Valentina Brancaleoni

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.

Porphyrias at a glance: diagnosis and treatment

Porphyrias are a group of eight rare inherited metabolic disorders of heme biosynthesis pathway. Porphyrias are still underdiagnosed, although examinations of urine and plasma are first-line tests for detecting excess of porphyrins or heme precursors in suspected patients. Diagnosis, particularly for the acute forms, is essential to avoid precipitating factors and the use of triggering drugs. Mutation screening of family members is recommended to identify presymptomatic carriers and to prevent acute attacks. The therapeutic approach should be appropriate regarding specific forms of porphyria and treatment should be started promptly.

Congenital erythropoietic porphyria linked to GATA1-R216W mutation: challenges for diagnosis.

Congenital erythropoietic porphyria (CEP) is a rare genetic disease that is characterized by a severe cutaneous photosensitivity causing unrecoverable deformities, chronic hemolytic anemia requiring blood transfusion program, and by fatal systemic complications. A correct and early diagnosis is required to develop a management plan that is appropriate to the patients needs. Recently only one case of X‐linked CEP had been reported, describing the trans‐acting GATA1‐R216W mutation. Here, we have characterized two novel X‐linked CEP patients, both with misleading hematological phenotypes that include dyserythropoietic anemia, thrombocytopenia, and hereditary persistence of fetal hemoglobin. We compare the previously reported case to ours and propose a diagnostic paradigm for this variant of CEP. Finally, a correlation between phenotype variability and the presence of modifier mutations in loci related to disease‐causing gene is described.

Laboratory diagnosis of thalassemia

The thalassemias can be defined as α‐ or β‐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 β‐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 β‐thalassemia carriers, whereas a slight to relevant reduction is usually observed in α‐carriers. HbA2 determination is the most decisive test for β‐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 β‐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.

Multiplex ligation-dependent probe amplification: a novel approach for genetic diagnosis of Porphyria.

Porphyrias are disorders caused by the genetic defects of enzymes of the heme pathway and are characterized by such a wide genetic heterogeneity that even the molecular analysis is not always decisive for a correct diagnosis. In the past few years, deletion with a size range of few kilobase pairs have been reported. These peculiar mutations, missed by both sequencing and cytogenetic techniques, have been identified by time consuming and technically demanding methods. To provide a rapid and sensitive method for the detection of deletions responsible for porphyria, we successfully designed and tested seven chemically synthesized probe sets specific for each heme gene and their flanking regions, to be used in multiplex ligation-dependent probe amplification technique.

Advances in understanding the pathogenesis of congenital erythropoietic porphyria

Congenital erythropoietic porphyria (CEP) is a rare genetic disease resulting from the remarkable deficient activity of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthetic pathway. This enzyme defect results in overproduction of the non‐physiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I. The predominant clinical characteristics of CEP include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. The severity of clinical manifestations is markedly heterogeneous among patients; and interdependence between disease severity and porphyrin amount in the tissues has been pointed out. A more pronounced endogenous production of porphyrins concomitant to activation of ALAS2, the first and rate‐limiting of the haem synthesis enzymes in erythroid cells, has also been reported. CEP is inherited as autosomal recessive or X‐linked trait due to mutations in UROS or GATA1 genes; however an involvement of other causative or modifier genes cannot be ruled out.

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‐chromsomal 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.

A novel large deletion and three polymorphisms in the FECH gene associated with erythropoietic protoporphyria.

Abstract Background: Erythropoietic protoporphyria (EPP) is known to be inherited in both autosomal dominant and recessive manners. A deleterious mutation in conjunction with a polymorphic wild type allele underlies the molecular basis of the dominant type. Methods: We report a patient with EPP who was found to have a novel large deletion [c.1-9628_67+2871del12566 bp] and three polymorphisms [c.1–251A>G, c.68-23C>T and c.315-48T>C] in trans to the deletion in his ferrochelatase (FECH) gene. Results: The combination of the deletion and the polymorphisms reduced his FECH activity level to 20% of control. Conclusions: It is conceivable that a homozygous state for this polymorphic haplotype might be sufficient to produce clinical phenotype of EPP. The boundary between autosomal dominant and autosomal recessive inheritance may not always be distinct. Clin Chem Lab Med 2009;47:44–6.

A puzzling mutation in congenital erythropoietic porphyria and an association with β-thalassaemia trait.

evidence base for PCA treatments. Furthermore, secondand third-line treatments were often not recorded, and when stated also varied greatly between individuals (see Table 1). Comments made by respondents confirm that PCAs are poorly understood and that clinicians face uncertainty in their diagnosis and management. Unfortunately, several dermatologists believed that PCAs are untreatable and histology is unhelpful. Trichologists generally offered supportive care and referred patients for diagnosis and treatment either to their GP or to a dermatologist. This is the first study to provide epidemiological data of PCA in U.K. general dermatology and trichology practice. These data highlight that PCAs are rare, and may even occur less frequently than reported, due to the possibility of nonresponse bias. Thus, gaining significant clinical experience in PCA management is limited outside the setting of a specialist hair clinic. Important investigations, such as diagnostic scalp biopsy, are often not performed despite recommendations to biopsy all cases of scarring alopecia. Our data suggest that PCAs are generally managed with difficulty. Prompt referrals to specialist hair clinics should be encouraged to enable the development of clinical expertise and promote recruitment into basic science and clinical research studies. Early diagnosis and effective management are essential to halt progression of these debilitating conditions, with further research urgently needed to identify effective treatment strategies. A national database of PCA cases would assist in gaining a better understanding of the natural history of each entity and help identify optimal management practices.

Molecular Basis of β-Thalassemia Intermedia in Erbil Province of Iraqi Kurdistan

Abstract β-Thalassemia intermedia (β-TI) is a clinical term describing a range of clinical phenotypes that are intermediate in severity between the carrier state and β-thalassemia major (β-TM). To characterize the molecular basis of β-TI in Erbil Province, Northern Iraq, 83 unrelated patients were investigated. Detection of β-globin gene mutations was carried out by reverse hybridization assay and direct gene sequencing. All patients were screened for the XmnI polymorphism by direct sequencing of HBG2 (Gγ promoter gene). Detection of α-globin gene deletions and triplication was carried out using the reverse hybridization assay. Four main molecular patterns were identified in association with the β-TI phenotype, namely: β+/β+ (38.5%), β+/β0 (21.6%), β0/β0 (31.3%), and β0/wild type (8.4%). IVS-I-6 (T > C) was the most frequently encountered mutation (55 alleles, 34.6%), followed by IVS-II-1 (G > A) and codon 8 (−AA); furthermore, we report for the first time from Iraq two β+ mutations, −87 (C > G) and 5′ untranslated region (5′UTR) +22 (G > A). The XmnI polymorphism was detected in 47.0% of patients, mainly in association with the β0/β0 genotype. The α-globin gene deletions were encountered in four cases, including one case with (– –FIL) double gene deletion, a report that is the first from our country. The α-globin gene triplication was detected in five of the seven heterozygous β-thalassemia (β-thal) patients. Similar to other Mediterranean countries, inheritance of mild β-globin mutations was the main molecular pattern underlying β-TI in our patients followed by the ameliorating effect of the XmnI polymorphism.