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High Prevalence of Germline STK11 Mutations in Hungarian Peutz

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High Prevalence of Germline STK11 Mutations in Hungarian Peutz Papp et al. BMC Medical Genetics 2010, 11:169 http://www.biomedcentral.com/1471-2350/11/169 RESEARCH ARTICLE Open Access High prevalence of germline STK11 mutations in Hungarian Peutz-Jeghers Syndrome patients Janos Papp1, Marietta Eva Kovacs1†, Szilvia Solyom1,2†, Miklos Kasler3, Anne-Lise Børresen-Dale4,5, Edith Olah1* Abstract Background: Peutz-Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disease characterized by gastrointestinal hamartomatous polyposis and mucocutaneous pigmentation. The genetic predisposition for PJS has been shown to be associated with germline mutations in the STK11/LKB1 tumor suppressor gene. The aim of the present study was to characterize Hungarian PJS patients with respect to germline mutation in STK11/LKB1 and their association to disease phenotype. Methods: Mutation screening of 21 patients from 13 PJS families were performed using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). Comparative semi-quantitative sequencing was applied to investigate the mRNA-level effects of nonsense and splice-affecting mutations. Results: Thirteen different pathogenic mutations in STK11, including a high frequency of large genomic deletions (38%, 5/13), were identified in the 13 unrelated families studied. One of these deletions also affects two neighboring genes (SBNO2 and GPX4), located upstream of STK11, with a possible modifier effect. The majority of the point mutations (88%, 7/8) can be considered novel. Quantification of the STK11 transcript at the mRNA-level revealed that the expression of alleles carrying a nonsense or frameshift mutation was reduced to 30-70% of that of the wild type allele. Mutations affecting splice-sites around exon 2 displayed an mRNA processing pattern indicative of co-regulated splicing of exons 2 and 3. Conclusions: A combination of sensitive techniques may assure a high (100%) STK11 mutation detection frequency in PJS families. Characterization of mutations at mRNA level may give a deeper insight into the molecular consequences of the pathogenic mutations than predictions made solely at the genomic level. Background 19p13.3 [7,8]. The gene spans 23 kb, consists of nine Peutz-Jeghers Syndrome (PJS) is a rare autosomal-domi- coding exons and a final noncoding exon [9,10]. The nant hereditary condition with incomplete penetrance, protein it codes for plays a role in cellular energy meta- characterized by hamartomatous polyps of the gastroin- bolism, cell polarization, p53-dependent apoptosis, testinal tract and pigmented lesions of the buccal VEGF regulation and Wnt signal transduction [11-14]. mucosa, perioral region and other sites [1,2]. PJS TheprevalenceofgermlinepathogenicSTK11 point patients have an increased risk of cancer of multiple mutations in PJS cases has been reported with very dif- locations, predominantly the colon, small intestine, sto- ferent frequencies, ranging from about 90% to only 10% mach, esophagus, pancreas, breast, ovary and uterine [9,10,15], depending on both patient selection criteria cervix [3-6]. and the screening methods used. The low point muta- This condition is primarily associated with germline tion rates reported in some studies raised the possibility mutations in the serine/threonine kinase 11 (STK11/ of the existence of another PJS susceptibility gene, a LKB1) gene, localized on the chromosomal segment notion investigated by genetic linkage analyses in PJS families [16,17]. An alternative hypothesis, offering an STK11 * Correspondence: [email protected] explanation for the absence of mutations in PJS † Contributed equally families in some studies, is the existence of alterations 1Department of Molecular Genetics, National Institute of Oncology, not detectable by the conventional mutation screening Budapest, Hungary Full list of author information is available at the end of the article methods used. Multiplex Ligation-dependent Probe © 2010 Papp et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Papp et al. BMC Medical Genetics 2010, 11:169 Page 2 of 9 http://www.biomedcentral.com/1471-2350/11/169 Amplification (MLPA) proved to be a powerful, robust using the Power SYBR Green PCR Master Mix on an and easy-to-perform approach to scan for large genomic ABI Prism 7900 HT Sequence Detection System deletions, and this type of mutation was indeed shown (Applied Biosystems). The conditions for thermal to have a significant contribution to the STK11 muta- cycling were 50°C for 2 min and 95°C for 10 min, fol- tion pattern [18-20]. Taken together, STK11 aberrations lowed by 40 cycles of 95°C