Large deletions and splicing site mutations inthe STK11 gene in Peutz Jeghers Chilean families
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(2) 366. Yes Yes No No No Yes Yes Yes Yes Yes Yes No Yes Gastric-Duodene-Colon Gastric-Ileum-Colon Unknown Unknown Unknown Small bowel-Colon Small bowel Small bowel-Colon Small bowel-Colon Gastric-Small bowel-Colon Small bowel Small bowel-Colon Colon Yes Yes Unknown Unknown Unknown Yes Yes Yes Yes Yes Yes Yes Yes 8 9 11 12 13. 4. 3. F, female; M, male; PJS, Peutz-Jeghers Syndrome.. Yes No No No Yes. Yes. Yes. Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes. 54 27 31 12 10 39 40 37 30 18 9 34 15 37 1 26 7 6 9 17 12 2 3 3 34 14. Mucocutaneous pigmentation Family history of PJS Current age Age at diagnosis Gender. A total of 13 patients (10 women and 3 men), from eight unrelated families were included in this study (Table 1). Five out of eight families, had relatives with PJS (1, 3, 4, 8, 13). The age at diagnosis in patients ranged from 1 to 37 years old. The late PJS diagnosis in three patients, only when signs of polyps, anemia and/or bleeding were evident, was due to the absence of family history. All young patients with family history were diagnosed by the presence of mucocutaneous. Patient. Patient enrollment (features). Family. Results. Table 1. Clinical characteristics of patients with Peutz-Jeghers syndrome. DNA was isolated from whole blood sample using the method described by Lahiri and Nurnberger (12). The entire coding region and splice junctions of the STK11 gene were amplified by polymerase chain reaction (PCR) (3, 13). Mutational screening was performed by SSCP analysis (14), followed by DNA sequencing. Mutations were confirmed by two independent PCR reactions. Mutations were named according to the nomenclature proposed by the Human Genome Variation Society (http://www.hgvs.org/mutnomen/) based on the cDNA reference sequence NM_000455.4, and the protein reference sequence NP_000446.1. Negative samples for SSCP analysis were further examined by the MLPA method. The MLPA kit P101 (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer’s recommendations was used. To determine the breakpoints for the STK11 deletion in PJS6 and PJS25 patients by MLPA analysis, long-range PCR was carried out using the KOD Xtreme Hot Start DNA Polymerase (Novagen, Toyobo, Japan) according to conditions recommended by the manufacturer. Breakpoints were verified by DNA sequencing of purified PCR products.. Diagnostic criteria of PJS. Mutational screening. Hamartomas polyps. Localizationof polyps. Patients were referred for genetic testing from different regions of our country, between 2008 and 2010. Research was carried out in agreement with the Institutional Ethical Board. Written and signed informed consents were obtained from each patient. All patients fulfilled the diagnostic criteria of PJS (the presence of hamartomatous polyps and/or mucocutaneous pigmentation and/or family history of PJS) (11). A total of 13 PJS patients from eight unrelated families were included in this study (Table 1).. F F F F M F M M F F F F F. Family recruitment. PJS 1 PJS 12 PJS 2 PJS 3 PJS 4 PJS 6 PJS 7 PJS 18 PJS 10 PJS 11 PJS 15 PJS 25 PJS 27. Patients and methods. 1. Intussusception. Cancer. from Colombia describing two mutations in PJS families (10). The aim of this study was to characterize the STK11 in Chilean families diagnosed with PJS, in search of possible point mutations and/or gene rearrangements.. No No No No No No No No No No No No No. Orellana et al..
