Genetic Studies in Malta

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A number of genetic studies have been performed in Malta, where different groups of scientists studied the genetics of a number of human disorders. Maybe the most studied molecule so far was haemoglobin together with the β-globin gene and the globin cluster, which were studied mostly in thalassaemic patients. On this page I am going to focus on studies done to identify genes and variants that might result in an increased susceptibility for osteoporosis and coeliac disease. Both disease, as many other common diseases such as atherosclerosis, diabetes and epilepsy, are complex which means that they are caused by interactions of genes with environment and also by interactions between different genes. These characteristics present a number of difficulties to the researcher because it makes it more difficult to identify causative variants.  Complex disorders are commonly found within populations and although sometimes they are observed to segregate in families usually they do not show classical patterns of Mendelian inheritance. 

 

Genetic studies of Osteoporosis

For the last eight years, I've been studying the genetics of osteoporosis using both linkage and association approaches. A total of 17 single nucleotide polymorphisms (SNPs) within six genes were studied for association with bone mineral density (BMD) in a group of osteoporotic and osteopenic postmenopausal women and controls (age and sex matched). These polymorphisms were analysed by PCR-RFLP using agarose and polyacrylamide gel electrophoresis. The genes chosen are known to be involved in bone physiology and include those coding for various receptors (vitamin D receptor, oestrogen receptor), regulators of osteoclastogenesis (TNFRSF11B) and structural genes such as COL1A1 gene. Allele frequencies and Hardy-Weinberg equilibrium for the SNPs studied are shown in the table below.

 

Gene

SNP

Genotype

N (%)

Allele Frequency

Het

c2

p Value*

 

 

 

VDR

FokI

CC

76 (60.8)

C : 0.77

0.35

1.30

0.25

 

CT

40 (32.0)

T : 0.23

 

 

 

 

TT

9 (7.2)

 

 

 

 

BsmI

GG

24 (19.2)

G : 0.44

0.49

0.16

0.69

 

GA

59 (47.2)

A : 0.56

 

 

 

 

AA

42 (33.6)

 

 

 

 

ApaI

TT

39 (31.7)

T : 0.56

0.49

0.10

0.76

 

TG

59 (48.0)

G : 0.44

 

 

 

 

GG

25 (20.3)

 

 

 

 

TaqI

TT

44 (35.8)

T : 0.61

0.48

0.18

0.67

 

TC

61 (49.6)

C : 0.39

 

 

 

 

CC

18 (14.6)

 

 

 

 

 

G261C

GG

96 (89.7)

G : 0.95

0.10

0.31

0.58

 

GC

11 (10.3)

C : 0.05

 

 

 

ER1

PvuII

TT

21 (16.5)

T : 0.42

0.49

< 0.01

0.95

 

TC

61 (48.0)

C : 0.58

 

 

 

 

CC

45 (35.4)

 

 

 

 

XbaI

AA

17 (13.4)

A : 0.38

0.47

0.01

0.94

 

AG

60 (47.2)

G : 0.63

 

 

 

 

GG

50 (39.4)

 

 

 

 

 

G2014A

GG

83 (67.5)

G : 0.83

0.29

0.23

0.64

 

 

GA

37 (30.1)

A : 0.17

 

 

 

 

 

AA

3 (2.4)

 

 

 

 

TNFRSF11B

A163G

AA

92 (78.6)

A : 0.88

0.20

0.17

0.68

 

AG

24 (20.5)

G : 0.12

 

 

 

 

GG

1 (0.9)

 

 

 

 

T950C

TT

24 (19.4)

T : 0.48

0.50

2.68

0.10

 

TC

71 (57.3)

C : 0.52

 

 

 

 

CC

29 (23.4)

 

 

 

 

G1181C

GG

30 (26.5)

G : 0.54

0.50

0.82

0.37

 

GC

61 (54.0)

C : 0.46

 

 

 

 

CC

22 (19.5)

 

 

 

 

COL1A1

G1546T (Sp1)

SS

51 (41.8)

S : 0.65

0.48

0.10

0.75

 

Ss

57 (46.7)

s : 0.45

 

 

 

 

Ss

14 (11.5)

 

 

 

 

G-1997T

TT

10 (8.1)

T : 0.26

0.38

0.84

0.36

 

TG

43 (35.0)

G : 0.74

 

 

 

 

GG

70 (56.9)

 

 

 

 

MTHFR

C677T

CC

49 (38.9)

C : 0.64

0.46

0.88

0.35

 

