Y-chromosome R-M343 African Lineages and Sickle Cell Disease reveal structured assimilation in Lebanon

Abstract

We have sought to identify signals of assimilation of African male lines in Lebanon by exploring the association of sickle cell disease in Lebanon with Y-chromosome haplogroups that are informative of the disease origin and its exclusivity to the Muslim community. A total of 732 samples were analyzed including 33 sickle cell disease patients from Lebanon genotyped for 28 binary markers and 19 short tandem repeats on the non-recombinant segment of the Y chromosome. Genetic organization was identified using populations known to have influenced the genetic structure of the Lebanese population, in addition to African populations with high incidence of sickle cell disease. Y-chromosome haplogroup R-M343 sub-lineages distinguish between sub-Saharan African and Lebanese Y chromosomes. We detected a limited penetration of sickle cell disease into Lebanese R-M343 carriers, restricted to Lebanese Muslims. We suggest that this penetration brought the sickle cell gene along with the African R-M343, probably with the Saharan caravan slave trade.

Introduction

Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the β-globin chain, widely distributed throughout equatorial Africa reaching heterozygote frequencies as high as 30-40% in parts of Uganda, West Africa, and Zaire¹ because it confers resistance to malaria. The sickle gene spread to North Africa and the Mediterranean along Saharan caravan trading routes, to North and South America, and the Caribbean by slave trade, and then to Northern Europe by immigration from the Caribbean². Linkage analysis of the β-globin sickle mutations suggest homoplasy,¹ with African and Asian origins distinguished by their haplotypes³. Previous studies of sickle mutations in the Lebanese population showed that 91.5% of the patients were Sunni and Shiite Muslims⁴ (Sunni and Shiites make 58% of the general Lebanese population⁵) and that 90% of SCD subjects carried an African haplotype⁶.

The origin, time of spread, or details of the association of SCD with Lebanese Muslims cannot be deduced from the genetic content of sickle haplotypes. The sickle mutation is estimated to have arisen 3000-6000 generations ago^{7, 8}, while the haplotypes surrounding the β-globin locus are even older. Specificity of geographic origin among SCD haplogroups has been noted⁹ along with some historical, sociological and genetic suggestions of possible migration routes into Lebanon^{10, 11}. Also, studies of heterozygosity have been used to impose limits on time of recent migration.¹²

We have previously used Y chromosome markers to study population structure and migration into Lebanon¹³. Since SCD is closely affiliated with specific Lebanese communities which correlate with Y-chromosome diversity, SCD and Y-chromosome polymorphisms could reveal structure in migrant assimilation. Indeed, the time of spread of the sickle mutation across the Sahel has been previously inferred from a Y-chromosome Single Nucleotide Polymorphism (SNP) that was co-integrated with the sickle gene by West African tribes migrating to the East¹⁴.

In this study, we investigated the penetration of the sickle-cell mutation into Lebanon, and explored association between certain African Y-chromosome lineages and the chromosome 11 sickle cell mutation in order to determine whether there is a sign of persistent population structure characterizing assimilation of Africans in Lebanon. We also investigated the sickle cell penetration into the broader Lebanese population through examination of a set of sickle cell patients bearing non-African Y haplogroups.

Materials and methods

Subjects and Comparative Datasets

The populations selected for this study were those who had potentially influenced the genetic structure of the Lebanese population¹⁵ in addition to African populations with high incidence of SCD. A total of 732 samples were analyzed including newly genotyped samples from Libya (33), Kuwait (8), and Lebanese SCD patients (28 Muslims, 5 Christians). The new samples were added to previously genotyped samples from our laboratory^{13, 16, 17}; Lebanon (250), Egypt (84), Iran (56), and Malta (20). In addition comparative data from Central Africa (46), Algeria (16), Italy (103), KSA (50), UAE (18), Uganda (10), and Yemen (5) were obtained from published sources^18-23. All samples had an unknown SCD status except for the 33 newly genotyped Lebanese samples. SCD male subjects were used from our database⁶ after they were anonymiszed and approved for this study by the IRB of the Lebanese American University. All other participants provided detailed information on their geographical origin, and gave informed consent approved by the IRB of the Lebanese American University.

Genotyping

DNA was extracted from blood using a standard Phenol-Chloroform method. Samples were genotyped using the Applied Biosystems 7900HT Fast Real-Time PCR System with a set of 28 custom Y-chromosomal binary marker assays (Applied Biosystems, Foster City, CA) from the non-recombining portion of the Y chromosome which define 21 haplogroups. The new samples were additionally amplified at 19 Y-chromosomal STR loci in two multiplexes and analyzed on an Applied Biosystems 3130xl Genetic Analyzer. The first multiplex contained the standard 17 loci of the Y-filer™ PCR Amplification kit (Applied Biosystems, Foster City, CA). The remaining two loci, DYS388 and DYS426, were genotyped in a separate custom multiplex provided by Applied Biosystems.

Statistical Analyses

From R-M343, J-M172, J-M267, and E-M35, we found that R-M343 had regionally differentiating Y STR haplotypes identified by Reduced Median Networks²⁴ with a reduction coefficient of 1, and validated with MDS²⁵ applied to Φ_ST computed in ARLEQUIN²⁶, and tested using AMOVA²⁷.

