Genetic screening for low-penetrance variants in protein-coding genes.

Genetic testing holds great promise as a screening tool to identify persons at risk for a disease at the presymptomatic stage. However, the complexities of gene-disease associations, even in single-gene diseases, pose important challenges. These challenges include defining the role of screening for mutations that have low penetrance, which cause disease in only a minority of patients with the genotype. On the basis of the high rate of false positives, medical expert panels to date have largely discouraged genetic testing for low-penetrance mutations for use in population-based screening, although official recommendations currently exist for only a few genes. We examine the relatively limited experience of population-based screening for low-penetrance mutations in clinical settings to date, including screening for glucose-6-phosphate dehydrogenase deficiency and a low-penetrance mutation for cystic fibrosis in newborns, type 1 Gaucher disease carrier screening, and screening for adults for hemochromatosis. The trend toward recommending restricting use of these tests by medical experts is contrasted with the growing availability of genetic tests, including those for low-penetrance mutations, through direct-to-consumer outlets.

Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6.

Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin (HFE2) that stimulates expression of the hepcidin encoding gene (HAMP). Iron attenuation is countered through a pathway involving the hepatocyte-specific plasma membrane protease matriptase-2 encoded by TMPRSS6, leading to suppression of HAMP expression. The non-redundant function of hemojuvelin and matriptase-2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6, which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase-2. To examine the relationship between hemojuvelin and matriptase-2 in vivo, we crossed mice lacking the protease domain of matriptase-2 with mice lacking hemojuvelin. Mice lacking functional matriptase-2 and hemojuvelin exhibited low Hamp (Hamp1) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin-deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase-2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase-2/TMPRSS6 protease activity.

Regulation of hepcidin and iron-overload disease.

Hepcidin, a 25-amino-acid antimicrobial peptide, is the central regulator of iron homeostasis. Hepcidin transcription is upregulated by inflammatory cytokines, iron, and bone morphogenetic proteins and is downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. The iron transporter ferroportin is the cognate receptor of hepcidin and is destroyed as a result of interaction with the peptide. Except for inherited defects of ferroportin and hepcidin itself, all forms of iron-storage disease appear to arise from hepcidin dysregulation. Studies using multiple approaches have begun to delineate the molecular mechanisms that regulate hepcidin expression, particularly at the transcriptional level. Knowledge of the regulation of hepcidin by inflammation, iron, erythropoiesis, and hypoxia will lead to an understanding of the pathogenesis of primary hemochromatosis, secondary iron overload, and anemia of inflammatory disease.

The global prevalence of glucose-6-phosphate dehydrogenase deficiency: a systematic review and meta-analysis.

Glucose-6-phosphate deficiency is the most prevalent enzyme deficiency, with an estimated 400 million people affected worldwide. This inherited deficiency causes neonatal hyperbilirubinemia and chronic hemolytic anemia. Although most affected individuals are asymptomatic, exposure to oxidative stressors such as certain drugs or infection, can elicit acute hemolysis. To characterize the global prevalence of G6PD deficiency, we conducted a systematic review of the G6PD deficiency literature, drawing studies from various databases, including MEDLINE/Pubmed and Biosis. Selected studies included cross-sectional and longitudinal studies published between 1960 and 2008. Additionally, meta-analytic procedures were employed to assess the degree of heterogeneity amongst prevalence estimates and, where appropriate, pool them. The searches yielded a total of 280 prevalence estimates, corresponding to 88 countries. The highest prevalence rates were reported among Sub-Saharan African countries, even after adjusting for assessment method. Meta-analysis revealed a high degree of heterogeneity for regional and global prevalence estimates. This heterogeneity in reported estimates appeared to be due to differences in G6PD deficiency assessment and diagnostic procedures. The magnitude and variation in global, regional, and country-level prevalence rates of G6PD deficiency are of public health import, particularly in planning programs to improve neonatal health and in the distribution of various medications, especially antimalarial drugs, as G6PD deficiency is most prevalent in malaria-endemic areas.

Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness.

