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Assessment of cardiovascular risk using established risk scores such as ESC SCORE2 or PROCAM insufficiently emphasise the role of genetic factors. We hypothesise that commercially available genetic assays may provide additional information on hereditary cardiovascular risk in a timely and cost-efficient manner.In a cohort of 51 patients treated for coronary artery disease (CAD) at University Hospital Heidelberg, Germany, a subgroup of patients with "unstable" CAD (i.e. recurrent acute coronary syndrome) was identified and compared to patients with "stable" disease (i.e. chronic coronary syndrome). Gene array analysis using a commercial assay for 15 potentially pathogenic polymorphisms revealed our cohort's genetic risk profile regarding atherosclerotic/thromboembolic events. Improvement of cardiovascular risk assessment based on established risk scores was analysed using net reclassification, logistic regression and receiver operating characteristic (ROC) analysis.Discriminatory capacity of traditional risk scores such as SCORE2 or PROCAM with regard to stable and unstable CAD groups was poor (ROC AUC0.5). Patients with "unstable" CAD exhibited a significantly increased frequency of pathogenic eNOS 894 T and MTHFR 1298 C polymorphisms compared to "stable" CAD patients, and information on these polymorphisms individually as well as combinations with additional polymorphisms included in the assay such as ACE D/D or PAI-1 5 G variants markedly improved risk prediction compared to SCORE2/PROCAM alone (ROC AUC ≥0.75).Commercially available assays for genetic polymorphisms may provide valuable information on individual genetic cardiovascular risk, potentially guiding future primary and/or secondary preventative therapies for coronary artery disease.

Objectives FMF shows considerable variability in severity and type of clinical manifestations by geographic region, which are attributed to Mediterranean fever (MEFV) gene allelic heterogeneity, additional genetic modifiers and environmental factors. Considering the severe impact of MEFV mutation M694V on the FMF phenotype, this work aimed at investigating a possible disease modifying role of the serum amyloid A1 (SAA1) genotype in a cohort of 386 Armenian FMF patients homozygous for MEFV mutation M694V. Methods A cohort of 386 Armenian patients diagnosed with FMF based on the Tel-Hashomer criteria and carrying two MEFV M694V mutant alleles were included in this study. Fifty-two (13.40%) of these patients experienced their first attack at the age of ≥20 years (i.e. adult-onset FMF). MEFV and SAA1 analyses were performed by a commercial reverse-hybridization assay, and resulting genotypes were matched against the patients’ clinicodemographic profiles. Results Genotypic distribution of SAA1 alleles was significantly different between patients with an age of onset Conclusion We have identified a significant relationship between the SAA1β/β genotype and the age of disease onset in M694V homozygous FMF patients.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics) for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7%) patients; 31 (50%) patients were homozygotes, 9 (14.5%) were heterozygotes, and 2 (3.2%) were compound heterozygotes with 3 mutations, while 20 (32.3%) patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4%) patients, followed by I2Splice and Q318X in 8 (12.9%) patients each, I172N in 5 (8.1%) patients, and V281L in 4 (6.5%) patients. Del 8 bp, P453S, and R483P were each found in one (1.6%) and complex alleles were found in 2 (3.2%). Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W) were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries.

Familial Mediterranean fever (FMF) is an autosomal-recessive, inflammatory disorder characterised by short, recurrent attacks of fever, accompanied by pain in the abdomen, chest, or joints and complications of amyloidosis. Recently, we observed a significant association between the serum amyloid A1 (SAA1) β/β genotype and a delayed disease onset in 386 M694V homozygous FMF patients. This follow-up study was conducted to additionally analyse MEFV genotypes other than M694V/M694V for a possible influence of the SAA1 genotype on the age of disease onset.A total of 700 Armenian patients diagnosed with FMF based on the Tel-Hashomer criteria and carrying two MEFV mutant alleles were included in this study. Patients were divided into three MEFV genotypic subgroups: M694V homozygotes (M694V/M694V), M694V compound heterozygotes (M694V/Other), and patients with genotypes excluding M694V (Other/Other). MEFV and SAA1 analyses were performed by a commercial reverse-hybridisation assay, and resulting genotypes were matched against the demographic and clinical characteristics of the patients.Within the subgroup of M694/M694 homozygotes, SAA1 genotype β/β could be identified in 115 (34.43%) and 32 (61.54%) patients with an age of onset20 and ≥20 years, respectively(p0.001). However, no such relationship could be observed for MEFV genotypic subgroups M694V/Other (p=0.465) and Other/Other (p=0.697).Our data suggest, that the influence of SAA1 genotypic variation on the age of disease onset restricts to FMF patients homozygous for MEFV mutation M694V.

Background Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. Methods The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. Results In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. Conclusions These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

This work aimed at investigating demographic, clinical, and genetic characteristics of individuals experiencing their first familial Mediterranean fever (FMF) attack at age ≥40 years in a very large cohort of Armenian FMF patients. In total, 10,370 Armenian patients diagnosed with FMF based on the Tel Hashomer criteria and carrying at least one MEFV mutant allele were included in this study. A total of 354 (3.40%) patients had late-onset FMF. Of these, 194 (54.80%) were female and 160 (45.20%) were male. The following genotypes were significantly associated with the late-onset variant: M680I/E148Q (P = 0.004), M694V/E148Q (P

INTRODUCTION Fetal hemoglobin (HbF) is the major modifier for sickle cell disease (SCD) severity. HbF is modulated mainly by three major quantitative trait loci (QTL) on chromosomes 2, 6, and 11. METHODS Five SNPs in the three QTLs (HBG2, rs7482144; BCL11A, rs1427407 and rs10189857; and HBS1L-MYB intergenic region, rs28384513 and rs9399137) were investigated by multiplex PCR and reverse hybridization, and their roles in HbF and clinical phenotype variability in Iraqi Kurds with SCD were assessed. RESULTS HBG2 rs7482144 with minor allele frequency (MAF) of 0.133 was the most significant contributor to HbF variability, contributing 18.1%, followed by rs1427407 (MAF of 0.266) and rs9399137 (MAF of 0.137) at 14.3% and 8.8%, respectively. The other two SNPs were not significant contributors. Furthermore, when the cumulative numbers of minor alleles in the three contributing SNPs were assessed, HbF% and hemoglobin concentration increased with increasing number of minor alleles (P