for 15 sec and 60°C for seem to date to account for almost all familial PJS cases, 1 min. The regions used for copy number testing were bringing the existence of genetic heterogeneity into 16.4 kb, 31.6 kb, 99.4 kb, 110.5 kb, and 120.1 kb question. upstream of the STK11 gene, all selected from non- We report here the clinicopathological manifestation repetitive regions. Primer sequences and localization and results of a comprehensive mutation analysis of the data are given in Additional file 3: Table S1. The ratio STK11 gene in 13 unrelated PJS families. We describe a changes between these 5’ and known two-copy regions ΔΔ number of novel mutations in PJS patients comprising were calculated by the 2- Ct method [22] using muta- the largest number of patients from the Central-Eastern tion-negative samples for calibrations after ensuring that European region reported so far. amplification efficiencies of the control and target amplicons were comparable. Methods The mutation nomenclature used here complies with Patients and samples the recommendations of den Dunnen and Antonarakis Individuals in this study were referred for genetic coun- [23,24], sequence variations are named in relation to the seling and testing to the Department of Molecular ATG codon in cDNA reference sequence NM_000455.4, Genetics at the National Institute of Oncology, Buda- and predicted changes at the protein level are given pest, Hungary between 1995 and 2008. All investigations according to the protein reference sequence NP_000446.1, have been carried out in compliance with internationally as detailed on the website of the Human Genome Varia- recognized guidelines. Study protocols have been tion Society [25]. approved by the Institutional Ethical Board. Written informed consent was obtained from each patient. Expression analysis Included in this study were 21 patients from 13 Peutz- We performed RNA extraction from blood leukocytes of Jeghers families. mutation carriers using the RNAqueous Kit, and carried out cDNA synthesis starting from 400 ng of total RNA Mutation analysis using the High-Capacity cDNA Reverse Transcription DNA was extracted from blood samples of all consent- Kit according to the manufacturer’s instructions ing subjects using the classic phenol-chloroform (Applied Biosystems). In order to assess the effects of method. The entire coding region and splice junctions the mutations at the mRNA level, we sequenced the of the STK11 gene were amplified by PCR (primer cDNA of carrier individuals and estimated the expres- sequences are available upon request). Systematic muta- sion levels of the mutant alleles by comparing the area- tion screening was performed using direct bidirectional under-the-curve ratios at heterozygous mutation posi- sequencing applying an ABI 3130 Genetic Analyzer tions, using the genomic DNA sample as calibrator. (Applied Biosystems). The presence of all mutations was confirmed using a different blood sample. Statistical analysis Additionally, the coding region of STK11 was Differences between groups were calculated using a Stu- screened for genomic aberrations using the MLPA Kit dent’st-test,withpvalueslessthan0.05considered P101 (MRC-Holland), according to the manufacturer’s significant. recommendations, and as described previously [21]. For the determination of the exact lengths of deletions Results having both breakpoints within the gene, we applied a Patient characteristics combination of XL-PCR and sequencing. Since the A total of 13 probands and 8 affected relatives with a various deletions detected all required individual history of Peutz-Jeghers syndrome (PJS) were included approaches to determine the exact length of the dele- in this study. The disease was diagnosed at an average tion, a detailed description is outlined for each of them age of 21.9 years (within a range of 3-51 years). All but in the legends of the figures (Additional files 1 and 2: one of the probands showed the classical PJS phenotype Figures S1 and S2). of buccal freckling and hamartomatous polyps; one pro- To assess the approximate length of deletions extend- band (HP09), diagnosed at 35 years of age, showed no ing over the 5’ gene boundaries, the sequence copy typical mucocutaneous pigmentation. From the 13 pro- number at multiple sites upstream of STK11 was deter- bands only 8 had a family history of the disease, while mined. Gene dosage assays were performed in triplicates the remaining 5 cases (38%) appeared to be the result of Papp et al. BMC Medical Genetics 2010, 11:169 Page 3 of 9 http://www.biomedcentral.com/1471-2350/11/169 Table 1 Clinical characteristics of patients diagnosed with Peutz-Jeghers syndrome Family Patient Gendera Ageb Localizationc Polyp count PPd
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