Pancreatic hemi-agenesis in MEN1: A clinical report.

We first describe a patient with multiple endocrine neoplasia type 1 (MEN1) and dorsal pancreatic hemi-agenesis. Previously, pancreas divisum has been reported in MEN1. Recent data in mice have elucidated the molecular mechanisms of pancreatic endoderm specification. Disinhibition of hedgehog signaling appears to be important in how Gata4 and Gata6 variants cause pancreatic agenesis. Disinhibition of hedgehog signaling has also been observed in Men1 knockout pancreatic islets. Although we cannot exclude a spurious association between dorsal pancreatic hemi-agenesis and MEN1 in our patient, we argue that developmental abnormalities of the pancreas may have to be considered as possibly related to the MEN1 phenotype.

From the set-up of a screening program of breast cancer patients to the identification of the first BRCA mutation in the DR Congo.

BACKGROUND: Breast cancer incidence in African population is low compared to western countries but the mortality rate is higher and the disease presents at a younger age and at a more advanced stage. The World Health Organisation and the Breast Health Global Initiative concluded that in low and middle income countries early breast cancer detection can be achieved by informing women on symptoms of breast cancer, on the practice of breast self-examination and clinical breast examination by trained health care workers. Based on these recommendations, we set up a breast cancer awareness campaign in Kinshasa, Democratic Republic of Congo (DRC). This paper describes the strategy that was established and the results that were achieved. METHODS: A breast cancer awareness campaign was started in 2010 and data were collected until the end of 2012. Clinicians (expert group) trained nurses and health care workers (awareness groups) on clinical, technical and social aspects of breast cancer. Different channels were used to inform women about the campaign and clinical data (on medical and family history) were collected. The participating women were investigated with clinical breast examination by the awareness group. Women in whom a palpable mass was detected were referred to the hospital: they received a mammography and ultrasound and--in case of suspicious findings--additionally a core needle biopsy. In case of a positive family history, a blood sample was taken for genetic investigation. RESULTS: In total, 4,315 women participated, resulting in 1,113 radiological breast examinations, performed in the General Hospital of Kinshasa of which 101 turned out to be malignant lesions. Fifty six percent of the women with breast cancer were less than 50 years old and 75% (65/87) were stage III tumors. A BRCA gene mutation was identified in a family with a severe history of breast cancer. CONCLUSIONS: Even without financial support, it was possible to start an awareness campaign for breast cancer in Kinshasa. This campaign increased the awareness on cancer of the women in Kinshasa. The results demonstrate that this campaign had an immediate impact on patients and their families.

Molecular analysis of the breast cancer genes BRCA1 and BRCA2 using amplicon-based massive parallel pyrosequencing.

The aim of this study was to implement the massively parallel sequencing technology for diagnostic applications. We evaluated an amplicon-based method for the analysis of the BRCA1 and BRCA2 genes on the Roche 454 GS-FLX sequencer, to identify disease-causing mutations in breast and/or ovarian cancer patients. A first evaluation relied on the analysis of DNA fragments containing known mutations. Secondly, the entire coding regions of the BRCA1 and BRCA2 genes were interrogated in more than 400 patient samples, using a multiplex PCR-based assay. Variants were filtered on the basis of their frequency (20%) and sequencing depth (>25×). Special attention was given to sequencing accuracy in homopolymers. In the initial evaluation, all known heterozygous mutations were detected. The percentage of mutant reads ranged from 22% to 62%. For the multiplex assay, 95% sensitivity and 91% specificity were obtained. In addition, we were able to reliably distinguish mutations from noise through the analysis of the raw signal intensities in homopolymers. This work presents an evaluation of the next-generation sequencing for use in diagnostics, based on a relatively high number of samples and experiments. We anticipate that the technique would further improve, and would allow reducing the costs per analysis and the turn-around time, to benefit patients who undergo BRCA molecular testing.

Recurrent copy number alterations in BRCA1-mutated ovarian tumors alter biological pathways.

Array CGH was used to identify recurrent copy number alterations (RCNA) characteristic of either BRCA1-related or sporadic ovarian cancer. After preprocessing, both groups of patients were modeled using a recurrent Hidden Markov Model to detect RCNA. RCNA with a probability higher than 80% were called. After removing RCNA present in both groups, the genes present in the remaining RCNA were investigated for enrichment of pathways from external databases. More RCNA were observed in the BRCA1 group, and they display more losses than gains compared to the sporadic group. When focusing on the type of RCNA, no significant difference in length was seen for the gains, but there was a statistically significant difference for the losses. In the sporadic group, a great proportion of the altered regions contain genes known to have a function in cell adhesion and complement activation, whereas the BRCA1 samples are characterized by alterations in the HOX genes, metalloproteinases, tumor suppressor genes, and the estrogen-signaling pathways. We conclude that BRCA1 ovarian tumors present a different type, number, and length of RCNA; a huge amount of the genome is lost, resulting in important genomic instability. Moreover, important biological pathways are altered differentially when compared to the sporadic group.

Assessment of endostatin gene therapy for familial adenomatous polyposis-related desmoid tumors.

