Multiple endocrine neoplasia 2 in Cyprus: evidence for a founder effect.

PURPOSE: Multiple endocrine neoplasia type 2 (MEN2) affects patients with RET proto-oncogene mutations. This cohort study refers to patients who were diagnosed with familial medullary thyroid carcinoma (MTC) and underwent RET genetic testing in Cyprus between years 2002 and 2017. METHODS AND PATIENTS: Forty patients underwent RET testing by Sanger sequencing of exons 10-11 and 13-16. Genotyping with STR genetic markers flanking the RET gene along with Y-chromosome genotyping and haplogroup assignment was also performed. RESULTS: RET mutations were identified in 40 patients from 11 apparently unrelated Cypriot families and two non-familial sporadic cases. Nine probands (69.2%) were heterozygous for p.Cys618Arg, one (7.7%) for p.Cys634Phe, one (7.7%) for the somatic delE632-L633 and two (15.4%) for p.Met918Thr mutations. The mean age at MTC diagnosis of patients carrying p.Cys618Arg was 36.8 ± 14.2 years. The age of pheo diagnosis ranged from 26 to 43 years and appeared simultaneously with MTC in 5/36 (13.9%) cases. The high frequency of the p.Cys618Arg mutation suggested a possible ancestral mutational event. Haplotype analysis was performed in families with and without p.Cys618Arg. Six microsatellite markers covering the RET gene and neighboring regions identified one core haplotype associated with all patients carrying p.Cys618Arg mutation. CONCLUSIONS: The mutation p.Cys618Arg is by far the most prevalent mutation in Cyprus followed by other reported mutations of variable clinical significance. The provided molecular evidence speculates p.Cys618Arg mutation as an ancestral mutation that has spread in Cyprus due to a possible founder effect.

Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species.

Oxidative stress in ß-thalassaemia and sickle cell disease.

Sickle cell disease and ß-thalassaemia are inherited haemoglobinopathies resulting in structural and quantitative changes in the ß-globin chain. These changes lead to instability of the generated haemoglobin or to globin chain imbalance, which in turn impact the oxidative environment both intracellularly and extracellularly. The ensuing oxidative stress and the inability of the body to adequately overcome it are, to a large extent, responsible for the pathophysiology of these diseases. This article provides an overview of the main players and control mechanisms involved in the establishment of oxidative stress in these haemoglobinopathies.

Alpha-thalassaemia prenatal diagnosis by two PCR-based methods.

In Cyprus all couples carrying alpha0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for alpha-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the psialpha1- and alpha2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two alpha-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for alpha-thalassaemia have been reported to date.

A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction.

The spectrum of the beta-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 beta-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (-A) and IVS-I-129 (A-->C), both found in Sri Lankan patients. Two beta-thalassemia mutations were found to coexist in one beta-globin gene: Sri Lankan patients homozygous for the beta0 codon 16 (-C) frameshift were also homozygous for the beta+ codon 10 (C-->A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C-->A) mutation is just a rare polymorphism on an ancestral allele, on which the beta0 codon 16 (-C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the beta-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.

Hb Bart's levels in cord blood and alpha-thalassemia mutations in Cyprus.

The purpose of this study was to examine the frequency of alpha-thalassemia in the population of Cyprus using cord blood samples. The levels of Hb Bart's were compared with the hematological indices and the results correlated with the presence of alpha-thalassemia mutations. The protocols for the polymerase chain reaction detection of the six most common alpha-globin mutations encountered in Cyprus were optimized, and the frequency of each mutation was determined through the screening of 495 random cord blood samples. The total allele frequency for the mutations examined was 10.6%, of which 1% is due to the triplication of the alpha-globin genes. The -alpha(3.7 kb) deletion accounts for 72.8% of all detectable mutations, while the--MED-I and -(alpha)-20.5 kb mutations account for 7.8%. The level of Hb Bart's and the MCV and MCH values in cord blood samples were found to correlate closely with the severity of alpha-thalassemia, although the -alpha(3.7 kb) deletion and perhaps other mild alpha-thalassemia mutations may not give detectable Hb Bart's levels. A reasonably accurate estimate of the alpha-thalassemia carrier frequency may be obtained from cord blood studies if Hb Bart's estimates are combined with hematological indices. When molecular methods are added, these give the best way to use cord bloods to survey populations for alpha-thalassemia.