Hepcidin plays a major role in the regulation of iron homeostasis. Several bone morphogenetic proteins (BMPs) are strong inducers of hepcidin (Hamp1, HAMP) expression. Hemojuvelin, a protein critical for maintaining appropriate levels of hepcidin, acts as a coreceptor for BMP2 and BMP4, thereby providing a link between iron homeostasis and the BMP-signaling pathway. We show that a robust BMP, hemojuvelin, and SMAD1 response by murine Hamp1 is dependent on a distal BMP responsive element (BMP-RE2), the adjacent bZIP, HNF4alpha/COUP binding sites, and plus or minus 50 bp of the flanking area within -1.6 to -1.7 kb of the Hamp1 promoter. Furthermore, the STAT site and the BMP responsive element (BMP-RE1) located in the proximal 260-bp region of the Hamp1 promoter are also indispensable for maximal activation of hepcidin transcription. The homologous motifs in the distal and proximal regions of the human HAMP promoter act in a manner similar to the murine Hamp1 promoter. Therefore, we propose that the regulation of hepcidin by the BMP pathway involves the formation of a complex of liver-specific and response-specific transcription factors bound to the distal BMP-RE2 /bZIP/HNF4alpha/COUP region and to the proximal BMP-RE1/STAT region possibly by physical association of the 2 regions.

Severe iron overload with a novel aminolevulinate synthase mutation and hepatitis C infection. A case report.

A 55 year old man with a history of chronic hepatitis C infection was found to have severe hemochromatosis: hepatic cirrhosis, cardiomyopathy, arrhythmia, hypogonadism, diabetes and bronzed skin color. After 50 phlebotomies, he underwent a combined heart and liver transplant. Genetic analyses identified a novel mutation in the iron responsive element of the ALAS2 gene. No mutations were found in other genes associated with adult or juvenile hemochromatosis including HFE, transferrin receptor-2 (TFR2), ferroportin (SLC40A1), hepcidin (HAMP) and hemojuvelin (HJV). We suggest that the ALAS2 mutation together with chronic hepatitis C infection may have caused the severe iron overload phenotype.

The anemia of ageing is not associated with increased plasma hepcidin levels.

It has been proposed that the anemia of ageing may be caused, at least in part, by elevated hepcidin levels, representing a response to increased IL-6 levels. Using a recently developed immunoassay, we have measured the plasma hepcidin levels of eight patients with the anemia of ageing and sex- and age-matched controls, and found that the levels of hepcidin were not increased in patients with the anemia of ageing. In contrast, patients with the anemia of inflammation have higher hepcidin levels than sex- and age-matched controls. In the overall group there was a strong correlation between serum ferritin levels and hepcidin levels, as has been found previously.

The serine protease TMPRSS6 is required to sense iron deficiency.

Hepcidin, a liver-derived protein that restricts enteric iron absorption, is the key regulator of body iron content. Several proteins induce expression of the hepcidin-encoding gene Hamp in response to infection or high levels of iron. However, mechanism(s) of Hamp suppression during iron depletion are poorly understood. We describe mask: a recessive, chemically induced mutant mouse phenotype, characterized by progressive loss of body (but not facial) hair and microcytic anemia. The mask phenotype results from reduced absorption of dietary iron caused by high levels of hepcidin and is due to a splicing defect in the transmembrane serine protease 6 gene Tmprss6. Overexpression of normal TMPRSS6 protein suppresses activation of the Hamp promoter, and the TMPRSS6 cytoplasmic domain mediates Hamp suppression via proximal promoter element(s). TMPRSS6 is an essential component of a pathway that detects iron deficiency and blocks Hamp transcription, permitting enhanced dietary iron absorption.

Glucose-6-phosphate dehydrogenase deficiency: a historical perspective.

Glucose-6-phosphate dehydrogenase deficiency serves as a prototype of the many human enzyme deficiencies that are now known. Since its discovery more than 50 years ago, the high prevalence of the defect and the easy accessibility of the cells that manifest it have made it a favorite tool of biochemists, epidemiologists, geneticists, and molecular biologists as well as clinicians. In this brief historical review, we trace the discovery of this defect, its clinical manifestations, detection, population genetics, and molecular biology.

