The Italian National External quality assessment program in molecular genetic testing: results of the VII round (2010-2011).

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

The deltaccr5 mutation conferring protection against HIV-1 in Caucasian populations has a single and recent origin in Northeastern Europe.

The chemokine receptor CCR5 is encoded by the CMKBR5 gene located on the p21.3 region of human chromosome 3, and constitutes the major co-receptor for the macrophage-tropic strains of HIV-1. A mutant allele of the CCR5 gene, Delta ccr5 , was shown to provide to homozygotes with a strong resistance against infection by HIV. The frequency of the Delta ccr5 allele was investigated in 18 European populations. A North to South gradient was found, with the highest allele frequencies in Finnish and Mordvinian populations (16%), and the lowest in Sardinia (4%). Highly polymorphic microsatellites (IRI3.1, D3S4579 and IRI3.2, D3S4580 ) located respectively 11 kb upstream and 68 kb downstream of the CCR5 gene deletion were used to determine the haplotype of the chromosomes carrying the Delta ccr5 variant. A strong linkage disequilibrium was found between Delta ccr5 and specific alleles of the IRI3.1 and IRI3.2 microsatellites: >95% of the Delta ccr5 chromosomes carried the IRI3.1-0 allele, while 88% carried the IRI3.2-0 allele. These alleles were found respectively in only 2 or 1.5% of the chromosomes carrying a wild-type CCR5 gene. From these data, it was inferred that most, if not all Delta ccr5 alleles originate from a single mutation event, and that this mutation event probably took place a few thousand years ago in Northeastern Europe. The high frequency of the Delta ccr5 allele in Caucasian populations cannot be explained easily by random genetic drift, suggesting that a selection advantage is or has been associated with homo- or heterozygous carriers of the Delta ccr5 allele.

Genotype of subjects with borderline hemoglobin A2 levels: implication for beta-thalassemia carrier screening.

In this study, we have defined by molecular analysis, the alpha, beta, and delta globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for beta-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the beta, in both the delta and the beta, or in the alpha globin gene. Specifically seven of these subjects were carriers of the -101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for delta and beta thal, and two had the triple alpha globin gene and two the single alpha globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical beta-thal carrier, should be extensively investigated in order not to miss heterozygous beta-thalassemia.

Thalassaemia in Azerbaijan.

beta thalassaemia is present throughout the southern regions of the former USSR. We have defined the clinical picture of the disorder, the spectrum of beta thalassaemia mutations, and the role of customary consanguineous marriage in Azerbaijan, where thalassaemia presents a public health problem of the same order as that in Greece. Contrary to earlier suggestions, we found that the common form of the disorder is typically severe. Typical Turkish, Mediterranean, Azeri, Kurdish, and Asian Indian mutations were found, consistent with the history of the region. The common Mediterranean beta 0 thalassaemia mutation (codon 39) was not found. Three mutations (codon 8-AA, IVS2-1 and IVS1-110) account for over 80% of beta thalassaemia genes. Consanguineous marriage appears to contribute relatively little to the frequency of affected births. These observations provide the basis for a thalassaemia prevention programme in Azerbaijan.

Early transabdominal chorionic villus sampling in couples at high genetic risk.

OBJECTIVE: The purpose of the study was to evaluate the feasibility and safety of transabdominal chorionic villus sampling before 9 weeks' gestation. STUDY DESIGN: Two hundred pregnancies at risk for beta-thalassemia (n = 198) or Duchenne muscular dystrophy (n = 2) underwent transabdominal CVS at 6 through 8 weeks. Sampling success and fetal loss are expressed in percentages. RESULTS: Sampling was successful in all cases (100%). Forty-eight fetuses were affected by beta-thalassemia and one by Duchenne muscular dystrophy. The percentage of fetal loss, expressed as a proportion of continuing pregnancies, was 4.0%. All women (n = 144) have been delivered, and no misdiagnoses have occurred. We observed one anencephalus and one mild limb defect consisting of absence of distal phalanges of index and little fingers of both hands and distal phalanges of both little toes. CONCLUSION: Transabdominal CVS before 9 weeks is a reliable and relatively safe method for prenatal diagnosis in patients at high risk for genetic diseases. However, further studies are necessary to assess the risk to the fetus.

Heterozygous beta-thalassemia: relationship between the hematological phenotype and the type of beta-thalassemia mutation.

