Lucia Perseu

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.

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Common variants in the SLCO1B3 locus are associated with bilirubin levels and unconjugated hyperbilirubinemia

Bilirubin, resulting largely from the turnover of hemoglobin, is found in the plasma in two main forms: unconjugated or conjugated with glucuronic acid. Unconjugated bilirubin is transported into hepatocytes. There, it is glucuronidated by UGT1A1 and secreted into the bile canaliculi. We report a genome wide association scan in 4300 Sardinian individuals for total serum bilirubin levels. In addition to the two known loci previously involved in the regulation of bilirubin levels, UGT1A1 (P = 6.2 x 10(-62)) and G6PD (P = 2.5 x 10(-8)), we observed a strong association on chromosome 12 within the SLCO1B3 gene (P = 3.9 x 10(-9)). Our findings were replicated in an independent sample of 1860 Sardinians and in 832 subjects from the Old Order Amish (combined P < 5 x 10(-14)). We also show that SLC01B3 variants contribute to idiopathic mild unconjugated hyperbilirubinemia. Thus, SLC01B3 appears to be involved in the regulation of serum bilirubin levels in healthy individuals and in some bilirubin-related disorders that are only partially explained by other known gene variants.

Amelioration of Sardinian β0 thalassemia by genetic modifiers

Sardinian beta-thalassemia patients all are homozygotes for the same null allele in the beta-globin gene, but the clinical manifestations are extremely variable in severity. Previous studies have shown that the coinheritance of alpha-thalassemia or the presence of genetic variants that sustain fetal hemoglobin production has a strong impact on ameliorating the clinical phenotype. Here we evaluate the contribution of variants in the BCL11A, and HBS1L-MYB genes, implicated in the regulation of fetal hemoglobin, and of alpha-thalassemia coinheritance in 50 thalassemia intermedia and 75 thalassemia major patients. We confirm that alpha-thalassemia and allele C of single nucleotide polymorphism rs-11886868 in BCL11A were selectively represented in thalassemia intermedia patients. Moreover, allele G at single nucleotide polymorphism rs9389268 in the HBS1L-MYB locus was significantly more frequent in the thalassemia intermedia patients. This trio of genetic factors can account for 75% of the variation differences in phenotype severity.

KLF1 gene mutations cause borderline HbA(2).

Increased hemoglobin A(2) (HbA(2); ie, levels > 3.9%) is the most important feature of β-thalassemia carriers. However, it is not uncommon to find persons with borderline HbA(2) (levels, 3.3%-3.8%), who pose a relevant screening problem. Several genotypes have been associated with borderline HbA(2), but sometimes the reasons for this unusual phenotype are unknown. In this paper, we report, for the first time, that mutations of KLF1 result in HbA(2) levels in the borderline range. Six different KLF1 mutations were identified in 52 of 145 subjects with borderline HbA(2) and normal mean corpuscular volume and mean corpuscular hemoglobin. Two mutations (T327S and T280_H283del) are here reported for the first time. The prevalent mutation in Sardinians is S270X, which accounts for 80.8% of the total. The frequent discovery of KLF1 mutations in these atypical carriers may contribute significantly to the thalassemia screening programs aimed at identification of at risk couples.

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.

Genetic modifiers of β-thalassemia and clinical severity as assessed by age at first transfusion

