Hypersensitive site 2 located in the f-globin locus control region confers high levels of expression to the genes of the 1-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (acti-

␤-Thalassemia and sickle cell disease both display a great deal of phenotypic heterogeneity, despite being generally thought of as simple Mendelian diseases. The reasons for this are not well understood, although the level of fetal hemoglobin (HbF) is one well characterized ameliorating factor in both of these conditions. To better understand the genetic basis of this heterogeneity, we carried out genome-wide scans with 362,129 common SNPs on 4,305 Sardinians to look for genetic linkage and association with HbF levels, as well as other red blood cell-related traits. Among major variants affecting HbF levels, SNP rs11886868 in the BCL11A gene was strongly associated with this trait (P < 10 ؊35 ). The C allele frequency was significantly higher in Sardinian individuals with elevated HbF levels, detected by screening for ␤-thalassemia, and patients with attenuated forms of ␤-thalassemia vs. those with thalassemia major. We also show that the same BCL11A variant is strongly associated with HbF levels in a large cohort of sickle cell patients. These results indicate that BCL11A variants, by modulating HbF levels, act as an important ameliorating factor of the ␤-thalassemia phenotype, and it is likely they could help ameliorate other hemoglobin disorders. We expect our findings will help to characterize the molecular mechanisms of fetal globin regulation and could eventually contribute to the development of new therapeutic approaches for ␤-thalassemia and sickle cell anemia.globin gene regulation ͉ polymorphism ͉ sickle cell anemia

Background Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. Methods Using case–control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus–specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. Results A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion–deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. Conclusions A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.)

Brief Reporthaematologica | 2011; 96 (5) 767 IntroductionPersistent expression of fetal hemoglobin (HbF) is of great clinical relevance given its role in the amelioration of the phenotype of beta-thalassemia and sickle cell anemia. Several studies have identified genes and genetic variants controlling HbF levels in adults (HBG1/HBG2, HBS1-MYB and BCL11A) able to improve the severity of the two major beta-hemoglobinopathies, beta thalassemia and sickle cell anemia. [1][2][3][4] Recently a nonsense mutation in the KLF1 gene, which ablates the DNA binding domain of this key erythroid transcriptional regulator, has been reported in a large Maltese family with hereditary persistence of HbF (HPFH). 5,6 Haploinsufficiency of KLF1 expression has been considered to be responsible for HPFH.In the Sardinian family described here, we found a marked increase of HbF only in compound heterozygotes for two KLF1 mutations and we did not confirm the KLF1 haploinsufficiency as a cause of HPFH. Moreover, we report, for the first time in humans, very high levels of zinc protoporphyrin associated with KLF1 mutations. Design and MethodsWe studied a Sardinian family with HPFH. Blood samples were obtained after informed consent. Hematologic and biochemical analyses were performed according to standard procedures. Zinc protoporphyrin in RBC was determined with ZPP hematofluorometer (AVIV Biomedical, Lakewood, NJ, USA) and blood protoporphyrin IX with a fluorometric method.Genomic DNA was obtained from peripheral blood by standard methods.Mutation analysis was performed by PCR amplification and DNA sequence analysis of the KLF1 gene using previously described primers.6 Genotyping of individual SNPs in the HBS1L-MYB (rs9399137) and BCL11A (rs11886868) loci was performed using Taqman genotyping assay (Applied Biosystem, Warrington, UK). Alpha globin and bilirubin UDP-glucuronosyltransferase (UGT1A1) gene genotyping was carried out as previously described. 7,8 The site directed mutation in K332Q in the KLF1 cDNA was obtained with the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA).Band shift, supershift, Western blots and transactivation analysisCompound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin ABSTRACT were performed as previously described. 9 The intensities of the KLF1 shifted bands were determined with the ImageQuant software after gel autoradiography on phosphoscreen and acquisition with PhosphoImager Storm 840 (GE Healthcare).The study was approved by the institutional review board of the hospital (ASL8 Ethics Commitee).

A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.