Constitutive activation of the Wnt signaling pathway is a hallmark of many cancers, including familial adenomatous polyposis (FAP)-related desmoid tumors. Endostatin is a well-known antiangiogenic protein that has been described recently as a potential inhibitor of this signaling pathway. Here, we show that endostatin directly induces apoptosis and inhibits the Wnt signaling pathway in colorectal cancer cell lines bearing mutations on the adenomatous polyposis coli (APC) gene as a model of FAP-related malignant cells. We then explore the relationship between apoptosis and inhibition of this pathway and show that they are not correlated. These results seem to contradict a well-recognized study, showing that reintroduction of the APC cDNA in APC-deficient cells leads to apoptosis. To reconcile our conclusions with the literature, we further show that a truncated fragment of APC capable of inhibiting the Wnt signaling pathway in SW480 cells is incapable of inducing apoptosis in these cells, confirming that APC-mediated apoptosis is uncoupled to the inhibition of the Wnt signaling pathway. Finally, we show that endostatin directly induces cell death on primary FAP-related desmoid tumor cells in culture. This phenomenon is also independent of the inhibition of the Wnt signaling pathway. Considering the current lack of effective treatment against desmoid tumors, we advocate that endostatin gene therapy represents an attractive new therapeutic approach for this disease.

Diagnostic DHPLC Quality Assurance (DDQA): a collaborative approach to the generation of validated and standardized methods for DHPLC-based mutation screening in clinical genetics laboratories.

Genetic testing in a clinical diagnostic environment must be subject to rigorous quality control procedures, in order to ensure consistency and accuracy of results. Denaturing high performance liquid chromatography (DHPLC) has become a standard prescreening tool for mutation detection, offering very high efficiency and sensitivity of detection. Despite the relatively simple software-assisted assay setup, DHPLC is a complex assay, and quality control is reliant on ensuring optimal instrument performance, excellent assay design and validation, and sufficient user training and proficiency to interpret results. We describe here a unique collaborative effort by a group of diagnostic clinical genetics laboratories with DHPLC expertise who, together with the manufacturer of one of the most widely used DHPLC platforms, have generated standard operating procedures (SOPs) for instrument operation and maintenance, and for mutation detection by DHPLC. We also describe the validation of a disease-specific SOP for DHPLC assisted mutation screening of the MECP2 gene associated with Rett syndrome. The proposed SOP was validated, and used independently in two laboratories to introduce MECP2 testing. In addition, we provide empirically derived normal ranges for the WAVE System Mutation Standards, which are essential for optimal instrument performance. This effort was initiated to try to standardize DHPLC-based mutation screening procedures across laboratories, and so increase the overall quality of this testing method. This endeavor will thus save each laboratory from having to generate SOPs on their own, which is a lengthy and laborious task. In this respect, we define "generic" SOPs as procedures that are easily adaptable to the individual laboratories' quality systems.

Large deletions of the APC gene in 15% of mutation-negative patients with classical polyposis (FAP): a Belgian study.

Germline mutations of the APC gene are responsible for familial adenomatous polyposis (FAP). Most of the mutations are protein truncating mutations and are spread over the coding region. Rare whole-gene deletions or exonic deletions have been described. From a series of 85 patients clinically diagnosed with FAP or attenuated FAP (AAPC) in our center, 30 (35%) were found to have truncating or missense mutations. We have now screened the remaining 55 patients for exonic deletions or duplications, first by semi-quantitative PCR and later by multiplex ligation-dependent probe amplification (MLPA). Three whole-gene deletions and one exon 14 deletion were found (5% of patients). The whole-gene deletions were confirmed by fluorescence in situ hybridization (FISH) analysis, and the breakpoints of the exon 14 deletion could be determined using long range PCR. Further characterization of the whole gene deletions was performed using extragenic polymorphic markers and/or semi-quantitative PCR. We could demonstrate that the deletions do not encompass the MCC gene. Interestingly, the phenotype of the deletion patients was not different from that of patients with truncating mutations. The polyp numbers ranged from attenuated to profuse polyposis and the interfamilial variability of disease phenotype was as in other FAP families. In none of the 28 AAPC patients included in this study, was a large deletion found, while 15% of the patients with classical polyposis had a genomic deletion. It corroborates recently published data, suggesting that large deletions may occur with a frequency higher than 10% in mutation-negative patients with a classical polyposis. In this article, we have included an overview of genomic rearrangements in the 5q21 region.

Pathogenic mutations and rare variants of the APC gene identified in 75 Belgian patients with familial adenomatous polyposis by fluorescent enzymatic mutation detection (EMD).

Familial adenomatous polyposis (FAP) is a dominant inherited colorectal cancer syndrome which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Enzymatic mutation detection (EMD) has potential advantages over the standard protein truncation test (PTT) that is currently used in screening the APC gene for mutations. First we wanted to validate the EMD technique in comparison to PTT. Secondly, we wanted to develop an efficient working protocol for EMD screening of APC. Seventy-five unrelated patients were screened for mutations. All mutations that had previously been detected by PTT were also identified by EMD; the sizes of the cleavage fragments were as expected according to the position of the mutations within the amplicons. A new screening strategy based on EMD allows the analysis of the APC gene in 31 overlapping PCR fragments. In total, EMD efficiently detected the 26 truncating mutations in this series. In addition, two rare variants were also detected: the first is the typical Ashkenazi missense mutation I1307K while the second variant, E1317Q, has been identifed in Belgian patients and controls, and should no longer be considered as a pathogenic mutation, but rather classified as a polymorphism.