Gene therapy. The legacy of Waclaw Szybalski.

Seminal demonstration of the possibility of stable genetic modification of mammalian cells performed by Waclaw and Elisabeth Szybalski opened the doors for gene therapy, the term coined by Waclaw Szybalski already in 1962. In the next 60 years, numerous tools for gene delivery have been developed and applied for clinical research, culminating in the registration of several genetic therapies in Europe and the USA. Some of these strategies, aimed to treat severe combined immunodeficiencies, inherited forms of blindness, spinal muscular atrophy, some cancers, and genetic anemias, are the real hope for patients suffering from previously incurable diseases or the ones whose treatment was not effective. On the approaching 60th anniversary of gene therapy, combined with the 100th anniversary of the birth of Professor Waclaw Szybalski (September 9th, 1921), who passed away on December 16, 2020, here I present the summary of the most important aspects of clinical applications of genetic therapies.

[The History of Beta Thalassaemia in Sardinia: The Contribution of the Italian Schools of Pediatrics] / La storia della beta talassemia in Sardegna: Il contributo delle Scuole Italiane di pediatria

Beta thalassaemia represents one of the most common autosomal recessive disorders worldwide. High prevalence is present in the Mediterranean, Middle East and Far East. The highest incidences are reported in Cyprus, South East Asia and Sardinia and are most likely related to the selective pressure from Pl. falciparum, the causative agent of malaria. In Sardinia, because of the health relevance of beta thalassaemia and haemoglobinopathies and after the publication of the first scientific research on Cooley's anaemia, important Schools of Paediatrics and Clinical Genetics have been set up, which have contributed to defining diagnostic criteria, therapeutic and preventive measures (especially, newborn screening). The aim of the present study is to examine the results of the first scientific research made by the Sardinian Schools of Paediatrics and Clinical Genetics, from 1929 to 1957.

2,000 Year old ß-thalassemia case in Sardinia suggests malaria was endemic by the Roman period.

OBJECTIVES: The island of Sardinia has one of the highest incidence rates of ß-thalassemia in Europe due to its long history of endemic malaria, which, according to historical records, was introduced around 2,600 years ago by the Punics and only became endemic around the Middle Ages. In particular, the cod39 mutation is responsible for more than 95% of all ß-thalassemia cases observed on the island. Debates surround the origin of the mutation. Some argue that its presence in the Western Mediterranean reflects the migration of people away from Sardinia, others that it reflects the colonization of the island by the Punics who might have carried the disease allele. The aim of this study was to investigate ß-globin mutations, including cod39, using ancient DNA (aDNA) analysis, to better understand the history and origin of ß-thalassemia and malaria in Sardinia. MATERIALS AND METHODS: PCR analysis followed by sequencing were used to investigate the presence of ß-thalassemia mutations in 19 individuals from three different Roman and Punic necropolises in Sardinia. RESULTS: The cod39 mutation was identified in one male individual buried in a necropolis from the Punic/Roman period. Further analyses have shown that his mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were U5a and I2a1a1, respectively, indicating the individual was probably of Sardinian origin. CONCLUSIONS: This is the earliest documented case of ß-thalassemia in Sardinia to date. The presence of such a pathogenic mutation and its persistence until present day indicates that malaria was likely endemic on the island by the Roman period, earlier than the historical sources suggest.

[Macciotta's disease and the biography of Giuseppe Macciotta]. / La malattia di Macciotta e la biografia di Giuseppe Macciotta.

Giuseppe Macciotta directed the Pediatric Clinic of Cagliari in Sardinia. He dedicated his work to the study of pathology present in Sardinia and the Mediterranean basin area, He identified a variant of the ß thalassemia major (homozygote Cooley's disease). He defined this variant as subchronic erythroblastosis, referred to by many as Macciotta's disease. Subchronic erythroblastosis, whose symptoms include earlier onset and a more difficult course of illness, was characterized by appearance generally at the beginning of the second trimester of the baby's life, and a course of illness between 5 and 10 months and a fatal outcome. The picture of the illness was dominated by hyperemolysis, erythroblastemia, medullar erythroblastosis and hyperbilirubinemia. The rapid course of the illness did not even allow time to damage the skeleton, and thus produced the formation of typical skeletal alterations and cardiomegalia. In the years which followed, transfusional and precocious and rational therapies were carried out which permitted the abeyance of the debilitating course of Macciotta's disease. The symptomatology was interrupted before any picture of greater or lesser seriousness could be drawn.

