Steve Best

A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15

F cells measure the presence of fetal hemoglobin, a heritable quantitative trait in adults that accounts for substantial phenotypic diversity of sickle cell disease and β thalassemia. We applied a genome-wide association mapping strategy to individuals with contrasting extreme trait values and mapped a new F cell quantitative trait locus to BCL11A, which encodes a zinc-finger protein, on chromosome 2p15. The 2p15 BCL11A quantitative trait locus accounts for 15.1% of the trait variance.

Intergenic variants of HBS1L-MYB are responsible for a major quantitative trait locus on chromosome 6q23 influencing fetal hemoglobin levels in adults.

Individual variation in fetal hemoglobin (HbF, α2γ2) response underlies the remarkable diversity in phenotypic severity of sickle cell disease and β thalassemia. HbF levels and HbF-associated quantitative traits (e.g., F cell levels) are highly heritable. We have previously mapped a major quantitative trait locus (QTL) controlling F cell levels in an extended Asian-Indian kindred with β thalassemia to a 1.5-Mb interval on chromosome 6q23, but the causative gene(s) are not known. The QTL encompasses several genes including HBS1L, a member of the GTP-binding protein family that is expressed in erythroid progenitor cells. In this high-resolution association study, we have identified multiple genetic variants within and 5′ to HBS1L at 6q23 that are strongly associated with F cell levels in families of Northern European ancestry (P = 10−75). The region accounts for 17.6% of the F cell variance in northern Europeans. Although mRNA levels of HBS1L and MYB in erythroid precursors grown in vitro are positively correlated, only HBS1L expression correlates with high F cell alleles. The results support a key role for the HBS1L-related genetic variants in HbF control and illustrate the biological complexity of the mechanism of 6q QTL as a modifier of fetal hemoglobin levels in the β hemoglobinopathies.

HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers

Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q that are associated with elevated fetal hemoglobin (HbF) levels and alterations of other clinically important human erythroid traits. It is unclear how these noncoding sequence variants affect multiple erythrocyte characteristics. Here, we determined that several HBS1L-MYB intergenic variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We found that several HBS1L-MYB intergenic variants reduce transcription factor binding, affecting long-range interactions with MYB and MYB expression levels. These data provide a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a target for therapeutic induction of HbF to ameliorate sickle cell and β-thalassemia disease severity.

α‐Haemoglobin stabilising protein is a quantitative trait gene that modifies the phenotype of β‐thalassaemia

It has been suggested that altered levels or function of α‐haemoglobin stabilising protein (AHSP), an erythroid‐specific protein that binds specifically to free α‐(haemo)globin, might account for some of the clinical variability in β‐thalassaemia. To assess the variation of AHSP expression, mRNA levels in circulating reticulocytes of 103 healthy individuals were measured by quantitative reverse transcription‐polymerase chain reaction. AHSP expression varied up to threefold, and did not correlate with age or sex. A systematic survey of the AHSP locus identified eight sequence variants, of which six were common. Four common variants, including the longer homopolymer (T18) in the putative promoter, are strongly associated with AHSP expression. Reporter assays in K562 cells showed that the activity of the shorter (T15) reporter was relatively lower than that of the T18 reporter. In a study of nine anaemic patients who were heterozygous for β‐thalassaemia and also heterozygous for the triplicated α‐globin gene (ααα/αα), frequency of the shorter homopolymer was higher than expected. AHSP expression is variable, with cis control accounting for some of its variance. In some families, the subtle altered levels in AHSP related to the AHSP genotype appears to be a relevant contributory factor in the haematological phenotype.

Binding patterns of BCL11A in the globin and GATA1 loci and characterization of the BCL11A fetal hemoglobin locus

BCL11A is a major regulator of fetal hemoglobin production. Reduced levels of BCL11A have been shown to delay switching from fetal to adult hemoglobin, suggesting that it acts as a stage-specific repressor of gamma globin expression. We have carried out a survey of BCL11A binding in the globin, BCL11A and GATA1 loci by ChIP-on-chip analysis in primary human erythroid cells. We found strong occupancy in both alpha and beta globin upstream regulatory regions as well as in regions involved in switching and hereditary persistence of fetal hemoglobin. Genetic studies have identified a restricted 14kb region in BCL11A intron 2 as being highly associated with HbF levels. Strong GATA-1 binding and acetylated histone H3 was found in this area, which could be indicative of a regulatory element, changes in which might be responsible for the overall regulation of BCL11A. We also observed BCL11A and GATA-1 binding in a known auto-regulatory promoter element of the GATA1 locus.

