C. Rosatelli

PRENATAL DIAGNOSIS OF BETA-THALASSAEMIA WITH THE SYNTHETIC-OLIGOMER TECHNIQUE

103 couples attending the antenatal clinic in Sardinia were screened for the beta o-39 (nonsense) mutation, which codes for beta-thalassaemia, with the oligonucleotide method. In 94 couples both members had the beta o-39 mutant and thus were eligible for antenatal testing with this method. These pregnancies were monitored with amniocentesis (61) or trophoblast biopsy (33). Prenatal diagnosis in those monitored with amniocentesis was carried out with DNA analysis of uncultivated amniocytes (19) or cultivated cells (38). In 4 pregnancies results were unsatisfactory, and prenatal diagnosis was repeated with fetal-blood analysis. Trophoblast biopsy was unsuccessful in 1 pregnancy and gave a misdiagnosis in another because of maternal contamination. In the latter case the genotype of the fetus was established with amniocyte DNA analysis and globin-chain-synthesis studies.

Autosomal dominant Alport syndrome: molecular analysis of the COL4A4 gene and clinical outcome

BACKGROUND Alport syndrome is a clinically and genetically heterogeneous nephropathy characterized by glomerular basement membrane lesions often associated with hearing loss and ocular anomalies. While the X-linked and the autosomal recessive forms are well known, the autosomal dominant form is not well acknowledged. METHODS We have clinically investigated 38 patients with a diagnosis of autosomal dominant Alport syndrome belonging to eight different families. The analysis of the COL4A4 gene was performed by denaturing high performance liquid chromatography and automated DNA sequencing. RESULTS In our cohort of patients, only 24.3% (9/37) reached end-stage renal disease, at the mean age of 51.2 years. Four patients had hearing loss (13.3%) and none ocular changes. Molecular analysis revealed eight novel private COL4A4 gene mutations: three frameshift, three missense and two splice-site mutations. CONCLUSIONS These data indicate autosomal dominant Alport syndrome as a disease with a low risk of ocular and hearing anomalies but with a significant risk to develop renal failure although at an older age than the X-linked form. We were unable to demonstrate a genotype-phenotype correlation. Altogether, these data make difficult the differential diagnosis with the benign familial haematuria due to heterozygous mutations of COL4A4 and COL4A3, especially in young patients, and with the X-linked form of Alport syndrome in families where only females are affected. A correct diagnosis and prognosis is based on a comprehensive clinical investigation in as many family members as possible associated with a broadly formal genetic analysis of the pedigree.

Beta thalassaemia mutations in Sardinians: implications for prenatal diagnosis.

In this study we have characterised by oligonucleotide hybridisation and direct restriction endonuclease analysis the beta thalassaemia mutation in 494 Sardinian beta thalassaemia heterozygotes. The most prevalent mutation, accounting for 95.4% of the cases, was the nonsense mutation at codon 39. The remainder, in decreasing order of frequency, were a frameshift at codon 6 (2.2%), beta + IVS-1, nt 110 (0.4%), and beta + IVS-2, nt 745 (0.4%). This information allows prenatal diagnosis by DNA analysis to be made in the great majority of Sardinian couples at risk for beta thalassaemia.

Hematological phenotype of the double heterozygous state for alpha and beta thalassemia.

In this study, we have correlated the hematological phenotype of 56 Sardinian beta o-thalassemia heterozygotes with their alpha-globin genotype as defined by restriction endonuclease mapping. We found that the coinheritance of the deletion of one alpha-globin and, more obviously, two alpha-globin genes tend to normalize the thalassemia-like hematological phenotype commonly associated with the beta o-thalassemia carrier state. On the other hand, the association of the deletion of three alpha-globin genes caused a more severe phenotype. By globin chain synthesis analysis, those beta o-thalassemia heterozygotes with the (-alpha/alpha alpha) alpha-globin genotype had less deficiency of beta-chain synthesis than did those with the normal alpha-globin genotype (alpha alpha/alpha alpha). In heterozygotes with the (-alpha/-alpha) and in those with the (--/-alpha) alpha-globin genotype the imbalance was actually reversed with a mild or marked alpha-chain synthesis excess respectively.

The prenatal diagnosis of thalassaemia.

