Angela Belsito

γ1- and γ2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3′ terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (α1) and two basic (β1 and β2) proteins of ∼57–60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named γ1- and γ2-syntrophins. The human γ1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human γ2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the γ1-syntrophin gene to human chromosome 8q11 and the γ2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes

Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Identification and characterization of a novel member of the dystrobrevin gene family

A new member of the dystrobrevin gene family was identified using a bioinformatics approach. Sequence analysis indicates that this gene, named DTN‐B, is highly homologous to the rabbit A0, the previously described dystrobrevin (DTN), Torpedo 87 kDa and to the C‐terminus of dystrophin. The coiled‐coil domain, shown to be the site of interaction between dystrobrevins and dystrophin, is highly conserved. Immunostaining studies indicate that DTN‐B and DTN expression is absent in affected muscle fibers from DMD patients and carriers.

THE FOURTH COMPONENT OF THE SARCOGLYCAN COMPLEX

© 1997 Federation of European Biochemical Societies.

Erythrocyte genotyping for transfusion-dependent patients at the Azienda Universitaria Policlinico of Naples

BACKGROUND AND OBJECTIVES Although minor erythrocyte antigens are not considered clinically significant in sporadic transfusions, they may be relevant for multi-transfusion patients. When serological assay is not conceivable, molecular genotyping allows predicting the red blood cell phenotype, extending the typing until minor blood groups. The aim of this study was to evaluate the utility of blood group genotyping and compare the molecular typing of erythrocyte antigens with the established serological methods. MATERIALS AND METHODS We selected 225 blood donors and 50 transfusion-dependent patients at the Division of Immunohematology of the Second University of Naples. Blood samples were analyzed with NEO Immucor automated system and genotyped for 38 red blood cell antigens and phenotypic variants with the kit HEA BeadChip™. The comparative study was conducted for RhCE and Kell antigens whose typing is available with both methods. RESULTS We observed a good correlation between serological and molecular methods for donors that were concordant for 99.5% (224/225) and discordant for 0.5% (1/225). Patients resulted concordant only for 46.0% (23/50) and discordant for 54.0% (27/50); discrepancies were 46.0% (23/50) and 8.0% (4/50) for RhCE and Kell systems respectively. Through molecular genotyping we also identified polymorphisms in RhCE, Kell, Duffy, Colton, Lutheran and Scianna loci in donors and patients. CONCLUSIONS Blood group genotyping is particularly useful for poly-transfused patients. Molecular analysis confirms and extends serological test data and then allows us to obtain a better match. This molecular assay can be used in the future to prevent alloimmunization in transfusion-dependent patients.

Emerging strategies of blood group genotyping for patients with hemoglobinopathies

Red cell alloimmunization is a serious problem in chronically transfused patients. A number of high-throughput DNA assays have been developed to extend or replace traditional serologic antigen typing. DNA-based typing methods may be easily automated and multiplexed, and provide reliable information on a patient. Molecular genotyping promises to become cheaper, being not dependent on serologic immunoglobulin reagents. Patients with hemoglobinopathies could benefit from receiving extended genomic typing. This could limit post transfusional complications depending on subtle antigenic differences between donors and patients. Patient/donor compatibility extended beyond the phenotype Rh/Kell may allows improved survival of transfused units of red blood cells (RBC) and lead to reduced need for blood transfusion and leading to less iron overload and reduced risk of alloimmunization. Here we discuss the advantages and limitations of current techniques, that detect only predefined genetic variants. In contrast, target enrichment next-generation sequencing (NGS) has been used to detect both known and de novo genetic polymorphisms, including single-nucleotide polymorphisms, indels (insertions/deletions), and structural variations. NGS approaches can be used to develop an extended blood group genotyping assay system.