The prevalence of intragenic deletions in patients with idiopathic hypogonadotropic hypogonadism and Kallmann syndrome

Idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) are clinically and genetically heterogeneous disorders caused by a deficiency of gonadotrophin-releasing hormone (GnRH). Mutations in three genes—KAL1, GNRHR and FGFR1—account for 15–20% of all causes of IHH/KS. Nearly all mutations are point mutations identified by traditional PCR-based DNA sequencing. The relatively new method of multiplex ligation-dependent probe amplification (MLPA) has been successful for detecting intragenic deletions in other genetic diseases. We hypothesized that MLPA would detect intragenic deletions in ∼15–20% of our cohort of IHH/KS patients. Fifty-four IHH/KS patients were studied for KAL1 deletions and 100 were studied for an autosomal panel of FGFR1, GNRH1, GNRHR, GPR54 and NELF gene deletions. Of all male and female subjects screened, 4/54 (7.4%) had KAL1 deletions. If only anosmic males were considered, 4/33 (12.1%) had KAL1 deletions. No deletions were identified in any of the autosomal genes in 100 IHH/KS patients. We believe this to be the first study to use MLPA to identify intragenic deletions in IHH/KS patients. Our results indicate ∼12% of KS males have KAL1 deletions, but intragenic deletions of the FGFR1, GNRH1, GNRHR, GPR54 and NELF genes are uncommon in IHH/KS

Chromosome 19p13.3 deletion in a child with Peutz-Jeghers syndrome, congenital heart defect, high myopia, learning difficulties and dysmorphic features: Clinical and molecular characterization of a new contiguous gene syndrome

The Peutz-Jeghers syndrome (PJS) is an autosomal-dominant hamartomatous polyposis syndrome characterized by mucocutaneous pigmentation, gastrointestinal polyps and the increased risk of multiple cancers. The causative point mutation in the STK11 gene of most patients accounts for about 30% of the cases of partial and complete gene deletion. This is a report on a girl with PJS features, learning difficulties, dysmorphic features and cardiac malformation, bearing a de novo 1.1 Mb deletion at 19p13.3. This deletion encompasses at least 47 genes, including STK11. This is the first report on 19p13.3 deletion associated with a PJS phenotype, as well as other atypical manifestations, thereby implying a new contiguous gene syndrome

A novel mutation in STK11 gene is associated with Peutz-Jeghers Syndrome in Indian patients

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare multi-organ cancer syndrome and understanding its genetic basis may help comprehend the molecular mechanism of familial cancer. A number of germ line mutations in the STK11 gene, encoding a serine threonine kinase have been reported in these patients. However, STK11 mutations do not explain all PJS cases. An earlier study reported absence of STK11 mutations in two Indian families and suggested another potential locus on 19q13.4 in one of them. METHODS: We sequenced the promoter and the coding region including the splice-site junctions of the STK11 gene in 16 affected members from ten well-characterized Indian PJS families with a positive family history. RESULTS: We did not observe any of the reported mutations in the STK11 gene in the index patients from these families. We identified a novel pathogenic mutation (c.790_793 delTTTG) in the STK11 gene in one index patient (10%) and three members of his family. The mutation resulted in a frame-shift leading to premature termination of the STK11 protein at 286(th )codon, disruption of kinase domain and complete loss of C-terminal regulatory domain. Based on these results, we could offer predictive genetic testing, prenatal diagnosis and genetic counselling to other members of the family. CONCLUSION: Ours is the first study reporting the presence of STK11 mutation in Indian PJS patients. It also suggests that reported mutations in the STK11 gene are not responsible for the disease and novel mutations also do not account for many Indian PJS patients. Large-scale genomic deletions in the STK11 gene or another locus may be associated with the PJS phenotype in India and are worth future investigation

Mutations in STK11 gene in Czech Peutz-Jeghers patients

<p>Abstract</p> <p>Background</p> <p>Peutz-Jeghers syndrome (PJS) is an autosomal dominant hereditary disease characterized by mucocutaneous pigmentation and gastrointestinal hamartomatous polyposis. The germline mutations in the serine/threonine kinase 11 (<it>STK11</it>) gene have been shown to be associated with the disease. Individuals with PJS are at increased risk for development of various neoplasms. The aim of the present study was to characterize the genotype and phenotype of Czech patients with PJS.</p> <p>Methods</p> <p>We examined genomic DNA of 8 individuals from five Czech families by sequencing analysis of <it>STK11 </it>gene, covering its promotor region, the entire coding region and the splice-site boundaries, and by multiplex ligation-dependent probe amplification (MLPA) assay designed for the identification of large exonic deletions or duplications of <it>STK11 </it>gene.</p> <p>Results</p> <p>We found pathogenic mutations in <it>STK11 </it>gene in two families fulfilling the diagnostic criteria of PJS and in one of three sporadic cases not complying with the criteria. The patient with the frameshift mutation in <it>STK11 </it>gene developed aggressive gastric cancer. No other studied proband has developed a carcinoma so far.</p> <p>Conclusion</p> <p>Our results showed that a germline mutation of <it>STK11 </it>gene can be found not only in probands fulfilling the PJS diagnostic criteria, but also in some sporadic cases not complying with the criteria. Moreover, we observed a new case of aggressive gastric cancer in a young patient with a frameshift mutation of <it>STK11 </it>gene.</p

High prevalence of germline STK11 mutations in Hungarian Peutz-Jeghers Syndrome patients