Reduced Median Network and AMOVA was performed to ascertain the variance characteristics among the Y-STR loci of R-M343 that were common to this study and the literature; DYS393, DYS390, DYS19, DYS391, DYS439, DYS389I, DYS392, DYS389b, DYS437, DYS438. AMOVA seeks to identify variance within populations due to drift by comparing variation among groups of similar populations via a nested analysis of variance. MDS was executed with SPSS16 in two dimensions with a minimum S-stress of 0.005. Likewise, non-African haplogroups were identified, marking penetration of African sickle cell mutations into the general Lebanese population. An extension to the Hardy-Weinberg²⁸ equilibration model was constructed analytically following standard arguments,²⁹ assuming complete replacement each generation, where children randomly select both parents (sexes treated distinctly), randomly select a diploid chromosome 11 from each parent, and inherit the haploid Y chromosome from the father. Deviations from such random selection imply population structure. The extended Hardy-Weinberg equilibration process showed the heterozygous diploid sickle cell markers among females equilibrating immediately, while the deviation from equilibrium for the African Y vs. sickle mutations reduced by a factor of 2 each generation. At equilibrium, the Y African markers and the sickle markers are independent. This implies that, if 7 or more generations have occurred since African input, the deviation from equilibrium will not exceed 0.8%. Prevalence of SCD among Lebanese in general, and among R-M343 in Lebanon specifically, are not available, so standard tests of linkage disequilibrium are not possible. However, it is possible to test whether P(SCD|R – M343 = African) = P(SCD|R – M343). Therefore, a Fisher test for dependence between the Y African expansion marker and the sickle cell was applied to test population structure.

Results

Y genotyping of the SCD patients revealed 6 haplogroups (14 J-M267, 5 J-M172, 8 E-M35, 4 R-M343, 1 T-M70, 1 N-LLY22g) all previously reported in the general Lebanese population.^{13, 16, 17}

RM networks of R-M343 (Figure 1) showed significant regional differentiation between Central Africans, Europeans, and Lebanese. The probability that random region assignments would have isolated 46 Africans out of the 48 in the segregated branches, being drawn from 46 out of 124 by chance has a Fisher p-value <0.001. Corollary is that the two Lebanese samples in those branches likely represent African descent. Both samples were identified as Lebanese Muslims. The SCD samples were also all contained in African branches. The chances that all 4 SCD samples would have been drawn from African branches by chance have a p-value of 0.017. This result is also supported by the MDS plot, as well as significant AMOVA variation between the groups (20.07%, p<0.001, Table1). R-M343 SCD subjects were all Muslims and clustered with Central African haplotypes. The SCD-Central African Φ_ST distances was the lowest (Φ_ST=0.048) with no significant differentiation (p= 0.33±0.03) compared to all other populations, including the Lebanese (Φ_ST=0.215) with significant segregation (p= 0.027±0.019). MDS of the Φ_ST’s showed strong clustering of the SCD with Central Africans outlying all other populations (Figure 2).

Figure 1.

Sample distribution and Reduced Median Network of R-M343. The map shows the location of the samples used in the Networks and the respective color for every population. A) Reduced Median Network of R-M343 from all indicated populations in the map. B) Reduced Median Network of R-M343 from Lebanon, Central Africa and Lebanese Sickle Cell disease patients.

Table 1.

AMOVA results comparing variations among Europeans, Central Africans, and Lebanese

	Va;FCT Among Groups	Vb;FSC Within Groups	Vc;FSR Within Populations
Value	0.896; 0.201	0.287; 0.080	3.280; 0.265
% Variation	20.07	6.43	73.49
p-value	<0.001	<0.001	<0.001

Figure 2.

MDS plot of Φ distances between populations derived from Y-STR data of R-M343.

Reduced-Median networks of haplogroups E-M35, J-M267 and J-M172 (Figure 3) show that SCD haplotypes are present only in the general Lebanese population indicating penetration of the sickle gene into the population at large.

Figure 3.

Reduced Median Networks of J-M267, J-M172, and E-M35.

Discussion

We have shown that R-M343 STR haplotypes clearly discriminate between Lebanese and African origins, unlike other common haplogroups which also are not represented in the African populations. This enables identification of African chromosomes that have migrated to Lebanon. Further, SCD among R-M343 Lebanese subjects is largely confined to African haplotypes. The strong association of the sickle gene with the African R-M343 would suggest the source populations carried a high frequency of both markers. The sickle mutation is very common to African populations unlike R-M343 which is rare in Africa and found mainly in Europe and Asia. However, R-M343 has been found in high concentration in some populations from Central-West Africa where it reaches very high frequencies (up to 95%) in populations in the central Sahel; northern Cameroon, northern Nigeria, Chad, and Niger.^30,31 For many centuries African slaves were drawn from those populations and driven across the central Sahara to the Mediterranean ports, a regular trade seems to have been established in the early Islamic era³² and continued until the 1840’s^{32, 33} confined then to the central and eastern basins of the Mediterranean still under Ottoman influence³². Deviations from Hardy-Weinberg equilibrium of diploid SCD in both genders with haploid male African markers in a Wright-Fisher model decay by a factor of 2 per generation. Therefore, an unstructured population would have SCD nearly uniformly distributed among Lebanese R-M343 haplotypes, however the R-M343 SCD subjects were exclusive to African haplotypes, and found only among Lebanese Muslims.

The probability that 28 or more out of 33 samples drawn from a population would be Muslim when drawn from a population with 58% Muslim is estimated to be by binomial test. Conversion to Islam provided a means to better treatment and possible later emancipation,³³ and prohibition of non-Muslim slave ownership promoting slave conversions to Islam solely,³⁴ explains SCD association with Muslim subjects. However, the limited penetration of SCD into Lebanese R-M343 contradicts a picture of many centuries of conversion and assimilation refreshed by caravan trade, implying a persistently structured population, even allowing for SCD introduction through the female line. Even though equilibration was not observed among R-M343 subjects, mixing was reflected by penetration to non-African haplogroups J-M267, J-M172, and E-M35. In all, this study reveals subtle population structure surviving for over two centuries in Lebanon, with significant medical consequences.