The sickle allele (HbS) of the beta-globin gene (HBB), which in the homozygous state gives rise to sickle cell disease (SCD), occurs commonly in populations of African ancestry as a result of its protective effect against severe malaria [1-4]. HbS is classically thought to have arisen multiple times around the same time in recent human history (~5,000 years ago), largely driven by observations of distinct haplotype backgrounds across sub-Saharan Africa, Arabia and India [5-7]. These classical ?s haplotypes are named according to their putative geographical origins – Benin, Bantu (Central African), Cameroon, Senegal and Arab. In general, in regions where the ?s allele is found at substantial frequency, a specific ?s haplotype usually predominates, although other haplotype backgrounds – both classical and ‘atypical’ - are also observed. For example, although the Senegal ?s haplotype is the most commonly observed in Senegal, the Benin haplotype is not uncommonly seen [8,9]; similarly, we have observed a commonality of Benin haplotypes among ?s haplotypes sampled in Yaoundé, Cameroon, as oppose to the expected ‘Cameroon-type’ (unpublished data). Combined with observations of broad diversity among even geographically isolated ?s haplotypes [8,10,11], there remains some uncertainty surrounding the origins and interpretation of classical ?s haplotypes, particularly the contribution of complex genomic rearrangements and ancestral population events [12-15].

 

 

Much of the conventional wisdom concerning the origin of ?s haplotypes was concluded from the patterns of restriction fragment length polymorphisms observed in the early 1980’s [5,6,16]. The authors observed a single major ancestral haplotype in each population as well as several less-common, but more diverse, ancestral haplotype backgrounds. It was postulated that the observed data reflected either a single (most-common) mutation followed by complex genomic rearrangements or three to four independent mutations occurring independently. The relative simplicity of the latter explanation subsequently gained traction, bolstered by subsequent population surveys [17]and more detailed haplotype inference [18]. Since those original descriptions, however, there have been significant technological advances that have informed our understanding of the genomic architecture of the beta-globin region and potentially have implications for the accepted dogma of ?s haplotypes. In addition to the aforementioned increase in DNA sequence diversity on classical RFLP haplotypes, a propensity for gene conversion/structural chromosomal events in the beta-globin locus has since been described [19-21], and new signatures of selection, punctuated by long-range ancestral haplotypes, have been described in association with the HbS allele [9,22-24]. The dynamics of how these more recent observations interplay with one-another remains uncertain [25], but taken together, they suggest that our understanding of the genetic context and recurrent nature of the HbS allele may be substantially more complex than hitherto thought, and not fully captured by the original ?s haplotype descriptions. Given reported associations between ?s haplotypes and variability in SCD severity [26-28], and the central contribution of HbS to our understanding of population genetics [29], a updated characterization of ?s haplotypes could have profound implications for our understanding of human history and population migration; as well as for the first molecularly understood genetic disease - SCD.

 

 

 

The primary purpose of this study is the technological re-evaluation and characterization of ?s haplotypes. The specific aims are:

 

1. Describe the distribution of ?s haplotypes across multiple populations of African Ancestry

 

We will provide a robust overview of African ?s haplotypes by evaluating the frequency and diversity of Beta-S haplotypes in a cohort of SCD patients from across Africa.

 

2. Evaluate the genomic evidence for multiple, simultaneous origins for HbS using ?s haplotypes

 

2a. Define traditional beta-globin haplotypes utilizing next-generation sequencing technologies

 

We will combine next-generation sequencing technology with traditional RFLP genotyping to characterize and define classical ?s haplotypes in the context of common sequence variation.

 

2b. Compare long-range- and classical- ?s haplotypes

 

We will compare extended ancestral ?s chromosomes derived from long-range sequencing with ancestral chromosomes inferred from classical ?s haplotypes (characterized as in 2a). This data will be evaluated against haplotype expectations consistent with the multi-origin hypothesis for HbS. 

 

 

 

We propose to obtain 500 DNA samples from SCD patients in Africa. For two-hundred of these patients, DNA will also be obtained from the patients’ mother and father. Relevant demographic information – age, gender, grandparents’ ethnic affiliation – will also be recorded. Samples will be genotyped using RFLP to determine classical beta-S haplotypes. The samples will be sequenced over a 400 kilobase region encompassing beta-globin.

