Sanger Sequencing Technique Research Articles

A retrospective analysis of 237 Chinese families with Duchenne muscular dystrophy history and strategies of prenatal diagnosis.

To offer 4-year clinical prenatal diagnosis experience of Duchenne muscular dystrophy (DMD). Denaturing high-performance liquid chromatography (DHPLC) and Sanger sequencing were used for molecular diagnosis of 237 DMD families. In the study, deletions, duplications, complex rearrangement and small mutations accounted for 47.3%, 8.4%, 1.7% and 42.6% of 237 families, respectively. Sixty-six different deletion patterns were identified in 112 families. Fourteen different duplication patterns were identified in 20 families and 4 complex rearrangements were identified. About 87.1% different small mutation patterns were identified, including 37.6% different nonsense mutation patterns, 24.8% different frameshift mutation patterns, 7.9% different missense mutation patterns, and 16.8% different splice site mutation patterns. There was no significant difference in the age of onset and mutation patterns (P>.05). The follow-up examinations revealed that the pregnancies of 14 cases were interrupted. Two cases were preterm births, 151 cases were delivered at term, 63 cases continued to pregnancy, and 7 cases were lost to follow-up. DHPLC and Sanger sequencing technique are efficient, sensitive, and specific in screening for DMD gene mutations. And pre-pregnancy DMD gene examination is an important step to assess mutation type of family with suspected DMD and guides exactly prenatal diagnosis in high-risk families.

Cross sectional study to determine chloroquine resistance among Plasmodium falciparum clinical isolates from Khartoum, Sudan.

Background: Malaria continues to present a global health threat; the World Health Organization (WHO) reported 214 million cases of malaria by the year 2015 with a death rate of 438000. Sudan is endemic to malaria with over 95% of malaria cases due to Plasmodium falciparum. Chloroquine is a well-established drug in the treatment of P. falciparum malaria although its use has declined since its introduction as the drug of choice in treatment of malaria in Sudan. The mechanism of resistance has been attributed to mutations in P. falciparum Chloroquine resistance transporter gene coding for a key food vacuole proteins. In current study we aimed at verifying the genetic cause of resistance to Chloroquine in field isolates of P. falciparum. Methods: Twenty P. falciparum cases were diagnosed from East Nile hospital in Khartoum and recruited in the investigation. Nested PCR was conducted to isolate mutation regionin the PfCRT gene and the amplicons were sequenced using Sanger sequencing technique (Macrogen, Soule Korea). Results: 16/20 (80%) of the field isolates contained base pair mutation of codon 76 in the pfcrt gene thus being resistant to chloroquine treatment and only 4/20 (20%) did not contain such mutation. Conclusions: High treatment failures associated with Chloroquine treatment is evident of the high prevalence of mutant strains of P. falciparum field isolates thus suggesting the reduced relevance of Chloroquine as a treatment choice in the management of P. falciparum malaria.

Novel BRAF mutation in melanoma: A case report.

In melanoma, a number of specific genetic and genomic aberrations have been identified to be important in tumorigenesis. In particular, the mutant B-Raf proto-oncogene, Serine/Threonine kinase (BRAF) gene is the target of tailored therapy with kinase inhibitor molecules. Identification of the array of mutations in patients with melanoma will be useful in determining a genetic profile of the tumor with potential implications for treatment decisions. A rare aminoacidic insertion in codon 599 of the BRAF gene (c.1797_1798insACA, T599insT) was detected by using both direct (Sanger) sequencing and pyrosequencing techniques in a metastatic melanoma of a female elderly patient. As suggested in other clinical contexts including pilocytic astrocytoma, papillary thyroid carcinomas and anaplastic thyroid carcinomas, this unusual mutation may be associated with a modified spatial structure of activated P-loop, resulting in a constitutional activation of the BRAF protein. The patient died shortly following the test, thus no biological therapy was performed. Comparable data regarding treatment of melanoma patients with rare BRAF mutations is lacking, and the response to BRAF inhibitors requires further investigation.

Identification of the complete mitochondrial genome of Monomia gladiator (Decapoda: Brachyura: Portunidae) and its phylogenetic relationship

The complete mitochondrial genome sequence plays an important role in phylogenetic studies. In this study, the complete mitochondrial genome of Monomia gladiator was obtained by Illumina and Sanger sequencing techniques. The circular genome was 15,878 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a putative control region. This whole mitogenome composition was 33.32% for A, 35.69% for T, 11.75% for G, and 19.24% for C, respectively. The phylogenetic analysis suggested that M. gladiator was genetically closest to three Portunidae species (Charybdis japonica, C. feriata and Thalamita crenata). The newly described mitogenome may facilitate the phylogenetic studies on Portunidae crabs.

