ALPHA- AND BETA-GLOBIN GENE MUTATIONS IN GEORGIA AND ARMENIA.

Georgia and Armenia are situated at the northern rim of the thalassemia belt and bordering to countries with a known high prevalence of thalassemias. In this study we assessed the carrier frequency and potential spectrum of alpha- and beta-globin mutations among 202 and 190 unselected Georgian and Armenian subjects, respectively. We found four alpha-globin mutations (-3.7del, -4.2del, anti-3.7 triplication, poly-A2) in 9 Armenians (4.74%) and 4 Georgians (1.78%). The heterozygous beta-globin codon 8 [-AA] mutation was detected in one individual from Armenia only. Overall, carrier frequencies seem to be low in both countries, supporting the notion that thalassemias are not a major health problem there.

BRAF mutation status in primary and metastatic melanomas in two regions with differing potential ultraviolet radiation exposure.

BACKGROUND: Melanoma is seen as a heterogeneous molecular entity, with solar ultraviolet radiation (UVR) and BRAF mutation status being important determinants. AIM: To study primary and metastatic melanomas from two UVR-distinct regions to elucidate correlations between prognostic predictors, UVR and BRAF mutation status. METHODS: Extended BRAF testing for 9 mutations was obtained for 95 primary melanomas [Lebanon (LB) n = 55, Pakistan (PK) n = 40)] and 65 metastatic melanomas (LB n = 36, PK n = 29). Collected data included patient age and sex, melanoma size and anatomical location, prognostic parameters and solar elastosis grade for primary melanomas. For metastatic melanomas, site of metastasis, magnitude of necrosis and degree of pigmentation were assessed. Cumulative 21-year averages of potential UVR exposure for Lebanon (110 kJ/m(2) /year) and Pakistan (128 kJ/m(2) /year) were derived from the National Center for Atmospheric Research databases. RESULTS: BRAF mutation status was obtained for 146/160 cases (91.3%). Overall mutation rate was 24/88 (27.3%) in primary and 25/58 (43.1%) in metastatic melanoma. V600E was the predominant mutation in 21/24 (87.5%) of primary and 23/25 (92%) of metastatic melanomas. A 60% discordant mutation rate was identified; of three patients, two lost the mutation in the metastasis and one gained it. The relative incidence of BRAF mutation with potential UVR exposure showed a similar trend in primary (low vs. high UVR: 32.1% vs. 20.0%) and metastatic (57.1% vs. 21.7%) melanomas (P < 0.05). Predictors of BRAF mutations were trunk location and epithelioid and mixed cytology for primary and subcutaneous metastasis, low UVR exposure and absence of pigmentation for metastatic melanomas (P < 0.05). BRAF-positive status in primary melanomas was predicted by multivariate binary logistic regression with reasonable accuracy (C-statistic = 0.67, 95% CI 0.530-0.81 with one independent predictor, namely, epithelioid cytology (OR = 5.11, 95% CI 1.38-8.88, P = 0.01). In metastatic melanomas, high UVR (OR = 0.21, 95% CI 0.06-0.07; P < 0.01) was an independent negative predictor of BRAF mutation. CONCLUSIONS: We have documented the rate of different BRAF mutation types in a Lebanese and Pakistani cohort, and assessed correlations with prognostic markers and potential UVR exposure.

BRAF mutational epidemiology in dysplastic nevi: does different solar UV radiation exposure matter?

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 < 0.05). A 43.8% discordant mutation rate (7/16 patients) was found in patients with multiple nevi, including 2 patients with different BRAF mutations. Microscopic examination subdivided the dysplasia into mild (n = 24), moderate (n = 60) and severe (n = 41) with trunk predominance (72.8%). Higher rates of pigment in the stratum corneum were identified in Saudi Arabia (P < 0.05). No statistical significant increase in BRAF mutation rate was noted with advanced architectural and cytological atypia. Parameters associated with a negative BRAF mutation status included upper extremity location, regression, cohesiveness and presence of suprabasal melanocytes (P < 0.05). Positive BRAF mutation status was reasonably predicted by multivariate binary logistic regression by 2 independent predictors: Geographic location and compound nevus type. CONCLUSIONS: In our Near Eastern cohort, the BRAF mutation rate varied significantly by geographic location. In patients with multiple dysplastic nevi examined, discordant BRAF mutation status potentially negates an underlying constitutional predilection.

Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.

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.

Prevalence of HFE mutations in upper Northern Italy: study of 1132 unrelated blood donors.