In patients with non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutations have been associated with the tumor response to targeted therapy with EGFR tyrosine kinase inhibitors. Although labor intensive and not very sensitive (ie, an analytical sensitivity of 20%), direct sequencing is widely used for mutation detection. This study aimed at evaluating the potential of a test strip-based reverse-hybridization assay (EGFR StripAssay), designed for the simultaneous detection of 16 mutations in exons 18 to 21 of the EGFR gene, to sensitively identify EGFR mutation in DNA from NSCLC tissue samples. Formalin-fixed paraffin-embedded (FFPE) DNA samples from 59 patients with a histologically confirmed primary NSCLC tumor were used to compare the performance of the EGFR StripAssay against that of the Sanger sequencing. The EGFR StripAssay analysis identified 7 (11.8%) of 59 FFPE samples to carry an EGFR mutation, of which 4 (57.1%) and 3 (42.8%) samples were positive for exon 19 and 21 mutations, respectively. Of note, no sample was identified with EGFR exon 18 or 20 mutation. All mutations were confirmed by DNA sequencing. Using 50 ng of template DNA, the EGFR StripAssay demonstrated a detection limit of 1% mutant sequence in a background of normal DNA. The EGFR StripAssay is a fast and robust platform for the sensitive detection of EGFR mutation in FFPE DNA. Therefore, this assay could be considered as an alternative protocol to Sanger sequencing for EGFR mutation testing on limited-quantity samples.

Background This study aimed to evaluate the diagnostic performance of a novel nonquantitative methylation-specific reverse hybridization (MSRH) assay to detect secreted frizzled-related protein 2 ( SFRP2) promotor methylation in fecal DNA. Methods SFRP2 promoter methylation was investigated in stool DNA isolated from 18 colorectal cancer (CRC) patients and 22 healthy controls using the MSRH assay based on methylation-specific DNA amplification followed by reverse hybridization of biotinylated amplicons to sequence-specific methylation detection probes, with MethyLight serving as a reference method. Results SFRP2 promotor methylation as determined by MSRH vs. MethyLight showed a sensitivity and specificity of 61.1% and 86.3% vs. 77.7% and 77.3%, respectively. Moderate agreement (κ = 0.54, 95% confidence interval [95% CI], 0.29-0.80, pConclusions Our findings, although limited by the small sample size, do not support the use of the MSRH assay for CRC screening in stool.

Concordance between mutations in the primary papillary thyroid carcinoma (PTC) and the paired x lymph node metastasis may elucidate the potential role of molecular targeted therapy in advanced stages. BRAF and NRAS mutations in primary PTC (n = 253) with corresponding metastatic lymph node (n = 46) were analyzed utilizing StripAssays (ViennaLab Diagnostics). Statistical analysis was performed using (SPSS, Inc.), version 24.0 with a p-value of BRAF mutation frequency in conventional PTC (cPTC): 56.8%, papillary thyroid microcarcinoma (PTMC): 36.5%, PTMC-FV: 2.7% and PTC-FV: 4.1%. NRAS mutation frequency in PTC-FV: 28.6%, PTMC: 28.6%, PTMC-FV: 23.8%, and cPTC: 19.0%. BRAF mutation correlation with older age in cPTC (42.6 versus 33.6) years (p BRAF mutations were concordant in the primary and its corresponding lymph node deposits in PTC with a kappa of 0.77 (p-value BRAF mutations are predominant in cPTC and PTMC while NRAS mutations in PTC-FV. BRAF mutation is conserved in metastatic lymph node deposits, thus BRAF is an early mutational pathogenetic driver. Therefore, targeted therapy is potential in recurrent and advanced stage disease.

Background: Despite a high prevalence of anemia among nonpregnant Cambodian women, current reports suggest that iron deficiency (ID) prevalence is low. If true, iron supplementation will not be an effective anemia reduction strategy.Objective: We measured the effect of daily oral iron with or without multiple micronutrients (MMNs) on hemoglobin concentration in nonpregnant Cambodian women screened as anemic.Design: In this 2 × 2 factorial, double-blind, randomized trial, nonpregnant women (aged 18-45 y) with hemoglobin concentrations ≤117 g/L (capillary blood) were recruited from 26 villages in Kampong Chhnang province and randomly assigned to receive 12 wk of iron (60 mg; Fe group), MMNs (14 other micronutrients; MMN group), iron plus MMNs (Fe+MMN group), or placebo capsules. A 2 × 2 factorial intention-to-treat analysis with the use of a generalized mixed-effects model was used to assess the effects of iron and MMNs and the interaction between these factors. Results: In July 2015, 809 women were recruited and 760 (94%) completed the trial. Baseline anemia prevalence was 58% (venous blood). Mean (95% CI) hemoglobin concentrations at 12 wk in the Fe, MMN, Fe+MMN, and placebo groups were 121 (120, 121), 116 (116, 117), 123 (122, 123), and 116 (116, 117) g/L, with no iron × MMN interaction (P = 0.66). Mean (95% CI) increases in hemoglobin were 5.6 g/L (3.8, 7.4 g/L) (P < 0.001) among women who received iron (n = 407) and 1.2 g/L (-0.6, 3.0 g/L) (P = 0.18) among women who received MMNs (n = 407). The predicted proportions (95% CIs) of women with a hemoglobin response (≥10 g/L at 12 wk) were 19% (14%, 24%), 9% (5%, 12%), 30% (24%, 35%), and 5% (2%, 9%) in the Fe, MMN, Fe+MMN, and placebo groups, respectively.Conclusions: Daily iron supplementation for 12 wk increased hemoglobin in nonpregnant Cambodian women; however, MMNs did not confer additional significant benefit. Overall, ∼24% of women who received iron responded after 12 wk; even fewer would be likely to respond in the wider population. This trial was registered at clinicaltrials.gov as NCT02481375.