Lamin B‐receptor mutations in Pelger–Huët anomaly

Summary. Pelger–Huët anomaly is an inherited abnormality of neutrophils, characterized by reduced nuclear segementation and an apparently looser chromatin structure. Following linkage studies in two families, the lamin B‐receptor (LBR) was sequenced and mutations found: CCG→CTG causing proline→leucine in codon 119 of exon 3, and IVS11‐9 A→G, disrupting the splice acceptor site. The LBR gene (LBR) was also sequenced from a single English man with Pelger–Huët anomaly and a heterozygous C→G mutation was found in codon 569 of exon 14, predicted to cause a proline→arginine. Our results confirm recently published findings that LBR mutations cause Pelger–Huët.

A novel 506kb deletion causing εγδβ thalassemia.

We describe a novel deletion causing εγδβ thalassemia in a Pakistani family. The Pakistani deletion is 506kb in length, and the second largest εγδβ thalassemia deletion reported to date. It removes the entire β globin gene (HBB) cluster, extending from 431kb upstream to 75kb downstream of the ε globin gene (HBE). The breakpoint junction occurred within a 160bp palindrome embedded in LINE/LTR repeats, and contained a short (9bp) region of direct homology which may have contributed to the recombination event. Characterization of the deletion breakpoints has been particularly challenging due to the complexity of DNA deletion, insertion and inversion, involving a multitude of methodologies, mirroring the changing DNA analysis technologies.

Two candidate genes for low platelet count identified in an Asian Indian kindred by genome-wide linkage analysis : glycoprotein IX and thrombopoietin

A genome-wide linkage analysis of platelet count was carried out in a large Asian Indian kindred. Linkage analysis showed one marker (D3S1309) on chromosome 3q with a lod score of 3.26 and another (D3S1282) approximately 30 cM centromeric, with a lod score of 2.52. Multipoint analysis of chromosome 3q identified two peaks with maximum multipoint lod scores of 3.52 and 4.11 under markers D3S1309 and D3S1282, respectively. Two strong candidate genes for platelet variation were identified in the linked region; thrombopoietin (THPO) and glycoprotein IX (GPIX). Resequencing of four individuals revealed five single-nucleotide polymorphisms (SNPs) in THPO and one mutation in the transmembrane region of GPIX. Analysis of variance showed that the GPIX mutation and one THPO SNP accounted for 6 and 4% of the variation in platelet count, respectively. The THPO SNP lies in the 3′ untranslated region of the gene and has not been previously reported. The G to A transition at nucleotide 653 resulted in an Ala 156 (GCC) to Thr (ACC) replacement in the GPIX protein. The GPIX mutation was recently identified in a Chinese patient with Bernard–Soulier syndrome (BSS), a rare recessive bleeding disorder characterized by thrombocytopenia and giant platelets. One copy of the GPIX mutation was found in 300 European individuals with platelet counts within the normal range. The results suggest that two QTLs on chromosome 3q influence platelet count variation in the Asian Indian kindred, with the GPIX transmembrane mutation and the 3′ UTR SNP in THPO being strong candidates.

Heterogeneity of the ɛγδβ-thalassaemias: characterization of three novel English deletions

We have characterized three novel ɛγδβ‐thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire β‐globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98‐kb deletion extended 90‐kb upstream of the ɛ gene to 8 kb upstream of the Gγ‐gene, leaving the γ,δ and β‐genes intact. The 439 kb deletion is the largest deletion reported so far to cause ɛγδβ‐thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.

Heterogeneity of the egammadeltabeta-thalassaemias: characterization of three novel English deletions

We have characterized three novel ɛγδβ‐thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire β‐globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98‐kb deletion extended 90‐kb upstream of the ɛ gene to 8 kb upstream of the Gγ‐gene, leaving the γ,δ and β‐genes intact. The 439 kb deletion is the largest deletion reported so far to cause ɛγδβ‐thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.