Post-GWAS Validation of Target Genes Associated with HbF and HbA2 Levels.

Genome-Wide Association Studies (GWASs) have identified a huge number of variants associated with different traits. However, their validation through in vitro and in vivo studies often lags well behind their identification. For variants associated with traits or diseases of biomedical interest, this gap delays the development of possible therapies. This issue also impacts beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease (SCD). The definitive cures for these diseases are currently bone marrow transplantation and gene therapy. However, limitations regarding their effective use restrict their worldwide application. Great efforts have been made to identify whether modulators of fetal hemoglobin (HbF) and, to a lesser extent, hemoglobin A2 (HbA2) are possible therapeutic targets. Herein, we performed the post-GWAS in vivo validation of two genes, cyclin D3 (CCND3) and nuclear factor I X (NFIX), previously associated with HbF and HbA2 levels. The absence of Ccnd3 expression in vivo significantly increased g (HbF) and d (HbA2) globin gene expression. Our data suggest that CCND3 is a possible therapeutic target in sickle cell disease. We also confirmed the association of Nfix with γ-globin gene expression and present data suggesting a possible role for Nfix in regulating Kruppel-like transcription factor 1 (Klf1), a master regulator of hemoglobin switching. This study contributes to filling the gap between GWAS variant identification and target validation for beta-hemoglobinopathies.

Krüppel-Like Factor 1: A Pivotal Gene Regulator in Erythropoiesis.

Krüppel-like factor 1 (KLF1) plays a crucial role in erythropoiesis. In-depth studies conducted on mice and humans have highlighted its importance in erythroid lineage commitment, terminal erythropoiesis progression and the switching of globin genes from γ to ß. The role of KLF1 in haemoglobin switching is exerted by the direct activation of ß-globin gene and by the silencing of γ-globin through activation of BCL11A, an important γ-globin gene repressor. The link between KLF1 and γ-globin silencing identifies this transcription factor as a possible therapeutic target for ß-hemoglobinopathies. Moreover, several mutations have been identified in the human genes that are responsible for various benign phenotypes and erythroid disorders. The study of the phenotype associated with each mutation has greatly contributed to the current understanding of the complex role of KLF1 in erythropoiesis. This review will focus on some of the principal functions of KLF1 on erythroid cell commitment and differentiation, spanning from primitive to definitive erythropoiesis. The fundamental role of KLF1 in haemoglobin switching will be also highlighted. Finally, an overview of the principal human mutations and relative phenotypes and disorders will be described.

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.).

ß-defensin CNV is not associated with susceptibility to Candida albicans infections in Sardinian APS I patients.

OBJECTIVE: The aim of this study was to investigate whether a variation in the genomic copy number (CNV) of the ß-defensin cluster could be associated with the pre-disposition to chronic mucocutaneous candidiasis (CMC) in Sardinian APECED patients. SUBJECTS AND METHODS: The ß-defensin copy number variation was determined by MLPA analysis in 18 Sardinian APECED patients with CMC and in 21 Sardinian controls. Statistical analyses were performed with one-way ANOVA test. RESULTS: No statistically significant results were observed between the patients and controls groups. CONCLUSIONS: According to the results we have obtained, it appears that either ß-defensin genomic CNV is not a modifier locus for CMC susceptibility in APECED patients, or any effect is too small for it to be detected using such sample size. An extensive study on APECED patients from different geographical areas might reveal the real implication of the ß-defensin CNV in the susceptibility to Candida albicans infections.

Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.

BACKGROUND: In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. METHODS: Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. RESULTS: We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. CONCLUSIONS: In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%).

A synonymous mutation in the CFTR gene causes aberrant splicing in an italian patient affected by a mild form of cystic fibrosis.

Mutations within exons are responsible for aberrant splicing of pre-mRNA in several human disease genes and in some viral systems. Nonsense, missense, and even synonymous mutations can induce aberrant skipping of the mutant exon, producing nonfunctional proteins. In this paper, we describe the effect on the splicing efficiency of the synonymous variant 2811 G>T [Gly893Gly] detected in a patient of Italian descent affected by a mild form of cystic fibrosis, until now mentioned as sequence variation with unknown functional consequences. The study, performed through DNA as well as RNA analyses, shows that this mutation creates a new 5' splice site within exon 15, resulting in a transcript lacking 76 amino acid residues. Although this aberrant splicing causes a shorter exon 15, the downstream exonic sequence from exon 16 to the end of the open reading frame is in frame. This study indicates that apparently neutral polymorphism, which may be erroneously classified as nonpathogenic, may indeed led to aberrant splicing thereby resulting in defective protein.

Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.

Neonatal onset of nephrogenic syndrome of inappropriate antidiuresis.

This paper describes the manifestation in a child of a new syndrome characterized by unusual, severe, persistent hyponatremia associated with hyposmolarity, euvolemia, inappropriately concentrated urine and elevated natriuresis. This is the fourth case of this syndrome reported to date, and the first to be reported in a neonate. The clinical features resemble those typically observed in patients with inappropriate antidiuretic hormone secretion, although high arginine vasopressin (AVP) levels are lacking. The findings led the authors to hypothesise a nephrogenic syndrome of inappropriate antidiuresis (NSIAD). The previously described R137C gain-of-function mutation was detected by means of mutation analysis of the V2R gene. Our results indicate that NSIAD is already present during the neonatal period and that molecular analysis of the V2R receptor should therefore be carried out, in all newborns with prolonged euvolemic hyponatremia with hypo-osmolarity, high urinary sodium and normal/low or undetectable AVP levels.