CT

63 (50.0)

T : 0.36

 

 

 

 

TT

14 (11.1)

 

 

 

 

LRP5

Rs901823

TT

45 (39.8)

T : 0.63

0.47

0.03

0.88

 

TC

52 (46.0)

C : 0.37

 

 

 

 

CC

16 (14.2)

 

 

 

 

Rs546803

TT

62 (53.9)

T : 0.73

0.39

0.41

0.52

 

TC

43 (37.4)

C : 0.27

 

 

 

 

CC

10 (8.7)

 

 

 

 

*p values are for HWE using chi squared test

        The A314G SNP in the LRP5 gene was completely absent from the Maltese population

       Het = heterozygosity

 

None of the variants studied was observed to significantly affect BMD when tested as a quantitative trait. Only trends were observed for some polymorphisms and most of the trends also agreed with other studies done in other populations. The fact that statistical significance was not reached does not exclude any possible involvement of the gene or else even of that particular SNP with disease. One reason for this lack of association might be due to the very small sample size and thus to lack of statistical power. Genetic variants involved in complex disorders are usually common variants found within populations each having only a modest effect on disease. Thus it is the collective effect between different variants, influenced by the environment that eventually result in disease. Since these are common variants with only a very small effect, one needs to test hundreds or even thousands of individuals in order to have enough statistical power to detect their effect.

Only one polymorphism (T950C) found within the promoter region of the TNFRSF11B gene, coding for osteoprotegerin, was found to be significantly associated and possibly increase the risk for low bone mineral density (Vidal et al., 2006). This association was observed from the distribution of genotype frequencies between affected individuals and normal controls, where 83.3% of affected individuals were TT homozygotes. This genotype was only found in 16.67% of controls (chi=9.7; p=0.01; df=2). When constructing haplotypes from alleles of this variant with those of another two nearby SNPs, it was found that the frequency of the A-T-G haplotype was significantly higher in the affected group as opposed to controls (51.7%). Further to that when calculating odds ratio for the TT genotype (assumed to be risk genotype) against the CC genotype, a highly significantly odds ratio was obtained (OR=7.1; 95% CI=2.2 - 22.5); p=0.002). This might indicate that variants within this gene might be responsible for an increased risk for osteoporosis in the Maltese population. However to confirm this further studies are needed. For further information about the role of OPG in osteoclastogenesis, you can go to the bone physiology section. To download published research article go to my profile or click here.

Linkage Study

A family study, also known as linkage study, was performed using two extended families with multiple affected individuals. In this study a low resolution genome-wide scan was initially performed using 400 microsatellite markers spaced across the genome with an approximately equal spacing between them. Since it was difficult to define the phenotype for the simple reason that it was only based on DEXA measurements and people who appear normal today might become osteoporotic in a couple of years, we tested multiple models. According to WHO criteria osteoporosis is defined by t-scores but these does not apply for younger generations and not even for men. So we performed the analysis using different criteria and using both t-scores and z-scores, searching for the most consistent loci. From the analysis of data, a locus on chromosome 11p12 gave the most significant results with an initial NPL score of 5.77 (p=0.0006) (Vidal et al., 2007). This was the first time that strong suggestive linkage was reported to 11p12 (OMIM: 611739; BMD8; http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=611739). There was also another region on chromosome 5 which gave a very significant result when tested using a recessive model in one of the families. These two regions were further investigated by increasing the density of markers and performing a high resolution scan. This approach is used in a way as if one is using a magnifying glass to look in more detail at the indicated region of the genome. The results were replicated and the NPL score increased to 7.23 (p=0.0014) to marker D11S935 on chromosome 11. Besides the NPL score, two different haplotypes were observed segregating within the two families. Both families contributed to the LOD and NPL scores obtained at this locus, but when taking a closer look at the position of the linkage peak there was a difference of 2cM between the linkage peaks of each individual family making it possible that two different genes might be contributing for the disease at the same locus.

So the next step was to look at the genes that are known to be found at this locus and to sequence any possible candidates. The gene coding for tumour necrosis factor receptor associated factor (TRAF)-6 was initially sequenced. All eight exons and promoter region were sequenced in affected and non-affected family members. Three sequence variants were identified, an A to T transversion at position -721 from the transcriptional start site, an already known deletion/insertion of a T (rs3830511) in the polyT region just ahead of the intron/exon junction, and a G to A transition in intron 6. None of these variants were linked with the inherited haplotype but the A to T variant in the promoter region was very rare in the population (1.1%) and was only observed in three affected family members. The other two variants were also found in some other affected individuals. Diagram of the TRAF6 structural gene including variants identified in this study is shown here.