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disorder caused by mutations in the MEFV gene. The disease is especially common among Armenian, Turkish, Jewish and Middle East Arab populations. To identify the frequency and the spectrum of common MEFV mutations in different Iranian populations, we investigated a cohort of 208 unselected asymptomatic individuals and 743 FMF patients. Nine hundred and fifty-one samples were analysed for the presence of 12 MEFV mutations by PCR and reverse-hybridization (FMF StripAssay, ViennaLab, Vienna, Austria). Confirmatory dideoxy sequencing of all MEFV gene exons was performed for 39 patients. Fifty-seven (27.4%) healthy individual carried mutant MEFV alleles. Three hundred and ninety-one (52.6%) FMF patients were found positive for either one (172/743; 23.1%), two or three MEFV mutations. Using dideoxy sequencing, three novel variants, A66P, R202W and H300Q, could be identified. Our analysis revealed an allele frequency and carrier rate of 15.6 and 27.4%, respectively, among healthy Iranians. Still moderate compared to neighbouring Armenia, but higher than in Turkey or Iraq, these data suggest that FMF is remarkably common among Iranian populations. E148Q was most frequent in the group of healthy individuals, whereas M694V was the most common mutation among FMF patients, thereby corroborating previous studies on MEFV mutational spectra in the Middle East. Accordingly, MEFV mutations are frequent in healthy Iranian individuals across different ethnic groups. Based on this finding, the awareness for FMF and the implementation of augmented carrier screening programmes considering the multiethnic nature of the Iranian population should be promoted.

Statin-induced myopathy is reported to be significantly associated with the SCLO1B1 c.521T>C polymorphism. To date, SLCO1B1 c.521T>C epidemiologic data for the Austrian population is still lacking. Therefore, this study aimed at assessing the genotype and allele frequencies of the SLCO1B1 c.521T>C variant in Austria and evaluating the clinical performance of 2 commercial real-time polymerase chain reaction (PCR) assays. Genomic DNA isolated from 181 healthy individuals was analyzed for the SLCO1B1 c.521T>C polymorphism in a comparative manner using the SLCO1B1 c.521T>C RealFastTM Assay and the BioPro SLCO1B1 Genotyping real-time PCR Kit. A total of 10 (5.5%) and 44 (24.3%) out of 181 individuals were SLCO1B1 c.521T>C C/C-homo- and ­C/T-heterozygotes, the genotypes indicative of high and increased risk of statin-induced myopathy, respectively. The SLCO1B1 c.521C allele frequency rate was 17.7%. In conclusion, the genetic predisposition of elevated statin-induced myopathy risk in the Austrian population is frequent. Both real-time PCR assays under investigation here are reliable and robust SLCO1B1 c.521T>C genotyping tools in clinical routine.

Background Selective BRAF inhibitors have shown dramatic results with regard to improving outcome in patients with melanoma. Testing the BRAF status in matched primary and metastatic melanomas to optimize individual targeted therapy is not well investigated. Methods Extended BRAF testing using PCR for 9 mutations and VE1 immunohistochemistry for BRAF V600E detection on 95 lesions including 40 primary melanomas with their matched metastases (n = 42), recurrences (n = 9) and second primaries (n = 4) was performed. Nine patients had multiple metastases. Results V600E was the only identified mutation type; 35.4% of primary vs. 18.9% of metastatic melanomas. The overall primary-metastatic BRAF status discordance rate was 32.3% using PCR and 27.5% with immunohistochemistry, and was significantly more frequent in primary lesions with mutant BRAF (67%). Males and patients with metastasis to lymph nodes were less likely to be discordant compared to females and those with metastasis to other sites (p = 0.023). Discordant BRAF mutation status was predicted by multivariate binary logistic regression: the presence of a mutant BRAF in the primary melanoma [OR (95% C.I.) = 23.4 (2.4–229.7)] and female gender [OR = 10.6 (1.08–95)]. Inter-metastases BRAF concordance was 100% (6 comparisons). Conclusion A high discordant rate implies the need for clinical trials addressing the response to targeted therapy in patients with discordant BRAF statuses between their primary and metastatic lesions.

Background: We have already described a significantly elevated overall risk for recurrent pregnancy loss (RPL) in women carrying the coagulation factor XIII (FXIII) Val34Leu and/or the plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism assuming that these polymorphisms contribute synergistically to RPL because of impaired hypofibrinolysis. Recent studies on FXIII indicate that the impact of the FXIII 34Leu genotype on fibrin structure and fibrinolysis is affected by fibrinogen concentration. Therefore, we reinvestigated the association between fibrinogen concentrations and FXIII Val34Leu with early RPL. Materials and Methods: In this case-control study, we enrolled 49 women with a history of two consecutive or three to six nonconsecutive pregnancy losses between the 8th and 12th week of gestation and 48 healthy controls. The risk for RPL in carriers of FXIII 34Leu at fibrinogen levels above or below the median and first tertile of controls was evaluated. Results: In carriers of the 34Leu allele, fibrinogen levels below the median (i.e., ≤300 mg/dl) and the first tertile (i.e., ≤284 mg/dl) of controls were associated with an increased risk for RPL [(2.9 (1.1-7.7), 3.9(1.0-15.0)]. Conclusions: The FXIII Val34Leu polymorphism may be associated with the development of early RPL in association with fibrinogen concentrations. At fibrinogen levels in the low normal range, FXIII 34Leu may modify fibrin structure toward an increased resistance to fibrinolysis. © 2013 Wiley Periodicals, Inc.