A new insertion/deletion of the cystic fibrosis transmembrane conductance regulator gene accounts for 3.4% of cystic fibrosis mutations in Sardinia: implications for population screening.

Previous studies performed on Sardinian patients affected by cystic fibrosis (CF) have led to the identification of molecular defects in 87 of 88 patients. Two mutations, the F508del and T338I, were quite prevalent and accounted for 50% and 20% of the molecular defects, respectively. T338I has been detected rarely in other populations, most likely because of the genetic isolation of Sardinians. In the present study, we have performed a molecular analysis of the CF gene in eight Sardinian patients in whom only a single mutation has been defined. Using DNA analyses (Southern blot, single nucleotide polymorphisms, microsatellite analyses, and Extra-Long polymerase chain reaction) selected to detect gross gene rearrangement and by using mRNA studies, we detected a novel mutation c.54-5811_164 + 2186del8108ins182 in six of the eight patients investigated. This mutation consists of a gross deletion of 8108 bp spanning exon 2 with an insertion of 182 bp at the deletion junction, between nucleotide 54-5811 of intron 1 (IVS1 nt16864) and nucleotide 164 + 2186 of intron 2 (IVS2 nt 2186). By including the novel mutation in our mutation panel we are now able to reach a 95% detection rate, thereby improving the process of carrier detection and genetic counseling in Sardinia.

Thalassaemia-like carriers not linked to the beta-globin gene cluster.

This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a beta-thalassaemia-like phenotype in whom the beta-globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent beta-thalassaemia-like phenotype or a typical beta-thalassaemia carrier-like phenotype in different families. Compound heterozygosity for both beta-thalassaemia-like determinant and typical beta-thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical beta-like determinants were found not to be linked to the beta-globin cluster. Sequence analysis of the hypersensitive site cores of locus control region and of the genes coding for the transcription factors erythroid Kruppel-like factor and nuclear factor (erythroid-derived 2) were normal. beta-globin mRNA levels determined by real-time polymerase chain reaction were reduced in both types of beta-like carriers. These results indicate the existence of causative genetic determinants not yet molecularly defined, but most likely, resulting from either the reduction or loss of function of a gene coding for unknown transcriptional regulator(s) of the beta-globin gene. The knowledge of these rare beta-thalassaemia-like determinants have implications for clinical and, especially, prenatal diagnosis of beta-thalassaemia.

Preimplantation genetic diagnosis for beta-thalassaemia: the Sardinian experience.

OBJECTIVES: To report the experiences on preimplantation genetic diagnosis (PGD) in couples at risk for beta-thalassaemia in Sardinia. METHODS: 23 couples at risk for beta-thalassaemia were included in the PGD programme with a total of 42 cycles performed. Among these, 11 couples were fertile, while the remaining 12 had associated fertility problems. In vitro Fertilization (IVF), PGD and prenatal genetic molecular confirmation protocols and results are reported. RESULTS: All the patients followed the protocol of ovarian stimulation, oocyte retrieval, intracytoplasmic sperm injection (ICSI), embryo biopsy and genetic analysis. A total of 272 oocytes were fertilized in the regular way, and embryo biopsy was performed on 202 embryos. Out of these 202 embryos, 192 (95%) were successful. The genetic diagnosis was performed on 150 embryos (78.1%). Ninety-eight were identified as unaffected and 75 were transferred in 31 cycles. In the infertile patient group, two biochemical pregnancies (11.1% per transfer), in the fertile patient group, four clinical pregnancies, two twin and two singleton pregnancies (30.8% per transfer), were obtained. The genetic molecular results were confirmed in all pregnancies by first-trimester chorionic villus sampling (CVS). CONCLUSION: Our study shows that PGD for beta-thalassaemia is an available procedure for couples who wish to avoid termination of pregnancy, except in cases where the IVF cycle efficiency is very poor.

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

The silent beta-thalassemia mutation, beta(+)-101C-->T, is the only mutation currently described in the distal beta-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 beta-globin gene. DNA-protein interaction and transactivation analysis correlated the decrease in the beta-globin synthesis with the reduced binding and transactivation of EKLF to the mutant promoter. These data predict that the beta-101C-->G mutation will display a silent thalassemia phenotype similar to that of the beta-101C-->T mutation.

Delineation of the molecular defects in the AIRE gene in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.

In this study, we have carried out molecular analysis of the AIRE (autoimmune regulator) gene in 11 patients (from 8 families) affected by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, originating from a restricted area of Southern Italy (the Salento peninsula in Puglia). Of the 16 mutant AIRE alleles from the 8 probands studied, 12 carried a missense mutation (W78R in 9, P539L in 2, and P252L in 1), 2 carried the Q358X nonsense mutation, and 2 carried the 1058delT frameshift mutation. All these mutations except the 1058delT are novel. Each of the detected mutations either predicts a premature termination of the protein or results in a nonconservative amino acid change, most likely adversely affecting the function of the protein. The W78R missense mutation is relatively common in these patients, having been detected (in homozygosity or compound heterozygosity) in 6 of the 8 probands tested, indicating the presence of a founder effect. The results of this study contribute to the delineation of the molecular pathology of the AIRE gene and enhance our ability to perform a molecular diagnosis in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.