Carmelilia De Bernardo

A novel missense mutation in SLC40A1 results in resistance to hepcidin and confirms the existence of two ferroportin-associated iron overload diseases

Ferroportin‐related iron overload disease differs from haemochromatosis in that it has a dominant mode of inheritance and is usually associated with macrophage iron sequestration. However, it is thought that mutations with opposite effects on protein functions, i.e. loss‐of‐function versus gain‐of‐function mutations, are responsible for variable phenotype presentations. The present study investigated the functional relevance of a novel ferroportin variant: the c.1502 A>G transition, which changes amino acid 501 from tyrosine to cysteine (p.Y501C). This novel variant was identified in a pedigree originating from Central Italy and, although an intra‐familial phenotype heterogeneity was observed, it co‐segregated with an iron overload picture similar to that of the HFE‐related typical haemochromatosis. In cultured cells, the p.Y501C mutant protein reached the plasma membrane and retained a full iron export ability. By contrast, it was resistant to inhibition by hepcidin. These findings confirm that certain ferroportin mutations compromise the activity of hepcidin in iron homeostasis, mimicking hepcidin deficiency as described in all types of hemochromatosis.

Clinical phenotype and molecular characterization of 6q terminal deletion syndrome: Five new cases

Mental retardation, facial dysmorphisms, seizures, and brain abnormalities are features of 6q terminal deletions. We have ascertained five patients with 6q subtelomere deletions (four de novo, one as a result of an unbalanced translocation) and determined the size of the deletion ranging from 3 to 13 Mb. Our patients showed a recognizable phenotype including mental retardation, characteristic facial appearance, and a distinctive clinico‐neuroradiological picture. Focal epilepsy with consistent electroencephalographic features and with certain brain anomalies on neuroimaging studies should suggest 6q terminal deletion. The awareness of the distinctive clinical picture will help in the diagnosis of this chromosomal abnormality.

Autosomal-dominant Retinitis Pigrnentosa Associated with an Arg-135-Trp Point Mutation of the Rhodopsin Gene: Clinical Features and Longitudinal Observations

Purpose: To report the clinical and functional characteristics of patients affected with autosomal-dominant transmitted retinitis pigmentosa (adRP) from a large Italian pedigree in which a point mutation predicting the Arg-135-Trp change of rhodopsin was identified by polymerase chain reaction-single-strand conformation polymorphism analysis. Methods: Seven patients, ranging in age from 6 to 41 years, underwent a full clinical ophthalmologic evaluation, kinetic visual field testing, and electroretinographic testing. Results: In agreement with previous reports, this rhodopsin mutation yielded a particularly severe phenotype, both clinically and functionally. The evaluation of patients from this pedigree in the first and second decade of life demonstrated that retinal function is still electroretinographically measurable at least until 18 years of age, although reduced to 2% to 4% of normal. Longitudinal measures showed that the rate of progression of the disease was unusually high, with an average 50% loss per year of electroretinographic amplitude and visual field area with respect to baseline. Later in the course of the disease, macular function is also severely compromised, leaving only residual central vision by the fourth decade of life. Conclusions: The phenotype associated with mutations in codon 135 of the rhodopsin molecule appears to have an unusually high progression rate and yields an extremely poor prognosis. These distinctive features make the Arg-135-Trp phenotype substantially different from the general RP population, and also from many of the other adRP pedigrees with known rhodopsin mutations reported to date. Ophthalmology 1996,-103:1443-1452

Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major

The liver peptide hepcidin is the key regulator of iron homeostasis. This report shows that liver iron content and serum ferritin levels are greater in thalassemia patients with the −582G polymorphic change in the hepcidin promoter as compared with patients with the wild-type (A) sequence. This observation suggests that this change might influence the iron status of irregularly chelated thalassemic patients. See related perspective article on page 1185. Hepcidin is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide produced predominantly by hepatocytes. This molecule, encoded by the hepcidin antimicrobial peptide (HAMP) gene shows structural and functional properties consistent with a role in innate immunity. Moreover, as demonstrated in mice and humans, hepcidin is a major regulator of iron metabolism, and acts by binding to ferroportin and controlling its concentration and trafficking. In this study we investigated the influence that mutations in HAMP and/or hemocromatosis (HFE) genes might exert on iron metabolism in a group of poly-transfused thalassemic patients in preparation for bone marrow transplantation. Our results showed that the presence of the c.-582 A>G polymorphism (rs10421768) placed in HAMP promoter (HAMP-P) might play a role in iron metabolism, perhaps varying the transcriptional activation that occurs through E-boxes located within the promoter.