BRAF mutations have been implicated in initiating promutagenic cellular melanocytic proliferation mostly based on homogeneous Western-based cohorts. Data addressing the possible interaction between exposure to different solar ultraviolet radiation (UVR) magnitudes and BRAF mutation rate (BMR) in melanocytic nevi are limited.Extended BRAF testing for 9 mutations in 225 melanocytic nevus (MN) cases derived from 211 patients from 4 different UVR regions: Lebanon (n = 95; 110 kJ · m(-2) · yr), Syria (n = 23; 93.5 kJ · m(-2) · yr), Kingdom of Saudi Arabia (n = 70; 139 kJ · m(-2) · yr), and Pakistan (n = 37; 118 kJ · m(-2) · yr) was performed. Data collected included age, gender, anatomic location, and lesion size. Histological parameters recorded were MN type (junctional, compound, intradermal, classical blue, cellular blue, compound and intradermal spitz, and congenital) solar elastosis grade, and nevus pigmentation degree. Cumulative 21-year erythemally effective UV averages were derived from The National Center for Atmospheric Research.BRAF mutation status was obtained in 210 cases (6.7% failed polymerase chain reaction). Overall, BMR was 62.4% (131/210) with V600E mutation accounting for 98.5% of cases. Discordant mutation status was found in 2 of 10 patients with multiple nevi. BMR differed significantly, yet nonsystematically, among UVR regions; the highest was detected in nevi coming from Syria (18/23 cases, 78%), followed by Pakistan (21/30 cases, 70%), Kingdom of Saudi Arabia (47/70 cases, 67%), and Lebanon (45/87 cases, 52%). Mutation rates varied significantly across MN type (P0.001); the highest rate was recorded in the intradermal nevus type (33/39 cases, 84.6%), followed by the compound (26/32 cases, 81.2%) and congenital (60/74 cases 81.0%) nevi. Stratified by anatomic location, nevi occurring on the face (61/82, 74%) and trunk (58/78, 74%) had more frequent BMRs compared with those occurring on the upper (7/26, 27%) and lower extremities (5/24, 21%, P0.001). Severe pigmentation was less frequent in BRAF mutation-positive nevi [5/131 (4%) vs. 34/79 (43%); P0.001]. Multivariate independent predictors of BRAF mutation in MN were age [odds ratio (95% confidence interval ) = 1.43 (1.13-1.74) per 10 years; P = 0.004], anatomic location [P = 0.043 overall], and nevus type [P0.001 overall]. UVR region was not an independent predictor of BRAF mutation.Increased BRAF mutation with age along with the lack of a UVR magnitude-BRAF mutation association suggests that duration of exposure rather than UVR exposure dose is the more likely link to acquiring the mutation.

Background Proto-oncogene B-Raf (BRAF) mutation rates have been reported in nevi and melanomas of homogeneous Caucasian cohorts. Objective To study the demographics of BRAF mutations in dysplastic nevi of populations with differing potential solar UV radiation exposure. Methods Extended BRAF testing for 9 mutations in 125 dysplastic nevi from 101 patients, derived from populations with differing potential UV radiation exposure rates (Lebanon and Saudi Arabia), was performed. Clinical and microscopic parameters were recorded. Results BRAF mutation status was carried out for 101/125 (80.8%) cases with an overall mutation rate of 62.4% (63/101). V600E (c.1799T > A) was the predominant mutation, found in 61/63 (96.8%) cases. BRAF mutation rate differed significantly by potential UV radiation exposure (Lebanon: 53.4%, Saudi Arabia: 74.4%, P

We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAFStripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of theKRASgene andBRAFmutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), theKRAS-BRAFStripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect toBRAFV600E, theKRAS-BRAFStripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry theBRAFV600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates theKRAS-BRAFStripAssay to be a robust and sensitive method for the detection of commonKRAS/BRAFmutations in genomic DNA isolated from FFPE tissue samples.

We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAF StripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of the KRAS gene and BRAF mutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), the KRAS-BRAF StripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect to BRAF V600E, the KRAS-BRAF StripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry the BRAF V600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates the KRAS-BRAF StripAssay to be a robust and sensitive method for the detection of common KRAS/BRAF mutations in genomic DNA isolated from FFPE tissue samples.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics) for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7%) patients; 31 (50%) patients were homozygotes, 9 (14.5%) were heterozygotes, and 2 (3.2%) were compound heterozygotes with 3 mutations, while 20 (32.3%) patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4%) patients, followed by I2Splice and Q318X in 8 (12.9%) patients each, I172N in 5 (8.1%) patients, and V281L in 4 (6.5%) patients. Del 8 bp, P453S, and R483P were each found in one (1.6%) and complex alleles were found in 2 (3.2%). Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W) were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries.

Thalassemia is the most common genetic disorder in Iran. Some α-globin genotypes leading to Hb H disease may cause severe anemia and dependence on regular blood transfusions. In this study, 40 patients were analyzed for the molecular basis and the genotype-phenotype correlation of Hb H disease in Iran. α-Globin molecular analysis was performed by polymerase chain reaction (PCR) followed by agarose gel electrophoresis, reverse hybridization test strips or DNA sequencing. The most frequently observed α-globin genotypes were -α(3.7)/- -(MED) in 10 patients (25%), - -(20.5)/α(-5nt)α in six patients (15%) and - -(20.5)/-α(3.7) in four patients (10%). A subset of the identified Hb H genotypes, including - -(MED)/α(CS)α, - -(MED)/α(PolyA2)α and α(CS)α/α(CS)α, was associated with a need for regular or irregular blood transfusions. Our findings provide a basis for predicting phenotype severity by identifying the Hb H genotype and making more selective decisions for prenatal diagnosis.

Background: Atherosclerosis and thrombosis are the major manifestations underlying cardiovascular diseases (CVD), which are the leading cause of mortality and morbidity worldwide. Both result from an interaction between genetic and environmental risk factors. The goal of our study was to evaluate several polymorphisms identified as predisposing factors to atherosclerosis and thrombosis. Material and Methods: A series of 155 healthy unrelated individuals of Azorean origin were analyzed using the CVD StripAssay (ViennaLab Diagnostics, Austria) for the most established polymorphisms involved in blood coagulation (F2, F5, F13A1, FGB), fibrinolitic system (SERPINE1), platelet adhesion (ITGB3), homocysteine metabolism (MTHFR), reninangio-tensin system (ACE) and lipid metabolism (APOE). Results: No significant differences were observed in allelic frequencies when comparing our data to mainland Portugal. Group stratification according to the number of “increased” risk alleles, demonstrated that 116/155 (75%) individuals belong to the moderate risk group (5 - 10 risk alleles). Conclusions: Although acknowledging the fact that the allelic states at the analysed loci lack predictive value, the fact that a high frequency of individuals presents at least 5 risk alleles (124/155; 80%) is important for the establishment of the appropriate preventive measures in the Azorean population.

