Isadora Asunis

Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of -thalassemia

β-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 β-thalassemia, and patients with attenuated forms of β-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 β-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 β-thalassemia and sickle cell anemia.

Overexpression of the Cytokine BAFF and Autoimmunity Risk

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.)

Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin

The persistence of high fetal hemoglobin level in adults may ameliorate the clinical phenotype of beta-thalassemia and sickle cell anemia. Several genetic variants responsible for hereditary persistence of fetal hemoglobin, linked and not linked to the beta globin gene cluster, have been identified in patients and in normal individuals. Monoallelic loss of KLF1, a gene with a key role in erythropoiesis, has been recently reported to be responsible for persistence of high levels of fetal hemoglobin. In a Sardinian family, high levels of HbF (22.1–30.9%) were present only in compound heterozygotes for the S270X nonsense and K332Q missense mutations, while the isolated S270X nonsense (haploinsufficiency) or K332Q missense mutation were associated with normal HbF levels (<1.5%). Functionally, the K332Q Klf1 mutation impairs binding to the BCl11A gene and activation of the γ- and β-globin promoters. Moreover, we report for the first time the association of KLF1 mutations with very high levels of zinc protoporphyrin.

A novel silent beta-thalassemia mutation in the distal CACCC box affects the binding and responsiveness to EKLF

The silent β‐thalassemia mutation, β+‐101C→T, is the only mutation currently described in the distal β‐globin CACCC box. We present a novel mutation, a C→G transversion, in the same position. Expression analysis in heterozygous subjects demonstrated that the mutation determines a 20% reduction in the output of the β‐globin gene. DNA–protein interaction and transactivation analysis correlated the decrease in the β‐globin synthesis with the reduced binding and transactivation of EKLF to the mutant promoter. These data predict that the β‐101C→G mutation will display a silent thalassemia phenotype similar to that of the β‐101C→T mutation.

Vitamin D Responsive Elements within the HLA-DRB1 Promoter Region in Sardinian Multiple Sclerosis Associated Alleles

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15∶01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15∶01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04∶05, *03∶01, *13∶01 and *15∶01 and non-MS-associated *16∶01, *01, *11, *07∶01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15∶01, *16∶01, *11 and in 45/73 *03∶01 and in heterozygous state in 28/73 *03∶01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13∶03, *04∶05 and *07∶01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04∶05 and *03∶01 alleles carrying the new mutated VDRE, while the *16∶01 and *03∶01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16∶01 and in the *15∶01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients.

Klf1 Affects DNase II-Alpha Expression in the Central Macrophage of a Fetal Liver Erythroblastic Island: a Non-Cell-Autonomous Role in Definitive Erythropoiesis

ABSTRACT 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.

Regulation of the human HBA genes by KLF4 in erythroid cell lines

KLF1/EKLF and related Krueppel‐like factors (KLFs) are variably implicated in the regulation of the HBB‐like globin genes. Prompted by the observation that four KLF sites are distributed in the human α‐globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four α‐globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4‐specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4‐DNA binding, were transactivated by over‐expression of KLF4. In K562 cells, induced KLF4 expression with a Tet‐off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh‐RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.

A novel splicing defect (IVS6+1G>T) in a patient with pseudovitamin D deficiency rickets

A 15-month-old boy with severe rickets, that by clinical analysis was diagnosed as affected by hereditary pseudovitamin D deficiency rickets (PDDR), was evaluated for mutations in the 25OHD3 1α-hydroxylase gene. Molecular analysis showed a double heterozygous state for a novel splicing mutation in the invariant dinucleotide of the donor site of IVS6 and a 7 nucleotide insertion in the exon 8, which is common in different ethnical backgrounds.

A Novel Missense Mutation (C84R) in a Patient with Type II Vitamin D-dependent Rickets

A 7-year-old boy with severe rickets that by clinical analysis was diagnosed as affected by type II vitamin D-dependent rickets, was evaluated for mutations in the vitamin D receptor gene (VDR). The molecular analysis showed a homozygous state for a novel missense mutation (C84R) in a highly conserved nucleotide in the second Zn finger of the DNA binding domain.

The distal β-globin CACCC box is required for maximal stimulation of the β-globin gene by EKLF

The transcription factor erythroid Kruppel‐like factor (EKLF) specifically activates the β‐globin gene by interacting with the proximal β‐globin CACCC box, a known hot spot for thalassaemia mutations. This study investigated whether EKLF could also bind to, and activate from, the distal CACCC, which is a rare site of thalassaemia mutations. Using band shift and transient expression analysis with wild type, single and double CACCC mutants, we established that the distal CACCC box is weakly bound by EKLF, but, when mutated, significantly impairs EKLF‐dependent β‐globin stimulation. Thus, EKLF requires both CACCC boxes to maximally stimulate the β‐globin gene.