DNMT3A (R882) mutation features and prognostic effect in acute myeloid leukemia in Coexistent with NPM1 and FLT3 mutations

Objective/BackgroundIn the absence of high-risk cytogenetic, DNMT3A (DNA Methyltransferase 3a) mutation status has an impact on outcome in the presence of FLT3 (FMS-like Tyrosine Kinase3) and/or NPM1 (Nucleophosmin). In this study, we focus on the features and effect of DNMT3A (R882) mutation in acute myeloid leukemia (AML) in the presence or absence of NPM1 and FLT3 mutations. MethodsA total of 174 cytogenetically normal (CN)-AML cases were analyzed for NPM1, FLT3, and DNMT3A mutations. For NPM1 mutation detection, we used the pyrosequencing technique; for FLT3 mutations, polymerase chain reaction and RFLP with ECO-RV techniques were used, and for DNMT3A mutation analysis, we used Sanger sequencing and RFLP (Restriction Fragment Length Polymorphism) techniques. ResultsNPM1 mutation was found in 40.80%, DNMT3A in 12.06%, and FLT3 mutation was found in 16.66% of 174 CN-AML patients. We also found seven cases which were (NPM1+, FLT3+), 10 cases which were (NPM1+, DNMT3A+), and two cases were found positive for (DNMT3A+, FLT3+) mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (72.22% vs. 16.66%; p = .020), whereas FLT3/ITD and DNMT3A mutation was associated with higher white blood count (p = .081). Immunophenotypically, NPM1 and DNMT3A mutations were significantly associated with the lack of CD34, whereas FLT3/ITD mutation was positively associated with the expression of CD7. We also assessed the overall survival and progression-free survival of DNMT3A mutation status among patients with CN-AML. Indeed, DNMT3A mutations within the CN-AML subset were associated with significantly shorter overall survival and progression-free survival compared to NPM1 and FLT3 mutated patients (p = .067 and p = .065, respectively). ConclusionDNMT3A R882 mutation plays an important role in CN-AML patients’ prognosis and clinical outcomes in the presence and absence of NPM1 and FLT3 mutations.

Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system

目的应用CRISPR-Cas9基因编辑技术来实现同一染色体片段上的多个基因共缺失。方法利用分子克隆技术构建能使小鼠染色体11B3上Aloxe3-Alox12b-Alox8基因簇缺失的CRISPR-Cas9慢病毒质粒；通过转染HEK293T细胞来包装带CRISPR或Cas9 cDNA的慢病毒，进而感染小鼠NIH3T3细胞，提取这些细胞的全基因组；通过PCR扩增缺失后的片段，TA克隆、Sanger测序等技术手段鉴定Aloxe3-Alox12b-Alox8基因簇缺失的情况。结果成功构建了能使Aloxe3-Alox12b-Alox8基因簇发生缺失的CRISPR-Cas9慢病毒质粒；PCR检测证实了目的染色体片段（Aloxe3-Alox12b-Alox8基因簇）的缺失；TA克隆和Sanger测序明确了Aloxe3-Alox12b-Alox8基因簇发生缺失，并且该CRISPR-Cas9系统介导的切割事件产生的断点被准确地连接在一起，在两个切割位点间没有发生插入突变。结论利用CRISPR-Cas9基因编辑技术可以有效实现小鼠染色体11B3上Aloxe3-Alox12b-Alox8基因簇的大片段缺失。

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii.

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.

Evaluation of a Panel of Single-Nucleotide Polymorphisms in miR-146a and miR-196a2 Genomic Regions in Patients with Chronic Periodontitis.