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

Purpose: To investigate the association between genetic cardiovascular risk factors and exudative age-related macular degeneration (AMD) in a White Austrian population. Methods: Seventy-five unrelated AMD patients and 75 unrelated healthy, sex- and age-matched control patients were genotyped for the following 19 single nucleotide polymorphisms (SNPs) in 14 different genes: blood coagulation factor V (FV) R506Q, factor II (prothrombin) G20210A and factor XIII (FXIII) V34L; 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; plasminogen activator inhibitor 1 (PAI-1) 4G/5G; endothelial protein C receptor (EPCR) 4600 A>G (A3 haplotype), 4678 G>C (A1 haplotype); apolipoprotein B (ApoB) R3500Q; apolipoprotein E (ApoE) E2/E3/E4; β-fibrinogen −455 G>A; human platelet antigen 1 (HPA1) a/b; angiotensin-converting enzyme (ACE) I/D; endothelial nitric oxide synthase (eNOS) 786 T>C, 894 G>T; lymphotoxin alpha (LTA) 804 C>A and 9p21 rs10757278. Genotyping was carried out by polymerase chain reaction (PCR) followed by reverse hybridization (CVD StripAssays; ViennaLab Diagnostics, Vienna, Austria). Results: No statistically significant association could be observed between AMD and the investigated genetic risk factors for cardiovascular disease (CVD). All factors seem to be uniformly distributed in the two groups of AMD patients and healthy controls. Two variables –β-fibrinogen: −455 G>A (p = 0.0786) and apolipoprotein E4 (p = 0.0636) – were not as far from association as the others. Conclusion: Our data show that the 19 tested CVD risk markers do not play a significant role in AMD. β-Fibrinogen and apolipoprotein E4 should be examined in a larger cohort.

Two hundred and fifty-five patients from Mazandaran Province, Iran, all presenting with hypochromic and microcytic anemia, were selected for alpha-thalassemia (alpha-thal) mutation screening. We detected a total of 274 alpha-globin mutations in 227 (89%) of these patients. Among the 21 different alpha-globin alleles found, the -alpha(3.7) (44.9%), polyadenylation signal 2 (poly A2) (AATAAA>AATGAA) (18.2%), -alpha(4.2) (9.1%), alpha(IVS-I(-5 nt)) (6.5%), - -(MED) (4.3%), and alpha(codon 19 (-G)) (4%) were the most frequent. The other 15 mutations included variants that had not yet been observed in Iran, such as Hb Bleuland [alpha108(G15)ThrAsn, ACC>AAC (alpha2)], as well as a novel mutation on the alpha2 gene, also not described to date [3 ' untranslated region (3 'UTR) nucleotide (nt) 46 (C>A)]. These comprehensive new data are useful for establishing a screening strategy for the effective control of alpha-thal in Mazandaran Province.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics) for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7%) patients; 31 (50%) patients were homozygotes, 9 (14.5%) were heterozygotes, and 2 (3.2%) were compound heterozygotes with 3 mutations, while 20 (32.3%) patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4%) patients, followed by I2Splice and Q318X in 8 (12.9%) patients each, I172N in 5 (8.1%) patients, and V281L in 4 (6.5%) patients. Del 8 bp, P453S, and R483P were each found in one (1.6%) and complex alleles were found in 2 (3.2%). Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W) were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries.

To determine the molecular basis of β-thalassemia intermedia (TI) and the contribution of the three hemoglobin F (HbF) quantitative trait loci (QTLs) on chromosomes 11, 2, and 6 to the milder phenotype, a total of 102 Iraqi Arab patients with TI were studied. The β and α genotypes as well as HBG2 g. 158 C>T (rs7482144), BCL11A (rs1427407 and rs10189857), and HBS1L-MYB (rs28384513 and rs9399137) by multiplex polymerase chain reaction and reverse hybridization were studied. A total of 21 different β-thalassemia mutations arranged in 35 different genotypes were identified. The genotypes encompassed β(+)/β(+) mutations in 33 cases, β(+)/β(0) in 17 cases, β(0)/β(0) in 47 cases, β(0)/wild type in 3 and β(0)/Hb E in 2 cases. The most common was IVS-II-1 (G>A)/IVS-II-1 (G>A), followed by IVS-I-6 (T>C)/IVS-I-6 (T>C) and IVS-I-110 (G>A)/IVS-I-110 (G>A), in 31.4%, 17.6%, and 6.9%, respectively. Alpha-thalassemia mutations were found in 15.2% of those homozygous for the β-mutations, while α gene triplication was identified in all three heterozygotes. Of the five QTLs tested, only rs7482144 and rs10189857 were significantly associated with β(0)/β(0) when compared to β(+)/β(+), with odds ratios of 6.4 (95% confidence interval [CI] 2.9-14.0) and 3.2 (95% CI 1.2-8.6), respectively. In conclusion, this study has demonstrated that among Iraqi patients with thal intermedia, the main contributors to the milder phenotype were β(+) alleles, XmnI polymorphism, and BCL11A (rs10189857), while other QTLs on chromosomes 2 and 6, as well as alpha-thalassemia, were not significantly relevant.

Familial Mediterranean fever (FMF) is an autosomal recessive inflammatory disorder predominantly affecting people living in or originating from areas around the Mediterranean Sea. It is caused by a number of mutations within the MEFV gene, which differently affect the severity of the disease phenotype. Because patients usually present with rather nonspecific clinical symptoms, MEFV genotyping can confirm and refine FMF diagnosis and improve treatment of affected individuals. We have performed a method comparison study on 100 Lebanese FMF patients to evaluate the potential of a rapid reverse-hybridization teststrip-based assay (FMF StripAssay) to serve as a first-line screening test for our population. When results obtained by reverse-hybridization and DNA sequencing of exons 2, 3, 5, and 10 were compared, the FMF StripAssay identified 144/149 mutations, and correctly typed all 12 different MEFV mutations covered. We conclude that reverse-hybridization provides a very rapid, accurate and easy-to-perform screening method, and, in combination with more comprehensive diagnostic methods, represents an efficient strategy for FMF genotyping.

