Rita Congiu

A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron

Previous studies have described a familial syndrome characterized by iron malabsorption, hypoferremia, and microcytic anemia that did not respond to oral iron and responded only partly to parenteral iron. In this work, Melis and coworkers studied a Sardinian family with this inherited condition. They found a homozygous causal mutation in the TMPRSS6 gene in affected members, who had inappropriately elevated levels of serum and urinary hepcidin. See related perspective article on page 1441. Background Hepcidin plays a key role in body iron metabolism by preventing the release of iron from macrophages and intestinal cells. Defective hepcidin synthesis causes iron loading, while overproduction results in defective reticuloendothelial iron release and iron absorption. Design and Methods We studied a Sardinian family in which microcytic anemia due to defective iron absorption and utilization is inherited as a recessive character. Five members showed iron deficiency anemia that was not responsive to oral iron and only partially responsive to parenteral iron administration. To investigate the involvement of known genes implicated in iron metabolism we carried out linkage analysis with microsatellite markers mapping close to these genes. Afterwards, a genome-wide search was performed. Results No linkage was found between the phenotype of the patients and several known human genes involved in iron metabolism (DMT1, TF, TFRC, ZIRTL, HAMP, HJV). Genome-wide scanning by microsatellites and single nucleotide polymorphisms showed a multipoint LOD score of 5.6 on chromosome 22q12.3–13.1, where the matriptase-2 (also known as transmembrane protease, serine 6 or TMPRSS6) gene is located. Its murine counterpart (Tmprss6) has recently been found to be an essential component of a pathway that detects iron deficiency and suppresses hepcidin production. Sequencing analysis of TMPRSS6 revealed a homozygous causal mutation, predicting a splicing error and a truncated TMPRSS6 protein in affected members. Homozygous subjects had inappropriately elevated levels of serum and urinary hepcidin. Conclusions The findings of this study suggest that the observed TMPRSS6 mutation leads to overproduction of hepcidin and, in turn, to defective iron absorption and utilization. More generally, they confirm in humans the inhibitory effect of matriptase-2 on hepcidin synthesis already demonstrated in mice.

Dystrophin analysis using a panel of anti-dystrophin antibodies in Duchenne and Becker muscular dystrophy.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in 19 patients with Xp21 disorders and in 25 individuals with non-Xp21 muscular dystrophy. Antibodies raised to seven different regions spanning most of the protein were used for immunocytochemistry. In all patients specific dystrophin staining anomalies were detected and correlated with clinical severity and also gene deletion. In patients with Becker muscular dystrophy (BMD) the anomalies detected ranged from inter- and intra-fibre variation in labelling intensity with the same antibody or several antibodies to general reduction in staining and discontinuous staining. In vitro evidence of abnormal dystrophin breakdown was observed reanalysing the muscle of patients, with BMD and not that of non-Xp21 dystrophies, after it has been stored for several months. A number of patients with DMD showed some staining but this did not represent a diagnostic problem. Based on the data presented, it was concluded that immunocytochemistry is a powerful technique in the prognostic diagnosis of Xp21 muscular dystrophies.

A locus for familial skewed X chromosome inactivation maps to chromosome Xq25 in a family with a female manifesting Lowe syndrome

AbstractIn mammals, X-linked gene products can be dosage compensated between males and females by inactivation of one of the two X chromosomes in the developing female embryos. X inactivation choice is usually random in embryo mammals, but several mechanisms can influence the choice determining skewed X inactivation. As a consequence, females heterozygous for X-linked recessive disease can manifest the full phenotype. Herein, we report a family with extremely skewed X inactivation that produced the full phenotype of Lowe syndrome, a recessive X-linked disease, in a female. The X chromosome inactivation studies detected an extremely skewed inactivation pattern with a ratio of 100:0 in the propositus as well as in five out of seven unaffected female relatives in four generations. The OCRL1 “de novo” mutation resides in the active paternally inherited X chromosome. X chromosome haplotype analysis suggests the presence of a locus for the familial skewed X inactivation in chromosome Xq25 most likely controlling X chromosome choice in X inactivation or cell proliferation. The description of this case adds Lowe syndrome to the list of X-linked disorders which may manifest the full phenotype in females because of the skewed X inactivation.