Screening for hemochromatosis by measuring ferritin levels: a more effective approach.

Because the penetrance of HFE hemochromatosis is low, traditional population screening measuring the transferrin saturation is unlikely to be cost-effective because the majority of subjects detected neither have clinical disease nor are likely to develop it. Three independent studies show that only patients with serum ferritin concentrations more than 1000 microg/L are at risk for cirrhosis, one of the main morbidities of hemochromatosis. Among 29,699 white subjects participating in the Scripps/Kaiser hemochromatosis study, only 59 had serum ferritin levels more than 1000 microg/L; 24 had homozygous mutant or compound heterozygous mutant HFE genotypes. In all but 5 of the other subjects, the causes of elevated ferritin were excessive alcohol intake, cancer, or liver disease. Screening for hemochromatosis with serum ferritin levels will detect the majority of patients who will be clinically affected and may detect other clinically significant disease in patients who do not have hemochromatosis genotypes. Because the ferritin level of the majority of adult homozygotes for HFE mutations does not rise over long periods of time, excluding subjects with serum ferritin levels less than or equal to 1000 microg/L should not result in missed opportunities for early treatment of patients who could benefit.

A new case of human atransferrinemia with a previously undescribed mutation in the transferrin gene.

Hereditary atransferrinemia is a very rare disorder characterized by microcytic anemia and iron overload. It has been reported in only 10 patients in 8 families. The molecular basis of atransferrinemia has been determined in only 3 human cases. We now report a new patient with this rare disorder, who is the first known case in Turkey, the 11th patient reported in the published literature and only the 4th case of human atransferrinemia characterized on a molecular basis. DNA analysis of the serum transferrin gene in the patient revealed a previously undescribed mutation in exon 4, a G-->A transition at cDNA 410(Cys137Tyr). A number of previously known polymorphisms and a previously undescribed mutation at IVS10(-23)C-->T, presumably a polymorphism, were also documented.

Glucose-6-phosphate dehydrogenase deficiency and antimalarial drug development.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is relatively common in populations exposed to malaria. This deficiency appears to provide some protection from this infection, but it can also cause hemolysis after administration of some antimalarial drugs, especially primaquine. The risk of drug-induced G6PD deficiency-related hemolysis depends on a number of factors including the G6PD variant, the drug and drug dosage schedule, patient status, and disease factors. Although a great deal is known about the molecular biology of G6PD, determining the potential for drug-induced hemolysis in the clinical setting is still challenging. This report discusses the potential strategies for assessing drug-induced G6PD deficiency-related hemolytic risk preclinically and in early clinical trials. Additionally, the issues important for conducting larger clinical trials in populations in which G6PD deficiency is prevalent are examined, with a particular focus on antimalarial drug development.

Different regulatory elements are required for response of hepcidin to interleukin-6 and bone morphogenetic proteins 4 and 9.

Hepcidin is a major regulator of iron homeostasis. Hepcidin expression is upregulated by inflammatory cytokines, particularly interleukin (IL)-6 and even more potently by the bone morphogenetic proteins 2, 4 and 9 (BMP-2, BMP-4 and BMP-9). This study showed that the regulation of hepcidin expression by IL-6 and BMPs occurs through distinct regulatory elements. The induction of hepcidin by BMPs requires at least two regions of the Hamp1 promoter, one between 140-260 bp and the other between 1.6-2.0 kb upstream of the start of translation. Reporter constructs including 1.6-2.0 kb of the Hamp1 promoter were induced >16-fold by BMPs whereas a 260 bp reporter Hamp1 promoter construct was induced only two- to threefold. The distal 1.6-2.0 kb region appeared to contain several different BMP-responsive elements, as incremental lengthening of the promoter construct in this region produced gradual escalation of BMP-responsiveness. In contrast, the IL-6 response required only the proximal 260 bp Hamp1 promoter region. Furthermore, there were no regulatory elements located in the non-coding or coding regions of Hamp1 and activation of the Hamp1 promoter was absent or markedly reduced in cells of non-hepatic origin.