A recently developed strip-assay for hemochromatosis provides a rapid method for simultaneous detection of multiple mutations, which among others includes the HFE gene mutations V53M, V59M, H63D, H63H, S65C, Q127H, E168Q, and C282Y, previously detected in the general South African population using gel-based mutation-screening methods. The objective of the study was to determine the frequency of the relatively rare mutations in samples selected for altered iron parameters or a family history of hereditary hemochromatosis (HH) as part of the validation process of the assay for routine diagnostic purposes. The study population consisted of 451 individuals previously screened for mutations C282Y and H63D by restriction enzyme analysis in order to confirm or possibly exclude a diagnosis of HH. These individuals were subjected to mutation screening using the commercially available hemochromatosis strip-assay. Previous positive results for mutations C282Y and H63D in 233 individuals confirmed the accuracy of the reverse-hybridization assay. Mutation S65C was detected in 13 Caucasians, including three compound heterozygotes. These constituted 2% (13/600) of the chromosomes without mutations C282Y or H63D. The African-specific HFE mutation V53M was detected in one out of 11 (9%) African subjects screened. Mutation E168Q was detected in a single Caucasian individual together with mutation H63D. Our data demonstrate the value of the strip-based technology in providing a rapid and reliable comprehensive test for simultaneous analysis of multiple mutations.

SummaryInherited and acquired thrombophilia are associated with recurrent pregnancy loss. Recently, an increased risk for thromboembolic disease was described for patients with elevated coagulation factor VIII, but it is unknown whether there is also an association to early pregnancy loss. We therefore evaluated the relation between recurrent early pregnancy loss and levels of coagulation factor VIII. We enrolled 49 unrelated Caucasian women with a history of 2 6 early pregnancy losses and 48 healthy controls, who had delivered at least one term infant and had never experienced pregnancy loss. We determined factor V Leiden-, G20210A prothrombin-, MTHFR C677Tand A1298Cgene mutations, levels of antithrombin, protein C, protein S, factor VIII, C-reactive protein and antiphospholipid antibodies. There was a significantly higher rate of pregnancy losses in women with Antiphospholipid Syndrome (p = 0.043). Furthermore, plasma levels of coagulation factor VIII were significantly higher in cases than in controls (130.5 IU/dl ± 25.4 vs 119.5 IU/dl ± 24.1; p = 0.032) and appeared independent of Creactive protein (R = 0.146, p = 0.323 in cases; R = 0.028, p = 0.850 in controls). The relative risk for recurrent pregnancy loss in women with factor VIII levels above 151 IU/dl (90th percentile of controls) was 2.5 (0.7 – 8.9, 95 percent confidence interval), for levels above 156 IU/dl (95th percentile of controls) 3.9 (0.8 – 20.0, 95 percent confidence interval). Elevated maternal plasma levels of coagulation factor VIII tend to be associated with an increased risk for recurrent early pregnancy loss.

DNA testing can provide a definitive diagnosis of familial hypercholesterolemia (FH), even in the absence of the clinical characteristics of this inherited cardiovascular disease (CVD) subtype. Our aim was to design a rapid diagnostic assay capable of simultaneously analyzing seven point mutations in the low-density lipoprotein receptor (LDLR) gene, which occur at high frequency in South African FH patients.The test is based on multiplex DNA amplification and hybridization to membrane strips presenting a parallel array of immobilized allele-specific oligonucleotide probes.A reverse-hybridization assay for genotyping LDLR point mutations D154N, D200G, D206E, C356Y, G361V, V408M, and P664L was set-up and validated using pretyped human DNA samples, as well as recombinant plasmid clones containing mutant alleles. The procedure is rapid (6 hours) and may be automated to a large extent.The new FH strip-assay forms an important part of the comprehensive cardiovascular genetic screen offered routinely to high-risk population groups in South Africa. A genetic approach based on FH testing in conjunction with other 'genetic' CVD risk factors is feasible and justified, since the spectrum of disease-related mutations have been defined to a large extent in the genetically distinct population groups of South Africa. Knowledge of a significantly increased CVD risk due to the presence of gene variations, which can be targeted for risk reduction by the avoidance of relevant environmental risk factors and the appropriate treatment, provides a powerful message to motivate people into implementing preventative measures based on their genetic profile.

Background: A successful outcome of pregnancy depends on proper placental formation. In the very beginning of this process, trophoblast invasion and fibrin deposition into the wall of the decidual veins play an important part. Two polymorphisms, coagulation factor XIII (FXIII) Val34Leu and plasminogen activator inhibitor 1 (PAI-1) 4G/5G, interfere with fibrin cross-linking and regulation of fibrinolysis and may therefore contribute to early pregnancy loss.Methods: We enrolled 49 unrelated Caucasian women with a history of two consecutive or three to six nonconsecutive early pregnancy losses and 48 unrelated parous healthy controls without a history of pregnancy loss and evaluated them for the following genetic variants: the factor V Leiden and prothrombin G20210A gene mutations, the methylenetetrahydrofolate reductase C677T and A1298C polymorphisms, and the PAI-1 4G/5G and FXIII Val34Leu polymorphisms.Results: For the isolated occurrence of PAI-1 4G/5G or FXIII Val34Leu, we found no statistically significant difference between cases and controls. For homozygosity of either or compound carrier status of both mutations, the overall relative risk for early pregnancy loss was significantly increased (odds ratio = 2.4; 95% confidence interval, 1.1–5.5; P = 0.032). We observed no statistically relevant association of any of the other tested mutations with early pregnancy loss.Conclusion: Homozygosity for PAI-1 4G or FXIII 34Leu polymorphisms as well as compound carrier status is associated with early pregnancy loss.