(3) Deletions and mutations in Peutz-Jeghers Chilean families pigmentation, most of them with polyps at 12 and 20 years old. None of the enrolled patients in this study has developed cancer up to now, maybe due to their young age (range 9–54 y.o.). Among relatives we found two PJS cases, one of them affected with colorectal and the other with pancreatic cancer, both deceased at 71 and 53 years old respectively. Other cases of cancer, such as breast and stomach, were present in some relatives; however information related to the development of PJS was not available. Mutational screening. We identified seven different mutations in STK11 gene, in seven out of the eight analyzed families. Patient PJS9/family 11, with no identified mutation, has no PJS family history but presents all the characteristic features of the syndrome. Point mutations. Two splice site mutations were found, one in intron 4 (c.597+2T>A) in patient PJS1/family 1 and one in intron 6 (c.862-2A>G) in PJS10/family 8. In addition, a frameshift mutation was detected in exon 1 (c.109delC) in patient PJS15/family 11 (Table 2). Mutation c.597+2T>A occurred at the second position of the splice donor site of intron 4. The RNA analysis by RT-PCR showed a skipping of exon 4 generating a frameshift and a premature stop at codon 227. The second splice mutation, c.862-2A>G, corresponded to a nucleotide substitution at the second position of intron 6 in the splice acceptor site. The RNA analysis showed a partial skipping of exon 7 and a full skipping of exons 7–8. The partial skipping of exon 7 produced a frameshift and a premature stop at codon 302. Lastly, the skipping of full exons 7 and 8 led into a deletion in frame of 83 aminoacids in the functional protein. Large deletions. In four different families, four genomic rearrangements were detected by MLPA including large deletions ranging from a single exon to the complete STK11 gene. In patient PJS27/family 13, a deletion comprising the promoter region, the 5 UTR and exon 1 was identified while in patient PJS2/family 3, a deletion of the entire STK11 gene was detected, including the promoter region. Furthermore, in patient PJS6/family 4 and in patient PJS25/family 12 deletions in exon 2 and exons 2 and 3 were found, respectively (Table 2). The exact position of nucleotides involved in the breakpoints leading into STK11 deletions was assessed by a long-range PCR and DNA sequencing. Sequence analysis of the deletion in patient PJS6, revealed the absence of 5816 bp corresponding to: 5330 bp of intron 1, 84 bp of exon 2 and 402 bp of intron 2. An insertion of 3nt, from unknown origin, was detected in the joining of introns 1 and 2. In patient PJS25, a deletion of 6364 bp was identified corresponding to: 5235 bp of intron 1, 84 bp of exon 2, 823 bp of intron 2,. 90bp of exon 3 and 132 bp of intron 3. In this case an insertion of 15nt occurred in the joining of introns 1 and 3 (Table 3). In this study, a co-segregation analysis in affected and non-affected relatives of four families was performed. In family 1, two children (one affected); in family 3, three sons (two affected); and in family 4, six siblings (two affected) were studied. This analysis revealed that the mutation was only present in affected relatives (Tables 1 and 2). In family 11, both healthy parents of PJS15 were studied, showing absence of the mutation, suggesting a de novo case. Relatives from families 8, 12 and 13 were not available for the genetic study. Discussion. The Chilean population is considered to be a mixture of Amerindian and European – mainly Spanish – populations, leading into an ethnic group with significant genetic differences from populations previously studied for PJS. Among the families from this study there were no ‘pure native’ families and all have Chilean ancestors as far as they know. The mutation frequency in STK11 in our group of patients was similar to the previous reports in the field (6–9). The use of two complementary techniques in the screening of gene alterations allowed us to identify mutations in 7 out of 8 families (Table 2). We were not able to find a mutation in patient PJS9/family 11, maybe due to alterations that escaped SSCP analysis or that are located in regions of the gene that were not evaluated (promoter region, introns and 5 or 3 untranslated regions). An alternative explanation for the lack of mutations in DNA from lymphocytes in some families is the occurrence of somatic mosaicism, which has been described in de novo cases of hereditary syndromes such as familial adenomatous polyposis. It is known that somatic mosaicism leads into an under-representation or absence of the mutation in some tissues. If that is the case, mutations in lymphocytes will not be detected by conventional approaches (15, 16). We must also consider the possibility of the existence of another gene involved, as it has been proposed in few reports, where linkage studies identified two different susceptibility loci at 19p13.4 and 6p11-cen (17, 18). Three novel point mutations were identified, in three families including one small deletion causing a frameshift and a premature stop and two splice site mutations. These changes resulted in the loss of the tyrosine kinase domain and three phosphorylation sites, Thr336, Ser325 and Thr366 in the C-terminus of the protein. It has been reported that auto-phosphorylation of Thr366 aminoacid is required to suppress cell growth, suggesting that it is relevant for STK11 function (19). Genomic rearrangements have been reported as the cause of mutations in different hereditary syndromes predisposing to cancer development (20, 21). In PJS, it is observed that about 31% of cases present major genomic deletions, instead of point mutations (6–9). In. 367.