Thalassaemia and Glucose-6-Phosphate Dehydrogenase Screening in 13- to 14-Year-Old Students of the Sardinian Population: Preliminary Findings

Objectives: In this paper we describe the outline and results of a 7-year screening programme for thalassaemias and glucose-6-phosphate dehydrogenase (G6PD) deficiency in 13- to 14-year-old students from the Sardinian population. Method: This programme had several steps: formal education on thalassaemia, request of informed consent by parents, blood testing and genetic counselling. Results:Out of 63,285 subjects tested, 6,521 (10.3%) were heterozygotes for β-thalassaemia, 16,175 (25.6%) for α-thalassaemia and 101 were carriers of a haemoglobin variant. One thousand four hundred and twenty (16.4%) males were hemizygotes for G6PD deficiency and 1,893 (20.6%) females were heterozygotes. Conclusion: The uptake of the programme was remarkably high and homogeneous across the island, indicating and confirming a great interest of the Sardinian population in any initiative directed at the prevention of homozygous β-thalassaemia.

Homozygous deletion of the major alpha-globin regulatory element (MCS-R2) responsible for a severe case of hemoglobin H disease

To the editor: Alpha-thalassemia commonly results from deletions or point mutations in one or both alpha-globin genes, located on chromosome 16p13.3. Rarely, alpha-thalassemia is caused by deletions in a region, located 30 to 70 kb upstream of the alpha-globin genes, containing 4 remote,

Variable dystrophin expression in different muscles of a Duchenne muscular dystrophy carrier

The majority of Duchenne muscular dystrophy (DMD) female carriers show dystrophin immunostaining abnormalities, although a significant proportion of clinically non‐manifesting carriers are normal following this analysis. We had the opportunity to study dystrophin immunostaining in two different muscles, the vastus lateralis and the rectus abdominis of a possible DMD carrier. While the vastus showed normal dystrophin immunostaining, pathological staining was detected in her rectus abdominis. These findings seem to indicate that dystrophin expression can vary in different muscle groups of a DMD carrier. The implications of these findings in DMD carrier detection and possible dystrophin function are discussed.

Iron-deficiency anemia secondary to mutations in genes controlling hepcidin

The discovery of the peptide hormone hepcidin in 2001 has shed light on the control of iron metabolism. Studies in animal models over the past few years have demonstrated its key role in regulating iron homeostasis. It was found that hepcidin deficiency leads to iron overload, and that its overexpression leads to severe iron-deficiency anemia. Since then, other genes regulating hepcidin expression have been discovered, and defects in them mostly resulted in iron overload. In 2008, a new gene, TMPRSS6, was identified that encodes a negative regulator of hepcidin expression. This discovery has been of great relevance because TMPRSS6 is the first gene regulating hepcidin, mutations in which cause chronic iron-deficiency anemia. Recently, genome-wide association studies identified common TMPRSS6 variants associated with hematological parameters, suggesting that TMPRSS6 is crucial in the control of iron homeostasis and normal erythropoiesis.