In this study we have correlated the severity of the hematological features to the type of the beta-thalassemia mutation [codon 39 (C----T), IVS-I nt 110 (G----A), IVS-I nt 1 (G----A), IVS-I nt 6 (T----C), IVS-II nt 745 (C----G), -87 (C----G) and beta 6 (-1 bp)], in a group of beta-thalassemia heterozygotes of Italian descent in whom we excluded the presence of iron deficiency or deletion alpha-thalassemia. The beta-thalassemia mutation was defined by dot blot analysis on amplified DNA with allelic specific oligonucleotide probes. We found that a) heterozygotes for beta+ IVS-I nt 6 and beta+ -87 mutations produce larger and better hemoglobinized red blood cells, and b) heterozygotes for beta+ IVS-I nt 6 and beta+ IVS-I nt 110 mutations have a less marked increase of Hb A2 levels as compared to heterozygotes for the other mutations investigated. These findings indicate that milder beta-thalassemia mutations such as the beta+ IVS-I nt 6 and beta+ -87, express also in the heterozygous state a milder phenotype as compared to beta o-thalassemia or severe beta+ thalassemia (beta+ IVS-I, nt 110). The Hb A2 levels, on the other hand, were not related to the severity of the mutation because of less marked increase was found in a mild (beta+ IVS-I nt 6) as well in a severe (beta+ IVS-I nt 110) mutation. From the practical point of view these findings should be adequately considered in carrier screening and genetic counselling.

Hemoglobin Cagliari (beta 60 [E4] Val----Glu): a novel unstable thalassemic hemoglobinopathy.

This report describes a patient with thalassemia intermedia-like phenotype born to normal parents in whom globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T to A substitution at codon 60 of the beta-globin gene arising as a de novo mutation. Normal sequences were detected at the homologous beta-globin locus. This mutation results in the substitution of a polar (glutamic acid) for a nonpolar (valine) residue near the corner of the heme pocket of the beta-globin chain. The novel variant has been designated Hb Cagliari, from the place of birth of the propositus. Kinetics of globin synthesis performed following splenectomy suggest that this new Hb variant is synthesized at a near normal rate but undergoes rapid breakdown. The extreme lability of the variant explains the clinical and hematologic picture characterized by marked ineffective erythropoiesis, thalassemia-like bone changes, iron overload, high proportion of Hb F in the peripheral blood, reduced beta/alpha-globin chain synthesis ratio in peripheral blood reticulocytes, and absence of the abnormal Hb in peripheral blood at extensive protein structural analysis before splenectomy. This case indicates that a thalassemic hemoglobinopathy should be suspected in the presence of a patient with a thalassemia intermedia-like phenotype born to normal parents, even when protein structural analysis fails to detect an abnormal Hb. DNA sequencing may allow to define the mutation, thus making the proper diagnosis.

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

This article reviews the molecular bases of alpha- and beta-thalassemias in Sardinia. In addition, it describes the characteristics and the effects of a genetic program designed to prevent homozygous beta-thalassemia. In the large majority of the cases (95.7%), beta-thalassemia is caused by the nonsense mutation at codon 39, followed by frameshifts at codon 6 (2.1%). Homozygous beta-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 G gamma, which is able to increase the G gamma 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 alpha-thalassemia in the form of two alpha-globin gene deletions of functional loss of the alpha 2-globin gene (homozygote for codon 39 nonsense mutation). The most prominent clinical form of alpha-thalassemia is hemoglobin H disease, which may result from the compound heterozygous state for deletion alpha 0- and alpha(+)-thalassemia (in 83.1% of cases) or deletion and nondeletion alpha-thalassemia (in 16.9%). Deletion Hb disease shows a milder clinical picture as compared to the nondeletion form. The most common nondeletion alpha-thalassemia is the ATG----ACG substitution at the initiation codon of the alpha 2 gene. Double heterozygotes of alpha (-alpha/-alpha) and beta-thalassemia or delta- and beta-thalassemia are relatively common and may cause confusion in carrier identification. The preventive program aimed to control beta-thalassemia is based on voluntary carrier screening., counselling, and prenatal diagnosis. Prenatal diagnosis is carried out by dot blot analysis with allelic specific oligonucleotides on amplified trophoblast DNA. This procedure gave very reliable results; in fact, no misdiagnosis has occurred so far. The preventive program was highly effective, resulting in a decline of the incidence of thalassemia major from 1:250 to 1:1,000 live births.

Molecular basis of beta-thalassemia intermedia in a southern Italian region (Puglia).

