La diagnostica molecolare della fibrosi cistica e delle malattie correlate a mutazioni del gene CFTR

Alberto Bonizzato*, Carlo Castellani
*Laboratorio Analisi Chimico-Cliniche ed Ematologiche, Azienda Ospedaliera di Verona

Biochimica Clinica: 2008; 32(2): 77-83 [Article in italian]

ABSTRACT
The molecular diagnosis of cystic fibrosis and CFTR-related disorders. Cystic fibrosis is the most frequent severe autosomal recessive disorder in Caucasian populations. There is great heterogeneity in the clinical manifestations of cystic fibrosis. Some patients may have a severe course with rapid progression of symptoms, while others have much milder or even atypical disease manifestations and still carry gene mutations. The gene responsible for the disease encodes for a chloride channel in the apical membrane of secretory epithelial cells. More than 1500 mutations have been identified in the cystic fibrosis gene, varying in their frequency and distribution. Very few mutations have a worldwide frequency above 0.1%, but some can reach high frequencies in selected populations. Current mutation screening tests range from the most commonly adopted mutation screening panels to extensive scanning protocols encompassing the whole coding region and exon-intron splice junctions. However, even the most extensive mutation screening tests fail to detect mutations in all cystic fibrosis alleles. Unidentified defects may lie in introns or in regulatory regions, which are not routinely investigated, or can be due to complex rearrangements difficult to detect at DNA level. Rearrangements can be optimally examined either by multiplex ligation-dependent probe amplification or quantitative multiplex polymerase chain reaction of short fluorescent fragments. The analysis of transcripts from nasal brushing is utilized to detect intronic mutations affecting RNA splicing.

Possible role of C-erb B-2 and transforming growth factor b1 in malignant transformation of hepatocytes
Olfat Shaker*, Ahmed Hashem, Lamia Mansour, Osama Taha
*Departments of Medical Biochemistry, Faculty of Medicine, University of Cairo, Egypt

Biochimica Clinica: 2008; 32(2): 84-88 [Article in english]

ABSTRACT
To evaluate the expression of C-erb B-2 oncoprotein and transforming growth factor (TGF) β1 in polymorphonuclear cells and sera of chronic hepatitis C (HCV) and hepatocellular carcinoma (HCC) patients compared to healthy control subjects, thirty chronic HCV patients, thirty HCC patients and thirty healthy control subjects were enrolled in the study. Measurements of TGF-β1 and C-erb B-2 were done by reverse transcription-polymerase chain reaction and sandwich enzyme immunoassays. A significant overexpression of TGF-β1 and neu-oncoprotein C-erb B-2 was detected in HCV and HCC patients compared to control subjects. Also, TGF-β1 and C-erb B-2 concentrations were higher in HCC than in HCV patients (P <0.001). The mean serum concentration of TGF-β1 was increased with increased degree of fibrosis (P = 0.003). Statistically significant differences (P <0.001) in mean serum concentrations of C-erb B-2 were observed between grade I vs grade III and grade II vs grade III in HCC patients. It is suggested that TGF-β1 and C-erb B-2 may be associated with the malignant transformation of hepatocyte or the progression of HCV-associated HCC.

Un approccio proteomico alla diagnosi molecolare delle amiloidosi sistemiche
Francesca Lavatelli*, Giovanni Palladini, Laura Obici, Gabriele Sarais, Vittorio Bellotti, Remigio Moratti, Riccardo Albertini, Giampaolo Merlini
*Centro per lo Studio e la Cura delle Amiloidosi Sistemiche, Laboratori di Biotecnologie Università di Pavia, Pavia

Biochimica Clinica: 2008; 32(2): 89-93 [Article in italian]

ABSTRACT
A proteomic approach to the molecular diagnosis of systemic amyloidoses.Protein deposition as amyloid is the basis of a group of diseases that have an enormous social and medical impact. In systemic forms, amyloid deposition is associated with dysfunction of vital organs and molecular typing of the deposits is necessary for diagnosis and treatment. More than 10 different proteins are known to be causative agents of systemic amyloidosis. The traditional diagnostic approach is multidisciplinary, but sometimes fails to identify the correct type. A proteomic approach could help in diagnosis, through the direct molecular characterization of fibrillar proteins in tissues, and cast insights into the mechanisms of tissue damage. We designed a proteomic strategy based on two-dimensional electrophoresis (2D-PAGE) followed by matrix assisted laser desorption-ionization mass spectrometry (MALDI-TOF MS) analysis and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients with different types of systemic amyloidosis. A rapid methodology to prepare samples for high-grade 2D-PAGE analysis from small amounts of fat tissue was developed. Significant differences in the proteome of adipose tissue were observed between controls and patients, consisting in novel spots in regions of patient gels compatible with the expected migration of amyloidogenic proteins, with a characteristic distinct appearance according to the amyloid type. In the AL and in the ATTR cases analyzed by MS, these spots proved to be formed by the predicted amyloidogenic proteins, thus confirming the diagnosis. This practical and feasible approach provides a reliable way to directly characterize protein deposits in patients with amyloid disease.

Il consolidamento della diagnostica proteica

Maria Stella Graziani*, Gabriella Righetti, Maddalena Marini, Lorena Zardo, Paolo Rizzotti
*Laboratorio di Analisi Chimico Cliniche ed Ematologiche, Ospedale Civile Maggiore, Azienda Ospedaliera di Verona

Biochimica Clinica: 2008; 32(2): 94-101 [Article in italian]

ABSTRACT
The consolidation of the protein measurementIn the last years, under the pressure of cost containment and greater efficiency, the clinical laboratory has evolved by consolidating a number of measurements on a single analytical platform. This laboratory area, named “corelab”, usually operates 24 h per day and is characterized by high throughput and short turnaround time. This organisation is considered very successful but, because of its capacity of performing very rapidly an elevated number of measurements, it seems less capable of assuring attention to the appropriateness of requests and to the clinical utilization of laboratory data. This paper is aimed to examine some of the consequences connected to the consolidation of specific protein measurement in the corelab area. These laboratory data typically require to be interpreted together with the results of protein electrophoresis pattern and reported with comments and/or suggestions especially when patients with monoclonal components are involved. For these patients a consultancy with clinicians is quite often necessary. From the point of view of consolidation, proteins can be divided into three groups: proteins that can be consolidated because they do not need to be interpreted on the basis of electrophoresis results and are easily measured by turbidimetry (albumin, haptoglobin); proteins that need to be interpreted on the basis of serum or urine electrophoretic patterns (i.e. immunoglobulins), the consolidation of which is not recommended because they can slow down the corelab turnaround time; and proteins with poor analytical performance on turbidimetry (i.e. low concentration proteins) that cannot be consolidated. In conclusion, the consolidation of specific protein measurements cannot always be recommended. The pros and cons of such a decision also depend on the amount of activity and on the type of laboratory as well.

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