Molecular characterization of beta-thalassemia in Syria.

This study concerns the determination of beta-thalassemia alleles and other hemoglobin variants in 82 patients from Syria. We have characterized 146 chromosomes and found 17 different beta-thalassemia mutations, and one beta-globin chain variant that gives rise to the abnormal Hb S. The eight most common beta-thalassemia mutations were the IVS-I-110 (G-->A), IVS-I-1 (G-->A), codon 5 (-CT), -30 (T-->A), codon 39 (C-->T), IVS-I-6 (T-->C), IVS-II-1 (G-->A), and codon 15 (TGG-->TAG). These mutations accounted for almost 75% of the total beta-thalassemia chromosomes. We identified 34 different genotypes with a high level of homozygosity. The various beta-thalassemia mutations were characterized using gene amplification with specific oligonucleotide primers, restriction enzyme analysis, denaturing gradient gel electrophoresis and direct sequencing. By combining these three approaches we were able to detect mutations in almost 90% of the chromosomes studied. Our findings provide a sound foundation on which to base a preventive program for thalassemia and we believe that the data that we present will facilitate the improvement of medical services such as carrier screening, genetic counseling, and prenatal diagnosis. Furthermore a detailed knowledge of the molecular pathology of beta-thalassemia will strongly improve the prenatal diagnosis services in Syria.

Comparative in vivo expression of beta(+)-thalassemia alleles.

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression.

alpha-Thalassaemia in the population of Cyprus.

We have determined the alpha-thalassaemia (alpha-thal) determinants in 78 patients with Hb H disease from Cyprus; 25 were Turkish Cypriots and 53 were Greek Cypriots. Four deletional and three non-deletional alpha-thal alleles were present; the -alpha(3.7 kb) alpha-thal-2 and the --MED-I alpha-thal-1 were most frequently seen; --MED-II and -(alpha)20.5 deletions occurred at considerably lower frequencies. About 15% of all chromosomes carried a non-deletional alpha-thal-2 allele; of these the 5 nucleotide (nt) deletion at the first intervening sequence (IVS-I) donor splice site was present in approximately 8% of all chromosomes. Two types of polyadenylation signal (poly A) mutations were observed. No striking frequency differences were seen between Greek and Turkish Cypriot patients. Combinations of the various types of alpha-thal resulted in eight different forms of Hb H disease. The phenotypes were comparable except for great variations in the level of Hb H which was highest (average approximately 22%) in the 12 patients with the alpha 5nt alpha/--MED-I combination. One patient with the same form of Hb H disease but with an additional beta-thal (IVS-I-110,G-->A) heterozygosity had a most severe microcytosis and hypochromia with < 1% Hb H. Variations in the level of Hb H might correlate with the severity of the disease, although this was not evident from the haematological data.

Detection of Duchenne and Becker muscular dystrophy carriers by quantitative multiplex polymerase chain reaction analysis.

We developed a method for the detection of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers. The method is based on the quantitative analysis of the products of standard multiplex polymerase chain reaction (PCR) from 18 different exons of the dystrophin gene, and is designated "QM-PCR." We detected deletions of one or more exons by standard multiplex PCR in DMD/BMD patients in 14 of 18 families examined (77.7%). The same deletions were readily demonstrated by QM-PCR in nine of 14 mothers (64.3%) and in another six of 22 possible carriers in these families. In five families where deletions were detectable in DMD/BMD patients, the mothers did not exhibit any deletions in their peripheral blood (35.7%). We obtained evidence for germinal mosaicism in at least two of these families and confirmed carrier identification by haplotype analysis using CA repeat polymorphisms at the 5' and 3' ends of the dystrophin gene. Furthermore, analysis of 17 coded DNA samples from normal females and obligatory carriers by QM-PCR showed that this technique could directly identify carriers of deletions in any of 18 different exons of the dystrophin gene. Its application in combination with existing techniques is expected to significantly improve the accuracy of carrier diagnosis in many families, and it may also be applicable to families in which pedigree and polymorphism information is insufficient for carrier diagnosis.