Periodontitis is an inflammatory disease caused by bacterial triggering of the host immune-inflammatory response, which in turn is regulated by microRNAs (miRNA). Polymorphisms in the miRNA pathways affect the expression of several target genes such as tumor necrosis factor-α and interleukins, which are associated with progression of disease. The objective of this study was to identify the association between the MiR-146a single nucleotide polymorphisms (SNPs) (rs2910164, rs57095329, and rs73318382), the MiR-196a2 (rs11614913) SNP and chronic periodontitis. Genotyping was performed for the MiR-146a (rs2910164, rs57095329, and rs73318382) and the MiR-196a2 (rs11614913) polymorphisms in 180 healthy controls and 190 cases of chronic periodontitis by the direct Sanger sequencing technique. The strength of the association between the polymorphisms and chronic periodontitis was evaluated using logistic regression analysis. Haplotype and linkage analyses among the polymorphisms was performed. Multifactorial dimensionality reduction was performed to determine epistatic interaction among the polymorphisms. The MiR-196a2 polymorphism revealed a significant inverse association with chronic periodontitis. Haplotype analysis of MiR-146a and MiR-196a2 polymorphisms revealed 13 different combinations, of which 5 were found to have an inverse association with chronic periodontitis. The present study has demonstrated a significant inverse association of MiR-196a2 polymorphism with chronic periodontitis.

SMOmutation status defines a distinct and frequent molecular subgroup in olfactory groove meningiomas

Meningiomas are the most common primary intracranial tumors in adults. Identification of SMO and AKT1 mutations in meningiomas has raised the hope for targeted therapies. It would be useful to know the precise frequency of these mutations in anatomical subgroups and clarify their prognostic value. We used the Sanger sequencing technique to characterize 79 samples of olfactory groove meningiomas for SMO (L412F and W535L) and AKT1E17K mutations. We reviewed clinical data to assess the prognostic value of these mutations in this anatomical subgroup. Out of the 79 patients with olfactory groove meningiomas, we identified targetable mutations in 34 patients (43%) (22 patients [28%] with SMO mutation-L412F almost exclusively-and 12 patients [15%] with AKT1 mutation). Meningiomas in the SMO-mutant group had an overall 36% recurrence rate, significantly higher than in the AKT1-mutant group (16%) and in the "SMO and AKT1 wildtype" group (11%) (χ2 test, P = .04). All late recurrences (after 5 y) occurred in the SMO-mutant group. Among grade I meningiomas, the SMO-mutant group was identified as having a significantly poorer prognosis. World Health Organization histological grade II (P = .006) and incomplete resection (P = .001) were independently associated with shorter recurrence-free survival. Molecular diagnosis of SMOL412F/W535L and AKT1E17K mutations improves prognostic evaluation in olfactory groove meningiomas and opens new therapeutic perspectives with SMO or AKT inhibitors for recurrent cases.

Molecular patterns of β-thalassemia mutations of Saudi patients referred to King Faisal Specialist Hospital and Research Center

Background: Beta thalassemias are a group of hereditary blood disorders that are characterized by reduction or complete absence of the β-globin chain synthesis due to mutations, affecting critical areas of the β-globin gene on the chromosome 11. The disease is inherited in an autosomal recessive manner, with severity ranging from asymptomatic individuals to transfusion dependent anemia, according to the nature of the mutation. The prevalence of the β-globin gene in various areas of Saudi Arabia, previously reported as 0.01 to 0.15% in general population. Materials And Methods: A cohort of 131 samples, which were submitted for β-globin gene mutation analysis in King Faisal Specialist Hospital and Research Center during 2008 to 2013, were tested for the entire genomic region (3-Exons and 2-Introns) of the HBB gene by direct Sanger sequencing technique. Results: Out of the total population tested, 28 (21%) were undetectable cases and 103 (79%) were detectable for β-globin chain mutations. Nineteen different mutations of the HBB gene were identified in all detectable cases (103 patients). Among these mutations c.315+1G>A, c.118C>T, and c.92+5G>C were detected in the majority of cases (66%) with five novel mutations (c.410G>A, c.-151C>T, c.68_74delAAGTTGG, c.316-3C>A, and c.-31C>T) that are reported for the first time in Saudi population. Discussion: The result of this retrospective study confirms the previously reported common β-thalassemia mutations among Saudi population.

The MEFV gene pathogenic variants and phenotype-genotype correlation in children with familial Mediterranean fever in the Çanakkale population.

