Laboratory diagnosis of thalassemia.

The thalassemias can be defined as α- or ß-thalassemias depending on the defective globin chain and on the underlying molecular defects. The recognition of carriers is possible by hematological tests. Both α- and ß-thalassemia carriers (heterozygotes) present with microcytic hypochromic parameters with or without mild anemia. Red cell indices and morphology followed by separation and measurement of Hb fractions are the basis for identification of carriers. In addition, iron status should be ascertained by ferritin or zinc protoporphyrin measurements and the iron/total iron-binding capacity/saturation index. Mean corpuscular volume and mean corpuscular hemoglobin are markedly reduced (mean corpuscular volume: 60-70 fl; MCH: 19-23 pg) in ß-thalassemia carriers, whereas a slight to relevant reduction is usually observed in α-carriers. HbA2 determination is the most decisive test for ß-carrier detection although it can be disturbed by the presence of δ-thalassemia defects. In α-thalassemia, HbA2 can be lower than normal and it assumes significant value when iron deficiency is excluded. Several algorithms have been introduced to discriminate from thalassemia carriers and subjects with iron-deficient anemia; because the only discriminating parameter is the red cell counts, these formulas must be used consciously. Molecular analysis is not required to confirm the diagnosis of ß-carrier, but it is necessary to confirm the α-thalassemia carrier status. The molecular diagnosis is essential to predict severe transfusion-dependent and intermediate-to-mild non-transfusion-dependent cases. DNA analysis on chorionic villi is the approach for prenatal diagnosis and the methods are the same used for mutations detection, according to the laboratory facilities and expertise.

X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria.

X-linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain-of-function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X-linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X-chromosomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc-finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X-chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild-type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin-containing peripheral blood fluorocytes. When the wild-type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP.

Clinical, biochemical and genetic characteristics of Variegate Porphyria in Italy.

Variegate Porphyria (VP) is an autosomal dominant disorder found worldwide but is rare in Italy. In this study we provide an overview of clinical, biochemical and genetic background of 33 Italian VP patients diagnosed in the last fifteen years. About 70% of patients had experienced clinical symptoms: 43.4% had photosensivity, 8.7% acute attacks and 47.8% both. Among the 33 patients, 14 different mutations were identified. Of these only 6 defects have been previously described in other countries and 8 are unique having been identified for the first time in Italy. Two of these, the c.851G>T and the c.1013C>G, were found in two and four unrelated families respectively. No mutation has been found in homozygosis and no significant correlation has been observed between specific clinical and biochemical manifestations and the type of mutation. In contrast, normal faecal protoporphyrin excretion was high predictive of silent phenotype. Normal urinary excretion of PBG and ALA, predicted absence of neurovisceral symptoms. This paper represents the first compilation of data on genotype-phenotype relation in Italian patients with VP.

Co-existence of two functional mutations on the same allele of the human ferrochelatase gene in erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an autosomal dominant disease with incomplete penetrance due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the final step of the heme biosynthetic pathway. The clinical phenotype of EPP results from co-inheritance of a mutated allele and a wild-type low-expressed allele of the FECH gene. To date, more than 88 different mutations have been identified in the FECH gene of patients with EPP. There are evidences suggesting that an entire haplotype (-251G, IVS1-23T and IVS3-48C) reduces allele expression. In this study, we searched for the -251A/G, IVS1-23C/T and IVS3-48T/C polymorphisms in two unrelated Italian families with EPP. In all the patients, carrying the -250G>C mutation in the promoter region, the IVS3-48C on the other allele showed apparent homozygosity and absence of Mendelian segregation. By RNA and long polymerase chain reaction analysis, we identified a deletion of 5576 bp (g12490_18067), including exons 3 and 4, in cis with the -250G>C mutation in the promoter.