TFR2-related hereditary hemochromatosis as a frequent cause of primary iron overload in patients from Central-Southern Italy

OBJECTIVE Hereditary hemochromatosis (HH) is a common Mendelian disorder of iron metabolism. Eighty percent of northern Europeans descendant HH patients carry the same mutation (p.C282Y) in the HFE gene. Simultaneously, due to a founder effect, its frequency varies considerably between different populations. In Central-Southern Italy the prevalence of p.C282Y mutation is low and in several patients the disease has different causes. Four additional rarer forms have been described. Type 3 HH has been reported in about 50 families and no more than 30 TFR2 pathogenic mutations have been globally identified. The aim of this study is to assess the TFR2 role in non-HFE HH pathogenesis. STUDY DESIGN AND SETTING TFR2 sequence analysis was performed on 45 Italian patients without HFE mutations. RESULTS This study revealed TFR2 biallelic pathogenic mutations in 7/45 (15.6%) individuals. Moreover monoallelic TFR2 deleterious defects (18%) or polymorphisms with unclear meaning (36%) were identified. Besides, we recognized 10 novel variants and 9 described changes. CONCLUSION We believe this to be the largest series of type 3 HH patients described so far. Present findings support the hypothesis of a main role of the TFR2 gene in HH pathogenesis in those regions, such as Central-Southern Italy, where the p.C282Y frequency is low.

Molecular analysis of sarcomeric and non-sarcomeric genes in patients with hypertrophic cardiomyopathy

BACKGROUND Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder characterized by unexplained left ventricle hypertrophy associated with non-dilated ventricular chambers. Several genes encoding heart sarcomeric proteins have been associated to HCM, but a small proportion of HCM patients harbor alterations in other non-sarcomeric loci. The variable expression of HCM seems influenced by genetic modifier factors and new sequencing technologies are redefining the understanding of genotype-phenotype relationships, even if the interpretations of the numerous identified variants pose several challenges. METHODS AND RESULTS We investigated 62 sarcomeric and non-sarcomeric genes in 41 HCM cases and in 3 HCM-related disorders patients. We employed an integrated approach that combines multiple tools for the prediction, annotation and visualization of functional variants. Genotype-phenotype correlations were carried out for inspecting the involvement of each gene in age onset and clinical variability of HCM. The 80% of the non-syndromic patients showed at least one rare non-synonymous variant (nsSNV) and among them, 58% carried alterations in sarcomeric loci, 14% in desmosomal and 7% in other non-sarcomeric ones without any sarcomere change. Statistical analyses revealed an inverse correlation between the number of nsSNVs and age at onset, and a relationship between the clinical variability and number and type of variants. CONCLUSIONS Our results extend the mutational spectrum of HCM and contribute in defining the molecular pathogenesis and inheritance pattern(s) of this condition. Besides, we delineate a specific procedure for the identification of the most likely pathogenetic variants for a next generation sequencing approach embodied in a clinical context.

Longitudinal hormonal evaluation in a patient with disorder of sexual development, 46,XY karyotype and one NR5A1 mutation

Steroidogenic factor 1 (encoded by the NR5A1 gene) is a critical regulator of reproduction, controlling transcription of key genes involved in sexual dimorphism. To date, NR5A1 variants have been found in individuals with a 46,XY karyotype and gonadal dysgenesis, as well as with a wide spectrum of genital anomalies and, in some patients, with adrenal insufficiency. We describe evolution of gonadal function, from the neonatal period to puberty, in a patient with a 46,XY karyotype, a disorder of sexual development, and a mutation (c.691_699dupCTGCAGCTG) in the NR5A1 gene. The patient, ascertained at birth due to ambiguous genitalia, showed normal values of plasma testosterone in the late neonatal period. Evaluation of the hormonal profile over time indicated severe tubular testicular hypofunction suggestive for a 46,XY disorder of gonadal development. A comprehensive review of published reports of 46,XY and disordered sexual development related to the NR5A1 gene confirmed the clinical and hormonal variability in patients with NR5A1 mutations. Analysis of multiple data allowed us to define the most common features associated with NR5A1 mutations. We further confirmed the indication to perform NR5A1 screening in patients with 46,XY karyotype and disordered sexual development even when Müllerian structures appear to be absent and plasma testosterone levels are within the normal range for age.