Congenital Gaucher disease with nonimmune hydrops/erythroblastosis, infantile arterial calcification, and neonatal hepatitis/fibrosis. Clinicopathologic report with enzymatic and genetic analysis.

The findings in a stillborn female fetus of 31 weeks' gestation with congenital Gaucher disease, nonimmune hydrops/erythroblastosis, infantile arterial calcification, and neonatal hepatitis/fibrosis are presented, the first report of this complete constellation. Prior reports describe two similar patients. One lacked the hepatocellular features of giant cell hepatitis although manifesting hepatic fibrosis; the second lacked hepatic pathology. The diagnosis of Gaucher disease herein was established by microscopic examination of the proband, enzymatic analysis of trophoblast, and enzymatic and genetic study of the parents. The father was heterozygous for a recombinant glucocerebrosidase gene; the mother demonstrated a unique frame shift mutation. Thus the fetus is a compound heterozygote for a null and a severe mutation. Studies of parental DNA were negative for the D409H mutation of type IIIc Gaucher disease. Genetic studies were not performed of the ENPP1 gene, mutations of which are associated with idiopathic infantile arterial calcification.

The role of STAT, AP-1, E-box and TIEG motifs in the regulation of hepcidin by IL-6 and BMP-9: lessons from human HAMP and murine Hamp1 and Hamp2 gene promoters.

Hepcidin, the principal regulator of the iron metabolism, is up-regulated in response to inflammatory stimuli, bone morphogenic proteins (BMPs) and iron excess. There are two murine hepcidin genes: hepcidin-1 (Hamp1) and hepcidin-2 (Hamp2). Hamp1 gene responds to both IL-6 and BMPs while Hamp2 responds to neither. We replaced the putative functional regulatory motifs of the Hamp1 promoter with the corresponding putative "non-functional" Hamp2 motifs and vice versa in reporter constructs. Conversion of the Hamp1 STAT site into the Hamp2 site reduced the basal level of reporter expression but did not affect IL-6 and BMP responsiveness; replacing Hamp2 site with the Hamp1 site only resulted in partial responsiveness. These data are in contrast to the role of the STAT site in the human hepcidin promoter which is important in both basal level and IL-6 inducible promoter activity. The murine AP1, E-box and TIEG motifs were found to neither influence the basal level of expression of Hamp1 and HAMP promoters nor play a critical role in the IL-6 and BMP-9 induced response. Our data suggest that the STAT site (nt -148 to -130) is important for the regulation of basal level expression of Hamp1 but there are additional regions that are responsible for the IL-6 and BMP-9 responsiveness within the Hamp1 promoter.

Human chitotriosidase polymorphisms G354R and A442V associated with reduced enzyme activity.

A common polymorphism in the chitotriosidase gene (CHIT1) exists in which a 24 bp duplication in exon 10 results in aberrant splicing and deletion of 87 nucleotides. In this study, the gene frequency was found to be 0.56 (n=2054) in subjects of Asian ancestry, 0.17 (n=984) in subjects of European ancestry and 0.07 (n=536) in subjects of African ancestry. Notably, the median enzyme activity in wild-type subjects (TT) was much higher in subjects of European ancestry (2.69 mU/ml, n=202 subjects), than subjects of African (2.57 mU/ml, n=230 subjects) (P<0.0001) and Asian ancestry (0.86 mU/ml, n=114 subjects) (P<0.0001). The question of why chitotriosidase deficiency exists at such a high frequency is a challenging one. We postulated that if there was a selective advantage for chitotriosidase deficiency then there would be polymorphisms that would be associated with reduced enzyme activity independent of the 24 bp duplication. We found that the G102S and the A442G polymorphisms found occurring in subjects of all ancestries were not significantly associated with a reduction of enzyme activity. In contrast, the G354R (P<0.0001) and the A442V (P=0.0013) polymorphisms occurring predominantly in subjects of African ancestry were significantly associated with reduced chitotriosidase activity. We also investigated the possibility that chitotriosidase deficiency was associated with tuberculosis or with atopy, including allergic rhinitis, contact dermatitis, food or drug allergies and asthma.