Diagnostic potential of hepcidin testing in pediatrics.

OBJECTIVES: Hepcidin, a peptide hormone released by hepatocytes into circulation is the main regulator of dietary iron absorption and cellular iron release. Although commercial tests are available, assay harmonization for hepcidin has not been yet reached, making reference intervals and consequent clinical decisions still elusive for each assay and specific population. The aim of this study is to set up hepcidin measurement in pediatric age and to investigate its potential usefulness in the diagnosis and management of iron disorders in children. METHODS: Serum hepcidin was measured by using an automated commercial immunoassay. Reference values were obtained from 86 healthy children. Hepcidin was then evaluated in 52 children with diseases where this hormone was expected to be differently regulated. RESULTS: Hepcidin values were 43.6 ng/mL median; 32-52.7 1-3 q: in males and 36.4 ng/mL median; 28.5-45.7 1-3 q: in females (P = 0.039). Hepcidin was significantly higher in postpubertal normal females than in normal males. Hepcidin resulted up-regulated in anemia of chronic disease of children affected by systemic Juvenile Idiopathic Arthritis and decreased after treatment with anakinra, an anti-interleukin-1 receptor antagonist. In iron deficiency anemia patients on oral iron supplementation and in ß-thalassemia subjects, hepcidin levels were similar to those found in healthy subjects. CONCLUSIONS: This study sets up reference values for pediatric population and shows that in normal controls serum hepcidin react differently to puberty in females vs. males. In addition, it suggests that serum hepcidin may discriminate microcytic inflammatory anemia of Juvenile Idiopathic Arthritis from iron deficiency anemia. Overall these findings may represent a helpful tool for future studies tailored to understand the role of hepcidin in management of iron disorders in children.

The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype.

BACKGROUND: The high mutational heterogeneity of hemophilia A is a challenge for the provision of genetic services. We plan to identify the mutation in patients with hemophilia A in order to create a confidential national database of mutations for the optimization of genetic services in Italy. DESIGN AND METHODS: The factor VIII gene (F8) was analyzed in 1296 unrelated patients with hemophilia A using screening methods for intron 22 and 1 inversions and rare mutations (denaturing high performance liquid chromatography, conformation sensitive gel electrophoresis) and/or direct sequencing. RESULTS: F8 mutations were identified in 874 (89%), 146 (89%), and 133 (94%) families with severe, moderate, or mild hemophilia A, respectively. Mutations predicting a null allele were responsible for 80%, 15%, and less than 1% of cases of severe, moderate, or mild hemophilia A, respectively. About 40% of missense and nonsense mutations occurred at a CpG site, arginines being most frequently affected. Of the small deletions or insertions, 29% occurred at one of two stretches of adenines, codons 1191-1194 (8As) and 1439-1441 (9As). Overall, these "hotspots" accounted for 31% of the point mutations in the patients with hemophilia A. Inhibitors developed in 22% of the patients with severe hemophilia A, 8% of those with moderate disease and in 4% of patients with mild hemophilia A. Patients who had severe hemophilia A and mutations predicting a null allele developed inhibitors more frequently (22 to 67%) than patients with missense mutations (5%). CONCLUSIONS: We report a wide spectrum of mutations in a large national database. The type of mutation was a strong predictor of the clinical phenotype. This database is expected to considerably improve the genetic counselling and medical care of families with hemophilia A in Italy.

Familial nonrandom inactivation linked to the X inactivation centre in heterozygotes manifesting haemophilia A.

A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common - often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

Exon skipping partially restores factor VIII coagulant activity in patients with mild hemophilia A with exon 13 duplication.

Ectopic mRNA was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in patients with duplication of F8 gene exon 13, a mutation which has been demonstrated to be a cause of mild hemophilia A in 32% of Northern Italian subjects. Two different transcripts originate from mutated genomic DNA, due to alternative splice processes. The larger-sized transcript contains both duplicated exons 13, the smaller one contains only one exon 13. The residual FVIII:C activity which accounts for the mild hemophilia A phenotype derives from the latter transcript.

Duplication of exon 13 causes one third of the cases of mild hemophilia A in northern Italy.

A rearrangement of exon 13 in the factor VIII gene has been identified as the causative mutation in 32% of Northern Italian patients with mild hemophilia A. We have demonstrated that all share a common haplotype, thus suggesting that the mutation likely occurred in a single ancestor. To date, no predominant mutation has been identified in mild hemophilia A, therefore it would be extremely useful to carry out more extensive studies to ascertain whether the mutation is confined to northern Italy.

Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity.

The interplay between impaired protein biosynthesis and/or function caused by missense mutations, particularly in relation to specific protein regions, has been poorly investigated. As model we chose the severe p.Y450C mutation in the carboxyl-terminal region of coagulation factor IX (FIX) and, by expression of a panel of recombinant variants, demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminal mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts.

Unusual presentation of haemophilia in two paediatric patients.