BACKGROUND: In the Italian general population, prevalence of C282Y is lower than in Northern European countries. We hypothesised a higher prevalence of C282Y in Northern than in Central and Southern Italy. We previously identified a nonsense mutation (W169X) in haemochromatosis probands originating from a Northern Italian region (Brianza). AIM: To define the prevalence of HFE mutations in that region. Subjects and methods. A total of 1132 unrelated blood donors from the Blood Banks of Monza and Merate were investigated for C282Y, H63D, S65C and W169X mutations by PCR-restriction assays. A total of 300 were also tested for rare HFE and TFR2 mutations by reverse-hybridization test strips. RESULTS: Two C282Y homozygotes, eight C282Y/H63D compound heterozygotes, 27 H63D homozygotes and one W169X heterozygote were found. The allele frequencies of C282Y, H63D, S65C, and W169X were 3.2, 13.4, 1.3, and 0.04%, respectively. CONCLUSIONS: Our results confirm the existence of a decreasing frequency of C282Y allele from upper to lower Northern Italy. This difference is probably related to the larger Celtic component of upper Northern Italian populations in which screening studies for haemochromatosis may even be cost effective. W169X, due to its severity, should be looked for in all haemochromatosis patients of Northern ancestry with an incomplete HFE genotype.

Comparative study of the two more frequent HFE mutations (C282Y and H63D): significant different allelic frequencies between the North and South of Portugal.

An earlier study of reference values of iron parameters in Portugal showed significant differences between populations from northern and southern villages. This study addresses the question of the geographical distribution in Portugal of the two main mutations (C282Y and H63D) of the hereditary hemochromatosis gene, HFE. For that purpose, a stratified sample of 640 anonymous dried blood spot samples was randomly selected from the major regions of Portugal: North, Center, Lisbon and the Tagus Valley, Alentejo and Algarve. Differences in the geographical distribution of these two mutations were observed thus confirming the presumed differences between the age of the two mutations which is compatible with the postulated Celtic/Nordic origin of the C282Y mutation. The finding of a significantly higher allelic frequency of the C282Y mutation in the North (0.058) than in the South (0.009) could also point to an effect of differential selective forces acting in the different geographical areas of the country. Data on archaeological, ethnographic and linguistic records and on the North/South distribution of Portuguese cattle breeds of European or African origin have also been reported. In addition to their interest for population genetics, the results represent a reminder of the need to take into account regional differences in the design of strategies for population screening of hereditary hemochromatosis.

A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations.

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.

HLA-B*27 typing by group-specific hybridization in microtiter plates.

We have developed and evaluated a test for HLA-B*27 based on PCR and DNA hybridization in microtiter plates. A region within exon 2 of the HLA-B gene is amplified and labeled by PCR and the amplification product is hybridized to a group-specific HLA-B*27 and a generic control oligonucleotide probe in two separate cavities of the plate. Bound sequences are detected using an ELISA-like protocol. The assay has been evaluated on 254 DNA samples routinely received for B27 testing in parallel with serological and SSP-PCR typing. Results were concordant in typing 102 HLA-B27-positive and 152 HLA-B27-negative individuals except for two samples containing HLA-B*73, which stained B27 positive in the microwell test. The new procedure is rapid and simple to perform, and the microwell format is particularly suitable for automation.

Ultrastructural localization of microtubule-associated proteins (MAP) 1 in cilia of the respiratory tract.

The presence and localization of high molecular weight microtubule-associated proteins of the MAP 1 class in ciliated cells of porcine and rat respiratory tract was studied by immunoblotting and immunoelectron microscopy. Ciliary shafts of the porcine tracheal epithelium were isolated using a method that minimizes contamination of the preparation by other cellular fragments and fat. Immunoblotting with rabbit antibodies to bulk MAP 1 from hog brain clearly revealed the presence of anti-MAP 1-immunoreactive high molecular weight proteins of the MAP 1 size in these preparations. To localize MAP 1 proteins at the ultrastructural level, rat and porcine tracheal epithelia were embedded in LR White and subjected to immunogold electron microscopy. Anti-MAP 1-immunoreactive material was found at ciliary shafts and basal bodies, but not at basal feet or ciliary rootlets. Interestingly, the necklace region between the shaft and the basal body of the cilium was hardly reactive with anti-MAP 1 antibodies. This may indicate a reduced stability of ciliary microtubules in this region and could be an explanation why ciliary shafts in general break more easily there than elsewhere.