Targeted next generation sequencing identifies functionally deleterious germline mutations in novel genes in early-onset/familial prostate cancer

<div><p>Considering that mutations in known prostate cancer (PrCa) predisposition genes, including those responsible for hereditary breast/ovarian cancer and Lynch syndromes, explain less than 5% of early-onset/familial PrCa, we have sequenced 94 genes associated with cancer predisposition using next generation sequencing (NGS) in a series of 121 PrCa patients. We found monoallelic truncating/functionally deleterious mutations in seven genes, including <i>ATM</i> and <i>CHEK2</i>, which have previously been associated with PrCa predisposition, and five new candidate PrCa associated genes involved in cancer predisposing recessive disorders, namely <i>RAD51C</i>, <i>FANCD2</i>, <i>FANCI</i>, <i>CEP57</i> and <i>RECQL4</i>. Furthermore, using <i>in silico</i> pathogenicity prediction of missense variants among 18 genes associated with breast/ovarian cancer and/or Lynch syndrome, followed by KASP genotyping in 710 healthy controls, we identified “likely pathogenic” missense variants in <i>ATM</i>, <i>BRIP1</i>, <i>CHEK2</i> and <i>TP53</i>. In conclusion, this study has identified putative PrCa predisposing germline mutations in 14.9% of early-onset/familial PrCa patients. Further data will be necessary to confirm the genetic heterogeneity of inherited PrCa predisposition hinted in this study.</p></div

“Likely/Potentially pathogenic” missense variants found in the 121 cases by targeted NGS panel.

<p>“Likely/Potentially pathogenic” missense variants found in the 121 cases by targeted NGS panel.</p

Pedigrees of patients carrying truncating/deleterious mutations in new candidate PrCa risk genes involved in Fanconi anemia.

<p>(A) Patient HPC186 harboring the <i>RAD51C</i> frameshift mutation c.890_899del. (B) Patient HPC447 harboring the <i>FANCD2</i> splicing mutation c.2494+2T>C. (C) Patient HPC150 harboring the <i>FANCI</i> frameshift mutation c.206del. Electropherograms of the Sanger sequencing validations are shown.</p

Pedigrees of patients harboring truncating/deleterious mutations in new candidate PrCa risk genes involved in other recessive disorders.

<p>(A) Patient HPC421 harboring the <i>CEP57</i> nonsense mutation c.791C>G. (B) Patient HPC455 harboring the <i>RECQL4</i> frameshift mutation c.2636del. Electropherograms of the Sanger sequencing validations are shown.</p

Pedigrees of patients carrying truncating/deleterious mutations in the known PrCa risk genes <i>ATM</i> and <i>CHEK2</i>.

<p>(A) Patient HPC177 harboring the <i>ATM</i> stop mutation c.652C>T. (B) Patient HPC395 harboring the <i>CHEK2</i> splicing mutation c.593-1G>T. Electropherograms of the Sanger sequencing validations are shown.</p

Samples investigated in the study.

1<p>All 38 investigated fusion genes are ranked based on the likelihood of being the correct fusion gene in the particular sample. Lower numbers indicate higher likelihood.</p>2<p>Information about RT-PCR protocols for fusion gene detection. In house: protocol not previously published. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070649#s2" target="_blank">Materials and Methods</a> for more details. NK: Normal karyotype.</p

Identification of Two Novel <i>HOXB13</i> Germline Mutations in Portuguese Prostate Cancer Patients

<div><p>The <i>HOXB13</i> germline variant G84E (rs138213197) was recently described in men of European descent, with the highest prevalence in Northern Europe. The G84E mutation has not been found in patients of African or Asian ancestry, which may carry other <i>HOXB13</i> variants, indicating allelic heterogeneity depending on the population. In order to gain insight into the full scope of coding <i>HOXB13</i> mutations in Portuguese prostate cancer patients, we decided to sequence the entire coding region of the <i>HOXB13</i> gene in 462 early-onset or familial/hereditary cases. Additionally, we searched for somatic <i>HOXB13</i> mutations in 178 prostate carcinomas to evaluate their prevalence in prostate carcinogenesis. Three different patients were found to carry in their germline DNA two novel missense variants, which were not identified in 132 control subjects. Both variants are predicted to be deleterious by different <i>in silico</i> tools. No somatic mutations were found. These findings further support the hypothesis that different rare <i>HOXB13</i> mutations may be found in different ethnic groups. Detection of mutations predisposing to prostate cancer may require re-sequencing rather than genotyping, as appropriate to the population under investigation.</p></div

Pedigrees of individuals with the <i>BRCA2</i> c.156_157insAlu mutation detected in FFPE tissue.

<p>Family of an individual with male breast cancer (A), an individual with peritoneal/fallopian tube cancer (B), and one individual with an ampulla of Vater carcinoma (C). The index case is indicated by an arrow.</p

<i>HOXB13</i> structure and distribution of coding non-synonymous variants reported in prostate cancer patients.

<p>The homeobox protein Hox1A3 N-terminal domain (PF12284) and the homeobox domain (PF00046) are represented as an orange box inside each of the corresponding exons (homeobox protein Hox1A3 N-terminal domain: residues 21–123; homeobox domain: residues 217–273). Variants located within these domains are shown above the corresponding domain. Both missense variants found in our patients [p.(Ala128Asp) and p.(Phe240Leu)] are shown in red. Variants described by other authors are shown in black.</p

Clustal W2 alignment of the human HOXB13 protein and its orthologues in selected species.

<p>The amino acids residues predicted to be changed by both missense mutations identified in this study [p.(Ala128Asp) and p.(Phe240Leu)] are highlighted by grey shaded boxes and residue 84 predicted to be affected by the previously described G84E mutation is highlighted by a grey box.</p