The variant within the promoter region was further studied for its possible effects on gene expression using a reporter assay system. Constructs were prepared from normal and affected alleles using pGL3 enhancer vector which were transfected into cultured HeLa and RAW264.7 cell lines. RAW264.7 cells were further stimulated by M-CSF and RANKL to differentiate into osteoclasts. Luciferase activity was measured and results showed that this variant actually increased gene expression, in such case this will result in an increased osteoclastogenesis and hence risk for osteoporosis. Further studies are needed to confirm these findings and to look in more detail at the promoter region and the control of gene expression.

Other genes were recently sequenced which include those coding for FGF18, MSX2, EXT2, IFNGR1, TNFAIP3 and CD44. Interesting variants were found in the CD44 gene also found on chromosome 11p12, where a synonymous variant within exon 9 was linked with the inherited haplotype in one of these families. This variant is being further investigated for possible functional effects that might increase the risk for osteoporosis.

These studies were funded by the Research Fund Committee, University of Malta.

Download complete PhD thesis from here.

 

Genetic studies in coeliac disease

A linkage study was also performed in an extended family with a high incidence of coeliac disease. This family consisted of 17 members with 4 individuals diagnosed as coeliac by biopsy and some others having an unclear phenotype some only showing high anti-gliadin antibodies. The same approach as for osteoporosis was taken where also different models were tested using different criteria for phenotype definition. Following linkage analysis using two-point and multi-point analysis two regions on chromosome 9q and 11p11 were identified. The HLA locus on chromosome 6 was not indicated by linkage so we performed HLA typing in this family showing results in the table below. It was concluded that in this family HLA was not the only causative factor but it was only causing disease in the presence of the CD59 A allele. Also most family members carried the HLA-DQ2.5 high risk HLA type in trans rather than in cis.

HLA typing in family members and the GA variant identified in CD59

Family Member

Status

Risk

DQA1

DQB1

 

DRB1

HLA type

CD59 G/A Variant

I : 1

Not known

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 11

DR7 / DQ2

DR11 / DQ7

GG

II : 1

Norm

No risk

05

ND

0301 / 04

ND

11 / 12

DR11 / DR12 / DQ7

GG

II : 2

High AGA

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 12

DR7 / DQ2

DR12 / DQ7

GA

II : 3

Affected

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 11

DR7 / DQ2

DR11 / DQ7

GA

II : 8

Norm

No risk

05

ND

0301 / 04

ND

11 / 12

DR11 / DR12 / DQ7

GG

II : 4

Norm

No risk

05

ND

0301 / 04

ND

11 / 12

DR11 / DR12 / DQ7

GG

II : 5

Symptomatic

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07

DR7 / DQ2

DQ7

GA

II : 6

Affected

Low (DQ2.2)

0201

ND

02

ND

07

DR7 / DQ2

GA

II : 7

Affected

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 12

DR7 / DQ2

DR12 / DQ7

GA

III : 1

Severely Affected

Low (DQ2.2)

0201

03

02

0302

07 / 04

DR7 / DQ2

DR4 / DQ8

GA

III : 2

High AGA

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 12

DR7 / DQ2

DR12 / DQ7

GA

III : 3

Normal

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 12

DR7 / DQ2

DR12 / DQ7

GG

III : 4

Normal

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 11

DR7 / DQ2

DR11 / DQ7

GG

III : 6

Normal

High (DQ2.5 in cis)

05

ND

02

0301 / 04

07 / 11 / 12

DR7 / DQ2

DR11 / 12 / DQ7

GG

III : 8

Normal

No risk

05

ND

0301 / 04

ND

11 / 12

DR11 / DR12 / DQ7

GA

III : 10

Normal

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 11

DR7 / DQ2

DR11 / DQ7

GA

III : 11

Symptomatic

High (DQ2.5 in trans)

0201

05

02

0301 / 04

07 / 11

DR7 / DQ2

DR11 / DQ7

GA

 

Sequencing of CD44, APAF1 and CD59 found on chromosome 11 was performed, from which a novel G to A transition was identified in exon 3 of the CD59 gene which was linked with the inherited haplotype observed by linkage. This variant does not result in an amino acid change so we tested the hypothesis that this could have an effect on pre-mRNA splicing. Until now this does not seem to be the case, but further studies are underway to confirm whether this could occur in a tissue specific manner since the RNA so far tested was obtained from peripheral blood and we need to look at expression in the duodenal mucosa where the main pathological changes are seen in coeliac disease. Analysis of the CD59 molecule derived from biopsies at the time of diagnosis from coeliac patients carrying the identified variant did not show any difference in the size of linear protein when compared to normal controls. This does not exclude possible effects of this variant on the conformation of the protein especially since it is known that a codon change from a common to a rarer one without changing the amino acid sequence, can affect co-translation folding. CD59 or protectin is a potent inhibitor of the complement system and so it is important to protect the intestinal mucosa from the membrane attack complex of complement during an inflammatory process.