The aim of the current study was to determine the frequency of the Mediterranean fever (MEFV) gene pathogenic variants in 60 children diagnosed with familial Mediterranean fever (FMF) and to compare the phenotype-genotype correlation. Genomic DNA was isolated by the spin-column method from peripheral blood samples (collected in vacutainers containing EDTA) and buccal smears. The MEFV gene profiles for the current FMF cohort were genotyped by pyrosequencing and direct Sanger sequencing techniques for the target pathogenic variants. The most prominent clinical symptoms were abdominal pain (53.4%), fever (23.4%) and arthritis (23.3%). Eighteen different pathogenic variants were identified and the most frequent were p.Met694Val (20.0%), p.Glu148Gln (13.3%), p.Met680 Ile (11.7%) and p.Arg202Gln (11.7%). Abdominal pain, fever and arthritis were the most common presenting clinical characteristics. Results showed that not only clinical characteristics, but also genotyping of the MEFV gene is needed to establish the correct diagnosis of FMF in children and other family members.

Bacterial communities associated with apical periodontitis and dental implant failure.

BackgroundPreviously, we demonstrated that bacteria reside in apparently healed alveolar bone, using culture and Sanger sequencing techniques. Bacteria in apparently healed alveolar bone may have a role in peri-implantitis and dental implant failure.ObjectiveTo compare bacterial communities associated with apical periodontitis, those colonising a failed implant and alveolar bone with reference biofilm samples from healthy teeth.Methods and resultsThe study consisted of 196 samples collected from 40 patients undergoing routine dental implant insertion or rehabilitation. The bacterial 16S ribosomal DNA sequences were amplified. Samples yielding sufficient polymerase chain reaction product for further molecular analyses were subjected to terminal restriction fragment length polymorphism (T-RFLP; 31 samples) and next generation DNA sequencing (454 GS FLX Titanium; 8 samples). T-RFLP analysis revealed that the bacterial communities in diseased tissues were more similar to each other (p<0.049) than those from the healthy reference samples. Next generation sequencing detected 13 bacterial phyla and 373 putative bacterial species, revealing an increased abundance of Gram-negative [Prevotella, Fusobacterium (p<0.004), Treponema, Veillonellaceae, TG5 (Synergistetes)] bacteria and a decreased abundance of Gram-positive [(Actinomyces, Corynebacterium (p<0.008)] bacteria in the diseased tissue samples (n=5) relative to reference supragingival healthy samples (n=3).ConclusionIncreased abundances of Prevotella, Fusobacterium and TG5 (Synergistetes) were associated with apical periodontitis and a failed implant. A larger sample set is needed to confirm these trends and to better define the processes of bacterial pathogenesis in implant failure and apical periodontitis. The application of combined culture-based, microscopic and molecular technique-based approaches is suggested for future studies.

Resolving phylogenetic relationships and species delimitations in closely related gymnosperms using high-throughput NGS, Sanger sequencing and morphology

Plastid genomes have been widely applied to elucidate plant evolution at higher taxonomic levels, but have rarely been considered useful for addressing close relationships. Here, we resolve the phylogeny and taxonomy of the Chinese lianoid Gnetum clade (Gnetales), using high throughput and Sanger sequencing techniques and studies of plant morphology. Despite previous efforts, relationships among taxa and the taxonomy within the clade have remained unclear. We generated 11 plastid genomes representing one arborescent and four lianoid species. Phylogenetic analyses were conducted using (a) the entire plastid genomes and (b) the protein-coding genes only. Sequence divergence among the lianoid species was substantial, with 9345 variable sites. Four variable regions were identified, targeted and sequenced for an additional 54 specimens and analyzed together with one nuclear ribosomal marker. Results from the phylogenetic analyses corroborate G. parvifolium as sister to the remaining lianoid species and support the presence of at least five additional species in the Chinese lianoid clade: G. catasphaericum, G. formosum, G. luofuense, G. montanum and G. pendulum. Following morphological investigations, G. giganteum and G. gracilipes are included in and synonymized with G. pendulum. Gnetum hainanense is included in and synonymized with G. luofuense. Two names, G. indicum and G. cleistostachyum, remain questionable. A taxonomic revision and a key to Chinese lianoid Gnetum are presented. Internal nodes in the Chinese lianoid Gnetum clade are from the Miocene and onwards and coincide with the expansion of tropical to subtropical forests in South China, which may have facilitated speciation in the clade.

