Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians

Abstract

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. At stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 x 10(-5); D17S1868, 2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).

Full text

BRIEF COMMUNICATION
Genome-wide scans for leprosy and tuberculosis
susceptibility genes in Brazilians
EN Miller
1
, SE Jamieson
1
, C Joberty
1
, M Fakiola
1
, D Hudson
1
, CS Peacock
1
, HJ Cordell
1
, M-A Shaw
2
,
Z Lins-Lainson
3
, JJ Shaw
3,4
, F Ramos
3
, F Silveira
3
and JM Blackwell
1
1
Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, University of Cambridge School of Clinical Medicine,
Addenbrookes Hospital, Hills Road, Cambridge CB2 2XY, UK;
2
Department of Biology, University of Leeds, Leeds LS2 9JT, UK;
3
Instituto Evandro Chagas, Caixa Postal 3, 66.001 Belem, Brazil
Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed
in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9
chromosomal regions respectively with provisional evidence (Po0.05) for linkage. At stage 2, 58 markers from positive regions
were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive
markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3,
20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P¼0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3
regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P¼5.8  10
5
; D17S1868, 2.38, P¼0.0005; D20S889, 1.51,
P¼0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).
Genes and Immunity (2004) 5, 63–67. doi:10.1038/sj.gene.6364031
Keywords: tuberculosis; leprosy; susceptibility genes; genome scans
Many studies suggest that susceptibility to Mycobacter-
ium tuberculosis and M. leprae are genetically regulated in
humans (reviewed in
1-4
). One approach to identifying the
genes involved is to use family-based linkage analysis to
undertake a genome scan. The first such study using
affected sib-pair analysis in tuberculosis families from
The Gambia and South Africa
5
identified regions on
chromosomes 15q and Xq as providing evidence (LOD
scores¼2.00 and 1.77; P¼0.001 and 0.002, respectively)
suggestive of linkage to pulmonary tuberculosis. Allelic
association studies have subsequently identified the gene
UBE3A encoding a ubiquitin ligase, that is expressed in
macrophages and plays a key role in the ubiquitination
and degradation of specific proteins, as the putative
candidate susceptibility gene on 15q11-q13 (P¼0.002).
6
For leprosy, genome scans for Indian
7
and Vietnamese
8
populations have been reported. In the Indian study,
7
a
major locus (LOD score 4.09; Po2  10
5
) controlling
susceptibility to leprosy in a region of southern India
where tuberculoid or paucibacillary leprosy predomi-
nates was identified on chromosome 10p13. Further
analysis of the same study population
9
identified a
second and independent locus controlling leprosy on
chromosome 20p12.3 (LOD score 3.48; P¼0.00003). In the
Vietnamese study,
8
chromosome 6q25 (LOD score 4.31;
P¼5  10
6
) was identified as carrying major suscept-
ibility loci for leprosy per se. In this case, analysis by
disease sub-type provided supportive evidence (LOD
score 1.74; P¼0.002) for a locus on chromosome 10p13
controlling susceptibility to paucibacillary leprosy, con-
sistent with the bias towards this disease sub-type in the
Indian study population.
7
To determine whether the same or different regions of
the genome carry susceptibility loci for pulmonary
tuberculosis and leprosy per se in Brazil we have carried
out genome scans using a two-staged analysis of
extended multicase pedigrees for each disease. A full
description of the study site and clinical criteria for
inclusion in the study have been reported elsewhere
10,11
and are described in brief in Table 1. For each disease an
initial set of families (Table 1, scan 1) were genotyped for
405 markers at B10 cM intervals across the genome. As
outlined in the legend to Table 2, nonparametric linkage
analysis in ALLEGRO
12
that reports maximum Z scores
for the likelihood ratio (Z
lr
) and allele-sharing LOD
scores
13
was used to analyse the genome scan linkage
data. This analysis provided evidence at Po0.05 for
linkage of susceptibility to 8 regions of the genome on
chromosomes 2, 3, 7, 10, 11, 20, 21 and X (LOD scores 0.60
to 2.43; 1-sided p values 0.0004oPo0.048) for tubercu-
losis, and to 9 regions on chromosomes 6, 9, 11, 12, 13, 15,
16, 17, 20 (LOD scores 0.74 to 2.50; 1-sided P values
0.0003oPo0.033) for leprosy per se. For tuberculosis, 58
markers from the positive regions were typed in a second
set of families (Table 1), with 22 additional markers typed
Received 04 July 2003; revised 14 August 2003; accepted 19 August
2003
Correspondence: Dr Blackwell, Cambridge Institute for Medical Research,
Wellcome Trust/MRC Building, University of Cambridge School of
Clinical Medicine, Addenbrookes Hospital, Hills Road, Cambridge CB2
2XY, UK. Email: jennie.blackwell@cimr.cam.ac.uk
4
Present address: Sao Paulo University, Institute of Biomedical
Sciences, Department of Parasitology, 05508-900, Brazil.
Genes and Immunity (2004) 5, 63–67
&
2004 Nature Publishing Group All rights reserved 1466-4879/04
$
25.00
www.nature.com/gene

