New sequence variants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis in an elderly healthy female population in Croatia

Cvok, Mirela Levačić; Čretnik, Maja; Musani, Vesna; Ozretić, Petar; Levanat, Sonja

doi:10.1515/cclm.2008.307

Cited by 18 publications

(14 citation statements)

References 53 publications

(48 reference statements)

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“…35,73 For the alleles harboring 5, 7 and 8 CGG repeats we used genomic DNA from healthy control samples having (CGG) 5 /(CGG) 7 , (CGG) 7 /(CGG) 7 and (CGG) 8 /(CGG) 8 genotypes, respectively. For the allele with 6 CGG repeats we used a genomic DNA sample collected from an ovarian cancer patient with the (CGG) 6 /(CGG) 8 heterozygous genotype.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

et al. 2015

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Inga (2015) Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements, RNA Biology, 12:3, 290-304, DOI: 10.1080/15476286.2015 PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG) n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

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Section: Plasmid Constructionmentioning

confidence: 99%

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

et al. 2015

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“…BRCA1 and BRCA2 mutation analysis 2.2.1. HRMA (high-resolution melting analysis) DNA was extracted from peripheral blood leukocytes, and the entire coding sequence and exon-intron boundaries were amplified using the polymerase chain reaction (PCR): 36 PCR products sized 150-437 bp for BRCA1 and 49 PCR products 179-500 bp long for BRCA2 (Cvok et al, 2008).…”

Section: Candidatesmentioning

confidence: 99%

“…PCR reactions were performed as described previously (Cvok et al, 2008). PCR conditions were optimized to Tm between 49°C and 69°C for each segment.…”

Section: Candidatesmentioning

confidence: 99%

“…Difference in MAF between different groups was assessed by Fisher's exact test and for significantly different groups presented by crude odds ratio with 95% confidence interval (CI). MAFs for variants found in healthy controls were previously published (Cvok et al, 2008). Two-tailed P values of b0.05 with Bonferroni correction for multiple testing (according the number of compared variants) were considered statistically significant.…”

Section: Haplotype Inference and Statistical Analysismentioning

confidence: 99%

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Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

Levanat

Musani

Cvok³

et al. 2012

Gene

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“…HRM assays have been developed and optimized for mutation screening of large genes, such as CFTR, 20,21 BRCA1, and BRCA2. [22][23][24][25][26] Protocols for HRM analysis, in the context of high-throughput testing of large genes, have been reported. 24 High-throughput HRM data analysis is advantageous because most tested samples do not carry sequence changes and generate a "reference range" together with the actual reference sample owing to minor variations in the HRM hardware/ software system.…”

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confidence: 99%

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

Chen

Tranebjærg

Rendtorff

et al. 2011

The Journal of Molecular Diagnostics

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Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss. Whereas Pendred syndrome is thought to be the most common cause of syndromic hearing loss, 1,2 DFNB4 is a nonsyndromic form of hearing loss caused by mutations in the same gene. 3 Clinically, both can be associated with temporal bone abnormalities. 4 In addition, an abnormal perchlorate discharge test result and/or euthyroid goiter can be present in patients with Pendred syndrome but not in those with DFNB4. 3,4 This may be due to allelic heterogeneity because differences in residual activity of the protein products render different phenotypes. 5

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New sequence variants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis in an elderly healthy female population in Croatia

Cited by 18 publications

References 53 publications

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

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