This study was funded by the National RTDI 2004 Grant - Malta Council for Science and Technology.

Besides this study, previous association studies were performed in a population of coeliac disease patients using SNPs found within the CTLA4 and ICOS genes and also recently within the CD44 and CD59 genes.

Power Point Presentations

CTLA4 Study

CTLA4/ICOS Study

Linkage Study of Coeliac

 

 

 
Other Genetic Studies

At the Laboratory of Molecular Genetics a number of genetic studies were carried out that revealed a number of known and also novel mutations in a number of genes. Among the most studied were the thalassaemias as well as other haemoglobinopathies which revealed abnormal haemoglobins such as Hb F Malta I and Hb Valletta together with Hb St Luke's. Other studies were done on gangliosidosis and phenylketonuria, where in Malta we have an atypical form caused by a mutation within the tetrahydropterin gene (BH4) and not within the phenylalanine hydroxylase gene.

Another recent study was a linkage study performed in an extended pedigree with high incidence of epilepsy, using a 250,000 SNPs, from which a causative mutation caused by a deletion which was linked with the inherited haplotype observed by linkage, is being further investigated.

 

Future studies

Until now most of the genetic studies were addressed to the actual DNA sequence and how different variants mostly within candidate genes, assumed to be involved in disease, could increase the susceptibility for the disease in question. The draft sequence derived from the human genome project together with the advance in technologies triggered and helped a lot in these studies. On the other hand, the actual DNA sequence itself does not say anything, as there are a number of biological processes that needs to be addressed. I feel that at the moment we are looking at only a small corner from the whole picture and we need to look in more detail at the functional role of genes and gene variants. We need to look at the genome in its whole context and how genes are regulated and expressed in different cells and thus in different organs. We have to look at epigenetic controls including methylation, acetylation and chromatin modification together with other factors such as RNA splicing and control by RNA interference. Probably in the long run other mechanisms will be discovered and so the story will complicate itself even more. But this is what science is all about. The aim of medical research is to understand better the pathophysiology of disease and how the human body functions at the molecular level. This will in turn helps us to develop better treatments to prevent, control or even cure human disease with the ultimate goal being that of giving a personalised treatment.

 
Frequency of the CCR5-Δ32 polymorphism in the Maltese population at birth

Both α and β chemokine receptors expressed on the surface of CD4 positive T-lymphocytes and macrophages are known to serve as cofactors for the entry of the human immunodeficiency virus (HIV) into these cells, thus leading to acquired immunodefiency syndrome (AIDS). The macrophage (M)-tropic HIV-1 uses the β-chemokine receptor CCR5 to enter into macrophages although some strains can also use other β-chemokine receptors such as CCR3 and CCR2b (Rana et al., 1997).

A 32 base-pair deletion (Δ32) within the CCR5 gene found on chromosome 3p21, results in a truncated protein leading to lack of integration into the cell membrane (Liu et al., 1996).  Individuals homozygous for this polymorphism were found to be highly resistant to HIV-1 infection with M-tropic HIV-1 strains but were not resistant to T-cell (T)-tropic viruses that use the α-chemokine receptor fusin (CXCR-4) (Samson et al., 1996; Rana et al., 1997). Heterozygous individuals were also observed to have partial resistance to AIDS by delaying its onset (Liu et al., 1996). A marked degree of geographic variation exists in the distribution of this polymorphism, where highest frequencies are found in northern European populations which decrease southwards (allele frequency ranging from 16% to 4%)  (Novembre et al., 2005). Recent studies performed on ancient-DNA obtained from 2,900-year-old skeletons from Italy and Germany showed that this polymorphism was already prevalent in Europe at that time (Hummel et al., 2005). This polymorphism was also found in other populations of European descent (Brazilian) (Pereira et al., 2000), but was absent in African and most Asian populations (Yudin et al., 1998; Lu et al., 1999). It is thought that strong selective pressures over long periods of time, and not the present HIV infection, structured this gradient in allele frequencies across European populations (Novembre et al, 2005).

