Stefania Murru

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction–restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

Illegitimate recombination produced a duplication within the FVIII gene in a patient with mild hemophilia A

We have characterized an unusual duplication of exon 13 within the factor VIII gene in a patient with a mild form of hemophilia A. This duplication was the result of a nonhomologous breakage and reunion event of two misaligned wild-type chromosomes. Sequence analysis of the breakpoint region revealed the presence of AT-rich sequences and possible topoisomerase I sites, whose involvement in several cases of illegitimate recombination has been postulated.

The C–T substitution in the distal CACCC box of the β-globin gene promoter is a common cause of silent β thalassaemia in the Italian population

This paper describes four families of Italian descent in each of which the propositus had the clinical phenotype of thalassaemia intermedia, resulting from the compound heterozygous state for high HbA2β thalassaemia and type I silent β thalassaemia. Direct sequencing on amplified DNA and/or oligonucleotide analysis detected, in all families but one, the compound heterozygous state for codon 39 nonsense mutation and the C‐T substitution at position – 101 in the distal CACCC box of the β‐globin gene promoter (βth–101). Members of these families who are heterozygous for high HbA2β thalassaemia showed the codon 39 nonsense mutation, while those with the clinical phenotype of silent β thalassaemia had the βth–101 mutation. In the remaining family, the propositus and one of his siblings had the compound heterozygous state for a molecularly undefined high HbA2β thalassaemia and the βth–101 mutation in combination with the triple α globin gene arrangement. These patients showed a more severe thalassaemia intermedia like clinical phenotype as compared to those with the same β‐globin genotype and a normal α‐globin gene arrangement. In the families investigated the βth–101 was always associated with haplotype I. A group of patients with thalassaemia intermedia from Southern Italy, either homozygous or heterozygous for haplotype I and in whom previous studies had failed to define the mutation in one of the β thalassaemia globin genes, were screened by oligonucleotide analysis for the presence of the βth–101. Three out of nine were positive. These results indicate that the βth–101 mutation is a common cause of the type I silent β thalassaemia phenotype in the Southern Italian population.

A β‐thalassaemia phenotype not linked to the β‐globin cluster in an Italian family

Summary. This paper describes a family of Central Italian origin in which three patients in two generations had either thalassaemia intermedia or a late presenting form of thalassaemia major. Sequence analysis of the patients’ UNA revealed that only one of the β‐globin genes was affected by a β‐thalassaemia mutation (the codon 39 nonsense mutation), the other being completely normal, apart from the complex rearrangement (‐T + ATA) at position −5305’ to the CAP site of the β‐globin gene, which has uncertain clinical significance. Haematologically, all these patients were characterized by unusually low HbF levels (1.8–7.3%) for a β‐thalassaemia major or intermedia phenotype. The mother of the two patients with thalassaemia intermedia was heterozygous for β‐thalassaemia (codon 39 nonsense mutation), while the father had thalassaemia‐like red cell indices, an increased α/nonα chain synthesis ratio, a slight increase of HbF and a low HbA2 level, but showed entirely normal β‐globin gene sequences, apart from the complex rearrangement (‐T +ATA) at position −530 5’ to the CAP site. One of the thalassaemia intermedia patients married a normal woman and they had a child with thalassaemia major who inherited only the codon 39 nonsense mutation but not the complex rearrangement at position −530. The clinical phenotype of thalassaemia‐intermedia or major in the patients from this family may be explained by postulating the inheritance of the double heterozygous state for β‐thalassaemia and for a mutation in a gene coding for an erythroid‐specific DNA binding protein which may impair the function of the normal β‐globin gene. Heterozygosity for this postulated mutation (father of the patients with thalassaemia intermedia) may result in the production of a β‐thalassaemia carrier state with normal HbA2 level.

Promoter mutations producing mild β‐thalassaemia in the Italian population

Summary. In this study we have investigated the molecular basis for a mild form of β‐thalassaemia in three patients of Italian descent. In two, belonging to different families and affected by a mild and late‐presenting form of thalassaemia major, direct sequencing of amplified DNA detected a C→T substitution at position −87 of the β‐globin gene in the compound heterozygous state either with codon 39 nonsense mutation or β+IVSI, nt 110 mutation. The −87 (C→T) mutation has been previously described, in combination with the β+IVSI, nt 110 mutation, in a single patient with thalassaemia intermedia. Both our patients showed a more severe phenotype as compared to that resulting from compound heterozygosity for a severe β‐thalassaemia mutation and another promoter mutation (−87, C→G) at the same position. In the third patient with the thalassaemia intermedia phenotype, we detected a novel promoter mutation, consisting in a C→A substitution at position −86, in combination with the codon 39 nonsense mutation. The results of this study indicate that different nucleotide substitutions affecting the proximal CACCC box of the β‐globin gene in combination with severe β‐thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late‐presenting thalassaemia major.

Rat tyrosine hydroxylase gene polymorphisms

The rat tyrosine hydroxylase gene (TH) from a panel of outbred and inbred rat strains has been analysed by Southern blotting, restriction-endonuclease mapping and direct sequencing of PCR-amplified products for detecting DNA polymorphisms. Five polymorphic sites have been characterized. This information may be used in pharmacogenetic studies to determine the influence of the TH gene in animal models of affective disorders and addictive behaviours.

A new ? chain variant hemoglobin A2-Corfu or ?2?2 116 Arg?Cys (G18), detected by ?-globin gene analysis in a Greek family

SummaryHemoglobin A2-Corfu or α2δ2116 Arg→Cys (G18) is a new δ chain variant detected in a family of Greek descent by direct sequencing of amplified DNA.

Affinities in the construction techniques of a unitary project: the coastal towers of the Asinara Island (Sardinia)

This study stems from the idea of highlighting the constructive affinities among the three towers of the Asinara Island, resulting from a unitary construction project. The research methodology identifies a multidisciplinary protocol based on a stratigraphic approach. With the aid of historical sources and petrographic and geochemical investigations, the sequence of the construction phases and stratigraphic units was identified for the towers. The results were compared in order to highlight common elements and significant differences in the typology and proportional relationships between the geometry and the constructive elements. The comparison also included material composition and masonry texture. The results highlight several similarities among the three towers and confirm the initial thesis: the imprint due to the unitary construction project was of major importance in terms of construction choices and binds the three towers in a common thread. DOI: http://dx.doi.org/10.4995/FORTMED2015.2015.1768