We investigated the molecular bases for a mild phenotype by alpha-, beta- and gamma-globin gene analyses in 22 patients with transfusion-independent thalassemia intermedia (15) or a late-presenting form of thalassemia major (7) originating from Puglia, a region of southern Italy. Twenty-two patients with thalassemia major served as controls. The beta+ IVS-I nt 6 of the beta-globin gene and the C----T substitution at position -158 5' of the G gamma-globin gene were detected more frequently in patients with thalassemia intermedia or late-presenting thalassemia major considered together as compared to those affected by typical transfusion-dependent thalassemia major. Three of 15 patients with thalassemia intermedia had the triple alpha-globin gene arrangement in the heterozygous (2) or homozygous state (1) in association with heterozygous beta zero-thalassemia. From these results, we may conclude that the inheritance of a mild beta-thalassemia allele such as the beta+ IVS-I nt 6 mutation, in the homozygous or heterozygous state, the coinheritance with homozygous beta zero-thalassemia of the -158 (C----T) G gamma gene promoter mutation and the presence of heterozygous beta-thalassemia/triple alpha-globin gene arrangement are the most common reasons accounting for the development of attenuated forms of beta-thalassemia in Puglia.

Fetal hydrops in Sardinia: implications for genetic counselling.

This paper describes the first case of Hb Bart's hydrops fetalis syndrome in the Sardinian population. Despite the high frequency of a-thalassemia, fetal hydrops is extraordinarily rare in the Sardinian population because a-thalassemia is more usually the result of the single a-thalassemia globin gene deletion and is very rarely produced by the deletion of two a-globin genes. The fetus, the product of a consanguineous marriage at risk for beta-thalassemia, was monitored by chorionic villi DNA analysis which detected the heterozygous state for the codon 39 nonsense mutation. Follow-up ultrasound examination showed fetal hydrops, which led us to carry out further investigation. Hemoglobin and a-globin gene analysis on cord blood obtained by cordocentesis revealed the homozygous state for the most common deletion ao-thalassemia in Mediterranean populations. Retrospective evaluation of the father's hematological features showed very low MCH-MCV for a beta-thalassemia carrier which may indicate co-inherited a-thalassemia. These findings indicate that careful evaluation of red cell indices of parents at risk for beta-thalassemia and adequate consideration of the consanguinity may point to co-inherited a-thalassemia and lead to the appropriate analysis.

Molecular bases for cystic fibrosis in the Sardinian population.

Cystic fibrosis (CF) is a relatively uncommon genetic disorder in the Sardinian population. In this study, we have defined the frequency of the most common CF mutation (delta F508) and carried out a genotype-phenotype correlation analysis in a group of 21 patients with CF and of Sardinian descent. We detected the delta F508 mutation in 24 (57%) out of the 42 CF chromosome investigated. This mutation was found in the homozygous state in 9 patients and in the heterozygous state in 6 patients. The remaining 6 patients had other mutations. The delta F508 mutation was associated only with the KM19/XV2c 2 1 haplotype. Genotype-phenotype correlation analysis did not give clear-cut results, probably because of the small number of patients investigated. However, out of the four patients with meconium ileus, three were homozygous and one was heterozygous for the delta F508 mutation, confirming that the presence of delta F508 or other severe mutations in the homozygous state is the prerequisite for the development of meconium ileus.

The prevention of thalassemia in Sardinia.

In this paper we review the characteristics and effectiveness of a program aimed at preventing homozygous beta-thalassemia in the Sardinian population. The target population for screening were couples at marriage, conception or early pregnancy. Awareness of the problem and the involvement of the population were achieved via the mass media or by personal approaches through lectures or discussions. Parents' Associations were consulted and have made themselves available to prospective couples in several critical areas. Education on thalassemias was introduced into the school curriculum. Counseling was based on private interviews at which the several options available were discussed with the individual carrier or the couples. Prenatal diagnosis was chosen by the large majority of couples counseled. The introduction of 1st trimester diagnosis resulted in a striking increase of the acceptance rate from 93.2 to 99.1%. Prenatal diagnosis was carried out initially by fetal blood analysis and thereafter by trophoblast or amniocyte DNA analysis. Direct detection of the mutation by oligonucleotide hybridization on agarose gel separated DNA fragments or by dot-blot analysis with allelic specific oligonucleotide probes on enzymatically amplified DNA was used. This program resulted in a decline in thalassemia major births of 90%. The reasons for residual cases were mostly lack of information and, less frequently, misdiagnoses or refusal of fetal diagnosis.