Prenatal diagnosis of /?-thalassaemia was accomplished for the first time in 1975 by the analysis of fetal blood obtained through placental aspiration at an advanced gestational age (18 weeks: Kan et al, 1975a). Thanks to a combination of spectacular progress in the molecular pathology of thalassaemias (see reviews: Nienhuis et al, 1984; Orkin & Kazazian, 1984; Collins & Weissman, 1984); continuous improvement in methodologies for DNA analysis(Conneretal, 1983; Pirastuetal, 1983;Robin&Kan, 1985; Saikietal, 1985; Myers et al, 1985): and the introduction of new methods for fetal tissue sampling (Williamson et al, 1981; Old et al, 1982; Goossens et al, 1983; Modell, 1985). prenatal diagnosis of thalassaemias may be accomplished now, in the large majority of cases, within the first trimester of gestation (Old et al, 1982). Fetal blood analysis. Fetal blood sampling is carried out at 18 weeks gestation either with fetoscopy or placental aspiration (Kan et al, 1975b) and it is still associated with relatively high fetal mortality (5.4%: for review see Alter, 1984). Therefore nowadays this approach should be limited to those couples at risk for /?-thalassaemia in whom DNA analysis is not practicable (see below). Fractionation of in vitro synthesized globin chains on carboxymethyl-cellulose columns remains the most commonly used method. HPLC may be a valuable alternative but it has been less extensively applied. The major problem encountered with these methods is the overlap in / ? / y ratios between /?-thalassaemia heterozygotes and !+-thalassaemia homozygotes, which has been the main cause for misdiagnosis occurring in approximately 0.8% of cases tested (Alter, 1984). DNA analysis. Fetal DNA may be obtained either from amniotic fluid cells or from chorionic villi. Amniocentesis is associated with a very low risk (0.3%) of fetal loss: while chorionic villi biopsy carries a risk of fetal loss around 4% (Modell, 198 5). However, the latter approach has the distinct advantage of making diagnosis possible as early as a t 9-1 0 weeks gestation. Diagnosis within the first trimester is obviously more acceptable to many couples who might object, for both psychological and ethical reasons, to termination of pregnancy in the second trimester. Those types of thalassaemias that are caused by relatively gross structural changes in DNA, such as deletions or inversions, may be directly detected by restriction endonuclease analysis and hybridization with appropriate globin gene probes (Dozy et al, 19 79). This group includes the majority of aand G/?-thalassaemias, the

Thalassemias in Sardinia: molecular pathology, phenotype-genotype correlation, and prevention.

/?-thalassaemias and a few /?thalassaemia lesions, among which the only relatively common one is that accounting for 30% of /?-thalassaemia alleles in Asian Indians (Indian deletion). By contrast, the vast majority of /?-thalassaemias are caused by single nucleotide substitutions, deletion or addition of a few nucleotides. A few of these mutations alter a recognition site for a restriction

Quality assessment in cytogenetic and molecular genetic testing: the experience of the Italian Project on Standardisation and Quality Assurance.

This article reviews the molecular bases of α- and β-thalassemias in Sardinia. In addition, it describes the characteristics and the effects of a genetic program designed to prevent homozygous β-thalassemia. In the large majority of the cases (95.7%), β-thalassemia is caused by the nonsense mutation at codon 39, followed by frame-shifts at codon 6 (2.1%). Homozygous β-thalassemia most commonly results in thalassemia major, but in a small proportion this genotype produces milder forms referred to as thalassemia intermedia. The reasons for the attenuated forms were determined only in a small proportion of the cases. The reduced clinical severity was due to either of two factors: (a) There could be the presence of cytosine-thymidine (C → T) substitution of position −158 Gγ, which is able to increase the Gγ chain output (as in homozygotes for frameshift at codon 6 or compound heterozygote for frameshift at codon 6 and codon 39 nonsense mutation), (b) Reduced clinical severity could be the result of co-inheritance of α-thalassemia in the form of two α-globin gene deletions or functional loss of the α2-globin gene (homozygote for codon 39 nonsense mutation). The most prominent clinical form of α-thalassemia is hemoglobin H disease, which may result from the compound heterozygous state for deletion α°- and α+-thalassemia (in 83.1% of cases) or deletion and nondeletion α-thalassemia (in 16.9%). Deletion Hb disease shows a milder clinical picture as compared to the nondeletion form. The most common nondeletion α-thalassemia is the ATG → ACG substitution at the initiation codon of the α2 gene. Double heterozygotes of α (-α/-α) and β-thalassemia or δ- and β-thalassemia are relatively common and may cause confusion in carrier identification.