Thirty-year experience in preventing haemoglobinopathies in Greece: achievements and potentials for optimisation.

OBJECTIVES: Beta thalassaemia major (ß-TM) and sickle-cell disease (SCD) are severe haemogobinopathies requiring life-lasting, advanced medical management. In the Mediterranean region, both conditions occur with high frequency. We assessed the efficacy of the National Program for the Prevention of Haemoglobinopathies in Greece during the last 30 yrs. METHODS: Data of affected births between 01/01/1980 and 31/12/2009 were collected in a nationwide scale, and expected vs. observed rates of new births were calculated and compared. In a subpopulation of affected births of Greek origin, the causes for occurrence of the new affected birth were also collected and analysed. RESULTS: Overall, the reduction in new cases was 81.1% and 84.6% for ß-TM and SCD, respectively. For ß-TM, a constant declining trend was recorded over the 30-yr period, whereas for SCD, a transient reversal was observed in the mid-1990s probably due to the significant influx of immigrants of African origin. Programme failure was 2.2 times more common among new ß-TM births of Greek origin compared to new SCD cases (P < 0.001). Unawareness and parental choice were more frequent in SCD compared to ß-TM (unawareness: OR = 1.4, P = 0.05, parental choice: OR = 1.9, P = 0.01). The main cause for programme failure was carrier misidentification and incorrect genetic advice for ß-TM and SCD, respectively. CONCLUSIONS: The ß-TM and SCD prevention programme in Greece has significantly reduced the numbers of new affected births. The outcomes could be optimised in groups of non-Greek origin, in carrier identification and by offering specialised genetic counselling.

[The problem of characterization of thalassemia intermedia in Italian medical research]. / Il problema della caratterizzazione della talassemia intermedia nella ricerca medica Italiana.

The paper reconstructs the investigations and the debates on thalassemia intermedia in Italy from 1925 to the end of the 1940s. It examines particularly the contribution of the studies of Ezio Silvestroni and Ida Bianco to the identification of clinical features and etiological bases of thalassemia intermedia and consequently the nature of its relationships with Cooley's disease.

[Prevention of mediterranean anemia in Latium: Ida Bianco archives]. / La prevenzione dell'anemia Mediterranea nel Lazio: i documenti dell'archivio Ida Bianco.

Mediterranean anemia or beta-thalassemia is a hereditary syndrome characterized by a severe defect in haemoglobin production and an altered morphology of red blood cells. Homozygous condition for beta-thalassemia is characterized by short survival. Heterozygous condition is clinically found in adolescence and is characterized by a less aggressive phenotype. Ida Bianco, with her husband Ezio Silvestroni, has conducted a long struggle for beta-thalassemia prevention in Italy. They were the first to draw up an accurate map of the distribution of thalassemia in Italy and to conceive and implement a campaign against this genetic disease by the development of annual screening on at-school teenagers and pre-marriage prevention. Here we focused on the analysis of Ida Bianco's archives concerning screenings conducted on middle-schools in the Latium by the "Centro Studi della Microcitemia" of Rome from 1975 up to today. The results of the thirty-years prevention work in the Latium will be described.

[Epistemology and b-thalassemia]. / Epistemologia dell'anemia mediterranea nella storia della pediatria.

In this work the author examines the epistemological pathway to the study, diagnosis and therapy of b-thalassemia, serious and very frequent genetic disease in the Italian and Sardinian population know to paediatricians since 1925. The author critically explores the historical approaches to the comprehension of the disease, the phenotype characteristics, firstly described in Italy in 1929, and its familiarity, also described from several authors in the same years. The frequency and the variability of the disease in the population were poorly understood, partly because haematology was still under development and partly for the presence in the patients and in the general population of confounding symptoms and diseases. The hereditary transmission according to Mendelian laws was applied only to the study of few phenotype characteristics, in the attempt to limit the familiar transmission from the long surviving patients. For over 50 years the disease was considered lethal and there were not studies on the real efficacy of the available treatments.

From splenic anemia in infancy to microcythemia. The Italian contribution to the description of the genetic bases of Thalassemia.