Prenatal diagnosis of inherited hemoglobinopathies.

This paper reviews the methodology available to make prenatal diagnosis of inherited hemoglobinopathies by DNA analysis and the strategy to be used for the large scale application of this procedure to high-risk populations. The most straightforward approach for prenatal diagnosis is nowadays based on the analysis of DNA enzymatically amplified by the polymerase chain reaction (PCR). The mutations, produced by gross structural rearrangement of the DNA and those affecting a restriction recognition site, are directly detected by visualization following ethidium bromide staining of the electrophoretic pattern resulting from enzymatic digestion of amplified DNA. The remaining ones are detected by dot blot analysis with allelic specific oligonucleotide probes. Because in each population a limited number of specific beta-thalassemia mutations are prevalent, prenatal diagnosis by DNA analysis may be carried out by a population-specific strategy based on the amplification of those regions of the beta-globin genes containing the mutations most frequently occurring in each population followed by dot blot analysis with allelic specific oligonucleotide probes. This approach has the great advantage of being very simple, because radioactive probes are not necessary, very rapid, the results being obtained within 24 hours from sampling and very sensitive, only a limited amount of DNA in the order of 50 ng being necessary.

Prenatal diagnosis of beta-thalassaemia in Mediterranean populations by dot blot analysis with DNA amplification and allele specific oligonucleotide probes.

In this study, we describe a simple strategy to detect beta-thalassaemia mutations in prospective parents and to make prenatal diagnosis in pregnancies at risk in the Mediterranean population. Screening of prospective parents is carried out by dot blot analysis on enzymatically amplified DNA with a set of oligonucleotide probes complementary to the most common mutations in this population. Prenatal diagnosis is accomplished by the same procedure on enzymatically amplified amniocyte or trophoblast DNA. The main advantages of this procedure are the simplicity, sensitivity (0.05 micrograms of DNA), and rapidity (12-24 h). Further simplification is obtained by amplification of the DNA from crude amniotic cell lysate. The very low amount of fetal material necessary for this analysis eliminates the need to culture amniotic fluid cells and may decrease the fetal loss rate associated with trophoblast sampling. The number of specific DNA sequences obtained by the amplification procedure allowed us to use non-radioactive labelled oligonucleotide probes, which have several advantages compared to radioactive probes.

Molecular analysis of beta zero-thalassemia intermedia in Sardinia.

In this study we have carried out alpha- and beta-globin gene analysis and defined the beta-globin gene polymorphisms in a group of patients with thalassemia intermedia of Sardinian descent. A group of patients (109) with thalassemia major of the same origin served as control. Characterization of the beta-thalassemia mutation showed either a frameshift mutation at codon 6 or a codon 39 nonsense mutation. We found that homozygotes for the frameshift mutation at codon 6 or compound heterozygotes for this mutation and for the codon 39 nonsense mutation develop thalassemia intermedia more frequently than thalassemia major. The frameshift mutation at codon 6 was associated with haplotype IX that contains the C-T change at position -158 5' to the G gamma globin gene implicated in high gamma chain production and thus the mild phenotype. In patients' homozygotes for codon 39 nonsense mutation, those with thalassemia intermedia more frequently had the two-gene deletion form of alpha-thalassemia, or functional loss of the alpha 2 gene as compared with those with thalassemia major. In a few siblings with thalassemia major and intermedia, the thalassemia intermedia syndrome correlated with the presence of the -alpha/-alpha genotype. No cause for the mild phenotype was detected in the majority of patients who had not inherited either haplotype IX or alpha-thalassemia.

Prenatal diagnosis of beta-thalassaemia by second-trimester chorionic villus sampling.

In this study we evaluated the feasibility of second-trimester transabdominal chorionic villus sampling for prenatal diagnosis of beta-thalassaemia in 80 pregnancies at risk presenting in the second trimester at the Antenatal Service. Sampling was carried out from 13 to 20 weeks and was successful in all cases. The amount of chorionic villi obtained varied from 10 to 40 mg, which was sufficient to make fetal diagnosis by oligonucleotide analysis within 10 days from sampling in all cases. No fetal losses occurred. From these results we conclude that transabdominal chorionic villus sampling is a useful procedure for prenatal diagnosis of beta-thalassaemia in those couples presenting after the first trimester.