Chorionic villus sampling by rigid forceps: Experience with 300 cases at risk for thalassemia major

Abstract The first Italian national trial of external quality assessment in genetic testing was organised within the framework of the “Italian National Project for Standardisation and Quality Assurance of Genetic Tests”. Sixty-eight Public Health Service laboratories volunteered for the trial, which involved molecular genetic tests (cystic fibrosis, β-thalassaemia, familial adenomatous polyposis coli and fragile-X syndrome) and cytogenetic tests (prenatal and postnatal, the latter included cancer cytogenetics). The response rate was high (88.2%). The level of analytical accuracy was good, i.e., the percentage of laboratories that correctly genotyped all samples was 89.3% for cystic fibrosis, 90.9% for β-thalassaemia, 100% for familial adenomatous polyposis coli (despite two laboratories did not complete the analysis because the amount of DNA was considered insufficient), and 90.5% for fragile-X syndrome. Written reports differed widely and were judged “inadequate” in over 50% of cases. Most laboratories from the present study already have experience in previous European external quality assessments for at least one genetic test; this can explain the higher analytical accuracy in the Italian external quality assessment with respect to quality control programmes in other countries. Collaborative networks are strongly suggested to improve the quality of the reports.

Antenatal Diagnosis of Thalassemia Major in Sardinia

In this article we report the results of chorionic villus sampling by a biopsy forceps inserted via the cervix under ultrasonic guidance in 300 pregnancies at risk for thalassemia major. A sufficient amount of chorionic villi for deoxyribonucleic acid analysis by oligonucleotide hybridization was obtained in all cases tested but one, with a success rate of 99.7%. The percentage of fetal loss, expressed as proportion of continuing pregnancies, was 4.8%. To verify the results, we carried out amniocyte deoxyribonucleic acid analysis in all the continuing pregnancies for the first 100 cases and in those in which trophoblast deoxyribonucleic acid analysis showed the heterozygous state for beta-thalassemia for the second 200 cases. At the beginning we had two cases of decidual contamination in such an amount to cause misdiagnosis. Successively more careful elimination of decidual tissue from villi avoided avoided this pitfall. These results indicate that chorionic villus sampling by a rigid forceps is a reliable and relatively safe method for fetal diagnosis of genetic diseases by deoxyribonucleic acid analysis.

Prenatal Diagnosis of β Thalassaemia by Fetal Red Cell Enrichment with NH4Cl-NH4HCO3 Differential Lysis of Maternal Cells

In this report, we summarized our experience, carried out in Sardinia, with antenatal diagnosis in one thousand pregnancies in which the fetus was at risk for homozygous beta-thalassemia. In the majority of these cases, the thalassemia lesion segregating in the family was the nonsense mutation at the codon corresponding to amino-acid 39. At the outset (976 cases) we used globin chain synthesis analysis by column chromatography on fetal blood obtained by placental aspiration, and recently (24 cases) we employed the synthetic oligonucleotide method on amniocyte DNA. Apart from 126 pregnancies still in progress, in all the other cases the diagnosis has been confirmed. In the majority of the cases (99%), we obtained sufficient fetal blood for the analysis. The fetal mortality associated with placental aspiration was 6.1%. The biochemical analysis gave reliable results. We had two misdiagnoses (0.2%): one due to a nonglobin protein comigrating with the beta chains and the other for a misclassification of the type of thalassemia segregating in the family. The oligonucleotide method gave clear-cut results in all the cases tested. The method was sensitive enough to detect the mutation directly in the DNA isolated from 20-25 ml of amniotic fluid in 75% of the pregnancies tested. In one case, we successfully employed this method for the analysis of the DNA isolated from chorionic villi. The oligonucleotide method seems to be the best procedure for monitoring the pregnancies at risk for beta-thalassemia in places where one or a few beta-thalassemia lesions are prevalent.