Homozygous deletion of HFE is the common cause of hemochromatosis in Sardinia

We recently characterized an Alu-mediated recombination causing the loss of the complete HFE gene sequence. Here, we describe the case of a novel homozygous patient. We further show that HFE deletion results from a founder effect and that it represents the common cause of hemochromatosis in Sardinia. We recently reported the case of a 47-year old woman with a moderate iron overload due to an Alu-mediated recombination causing the loss of the complete HFE gene sequence.1 The same chromosomal alteration was identified by Pelucchi and co-workers in another woman. Despite a younger age at diagnosis (29 years), their patient showed a more impressive iron overload.2 A third case, a man, came to our attention. At the age of 44 he showed a transferrin saturation level of 80% and a serum ferritin level of 2080 μg/L. He was not genotyped for the p.C282Y and p.H63D variations because of an inability to amplify the HFE exons 2 and 4. We confirmed absence of the HFE gene in this patient but we were also interested in its origins. Indeed, the patient and both previously reported women were of Sardinian descent. The Sardinian population is genetically differentiated from the other Caucasian populations.3 It represents a genetic isolate where the p.C282Y mutation is considered as rare or even absent.4 This led us to assume that the HFE deleted allele was present at the population level and, related to a founder effect, was the common cause of hemochromatosis in this Mediterranean island. To characterize the contribution of the HFE deletion in the Sardinian population, we first established the frequency of this mutation in a sample set of 198 controls who originated from different districts of the island (90% of them were from South Sardinia, while the others originated from central and Northern parts of the island). Genotype analysis was performed using a rearrangement specific PCR, as previously described.1 The HFE deletion was detected at the heterozygous state in 4 subjects, giving a carrier frequency of 2.02% and an estimated homozygous frequency of 0.01% (one person in 10,000). We also looked for the p.C282Y mutation. It was identified at the heterozygous state in one of 193 subjects (0.52%). We next proceeded to analyze polymorphic microsatellite repeat markers flanking HFE. Based on results from the consanguineous family reported in our initial study,1 we selected a set of informative markers and defined a first chromosomal region of 12.049 megabases (Mb). This chromosomal region encompassed two HLA-class I loci which were added to the haplotypes analysis. Final results are summarized in Table 1 and Figure 1. A conserved haplotype of 3.150 Mb was deduced from the consideration of alleles shared by the 2 homozygous cases identified by us (P1, P2), and the 4 mutation carriers (MC 1–4). However, a recombination event could account for differences in HLA-A, HLA-B and D6S273 alleles of one of the study subjects (MC 3 in Table 1). This allowed us to consider a common haplotype of 7,359 Mb. Figure 1. Physical map of the chromosome 6 region flanking HFE. Distance between the D6S1621 and D6S2414 marker is indicated in grey; the arrow marks a possible ancestral haplotype. Distance between the D6S1621 and D6S1022 markers is indicated in black; the arrow ... Table 1. Allelic distribution of markers surrounding HFE. *The parentheses mark differences between the 6 individuals; these differences are probably related to a recombination event on chromosome 6 of the mutation carrier number 3. By their position on the same maritime routes, the histories of the West Mediterranean islands are very similar. Studies based on HLA polymorphisms have confirmed singularity of these populations, and have further revealed a close relationship between the Sardinian and Corsican populations. However, it must be pointed out that the HLA-A*2-B*58 haplotype is frequent in Sardinia (4.9%) and almost absent in the other West Mediterranean islands (Corsica and Balearic Islands). In fact, this haplotype could be of African origin.5 If so, comparing frequency of the HFE deleted allele (2%) to that of the HLA-A*2-B*58 haplotype (4.9%), one may assume that deletion of the HFE gene arose in the Sardinian population after contacts with African populations. But an African ancestry for the HFE deletion cannot be completely excluded. To conclude, we show that HFE deletion results from a founder effect rather than that of a mutational hotspot. We also demonstrate that, with an estimated homozygous frequency of one person in 10,000, HFE deletion is the common cause of hemochromatosis in Sardinia.

Germinal mosaicism in a Duchenne muscular dystrophy family : implications for genetic counselling

Melis MA, Cau M. Congiu R, Puddu R, Muntoni F, Cao A. Germinal mosaicism in a Duchenne muscular dystrophy family: implications for genetic counselling.

Frequency of Hemochromatosis C282Y and H63D Mutations in Sardinia

Hereditary hemochromatosis (HH) is one of the most common autosomal recessive disorders of iron metabolism among Caucasians, and it is associated with C282Y mutation of the HFE gene in populations of Celtic origins. A second mutation, H63D, shows a very high widespread frequency, although its role in iron metabolism is still inconclusive. There are no data on the frequencies of these two mutations in Sardinia, an island in the Mediterranean sea that has not been invaded by Celtic peoples. We examined 836 chromosomes from Sardinian subjects and tested for the mutation by restriction enzyme digestion of PCR products. Among the 836 analyzed chromosomes, we found a C282Y allele frequency of 0.0036 and an H63D allele frequency of 0.173. These data could explain the observed rarity of HH in Sardinia. The high allele frequency of H63D and the rarity of HH in Sardinia is suggestive that this mutation is not a major contributor to this disease.