Identification of a novel frameshift heterozygous deletion in exon 8 of the PAX6 gene in a pedigree with aniridia

Aniridia is a congenital, panocular abnormality which is characterized by partial or complete absence of iris and various degrees of iris hypoplasia. Mutations in the PAX6 gene are found in ~90% of cases with aniridia. The human PAX6 gene is located at chromosome 11p13 and encodes a transcriptional regulator that has crucial roles in the development of the eyes, central nervous system and pancreatic islets. The present study performed a clinical and genomic analysis of two families containing multiple cases of aniridia. All exons of the PAX6 gene of the probands were sequenced using the Sanger sequencing technique. A heterozygous non‑stop mutation in exon14 was identified in the first family, which has been previously reported for a different ophthalmological pathology. This mutation causes on‑going translation of the mRNA into the 3'‑untranslated region. In the second family, a novel frameshift heterozygous deletion in exon8 was identified.

Expanding the mutation spectrum in 130 probands with ARPKD: identification of 62 novel PKHD1 mutations by sanger sequencing and MLPA analysis.

Autosomal recessive polycystic kidney disease (ARPKD) is a rare severe genetic disorder arising in the perinatal period, although a late-onset presentation of the disease has been described. Pulmonary hypoplasia is the major cause of morbidity and mortality in the newborn period. ARPKD is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene that is among the largest human genes. To achieve a molecular diagnosis of the disease, a large series of Italian affected subjects were recruited. Exhaustive mutation analysis of PKHD1 gene was carried out by Sanger sequencing and multiple ligation probe amplification (MLPA) technique in 110 individuals. A total of 173 mutations resulting in a detection rate of 78.6% were identified. Additional 20 unrelated patients, in whom it was not possible to analyze the whole coding sequence, have been included in this study. Taking into account the total number (n=130) of this cohort of patients, 107 different types of mutations have been detected in 193 mutated alleles. Out of 107 mutations, 62 were novel: 11 nonsense, 6 frameshift, 7 splice site mutations, 2 in-frame deletions and 2 multiexon deletion detected by MLPA. Thirty-four were missense variants. In conclusion, our report expands the spectrum of PKHD1 mutations and confirms the heterogeneity of this disorder. The population under study represents the largest Italian ARPKD cohort reported to date. The estimated costs and the time invested for molecular screening of genes with large size and allelic heterogeneity such as PKHD1 demand the use of next-generation sequencing (NGS) technologies for a faster and cheaper screening of the affected subjects.

Diversity of Pneumocystis jirovecii during Infection Revealed by Ultra-Deep Pyrosequencing.

Pneumocystis jirovecii is an uncultivable fungal pathogen responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, the physiopathology of which is only partially understood. The diversity of the Pneumocystis strains associated with acute infection has mainly been studied by Sanger sequencing techniques precluding any identification of rare genetic events (< 20% frequency). We used next-generation sequencing to detect minority variants causing infection, and analyzed the complexity of the genomes of infection-causing P. jirovecii. Ultra-deep pyrosequencing (UDPS) of PCR amplicons of two nuclear target region [internal transcribed spacer 2 (ITS2) and dihydrofolate reductase (DHFR)] and one mitochondrial DNA target region [the mitochondrial ribosomal RNA large subunit gene (mtLSU)] was performed on 31 samples from 25 patients. UDPS revealed that almost all patients (n = 23/25, 92%) were infected with mixtures of strains. An analysis of repeated samples from six patients showed that the proportion of each variant change significantly (by up to 30%) over time on treatment in three of these patients. A comparison of mitochondrial and nuclear UDPS data revealed heteroplasmy in P. jirovecii. The recognition site for the homing endonuclease I-SceI was recovered from the mtLSU gene, whereas its two conserved motifs of the enzyme were not. This suggests that heteroplasmy may result from recombination induced by unidentified homing endonucleases. This study sheds new light on the biology of P. jirovecii during infection. PCP results from infection not with a single microorganism, but with a complex mixture of different genotypes, the proportions of which change over time due to intricate selection and reinfection mechanisms that may differ between patients, treatments, and predisposing diseases.

Efficacy of clinical diagnostic criteria for familial hypercholesterolemia genetic testing in Poland