Background The clinical and hematologic features of β-thalassemia are modulated by different factors, resulting in a wide range of clinical severity. The main factors are the type of disease-causing mutation and the ability to produce α-globin and γ-globin chains. In the present study we investigated the respective contributions of known modifiers to the prediction of the clinical severity of β-thalassemia as assessed by the patients’ age at first transfusion. Design and Methods We studied the effect of seven loci in a cohort of 316 Sardinian patients with β0-thalassemia. In addition to characterizing the β-globin gene mutations, α-globin gene defects and HBG2:g.−158C>T polymorphism, we genotyped two different markers in the BCL11A gene and three in the HBS1L-MYB intergenic region using single nucleotide polymorphism microarrays, imputation and direct genotyping. We performed Cox proportional hazard analysis of the time to first transfusion. Results According to the resulting model, we were able to explain phenotypic severity to a large extent (Harrell’s concordance index=0.72; Cox & Snell R2=0.394) and demonstrated that most of the model’s discriminatory ability is attributable to the genetic variants affecting fetal hemoglobin production (HBG2:g.−158C>T, BCL11A and HBS1L-MYB loci: C-index=0.68, R2=0.272), while the remaining is due to α-globin gene defects and gender. Consequently, significantly distinct survival curves can be described in our population. Conclusions This detailed analysis clarifies the impact of genetic modifiers on the clinical severity of the disease, measured by time to first transfusion, by determining their relative contributions in a homogeneous cohort of β0-thalassemia patients. It may also support clinical decisions regarding the beginning of transfusion therapy in patients with β-thalassemia.

Association of α globin gene quadruplication and heterozygous β thalassemia in patients with thalassemia intermedia

The degree of the globin chain imbalance is the pathogenetic clue to the clinical phenotype of thalassemia syndromes. This paper reports a duplication of the α globin gene locus in a group of hetereozygous β-thalassemia patients with the unexplained phenotype of thalassemia intermedia. Ten patients with thalassemia intermedia with variable severity and apparent simple heterozygosis for β0 39 C>T nonsense mutation were submitted to clinical, hematologic and molecular studies. The presence of an unknown molecular defect (silent β-thalassemia) unlinked to the β cluster interacting with the heterozygous β thalassemia, was previously postulated in these families. Analysis of the α globin gene cluster with PCR-based methods (MLPA, GAP-PCR, digestion with restriction enzymes) detected complex rearrangements in the α cluster. A duplication of the α globin gene locus, including the upstream regulatory region, was present in all the patients, associated in some of them with deletion or non-deletion α thalassemia. The variability of the clinical phenotype correlates with the degree of the globin chain imbalance. The presence of α globin cluster duplication should be considered in patients heterozygote for β-thalassemia with thalassemia intermedia phenotype and in the carriers of suspected silent β thalassemia.

New analytical tools and epidemiological data for the identification of HbA2 borderline subjects in the screening for beta-thalassemia

The increase of HbA(2) is the most important feature in the identification of beta-thalassemia carriers. However, some carriers are difficult to identify, because the level of HbA(2) is not in the typical range. Few data are available concerning the prevalence of such unusual phenotypes, and knowing their expected prevalence could be helpful in detecting systematic drifts in the analytical systems for HbA(2) quantification. In this study we report a retrospective investigation in two centres with high prevalence of beta-thalassemia. The prevalence of borderline subjects was found to be 2.2 and 3.0%, respectively. The genotypes of a subgroup of these subjects were then analyzed and in about 25% of cases a mutation in the globin genes was identified. We conclude that the occurrence of HbA(2) borderline phenotypes is not a rare event. In order to obtain more accurate HbA(2) measurements the development of an international reference measurement system for HbA(2), based on quantitative peptide mapping, has been recently started. We believe that the innovative approach of our method could also be used as a model to develop accurate quantitative methods for other red cell proteins relevant to the biodynamic properties and the surface electrochemistry of erythrocytes.

Genome-wide association analyses based on whole-genome sequencing in Sardinia provide insights into regulation of hemoglobin levels

We report genome-wide association study results for the levels of A1, A2 and fetal hemoglobins, analyzed for the first time concurrently. Integrating high-density array genotyping and whole-genome sequencing in a large general population cohort from Sardinia, we detected 23 associations at 10 loci. Five signals are due to variants at previously undetected loci: MPHOSPH9, PLTP-PCIF1, ZFPM1 (FOG1), NFIX and CCND3. Among the signals at known loci, ten are new lead variants and four are new independent signals. Half of all variants also showed pleiotropic associations with different hemoglobins, which further corroborated some of the detected associations and identified features of coordinated hemoglobin species production.