Genotype frequencies observed in the Maltese population (n = 232) were 97.8% WT/WT homozygotes, 2.2% WT/CCR5Δ32 heterozygotes, while no homozygotes (CCR5Δ32/CCR5Δ32) were identified. The minor allele frequency observed in the Maltese population was 1.1% (0.011) and allele frequencies were in Hardy-Weinberg equilibrium (χ2 = 0.028; p = 0.87). Calculated heterozygosity was of 0.0213 (95% confidence interval: 0.0030 - 0.0397).

For the first time, the frequency of the CCR5-Δ32 polymorphism in the Maltese population is being reported. To our knowledge, it is also the first time that this frequency is being estimated in newborns, thus reflecting the frequency in the general population at birth and minimising any possible effects that this variant might have on longevity when using older populations.  Until now importance was given to the protective role of the CCR5Δ32 allele against HIV but little was said about its possible negative effects. Recently, an increased risk of symptomatic infection with the West Nile virus in CCR5-Δ32 homozygotes was reported (Glass et al., 2006).  The CCR5-Δ32 polymorphism results in a completely non-functional receptor and thus could lead to a weakened immune response if the host was to be attacked by other organisms where the biological role of the CCR5 receptor cannot be outweighed by that of other receptors. Also there were conflicting reports about the role of the CCR5-Δ32 polymorphism in various diseases such as juvenile idiopathic arthritis (Scheibel et al., 2008), hepatitis C (Ahlenstiel et al., 2004) and systemic lupus erythematosus (Mamtani et al., 2008). All these factors could affect allele frequencies of this polymorphism when using an adult population, so using a random sample of newborns minimises such influences on frequency estimation. Differences in allele frequencies between newborns and adult populations were observed for variants within the klotho and methylenetetrahydrofolate reductase genes, both thought to affect longevity (Arking et al., 2002; Gueant-Rodriguez et al., 2006).    

The allele frequency observed in this study conforms with the European north to south gradient (Novembre et al., 2005). Malta is an island located about 60 miles to the southeast of Sicily, and a frequency of 1.1% for the CCR5Δ32 allele conforms well to its geographic position. This frequency is less than that observed in other island populations, including the Croatian island of Vis (1.5%) (Smaljanovic et al., 2006), Sardinia (2.1%) (Battiloro et al., 2000) and the Basque region (1.8%) (Lucotte, 2002), but is slightly higher than that observed in Corsica (0.9%) (Lucotte, 2002). Isolated populations have the lowest frequency even when compared to that of other European populations which ranges from 4.2% in Greeks (Novembre et al., 2005), 7.1% in mainland Croatians (Ristic etal., 2005), and increasing to 16% in northern European populations (Novembre et al., 2005). The only exceptions to this gradient effect are the Ashkenazi Jews, where the overall frequency of the CCR5Δ32 allele is 13.7%, probably due to a founder effect and/or genetic drift as a result of their historical background (Lucotte, 2003).

It is highly debatable as to what were the selective pressures that led to this geographical gradient across European populations. The deadly pandemics that struck Europe throughout the Middle Ages were among the reasons most usually given, although today these are highly disputed (Stephens et al., 1998; Cohn & Weaver, 2006). Comparisons of frequencies between skeletal remains of victims from the 14th century plague with those from 2,900 years ago showed similar results, suggesting that the Black Death was not the major selective force causing a rapid increase in CCR5-Δ32 gene frequencies (Hummel et al., 2005). If the Black Death were the major selective pressure, then one could assume that mortality was much higher in northern European countries when compared to southern Europe but historical evidence about the Black Death of 1346 – 53 shows the opposite (Cohn & Weaver, 2006). The plague struck Malta several times between the fourteenth and nineteenth centuries with the latest epidemic being that of 1813 (Savona-Ventura, 1997). Every outbreak resulted in the death of a significant percentage of the population ranging from 9% during the 1592 outbreak to 4% in the latest outbreak of 1813, where in the latter the population was of nearly 116,000. Other sources mentioned that during the outbreak of 1675 approximately 8,000 to 11,000 individuals perished from a population of approximately 50,000 (Blouet, 1967). The frequency of the CCR5-Δ32 allele in Malta is low, indicating that the plague hypothesis does not hold. If it did, then a higher frequency would be expected in a population that expanded following several population bottlenecks with expected high proportion of survivors being those carrying the CCR5-Δ32 allele. A reason why these several plague outbreaks did not have an effect on the frequency of the CCR5-Δ32 allele might be due to all these epidemics described as plagues, were actually caused by different etiological agents, thus exerting different selective pressures on the allele.

