Search

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.

Highlights • Disease-causing missense mutations mainly impair protein biosynthesis and/or function. • The p.Y450C mutation in factor IX (FIX) provided a model to study their interplay. • The mutation in the carboxyl-terminus impairs both FIX protein secretion and activity. • The phenyl group at this relatively conserved position (c234) has a key role. • The differential effects have pathophysiological and evolutionary implications., 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.

Summary. Deficiency or dysfunction of factor IX FIX leads to haemophilia B (HB), an X-linked, recessive, bleeding disorder. On a molecular basis, HB is due to a heterogeneous spectrum of mutations spread throughout the F9 gene. In several instances, a cause-effect relation has been elucidated, in others predicted possibilities have been offered by crystallography inspection and by software-constructed models of the protein. The aim of this study was to contribute to the understanding of HB molecular pathology. The F9 missense mutations we identified in 21 unrelated Italian HB patients by direct sequencing of the whole F9 coding regions were inspected for the causative effect they provoked on the ensuing transcript, and on the protein structure. Each alteration was studied in order to: (i) characterize the defect on the basis of the nature of the mutation; (ii) identify the predicted defect that is induced in the gene and (iii) speculate about the potential, detrimental effects which upset the protein functionality through an idealized FIX model. The resulting data may further contribute to the comprehension of the mechanisms underlying the disease.

Summary. Factor VIII gene inversion of intron 1 has recently been reported to be the mutation responsible for haemophilia A in about 5% of severe cases. In our series of patients, which is made up of 77 Italian cases negative for intron 22 inversion, the mutation was found in three sporadic and in one familial patients, with an overall frequency of 5.2%. The carrier status of the patients’ female relatives was assessed by mutation analysis and showed that only two-thirds of cases could be considered truly sporadic. The germ-line origin of the mutation was investigated in the two sporadic families by haplotype analysis on genomic DNA of the patients’ maternal grandparents. These studies indicated that both mutation events had occurred in the germ cell lines of the patients’ healthy grandfather, suggesting that, as already demonstrated for the inversion of intron 22, the male germ cell line is more susceptible to the intrachromosome recombination which leads to the inversion of intron 1.

Summary. We describe 18 novel mutations, unreported in the Haemophilia A mutation Databases, that have been identified in a cohort of unrelated, Italian patients affected with haemophilia A (HA). Screening of the factor VIII gene (FVIII) was performed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. Eight mutations were characterized as non-missense alterations, and the remaining 10 were missense mutations. Heterozygosity for the identified mutations was observed in the female relatives of patients belonging to eight families with sporadic cases. In an attempt to understand better the causative effect of the mutations and the clinical variability of the patients, missense mutation consequences were investigated for: (1) the nature of the new amino acid; (2) the location of the substituted amino acid within crystallographic and theoretical models; and (3) the degree of conservation of the native residue in factor VIII (FVIII) protein and FVIII-related protein family aligned sequences. These research tools have provided evidence that the mutations we describe involve residues that were conserved, at least in FVIII proteins, in all the species we compared.

Summary This article describes prenatal diagnosis (PND) of haemophilia B (HB) within the framework of Italian haemophilia centres and genetics laboratories. The study details the experience from six haemophilia genetic centres (three in the North, one in the Centre and two in the South of Italy) and summarizes the different techniques used to perform PND of HB during the last 15 years. To date, the Italian HB database includes 373 characterized unrelated patients and their genetic information has permitted the identification of 274 carriers of childbearing age. This database represents the main instrument for timely and precise PND. Sixty-six prenatal diagnoses were performed on 52 HB carriers whose average age at the time was 34 (ranging from 24 to 44 years). In 44 cases, genetic counselling for carrier status determination was performed before pregnancy, while eight were not studied prior to pregnancy. Foetal samples were obtained by chorionic villus sampling in 52 cases, by amniocentesis in 12 while two were diagnosed by analysis of free foetal DNA obtained from maternal peripheral blood. In 35 (53%) pregnancies the foetus was female. For 31 men (47%), haemophilia status was determined by analysis of previously determined informative markers or familial mutations (12 affected and 19 unaffected). There may be more than one laboratory involved in the PND diagnostic pathway (providing DNA extraction, karyotype analysis, gender determination, maternal contamination detection, molecular diagnosis and sequencing). Good communication between all the parties, coordinated by the haemophilia centre, is essential for a successful and rapid process.

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– 3q : 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 b-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.

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.

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.

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.

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.

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.

Ectopic mRNAanalysis andmolecular modelling substantiate severe haemophilia in a patient with a FVIII gene splice mutation

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.

We present the case of a patient with total colonic aganglionosis and small-bowel involvement (TCSA) associated with a 46, XX, del 10 (q11.21 q21.2) karyotype. Seromuscular biopsies were taken from the rectum, colon, cecum, and terminal ileum. The alpha-naphthylesterase technique was applied to demonstrate intestinal ganglia of the myenteric plexus. The patient did not have associated anomalies. The association of TCSA with 10 long-arm interstitial deletion has not been reported in the literature. Molecular studies in cases of TCSA might reveal whether chromosome 10 plays a role in the pathogenesis of the disease.