 

 

 

 

 

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2.   Hexter, A., Selective advantage of the sickle-cell trait. Science, 1968. 160(826): p. 436-7.

 

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5.   Wainscoat, J.S., et al., Multiple origins of the sickle mutation: evidence from beta S globin gene cluster polymorphisms. Mol Biol Med, 1983. 1(2): p. 191-7.

 

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9.   Hanchard, N., et al., Classical sickle beta-globin haplotypes exhibit a high degree of long-range haplotype similarity in African and Afro-Caribbean populations. BMC Genet, 2007. 8: p. 52.

 

10.  Patrinos, G.P., et al., Evidence for the molecular heterogeneity of sickle cell anemia chromosomes bearing the betas/Benin haplotype. Am J Hematol, 2005. 80(1): p. 79-80.

 

11.  Labie, D., et al., Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India. Hum Biol, 1989. 61(4): p. 479-91.

 

12.  Chakravarti, A., et al., Nonuniform recombination within the human beta-globin gene cluster. Am J Hum Genet, 1984. 36(6): p. 1239-58.

 

13.  Bouhassira, E.E., et al., A gene conversion located 5' to the A gamma gene in linkage disequilibrium with the Bantu haplotype in sickle cell anemia. J Clin Invest, 1989. 83(6): p. 2070-3.

 

14.  Currat, M., et al., Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta(S) Senegal mutation. Am J Hum Genet, 2002. 70(1): p. 207-23.

 

15.  Magana, M.T., et al., 3' haplotypes of the beta-globin gene in beta(S)-chromosomes of Mexican individuals. Blood Cells Mol Dis, 2005. 34(1): p. 48-52.

 

16.  Antonarakis, S.E., et al., Origin of the beta S-globin gene in blacks: the contribution of recurrent mutation or gene conversion or both. Proc Natl Acad Sci U S A, 1984. 81(3): p. 853-6.

 

17.  Oner, C., et al., Beta S haplotypes in various world populations. Hum Genet, 1992. 89(1): p. 99-104.

 

18.  Chebloune, Y., et al., Structural analysis of the 5' flanking region of the beta-globin gene in African sickle cell anemia patients: further evidence for three origins of the sickle cell mutation in Africa. Proc Natl Acad Sci U S A, 1988. 85(12): p. 4431-5.

 

19.  Borg, J., et al., Genetic recombination as a major cause of mutagenesis in the human globin gene clusters. Clin Biochem, 2009. 42(18): p. 1839-50.

 

20.  Holloway, K., V.E. Lawson, and A.J. Jeffreys, Allelic recombination and de novo deletions in sperm in the human beta-globin gene region. Hum Mol Genet, 2006. 15(7): p. 1099-111.

 

21.  Neumann, R., V.E. Lawson, and A.J. Jeffreys, Dynamics and processes of copy number instability in human gamma-globin genes. Proc Natl Acad Sci U S A, 2010. 107(18): p. 8304-9.

 

22.  Hanchard, N.A., et al., Screening for recently selected alleles by analysis of human haplotype similarity. Am J Hum Genet, 2006. 78(1): p. 153-9.

 

23.  Ghansah, A., et al., Haplotype analyses of haemoglobin C and haemoglobin S and the dynamics of the evolutionary response to malaria in Kassena-Nankana District of Ghana. PLoS One, 2012. 7(4): p. e34565.

 

24.  Liu, X., et al., Detecting and characterizing genomic signatures of positive selection in global populations. Am J Hum Genet, 2013. 92(6): p. 866-81.

 

25.  Jones, D.A. and J. Wakeley, The influence of gene conversion on linkage disequilibrium around a selective sweep. Genetics, 2008. 180(2): p. 1251-9.

 

26.  Powars, D.R., L. Chan, and W.A. Schroeder, Beta S-gene-cluster haplotypes in sickle cell anemia: clinical implications. Am J Pediatr Hematol Oncol, 1990. 12(3): p. 367-74.

 

27.  Powars, D.R., Sickle cell anemia: beta s-gene-cluster haplotypes as prognostic indicators of vital organ failure. Semin Hematol, 1991. 28(3): p. 202-8.

 

28.  Bean, C.J., et al., Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia. Br J Haematol, 2013. 163(2): p. 268-76.

 

29.  Flint, J., et al., The population genetics of the haemoglobinopathies. Baillieres Clin Haematol, 1998. 11(1): p. 1-51.