Another hypothesis states that it is more probable that a positive selective pressure was due to smallpox rather than the Black Death (Cohn & Weaver, 2006; Galvani & Slatkin, 2003). There are several reasons why this could be more probable including the fact that smallpox wiped out more of the population than the Black Death but over longer periods of time, affected younger people with a reproductive potential and also affected Scandinavian countries much more than the bubonic plague (Galvani & Slatkin, 2003). Conversely, population differentiation and long-range linkage disequilibrium at the CCR5-Δ32 locus were not different from the rest of the genome, showing that probably there isn’t a strong recent selection for CCR5-Δ32 as proposed by the previous hypotheses, and so its genetic variation is consistent with neutral evolution (Sabeti et al., 2005)

An alternative hypothesis for the north to south gradient could be that the CCR5-Δ32 allele gives little resistance to certain organisms that are more prevalent in the south due to weakening of the immune systems as discussed above.  If this happens, then there is a greater chance of survival for individuals carrying the wild-type allele than for those carrying the CCR5-Δ32 allele. This study continues to add to the existing knowledge about the geographical distribution of the CCR5-Δ32 polymorphism in Europe and the Mediterranean region.

 

Published article:

Vidal, C., and Xuereb-Anastasi, A. (2009) Frequency of the CCR5-delta 32 polymorphism in the Maltese population. International Journal of Immunogenetics 36: 301 – 304 (PMID: 19744036) (Impact factor: 1.16)

 

 

References

Ahlenstiel, G., Woitas, R.P., Rockstroh, J., & Spengler, U. (2004) CC-chemokine receptor 5 (CCR5) in hepatitis C – at the crossroads of antiviral immune response. Journal of Antimicrobial Chemotherapy, 53, 895 – 898.

Arking, D.E., Krebsova, A., Macek, M., Macek, M., Arking, A., Mian, I.S., Fried, L., Hamosh, A., Dey, S., McIntosh, I., & Dietz, H.C. (2002) Association of human aging with a functional variant of klotho. Proceedings of The National Academy of Sciences, 99, 856.

Battiloro, E., Andreoni, M., Parisi, S.G., Mura, M.S., Sotgiu, G., Aceti, A., Cossu, G., Concia,E., Verna, R, & D’Ambrosio, E. (2000) Distribution of the CCR5 delta32 allele in Italian HIV type 1 infected and normal individuals. AIDS Research and Human Retroviruses, 16, 181.

Blouet, B. (1967) The story of Malta. Malta Progress Press Ltd, Malta.

Cohn, S.K., & Weaver, L.T. (2006) The Black Death and AIDS: CCR5-Δ32 in genetics and history. Q J Med, 99, 497.

Galvani, A., & Slatkin, M. (2003) Evaluating plague and smallpox as historical selective pressures for the CCR5-Δ32 HIV resistance allele. Proceedings of the National Academy of Sciences, 100, 15276.

Glass, W.G., McDermott, D.H., Lim, J.K., Lekhong, S., Yu, S.F., Frank, W.A., Pape, J., Cheshier, R.C., & Murphy, P.M. (2006) CCR5 deficiency increases risk of symptomatic West Nile virus infection. The Journal of Experimental Medicine, 23, 35.

Gueant-Rodriguez, R.A., Gueant, J.L., Debard, R., Thirion, S., Hong, L.X., Bronowicki, J.P., Namour, F., Chabi, N.W., Sanni, A., Anello, G., Bosco, P., Romano, C., Amouzou, E., Arrieta, H.R., Sanchez, B.E., Romano, A., Herbeth, B., Guilland, J.C., & Mutchinick, O.M. (2006) Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations. American Journal of Clinical Nutrition, 83, 701.

Hummel, S., Schmidt, D., Kremeyer, B., Herrmann, B., & Oppermann, M. (2005) Detection of the CCR5-Δ32 HIV resistance gene in Bronze age skeletons. Genes and Immunity, 6, 371.

Liu, R., Paxton, W.A., Choe, S., Ceradini, D., Martin, S.R., Horuk, R., MacDonald, M.E., Stuhlmann, H., Koup, R.A., & Landou, N.R. (1996) Homozygote defect in HIV-1 co-receptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell, 86, 367.

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