Neuroendocrine differentiation of tumor tissue has been recognized as an important prerequisite for new targeted therapies. To evaluate the suitability of colorectal cancer (CRC) tissue for these treatment approaches and to find a possible link to pretherapeutic conditions of other targeted strategies, we compared neuroendocrine differentiation and KRAS/NRAS/BRAF/PIK3CA/TP53 mutational status in primary and metastatic CRC. Immunohistochemical expression analysis of neuroendocrine markers chromogranin A and synaptophysin was performed on archival CRC tissue, comprising 116 primary tumors, 258 lymph node metastases and 72 distant metastases from 115 patients. All CRC samples but 30 distant metastases were subjected to mutation analysis of KRAS, NRAS, BRAF, PIK3CA, and TP53. Neuroendocrine marker expression was found significantly less frequently in lymph node metastases compared to primary tumors and distant metastases (20%, 31%, 28%, respectively, P = 0.044). KRAS mutation rates increased significantly from primary tumors to lymph node metastases and distant metastases within the neuroendocrine negative CRC group (44%, 53%, 64%, respectively, P = 0.042). Neuroendocrine differentiation was significantly less concordant than KRAS/NRAS/BRAF/PIK3CA/TP53 mutational status in primary tumor/lymph node metastases pairs (65% versus 88%-99%; P < 0.0001) and primary tumor/distant metastases pairs (64% versus 83%-100%; P = 0.027 and P < 0.0001, respectively). According to these data, therapeutic targeting of neuroendocrine tumor cells can be considered only for a subset of CRC patients and biopsies from the metastatic site should be used to guide therapy. A possible importance of lacking neuroendocrine differentiation for progression of KRAS mutant CRC should be further investigated.

Our study aimed to determine the number of couples with normal hemoglobin (Hb) electrophoresis and low-borderline hematological values, which may come up with a clinically critical status in their offspring. The number of couples at risk for severe α-thalassemia (α-thal) needed to be estimated before recommending genetic counseling and prenatal diagnosis (PND). During the past 14 years, from at least 7000 referrals, 754 couples were investigated for α-thal by direct mutation detection methods followed by reverse strip assay and α-globin gene sequencing for inconclusive cases. Detection of silent β-thalassemia (β-thal) mutations was done in suspected cases by complete β-globin gene sequencing. We were able to provide a molecular diagnosis in 87.3% (658/754) of couples. A total of 9.1% (60/658) may have a clinically significant hemoglobinopathy in their offspring. Significant conditions included hydrops fetalis (20.0%; 12/60), certain Hb H (β4) genotypes (78.3%; 47/60) and β-thal intermedia (β-TI) (1.7%; 1/60). The diagnostic flowchart for couples with microcytic hypochromic anemia in countries with a high prevalence of hemoglobinopathies should include α and β gene sequencing. As our results indicate, every nine out of 100 of these couples will face significant hemoglobinopathies and every two out of 100 can carry Hb Bart's (γ4) hydrops fetalis in their pregnancies. For such cases, PND should be utilized to allow the carrier couples to decide whether or not to abort the fetus.

Hereditary hemochromatosis (HH) is a very common autosomal recessive disorder of iron metabolism and frequently associated with mutations in the HFE gene. Molecular genetic testing for HFE mutations is considered valuable for carrier identification, as well as for early diagnosis of the disease, allowing simple treatment by phlebotomy and normal survival of patients. We have developed a reverse-hybridization assay for the routine diagnosis of eight previously described and one novel (E168Q) HFE point mutations. The test is based on multiplex DNA amplification and ready-to-use membrane teststrips, which contain oligonucleotide probes for each wild-type and mutated allele immobilized as an array of parallel lines. The procedure is rapid and accessible to automation on commercially available equipment, and by adding new probes the teststrip can easily be adapted to cover an increasing number of mutations.

Objectives We evaluated whether the endothelial protein C receptor (EPCR) haplotypes A1 and A3 exert effects on the development of recurrent pregnancy loss (RPL) in association with factor V Leiden. Design and methods We determined the EPCR haplotypes A1 and A3 and factor V Leiden in 49 women with a history of RPL and 48 parous controls. Results In carriers of factor V Leiden the A1 haplotype decreased the relative risk for RPL from 2.2 to 1.0. Conclusions The EPCR A1 haplotype tends to modulate the risk for RPL in carriers of factor V Leiden.

BRAF mutation has been linked to the development of melanocytic tumors in homogeneous Caucasian cohorts. The role of solar UV radiation (UVR) in BRAF mutation status is poorly understood. We studied the epidemiology of BRAF mutation across a spectrum of melanocytic neoplasms in populations with differing UVR rates. Extended testing for 9 mutation types was attempted on 600 melanocytic neoplasms including banal nevi (n = 225), dys- plastic nevi (n = 113), primary (n = 172), and metastatic melano- mas (n = 90). Specimens were collected from 4 countries with increasing UVR rates (in kJ/m 2 /yr): Syria (n = 45; UVR = 93.5), Lebanon (n = 225; UVR = 110), Pakistan (n = 122; UVR = 128), and Saudi Arabia (n = 208; UVR = 139). UVR was estimated from 21-year averages from The National Center for Atmospheric Research database. The overall BRAF mutation rate was 49% (268 of 545) and differed significantly by the geographic location (34% Pakistan, 49% Lebanon, 67% Syria, and 54% Saudi Arabia; P = 0.001), neoplasm type (P , 0.001), and anatomical location (P , 0.001) but not with age (P = 0.07) and gender (P = 1.0). V600E was the predominant mutation type, found in 96.3% of the cases. Incidence of melanoma was significantly greater in BRAF- negative (39%) versus BRAF-positive (17%) groups. For BRAF- positive cases, less severe lesions were systematically more frequent (P , 0.001). Multivariate analysis indicated that BRAF mutation is predicted by neoplasm type, anatomical site, and geographic location. In our Near East cohort, BRAF mutation rates varied by geographic location but not based on UVR. BRAF-positive status was associated with less severe lesions.