Background and AimsFamilial hypercholesterolemia (FH), which leads to premature cardiovascular events, still remains underrecognized and undertreated in most countries. Untreated FH individuals aged 20–39 years are at 100-fold higher risk of mortality from coronary heart disease compared to those of a general population. Therefore, special efforts should be implemented to diagnose FH patients at early stages of life.The aim of this study was to evaluate the efficacy of the revised Dutch Lipid Clinic Network (DLCN) criteria proposed by the Polish Lipid Experts Forum to select index FH patients for DNA mutational analysis in Poland. MethodsThe study included 193 unrelated adult patients (mean age 48 ± 13 years) with clinical diagnosis of FH based on the revised DLCN score, tested sequentially for mutations in LDLR and APOB genes using bidirectional Sanger sequencing and MLPA techniques. The cut-off points of the clinical FH criteria score were assessed by ROC statistics to identify patients with the highest probability of carrying an FH-causing mutation. ResultsThe causal heterozygous LDLR or APOB mutation was identified in 41% (80/193) of probands. Adults aged <40 years were more likely to carry an FH-causing mutation compared to subjects aged ≥40 years (65% vs. 33%; p < 0.001). LDL-C thresholds for the molecular diagnosis of FH were 5.79 mmol/l for individuals aged<40 and 6.7 mmol/l for subjects ≥40 years old. The threshold values of the clinical diagnostic score for efficient selection of patients for genetic testing were 5 and 7 points for individuals aged <40 and ≥40 years, respectively. ConclusionsThe study validated the efficacy of proposed clinical FH criteria for the disease diagnosis in Poland. The clinical criteria score thresholds for positive FH molecular diagnosis differ depending on age (<40 and ≥40 years). We propose that in the healthcare systems with limited genetic testing resources individuals younger than 40 years, who fulfill the clinical criteria for possible, probable or definite FH should qualify for the FH mutation testing. The index patients aged ≥40 years with clinical diagnosis of probable or definite FH should also qualify for the genetic testing.

Aquaporin 1 protein expression is associated with BRAF V600 mutation and adverse prognosis in cutaneous melanoma.

Despite experimental findings suggesting the prognostic significance of Aquaporin 1 (AQP1) in human melanoma, no published clinical data are available. We studied the expression of AQP1 protein in cutaneous melanoma, correlated our findings with standard histological and genetic markers, and long-term clinical follow-up. Our study evaluated the AQP1 protein expression in 78 melanoma patients, representing two predefined risk cohorts using the immune labeling technique with commercially available anti-AQP1 antibodies on routinely formalin-fixed and paraffin-embedded tumor tissue samples. BRAF V600E mutation analyses were carried out successfully in 70 patients using PCR and restriction fragment length polymorphism analyses, followed by confirmatory analysis with the Sanger sequencing technique. AQP1-expressing melanoma cells were found in 52 cases (66.7%, median H-score=124.24). Significantly higher AQP1 H-scores (P=0.047) were found in the 'high-risk' patients. No correlations were found with the established histological markers, such as mitotic index (P=0.42), Clark level (P=0.95), and Breslow thickness (P=0.51). BRAF V600 mutation analyses were successful in 89%, and showed a two times higher mutation frequency in the 'high-risk' group. The BRAF V600 mutations were significantly associated with AQP1 expression (P=0.014). Long-term follow-up indicated a reduced progression-free survival (P=0.036) and overall survival (P=0.017) for the AQP1-positive cutaneous melanoma patients. AQP1 expression is likely to be associated with an adverse prognosis in cutaneous melanoma.

Improving the identification of mody mutations by using mlpa technique in the molecular diagnostics routine

Background Maturity-onset diabetes of the young (MODY) represents about 3-5% of cases of diabetes mellitus (DM). Searching for mutations can be performed either by Sanger sequencing or Multiplex Ligation-dependent Probe Amplification (MLPA) technique. MLPA is a powerful molecular tool that identifies large genetic rearrangements such as deletions and insertions, even though these kinds of mutations seem to be rare in the majority of MODY subtypes.

Impact of mutational status on survival in low-grade serous carcinoma of the ovary or peritoneum.

Background:Low-grade serous carcinoma of the ovary or peritoneum is a distinct, well- recognized histologic subtype characterized by young age at diagnosis, relative chemoresistance, and prolonged overall survival. Common mutations reported to be found within this subtype include KRAS and BRAF.Methods:Using clinical information of patients from our IRB-approved registry and tissue from a subset of these patients, we performed mutational analysis for KRAS and BRAF using the direct Sanger sequencing technique and correlated findings with the clinical outcome, overall survival (OS).Results:In 79 cases, patients with KRAS or BRAF mutations (n=21) had a significantly better OS than those with wild-type KRAS or BRAF (n=58) (106.7 months (95% CI, 50.6, 162.9) vs 66.8 months (95% CI, 43.6, 90.0)), respectively (P=0.018).Conclusions:Mutational status appears to be a potential prognostic factor in low-grade serous carcinoma of the ovary or peritoneum.