(4) 368. Patients. Family. PJS 27. Unknowna. FS, frameshift mutation. a Origin/parents/relatives unavailable or not investigated.. 13. Genomic rearrangements detected by MLPA 3 PJS 2 Inherited PJS 3 PJS 4 4 PJS 6 Inherited PJS 7 PJS 18 12 PJS 25 Unknowna. Co-segregation analysis. de novo. PJS 15. 11. Exon 1. Del exon 1 (c.-1120-?_ 76+?del). Del exons 2 and 3 (c.317-?_424+?del). Del exon 2 (c.317-?_334-?del). SSCP. SSCP and RT-PCR SSCP and RT-PCR. Technique. 58 aminoacid deleted, with no frameshift Absence of initiation codon, no translation of one allele. 28 aminoacid deleted, with no frameshift. Absence of initiation codon, no translation of one allele. Protein change. Exon 7 partial skipping (p.Gly288AspfsX302); Exon 7 and 8 skipping (deletion in frame) p.Gln37SerfsX50. Exon 4 skipping p.Gly155GlyfsX227. Aminoacid change. Whole gene deletion (c.-1120-?_1373+?del). Nucleotide change. c.109delC. c.862-2A>G. c.597+2T>A. Nucleotide change. Exons 2 and 3. Exon 2. Exons 1–10. Exon/intron. Exon 1. Intron 6. Unknowna. Exon/intron Intron 4. Co-segregation analysis Inherited. Patients. Point mutations 1 PJS 1 PJS 12 8 PJS 10. Family. Table 2. STK11 germline mutations detected in our cases series. Yes (6). Yes (6, 22, 23). Yes (24). Yes (6, 24). Described. Novel. Novel. Novel. Described. Orellana et al..
(5) Deletions and mutations in Peutz-Jeghers Chilean families Table 3. Breakpoints and deletion sizes in two PJS families Family. Distal BP. Proximal BP. Size (bp). Repeats. 4 12. 1,213,086 1,213,168. 1,218,902 1,219,550. 5816 6364. AluY/AluY/-. BP, breakpoints. The position of each breakpoint is indicated in base pairs (bp) from the telomere short arm. Sequence coordinates are based in the UCSC Human Genome Browser, February 2009.. this study four out of seven families showed deletions in the STK11 . Two of these are in frame deletions involving exon 2, and exons 2 and 3, leading to the absence of the kinase domain of the protein. The remaining two deletions involve the promoter region and exon 1, or the entire gene, both affecting the transcription of the STK11. The deletion of exons 2 and 3 in STK11, found in PJS25, has been previously described in two studies that also determined the breakpoints of the rearrangements (22, 23). These reports determined that the deletion is caused by a recombination between one of the AluI elements localized in intron 1 and another in intron 3. In patient PJS25 the breakpoints in introns 1 and 3 differed to the cases previously described. The breakpoint at intron 1 involved the same AluI element described (22, 23), but the breakpoint in intron 3 is localized in non-repetitive sequences. Deletion of exon 2 identified in patient PJS6, has been described previously (24), but no breakpoints have been determined. Our study revealed that the 5 end of the breakpoint also involved a different AluI element from the one involved in exon 2 and 3 deletions. Overall, our combined screening strategy for mutations was of 88%. In this study of Chilean PJS families, whole gene or exonic deletions appear to account for approximately 57% of all causative changes identified in the STK11. This is the second Latin-American study in the screening of mutations in PJS, and the first one combining two different approaches, point mutation detection and genomic rearrangements. Acknowledgements Supported by Clínica Las Condes. Author contributions: P. O., C. H., C. S., E. P., K. A. did substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, drafting the article or revising it critically for important intellectual content and final approval of the version to be published. Whereas F. L.K., J. C. P. C. have contributed only to conception and design, analysis and interpretation of data, drafting the article or revising it critically for important intellectual content and final approval of the version to be published.. References. 