimated to be 30.1 (95% confidence interval (CI), 7.5 to 120; P < 0.0001), and the corresponding cumulative risk estimates were 49% (95% CI, 15 to 93) to age 50 and 91% (95% CI, 44 to 100) to age 70. We found another eight families carrying this mutation in 779 families with multiple cases of breast cancer ascertained through family cancer clinics.ConclusionsThe PALB2 c.3113G > A mutation appears to be associated with substantial risks of breast cancer that are of clinical relevance.

Exploring the Association Between DICER1 Mutations and Differentiated Thyroid Carcinoma

CONTEXT Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome. Thyroid abnormalities are a common finding in DICER1 syndrome with multinodular goiter frequently present in many families in which a germline DICER1 mutation is segregating. Differentiated thyroid carcinoma (DTC) is infrequently seen in such pedigrees. In addition to germline DICER1 mutations, specific somatic mutations have been identified in the DICER1 ribonuclease IIIb catalytic domain in several tumor types. OBJECTIVE We aimed to determine whether such characteristic somatic DICER1 mutations are present in DTCs that arise within germline DICER1 mutation carriers. DESIGN AND SETTING The study involved an opportunistic collection of 3 cases of DTC arising in individuals suspected to have DICER1 syndrome and hospital-based ascertainment and testing was implemented. RESULTS We identified somatic DICER1 mutations in 3 DTCs arising in unrelated germline DICER1 mutation carriers, all of whom had been diagnosed in infancy with pleuropulmonary blastoma (PPB), were treated with chemotherapy, exposed frequently to diagnostic radiation, and subsequently developed DTC. The somatic mutations occurred within the DICER1 ribonuclease IIIb domain, affecting highly conserved amino acid residues central to the catalytic activity of the domain. CONCLUSION This report of somatic DICER1 mutations in DTC strengthens the association between DTC and the DICER1 syndrome. The possible association between germline DICER1 mutations, PPB treatment, and the risk of subsequent DTC must be considered by clinicians when treating PPB.

Germ-line and somatic DICER1 mutations in pineoblastoma

Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.

Rare germline mutations in PALB2 and breast cancer risk: a population-based study.

Germline mutations in the PALB2 gene are associated with an increased risk of developing breast cancer but little is known about the frequencies of rare variants in PALB2 and the nature of the variants that influence risk. We selected participants recruited to the Womens Environment, Cancer, and Radiation Epidemiology (WECARE) Study and screened lymphocyte DNA from cases with contralateral breast cancer (n = 559) and matched controls with unilateral breast cancer (n = 565) for PALB2 mutations. Five pathogenic PALB2 mutations were identified among the cases (0.9%) versus none among the controls (P = 0.04). The first‐degree female relatives of these five carriers demonstrated significantly higher incidence of breast cancer than relatives of noncarrier cases, indicating that pathogenic PALB2 mutations confer an estimated 5.3‐fold increase in risk (95% CI: 1.8–13.2). The frequency of rare (<1% MAF) missense mutations was similar in both groups (23 vs. 21). Our findings confirm in a population‐based study setting of women with breast cancer the strong risk associated with truncating mutations in PALB2 that has been reported in family studies. Conversely, there is no evidence from this study that rare PALB2 missense mutations strongly influence breast cancer risk. Hum Mutat 33:674–680, 2012.