Analysis of the miR-34a locus in 62 patients with familial cutaneous melanoma negative for CDKN2A/CDK4 screening

MicroRNAs are small non-coding RNAs, which inhibit expression of specific target genes at the post-transcriptional level and are often misregulated in human cancer. Among them, miR-34a is considered a tumor suppressor with a hypothetical role in melanoma tumorigenesis. In this work, 62 Italian index patients with familial melanoma and negative for CDKN2A/CDK4 screening were investigated for miR-34a germline mutations. Eight novel miR-34a sequence variants were identified at both the heterozygous (c.+259G>A, c.+424G>A, c.+1465C>T, c.+1769C>T, c.+2456T>G, c.+2603C>T, c.+2972T>A, c.+3069T>C) and homozygous (c.+424G>A, c.+1465C>T, c.+1769C>T) states. Molecular screening identified all nucleotide changes in a healthy population of 150 controls and demonstrated that they are common polymorphisms. However, statistically significant differences of allele and genotype frequencies were detected for c.+1465C>T and c.+1769C>T, and borderline values for c.+2456T>G. By stratifying patients by relevant clinical features (presence/absence of multiple primary melanoma, Breslow’s thickness, phototype and number of nevi), no significant findings were noted except for an association between the c.+424G>A (heterozygous individual GA) and multiple primary melanoma and phototype III–IV. Our preliminary study suggests that miR-34a, although having a role in late tumorigenesis, does not contribute to the inherited susceptibility to cutaneous melanoma. A function as phenotypic modulator in familial melanoma cannot be excluded.

Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP, BMP2, FTL and SLC40A1 genes.

Hereditary hemochromatosis (HH) is a heterogeneous disorder of iron metabolism. The most common form of the disease is Classic or type 1 HH, mainly caused by a biallelic missense p.Cys282Tyr (c.845G>A) mutation in the HFE gene. However, the penetrance of p.Cys282Tyr/p.Cys282Tyr genotype is incomplete in terms of both biochemical and clinical expressivity. Lack of penetrance is thought to be caused by several genetic and environmental factors. Recently, a lot of evidences on HH genetic modifiers were produced, often without conclusive results. We investigated 6 polymorphisms (rs10421768 in HAMP gene, rs235756 in BMP2 gene, rs2230267 in FTL gene, rs1439816 in SLC40A1 gene, rs41295942 in TFR2 gene and rs2111833 in TMPRSS6 gene) with uncertain function in order to further evaluate their role in an independent cohort of 109 HH type 1 patients. Our results make it likely the role of rs10421768, rs235756, rs2230267 and rs1439816 polymorphisms, respectively in HAMP, BMP2, FTL and SLC40A1 genes in HH expressivity. In addition, previous and our findings support a hypothetical multifactorial model of HH, characterized by a principal gene (HFE in HH type 1) and minor genetic and environmental factors that still have to be fully elucidated.

Disorders of sex development: a genetic study of patients in a multidisciplinary clinic

Sex development is a process under genetic control directing both the bi-potential gonads to become either a testis or an ovary, and the consequent differentiation of internal ducts and external genitalia. This complex series of events can be altered by a large number of genetic and non-genetic factors. Disorders of sex development (DSD) are all the medical conditions characterized by an atypical chromosomal, gonadal, or phenotypical sex. Incomplete knowledge of the genetic mechanisms involved in sex development results in a low probability of determining the molecular definition of the genetic defect in many of the patients. In this study, we describe the clinical, cytogenetic, and molecular study of 88 cases with DSD, including 29 patients with 46,XY and disorders in androgen synthesis or action, 18 with 46,XX and disorders in androgen excess, 17 with 46,XY and disorders of gonadal (testicular) development, 11 classified as 46,XX other, eight with 46,XX and disorders of gonadal (ovarian) development, and five with sex chromosome anomalies. In total, we found a genetic variant in 56 out of 88 of them, leading to the clinical classification of every patient, and we outline the different steps required for a coherent genetic testing approach. In conclusion, our results highlight the fact that each category of DSD is related to a large number of different DNA alterations, thus requiring multiple genetic studies to achieve a precise etiological diagnosis for each patient.