2. Hemminki A, Markie D, Tomlinson I et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 1998: 391: 184–187. 3. Jenne DE, Reimann H, Nezu J et al. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat Genet 1998: 18: 38–43. 4. Alessi DR, Sakamoto K, Bayascas JR. LKB1-dependent signaling pathways. Annu Rev Biochem 2006: 75: 137–163. 5. Karuman P, Gozani O, Odze RD et al. The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death. Mol Cell 2001: 7: 1307–1319. 6. Aretz S, Stienen D, Uhlhaas S et al. High proportion of large genomic STK11 deletions in Peutz-Jeghers syndrome. Hum Mutat 2005: 26: 513–519. 7. Chow E, Meldrum CJ, Crooks R et al. An updated mutation spectrum in an Australian series of PJS patients provides further evidence for only one gene locus. Clin Genet 2006: 70: 409–414. 8. Hearle NC, Rudd MF, Lim W et al. Exonic STK11 deletions are not a rare cause of Peutz-Jeghers syndrome. J Med Genet 2006: 43: e15. 9. Volikos E, Robinson J, Aittomaki K et al. LKB1 exonic and whole gene deletions are a common cause of Peutz-Jeghers syndrome. J Med Genet 2006: 43: e18. 10. Velez A, Gaitan MH, Marquez JR et al. Two novel LKB1 mutations in Colombian Peutz-Jeghers syndrome patients. Clin Genet 2009: 75: 304–306. 11. Aaltonen LA. Hereditary intestinal cancer. Semin Cancer Biol 2000: 10: 289–298. 12. Lahiri DK, Nurnberger JI Jr. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991: 19: 5444. 13. Dong SM, Kim KM, Kim SY et al. Frequent somatic mutations in serine/threonine kinase 11/Peutz-Jeghers syndrome gene in left-sided colon cancer. Cancer Res 1998: 58: 3787–3790. 14. Alvarez K, Hurtado C, Hevia MA et al. Spectrum of MLH1 and MSH2 mutations in Chilean families with suspected Lynch syndrome. Dis Colon Rectum 2010: 53: 450–459. 15. Baert-Desurmont S, Piton N, Bou J et al. A remarkable APC mosaicism with two mutant alleles in a family with familial adenomatous polyposis. Am J Med Genet A 2011: 155A: 1500–1502. 16. Necker J, Kovac M, Attenhofer M et al. Detection of APC germ line mosaicism in patients with de novo familial adenomatous polyposis: a plea for the protein truncation test. J Med Genet 2011: 48: 526–529. 17. Mehenni H, Blouin JL, Radhakrishna U et al. Peutz-Jeghers syndrome: confirmation of linkage to chromosome 19p13.3 and identification of a potential second locus, on 19q13.4. Am J Hum Genet 1997: 61: 1327–1334. 18. Tomlinson IP, Olschwang S, Abelovitch D et al. Testing candidate loci on chromosomes 1 and 6 for genetic linkage to Peutz-Jeghers’ disease. Ann Hum Genet 1996: 60: 377–384. 19. Sapkota GP, Boudeau J, Deak M et al. Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome. Biochem J 2002: 362: 481–490. 20. Michils G, Tejpar S, Thoelen R et al. Large deletions of the APC gene in 15% of mutation-negative patients with classical polyposis (FAP): a Belgian study. Hum Mutat 2005: 25: 125–134. 21. Taylor CF, Charlton RS, Burn J et al. Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA. Hum Mutat 2003: 22: 428–433. 22. De Rosa M, Galatola M, Quaglietta L et al. Alu-mediated genomic deletion of the serine/threonine protein kinase 11 (STK11) gene in Peutz-Jeghers syndrome. Gastroenterology 2010: 138: 2558–2560. 23. Resta N, Giorda R, Bagnulo R et al. Breakpoint determination of 15 large deletions in Peutz-Jeghers subjects. Hum Genet 2010: 128: 373–382. 24. de Leng WW, Jansen M, Carvalho R et al. Genetic defects underlying Peutz-Jeghers syndrome (PJS) and exclusion of the polarity-associated MARK/Par1 gene family as potential PJS candidates. Clin Genet 2007: 72: 568–573.. 1. van Lier MG, Westerman AM, Wagner A et al. High cancer risk and increased mortality in patients with Peutz-Jeghers syndrome. Gut 2011: 60: 141–147.. 369.
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