BACKGROUND: We have already described a significantly elevated overall risk for recurrent pregnancy loss (RPL) in women carrying the coagulation factor XIII (FXIII) Val34Leu and/or the plasminogen activator inhibitor‐1 (PAI‐1) 4G/5G polymorphism assuming that these polymorphisms contribute synergistically to RPL because of impaired hypofibrinolysis. Recent studies on FXIII indicate that the impact of the FXIII 34Leu genotype on fibrin structure and fibrinolysis is affected by fibrinogen concentration. Therefore, we reinvestigated the association between fibrinogen concentrations and FXIII Val34Leu with early RPL. MATERIALS AND METHODS: In this case‐control study, we enrolled 49 women with a history of two consecutive or three to six nonconsecutive pregnancy losses between the 8th and 12th week of gestation and 48 healthy controls. The risk for RPL in carriers of FXIII 34Leu at fibrinogen levels above or below the median and first tertile of controls was evaluated. RESULTS: In carriers of the 34Leu allele, fibrinogen levels below the median (i.e., ≤300 mg/dl) and the first tertile (i.e., ≤284 mg/dl) of controls were associated with an increased risk for RPL [(2.9 (1.1–7.7), 3.9(1.0–15.0)]. CONCLUSIONS: The FXIII Val34Leu polymorphism may be associated with the development of early RPL in association with fibrinogen concentrations. At fibrinogen levels in the low normal range, FXIII 34Leu may modify fibrin structure toward an increased resistance to fibrinolysis.

Familial Mediterranean fever (FMF) is an autosomal-recessive, inflammatory disorder characterized by short, recurrent attacks of fever, accompanied by pain in the abdomen, chest, or joints and erysipelas-like erythema. Its most severe complication is progressive amyloidosis, leading to end-stage renal failure. FMF predominantly affects Turks, Arabs, Armenians, and Sephardic Jews, with carrier rates reported as high as 1 in 5, but it has been observed in lower frequencies throughout the Mediterranean area (1). It is caused by several mutations within the marenostrin/pyrin-encoding gene MEFV on chromosome 16p13.3, which differently affect the severity of the disease phenotype and the risk of developing renal amyloidosis (2)(3)(4). Although established clinical criteria for FMF exist (5), many patients remain undiagnosed because of rather nonspecific symptoms; therefore, molecular genetic analysis could substantially improve early and correct diagnosis of FMF and allow initiation of lifelong prophylactic treatment of affected individuals with colchicine (6). Aiming at a simple but powerful first-line screening tool for FMF genotyping, we have set up a reverse-hybridization, teststrip-based assay (FMF StripAssay) for the simultaneous detection of 12 MEFV mutations: E148Q in exon 2, P369S in exon 3, F479L in exon 5, and M680I (G/C), M680I (G/A), I692del (2076–2078), M694V, M694I, K695R, V726A, A744S, and R761H in exon 10. For this purpose, we collected DNA samples from individuals who had previously been typed positive for one of these mutations and used them to generate recombinant plasmid clones for the 12 mutant alleles (TOPO TA Cloning Kit; Invitrogen). After confirming the presence of mutations by DNA sequencing, we used these plasmid clones as homozygous reference samples to determine suitable reverse-hybridization probes. We synthesized a series of candidate 15- to 25mer oligonucleotides, selected from the FMF databank sequence (GenBank accession no. AF111163) and encoding all wild-type or mutant alleles, and immobilized …

Background Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder most commonly caused by defects in the CYP21A2 gene. Neonatal CAH-screening based on 17-hydroxyprogesterone (17-OHP) measurements prevents life-threatening salt wasting conditions in newborns, but results in a considerable false-positive rate. Therefore, efficient second tier tests are required. Methods We developed a reverse-hybridization test strip-based assay (CAH StripAssay) covering the most prevalent CYP21A2 point mutations/small insertions/deletions occurring in Middle European populations. Assay specificity was validated using plasmid clones, and wild-type and mutant reference DNAs. Its practicability was evaluated in 271 samples from patients with CAH, suspected CAH, and dried blood spots from screening-positive newborns. Results All eleven point mutations and 51% of large deletions/conversions could be unambiguously identified when compared to reference methods (DNA sequencing, MLPA). After exclusion of rare mutations (6.4%) not covered by the StripAssay, the overall detection rate was 85%. Undetected heterozygous deletions/conversions caused a lack of information, but did not result in an incorrect prediction of phenotypes. Conclusions Our novel CAH StripAssay proved to be a fast (7 h) and reliable method for detection of common CYP21A2 mutations. Implemented as a second-tier test in CAH newborn screening, it has the potential to significantly reduce recall rates.

AimsTo evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.MethodsThe strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.ResultsThe strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.ConclusionsThe strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.

Our data on 114 Iranian individuals with thalassemia intermedia phenotype revealed homozygous or compound heterozygous beta-globin mutations to be the predominant disease factor in 86.2% of cases. However, 8.2% of these individuals were found to be heterozygous or wild type for beta-globin mutations. In search for determinants outside of the beta-globin gene, which could be responsible for the unexpected thalassemia intermedia phenotype in these subjects, we screened the alpha-globin genes, the 5′HS3 and 5′HS4 regions of the beta-globin LCR, and the NF-E2 transcription factor for sequence variations in selected individuals. The -3.7 deletion was the only alpha-globin mutation detected, and no alterations were found in 5′HS3 and NF-E2. Sequence analysis of the 5′HS4 LCR core region identified three known SNPs in a single patient, who required irregular blood transfusions. The A/G polymorphism in the 5′HS4 palindromic region was also observed to be variable. Family studies were carried out on a female G/G homozygous patient, who received irregular blood transfusions. Her father, who had the same heterozygous IVSII-1 beta-globin mutation but the A/G genotype at the 5′HS4 palindromic site, presented with mild anemia and no requirement for blood transfusions. This suggests an impact of SNPs in the 5′HS4 LCR core region on the thalassemia phenotype and offers an interesting subject for further investigations in the Iranian population.