Haemophilia A is a rare X-linked recessive bleeding disorder caused by deficiency or functional defects in coagulation factor VIII (FVIII). Here, we report two cases of challenging diagnosis of haemophilia A because of unusual presentation. The first case is a 10-month-old female, admitted to our hospital because a neck mass appeared within the previous 24 h, who had a past medical history consistent with recurrent spontaneous haematomas but no family history of bleeding disorders. Despite several radiological evaluations, only the histology of the mass defined the presence of a haematoma. Chromosomal analysis revealed a normal female karyotype and a de-novo mutation into the FVIII intron 22 associated with a skewed X chromosome inactivation. The second case is a male neonate with a history of seizures who underwent brain MRI that showed a suspicious vascular malformation on the quadrigeminal cistern, causing cerebellum compression and hydrocephalus. The clinical conditions of the child progressively worsened and blood tests revealed a severe deficit of FVIII levels. The radiological images were re-evaluated; vascular anomalies were excluded and the diagnosis of haematoma was made. Family history was negative for coagulation disorders. Molecular studies revealed a rearrangement of the FVIII gene involving intron 22. The haemophilia A diagnosis can be challenging. Lack of family history, difficulties in detecting haematomas by imaging techniques, female sex and neonatal age represent misleading factors that can delay the diagnosis.

Molecular cytogenetic definition of a translocation t(X;15) associated with premature ovarian failure.

OBJECTIVE: To characterize the breakpoints of a t(X;15) found in a woman with premature ovarian failure (POF). DESIGN: Case report. SETTING: Molecular and cytogenetics unit in a university-affiliated hospital. PATIENT(S): A 19-year-old infertile woman presenting with a normal female phenotype but primary amenorrhea. INTERVENTION(S): Molecular cytogenetic analyses and genetic counseling. MAIN OUTCOME MEASURE(S): Translocation t(X;15) defined by fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). RESULT(S): Chromosome and FISH analysis revealed 46,XX, t(X;15)(Xq22.1;p11); the active X was translocated and had been inherited from her mother. Detailed molecular characterization by FISH showed that the NXF5 (nuclear RNA export factor 5) gene was contained in the clone spanning the breakpoint on the X chromosome. CONCLUSION(S): The NXF5 gene is an appealing candidate for POF because it shows functional homology with the FMR1 (fragile X mental retardation 1) gene. Further analyses of its expression as well as mutation screening in other POF patients will help to elucidate its role.

Mosaic ring Y chromosome in two normal healthy men with azoospermia.

OBJECTIVE: To better define by molecular and cytogenetic techniques ring Y chromosomes detected in 2 infertile men. DESIGN: Case report. SETTING: Molecular genetics/cytogenetics unit in a university hospital. PATIENT(S): Two infertile men with azoospermia, presenting a normal male phenotype with complete masculinization. INTERVENTION(S): Karyotype and genetic counseling. MAIN OUTCOME MEASURE(S): Metaphases were studied by standard G- and Q-banding; fluorescent in situ hybridization and PCR were performed to analyze specific Y chromosome regions. RESULT(S): Chromosomal analysis detected a mosaicism with a Y chromosome ring cell line in 92% (patient 1) and 95% (patient 2) of the metaphases, coexisting with a 45,X cell line in the remaining metaphases. In patient 1, PCR analysis showed the presence of AZFa region and a partial deletion of AZFb region; AZFc region was deleted. In patient 2 all three AZF regions were deleted. CONCLUSION(S): A 45,X/46,X,r(Y) mosaicism can be detected not only in patients with Ullrich-Turner syndrome and in patients with various degrees of genital ambiguity but also in men presenting a normal phenotype. Their azoospermia can be explained by partial or total deletion of AZF regions.

Small FVIII gene rearrangements in 18 hemophilia A patients: five novel mutations.

Hemophilia A (HA) is a disorder caused by mutations of the FVIII gene, which is located on the tip of the long arm of the X chromosome. In a cohort of 18 unrelated Italian patients affected with HA of varying severity, we performed mutational screening of the gene by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing of abnormal peaks. We identified five novel mutations and 9 previously reported DNA alterations. Two of the 9 previously reported alterations were each common to 3 unrelated patients. Six different mutations were characterized as missense alterations, while 8 were non-missense mutations. Among the new gene alterations, one created a stop codon, one consisted of an out-of frame deletion, and one was a splice-site mutation. The last two were missense alterations. In an attempt to better understand the causative effect of the mutations and the clinical variability of the patients, we investigated the consequences of each missense mutation and visualized the effect of the amino acid change on structural FVIII models.

Mutation analysis impact on the genetic counseling of sporadic hemophilia B families.

Previous studies have shown that hemophilia B (HB) is the result of several different mutations, mostly single nucleotide substitutions, in the factor IX (FIX) gene. In order to evaluate the impact of mutation analysis on genetic counseling in sporadic and uninformative HB familial pedigrees, we re-analyzed by the conformation sensitive gel electrophoresis (CSGE) technique 14 patients, previously studied by restriction fragment length polymorphisms (RFLPs). A single mutation was present within the FIX gene of each patient: 12 mutations were single base substitutions, 1 was a base insertion, and 1 was a four nucleotide deletion; 4/12 mutations have not been described so far. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied; 3/12 de novo events were found in maternal meioses with a 25% mutation rate. Identification of the genetic defect was also successfully applied to three prenatal diagnoses.