<p>Abstract</p> <p>Background</p> <p>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 <it>STK11</it>/<it>LKB1 </it>tumor suppressor gene. The aim of the present study was to characterize Hungarian PJS patients with respect to germline mutation in <it>STK11</it>/<it>LKB1 </it>and their association to disease phenotype.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>Thirteen different pathogenic mutations in <it>STK11</it>, 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 (<it>SBNO2 </it>and <it>GPX4</it>), located upstream of <it>STK11</it>, with a possible modifier effect. The majority of the point mutations (88%, 7/8) can be considered novel. Quantification of the <it>STK11 </it>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.</p> <p>Conclusions</p> <p>A combination of sensitive techniques may assure a high (100%) <it>STK11 </it>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.</p

Gene Expression of the Tumour Suppressor LKB1 Is Mediated by Sp1, NF-Y and FOXO Transcription Factors

The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 5′-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides −345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription

Mutations in the LKB1 tumour suppressor are frequently detected in tumours from Caucasian but not Asian lung cancer patients

Somatic mutations of LKB1 tumour suppressor gene have been detected in human cancers including non-small cell lung cancer (NSCLC). The relationship between LKB1 mutations and clinicopathological characteristics and other common oncogene mutations in NSCLC is inadequately described. In this study we evaluated tumour specimens from 310 patients with NSCLC including those with adenocarcinoma, adenosquamous carcinoma, and squamous cell carcinoma histologies. Tumours were obtained from patients of US (n=143) and Korean (n=167) origin and screened for LKB1, KRAS, BRAF, and EGFR mutations using RT—PCR-based SURVEYOR-WAVE method followed by Sanger sequencing. We detected mutations in the LKB1 gene in 34 tumours (11%). LKB1 mutation frequency was higher in NSCLC tumours of US origin (17%) compared with 5% in NSCLCs of Korean origin (P=0.001). They tended to occur more commonly in adenocarcinomas (13%) than in squamous cell carcinomas (5%) (P=0.066). LKB1 mutations associated with smoking history (P=0.007) and KRAS mutations (P=0.042) were almost mutually exclusive with EGFR mutations (P=0.002). The outcome of stages I and II NSCLC patients treated with surgery alone did not significantly differ based on LKB1 mutation status. Our study provides clinical and molecular characteristics of NSCLC, which harbour LKB1 mutations

Oxidative Activity of the Type 2 Isozyme of 17β-Hydroxysteroid Dehydrogenase (17β-HSD) Predominates in Human Sebaceous Glands

Sebum production is regulated by the opposing effects of androgens and estrogens. The intracrine activity of steroid metabolizing enzymes is important in regulating sebum production because these enzymes can convert weak steroids from the serum into potent androgens and estrogens within the sebaceous gland (SG). 17β-hydroxysteroid dehydrogenase (17β-HSD) interconverts weak and potent sex steroids via redox reactions. In this regard, it may function as a gatekeeping enzyme regulating the hormonal milieu of the SG. Six isozymes of 17β-HSD have been identified that differ in their substrate preference and their preference to produce weak or potent sex steroids via oxidation or reduction, respectively. The goals of this study are: (i) to identify which isozyme (s) of 17β-HSD is active in SG; (ii) to determine if its activity differs in facial skin compared with nonacne-prone skin that may account for the regional differences in sebum production; (iii) to compare the activity of 17β-HSD in intact glands and in SG homogenates; and (iv) to determine if 13-cis retinoic acid inhibits 17β-HSD activity. Human SG were assayed for 17β-HSD activity using estrogens, androgens, and progestins as substrates. Oxidative activity of the type 2 isozyme predominated in all samples tested. Although transcripts for the types 1, 2, 3, and 4 isozymes were detected using reverse transcriptase-polymerase chain reaction, only mRNA for the predominant type 2 isozyme and the type 4 isozyme were detected in northern analysis. Greater reductive activity of 17β-HSD was noted in SG from facial areas compared with nonacne-prone areas, suggesting an increased net production of potent androgens in facial areas. Oxidation was more predominant over reduction in intact SG compared with homogenized SG, thus supporting the hypothesis that 17β-HSD protects against the effects of potent androgens in vivo. Activity of the type 2 17β-HSD was not inhibited by 13-cis retinoic acid. In conclusion, SG possess the cellular machinery needed to transcribe the genes for the type 1–4 isozymes of 17β-HSD. At the protein level, however, oxidative activity of the type 2 isozyme predominates, suggesting that 17β-HSD isozyme activity may be translationally regulated

A novel exon duplication event leading to a truncating germ-line mutation of the APC gene in a familial adenomatous polyposis family.

Familial Adenomatous Polyposis (FAP) is an autosomal dominant condition predisposing to multiple adenomatous polyps of the colon. FAP patients frequently carry heterozygous mutations of the APC tumour suppressor gene. Affected individuals from a cohort of FAP families (n=22), where no germ-line APC mutation was detected by direct sequencing, were analysed by Multiplex Ligation-dependent Probe Amplification (MLPA). MLPA identified a previously unreported APC mutation involving duplication of exon 4. Subsequent analysis of cDNA from affected family members revealed expression of mutant mRNA species containing two copies of exon 4, resulting in a frameshift and premature stop codon. Bioinformatic analysis of the relevant APC genomic segment predicted a role for homologous recombination possibly involving Alu repeats in the generation of this genotype. Our results highlight the importance of MLPA as an adjunct to exon-by-exon sequencing in identifying infrequent mutational events in cancer predisposing genes