Joanne Traeger-Synodinos

Cystic fibrosis in Greece: molecular diagnosis, haplotypes, prenatal diagnosis and carrier identification amongst high-risk individuals

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high‐risk groups – with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

Rapid and accurate quantitation of Hb Bart's and Hb H using weak cation exchange high performance liquid chromatography: correlation with the alpha-thalassemia genotype.

The Bio-Rad Variant Hemoglobin testing system is an automated high performance liquid chromatography analyzer marketed with a beta-thalassemia short program to quantify Hbs F and A2, and assist in detecting Hbs A, S, D, C, and E. Although the two hemoglobins present in Hb H disease, Hb Barts and Hb H, are separated by the system, they are not quantitated. In this study we modified the beta-thalassemia short program in order to facilitate quantitation of Hb Barts and Hb H. Blood samples from 60 patients with Hb H disease, with various underlying genotypes, were studied. Analyses were performed on the day of blood collection or on hemolysates stored at -80 degrees C in cyanide or carbomonoxy forms. The mean sum of Hb Barts and Hb H levels in all patients was found to be 12% (range 1.8-35%). Patients with nondeletional mutations (or association of alpha(0) deletion and nondeletional mutations) had notably higher Hb Barts and Hb H levels when compared to patients with deletional genotypes.

Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation.

To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (AF 508, G542X, G551D, 621+1 G→T, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the ΔF 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4A→G) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.

Hb Aghia Sophia [α62(E11)Val→0 (α1)], an “In-Frame” Deletion Causing α-Thalassemia

In this report we describe a case of Hb H disease due to the interaction of the --(MED 1) deletion with a new alpha(+)-thalassemia determinant. The molecular analysis of the probands genomic DNA was carried out by polymerase chain reaction amplification and sequencing of both alpha genes of the alpha(+)-thalassemia chromosome and revealed a deletion of codon 62 of the alpha1 gene. This DNA triplet codes for a valine residue at the E11 alpha helix, which is located in the interior of the heme pocket. Substitutions of valine E11 with other amino acid residues in the alpha as well as beta polypeptide chains lead, in the heterozygous carrier, either to Hb M disease or to congenital non-spherocytic hemolytic anemia. We assume that the deletion of valine at alpha62(E11) disrupts the conformation of the alpha chain to such an extent that the mutated subunit is rapidly removed by proteolysis. The final result is an alpha-thalassemia phenotype rather than an unstable hemoglobin syndrome. This conclusion is supported by the apparent absence of an abnormal alpha chain in the peripheral blood of the patient.

Association of unstable hemoglobin variants and heterozygous β-thalassemia: Example of a new variant Hb acharnes or [β53(D4) Ala Thr]

We report here the functional and structural characterization of Hb Acharnes [β53(D4) Ala → Thr], an unstable and electrophoretically silent variant, that was found associated in trans with a β0‐thalassemic mutation (IVSI‐1 G → A), in a patient with thalassemia intermedia syndrome. This case is discussed in comparison with other sporadic cases that we have previously investigated, resulting from the co‐inheritance of a β0‐thalassemic mutation (CD39 C → T) with two other types of unstable hemoglobins, Hb Köln [β98(FG5) Val → Met], and Hb Arta [β45(CD4) Phe → Cys]. It may be concluded that, in these associated forms, both the degree of instability of the variant and the altered oxygen binding properties (affecting the degree of tissue hypoxia) are major determinants of their clinical expression. Am. J. Hematol. 62:186–192, 1999.

Preliminary mutation analysis in the phenylanaline hydroxylase gene in Greek PKU and HPA patients

The presence of nine mutations in the phenylalanine hydroxlase (PAH) gene, previously described in phenylketonuria (PKU) patients of other Mediterranean and European populations, was assessed in 47 Greek PKU and 3 hyperphenylalaninaemia (HPA) patients. Of the nine mutations investigated, only five were detected, characterizing 31 % of the PKU alleles in our patients.

Unusual phenotypic observations associated with a rare HbH disease genotype (- -Med/αTSaudiα): implications for clinical management

Summary.  A single patient with a rare Haemoglobin H (HbH) disease genotype (‐ ‐Med/αTSaudiα) was observed to have exceptionally high levels of HbH (> 60%) and paradoxically high total haemoglobin levels. Studies of haematological parameters, blood biochemistry and oxygen transport properties revealed a severe functional anaemia, associated with marked erythropoietic stimulation and a markedly raised cardiac output. This rare case illustrates the complexity of interactions that may be associated with the clinical course of HbH disease, highlighting that haematological parameters alone may lead to spurious evaluation of clinical status. Issues related to the therapeutic management of unusual cases are raised.

Identification of two novel mutations (296 + 1G − C and A46D) in exon 2 of the CFTR gene in Greek cystic fibrosis patients

Two novel CFTR mutations were detected in Greek cystic fibrosis patients. One was a missense mutation, A46D, and the other a splice mutation, 296 + 1G-C. Neither was detected on normal chromosomes.