Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTPbinding 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.

These data show encouraging results for AMI in comparison to previous failed IVF cycles.

Three quantitative trait loci (QTLs) modifying fetal hemoglobin (HbF) levels have been identified, and these have been shown to have a predictive value of disease severity in β thalassemia and sickle cell disease in diverse ethnic groups. One of the HbF QTLs which consists of a set of common intergenic single nucleotide polymorphisms (SNPs) in the HBS1L-MYB intergenic region on chromosome 6q23, has also been consistently identified as highly associated with clinically important human erythroid traits. Despite extensive genetic evidence, a clear mechanistic basis for the association between the intergenic SNPs and erythroid biology has remained elusive, although the two flanking genes (HBS1L and MYB) are candidate target genes. Here, we set out to characterize the regulatory potential of the human HBS1L-MYB intergenic region in detail and to investigate its functional impact on the erythroid phenotype-associated variants. We profiled chromatin occupancy of the key erythroid LDB1 transcription factor (TF) complex in primary human erythroid progenitors (HEPs) using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) and quantitative PCR (ChIP-qPCR). We detected an intergenic cluster containing 7 binding sites for the LDB1–complex, characterized by strong binding and co-occupancy of core complex proteins LDB1, GATA1, TAL1 and ETO2. One of these sites was co-occupied by the erythroid-specific TF KLF1, a protein which was also found to bind the murine intergenic region. Depletion of LDB1, TAL1 and KLF1 in K562 cells using RNA interference resulted in a specific downregulation of MYB expression while leaving HBS1L levels unaffected, demonstrating that the erythroid TFs occupying the intergenic enhancers are required for MYB expression. Using chromosome conformation capture (3C) coupled to high-throughput sequencing (3C-Seq), we profiled higher order chromatin structure within the locus, and detected several strong chromatin co-associations between the MYB promoter and intergenic sequences, almost all of which correlated with TF binding. The binding activities were distributed over a conserved core region identical to a 24-kb interval containing genetic variants in strong genetic association with erythroid traits in human populations. This block of SNPs is referred to as HBS1L-MYB intergenic polymorphisms block-2 (HMIP-2). The SNPs within HMIP-2 clustered in 2 regions positioned directly under the 2 LDB1-complex ChIP-Seq peaks, at -84 and -71 kb from the MYB transcription start site. Using allele-specific ChIP in K562 cells, we observed diminished (25-50%) binding of LDB1, GATA1, TAL1 and KLF1 to the minor rs66650371 allele, showing that rs66650371 affects local TF binding. An allele-specific 3C analysis also showed reduced interactions between the minor rs66650371 allele at -84 and MYB. We validated and expanded observations made in K562 cells using primary human cells: 1. HEPs from high HbF individuals homozygous for all minor alleles of the phenotype-associated -84kb and -71kb intergenic variants in the conserved core (‘SNP/SNP’) showed consistently lower MYB levels throughout phase II of the culture as compared to wildtype control cells (‘WT/WT’); 37% lower MYB on average; 2. ChIP experiments on SNP/SNP and WT/WT HEPs showed reduced binding of GATA1 and KLF1 to the -84 and -71 regulatory elements (containing the associated variants), the results were further confirmed by allele-specific ChIP assays in SNP/WT HEPs; 3. 3C-qPCR assays on cultured SNP/SNP and WT/WT cells demonstrated diminished looping between the -84 element and the MYB promoter in SNP/SNP individuals; 4. SNP/WT HEPs also showed allelic imbalance of MYB but not HBS1L transcripts when compared to controls (WT/WT and SNP/SNP HEPs). In conclusion, we show that the HBS1L-MYB gene-free interval contains distal enhancer elements that interact with MYB, a critical regulator of erythroid development and HbF levels. Key variants in the intergenic interval affect MYB expression by reducing TF binding to its regulatory elements and disrupting long-range enhancer-gene interaction. Our study identifies the first causal link between the 6q23 HbF QTL and MYB regulation, provides novel insights into the molecular control of clinically important haematological traits and adds another layer of complexity to the regulation of MYB, suggesting potential targets for therapeutic intervention. Disclosures: Thein: Sangart: Consultancy; Shire: Research Funding; Novartis: Speakers Bureau.

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Background14q duplications caused by parental pericentric inversion of chromosome 14 are rarely reported and no clear genotype-phenotype correlation has been determined yet.Case PresentationHere we reported a 7 years old female patient with recombinant chromosome characterized by 14 q duplication and originated from maternal pericentric inversion of chromosome 14. Principal clinical findings of the child include developmental delay, microcephaly, hypertelorism, low set ears, clinodactyly of fifth fingers, hypotonia, telecanthus and cardiac malformation.ConclusionsHer final karyotype was 46,XX,rec(14)dup(14q)inv(14)(p11.2q24)mat,arr14q24.1-qter(64,800,000-108,350,000 bp)x3.