This article traces out part of the history of studies of the genetic bases of thalassemia carried out in Italy. In particular it illustrates the research and discussions that between the late 1920s and the second half of the 1940s led to the description of the genetic basis of beta-thalassemia. The article encountered by Italian research, explaining why, despite the large number of thalassemia cases and data collected for this disease, Italian researchers succeeded in demonstrating its Mendelian transmission only at the same time as the US researchers.

The beta+-IVS-I-6 (T-->C) mutation accounts for half of the thalassemia chromosomes in the Palestinian populations of the mountain regions.

A study of the spectrum of beta-thalassemia mutations in the southern part of the West Bank of the Palestinian Authority revealed the presence of 10 different beta-globin mutations. The study included 41 patients and 54 carriers of beta-thalassemia and sickle cell anemia. The spectrum of mutations observed was typically Mediterranean. However, their relative frequencies was unique. The predominant allele was IVS-I-6 (T-->C), with an exceptionally high frequency of 48.5% for this mutation. The homozygous IVS-I-6 patients had widely variable clinical presentations, from typical transfusion-dependent thalassemia major to non-transfusion-dependent thalassemia intermedia phenotype. Since it is so widespread in these West Bank populations, the IVS-I-6 mutation may date back to ancient times. The nonsense mutation at codon 37 (G-->A) was found at a relatively high frequency of 11.3%, supporting the hypothesis that it originated in this region. The other mutations, at decreasing frequencies ranging from 9.5-1.5%, were: IVS-I-110 (G-->A), frameshift codon 5 (- CT), IVS-I-1 (G-->A), IVS-II-1 (G-->A), Hb S [beta6(A3)Glu-->Val], frameshift codons 8/9 (+G), codon 39 (C-->T), and -30 (T-->A). Our findings will improve health care for the Palestinian population, and also has implications for the study of the origin and spread of thalassemia in the Middle East.

History and origin of beta-thalassemia in Turkey: sequence haplotype diversity of beta-globin genes.

In the present study we report the sequence haplotypes associated with 22 beta-globin gene mutations present in Turkey. Nine nucleotide polymorphisms and an (AT)xTy motif located at the 5' end of the beta-globin gene form the sequence haplotypes that were investigated in 204 unrelated beta-thalassemia and wild-type chromosomes from Turkey. Twelve sequence haplotypes were observed in the chromosomes analyzed and haplotypic heterogeneity was found in the wild-type beta-globin genes. Samples from the Black Sea region demonstrated a remarkable level of haplotypic heterogeneity in contrast to the homogeneity present in Central Anatolian samples. Of the 22 beta-globin mutations analyzed, 18 were related with single sequence haplotypes. This simple association led to the attempt to determine the origin of these mutations by comparing their frequencies in Turkey with those in other countries and/or the world distribution of the haplotypes carrying them. However, the presence of several exceptions for the "one haplotype/one mutation" rule showed that the beta-globin gene cluster is far from static. Each of the IVS-I-110 (G-->A), Cd 39 (C-->T), IVS-I-6 (T-->C), and -30 (T-->A) beta-globin mutations was associated with a minimum of two sequence haplotypes. This fact is best explained by the likelihood of strong recombination mechanisms taking place, rather than by assuming multiple origins for each of these alleles. According to our results, malarial selection for the oldest beta-thalassemia allele in Anatolia (i.e., IVS-I-110 G-->A) may have occurred between 6500 and 2000 B.C. From that date on, most of the common beta-thalassemia mutations in Turkey were established, and by the 13th century A.D. most of them were brought to frequencies close to those observed at present.

Sequence analysis reveals a beta-thalassaemia mutation in the DNA of skeletal remains from the archaeological site of Akhziv, Israel.

beta-Thalassaemia is manifested by severe anaemia and extensive bone pathology. Similar pathology may also result from other forms of anaemia. To clarify the precise cause, we performed DNA analyses on archaeological remains of a child with severe bone pathology. We found homozygosity for frameshift in codon 8 of beta-globin, causing a beta-null phenotype. Paradoxically, the child died when eight years old, whereas such patients are transfusion dependent from early infancy. An infrequent polymorphic marker in the child's DNA, and information from present-day patients, indicated that amelioration of the clinical condition was due to elevated fetal haemoglobin production. Thus this analysis provided not only precise diagnosis of a genetic disease but also allowed clarification of the molecular mechanism underlying the clinical presentation.