in both family sets. For leprosy, 42 markers from positive
regions were typed in a second set of families, with 30
additional markers typed in both family sets. Results
from the two independent sets of families for each
disease indicate true replication of linkage for chromo-
somes 10 and 20 for tuberculosis, and for chromosomes
6, 17 and 20 (lepromatous or multibacillary) for leprosy
(Table 1). Although replication was not achieved at
chromosome 11 in the second set of families for
tuberculosis, the combined analysis shows an increase
in peak LOD score from 1.58 (P¼0.004) in stage 1 to 1.85
(P¼0.002), indicating that the stage 2 families and grid
tightening stage 1 families have contributed to an
increase in the significance for linkage. Overall, the
combined analysis (Table 2) showed that three regions
retained evidence for linkage for each disease. For
tuberculosis these equated to peak nonparametric multi-
point allele sharing LOD scores of 1.31 (P¼0.007) for
D10S1656 at Genethon map position 158.3 cM on
10q26.13, of 1.85 (P¼0.002) for D11S4205 at position
67.4 cM on 11q12.3, and of 1.78 (P¼0.002) for D20S898 at
position 35.8 cM on 20p12.1. For leprosy per se, peak LOD
scores were obtained for HLA-DQA (LOD 3.23;
P¼5.8  10
5
) on 6p21.32, for D17S1868 (LOD 2.38;
P¼0.0005) at position 75.7 cM on 17q22, and for
D20S889 (LOD 1.51; P¼0.004) at position 11 cM on
20p13. The regions 6p21.32 and 17q22 had each already
been highlighted as carrying susceptibility genes for
leprosy by our earlier analyses of candidate gene regions
in this Brazilian population.
10,14
In each case, more
detailed analysis has indicated that more than one locus
in each of these immune response gene dense regions
might affect susceptibility to leprosy, including genes
that contribute to leprosy per se and to lepromatous
versus tuberculoid disease sub-types. For chromosome
20, the peak of linkage (D20S889) we observe for leprosy
per se in Brazil (Figure 1) lies B3.5 Mb distal to that
observed at D20S115 for leprosy in India.
9
Because of this
earlier observation,
9
we typed all of the additional
markers across this region that had been used in the
Indian study. Stratification by disease sub-types (Figure
1) indicates that most of the peak at D20S889 is
contributed by the lepromatous leprosy families,
although there is a peak of linkage for tuberculoid
leprosy 1.4 Mb proximal to D20S889 at D20S835. If there
is a common disease susceptibility locus affecting
susceptibility to leprosy in both Brazil and India, our
data indicate that the gene might lie more distally on
20p13. The predominance of lepromatous leprosy in
Brazil and tuberculoid leprosy in India also indicates that
a single locus would have to control susceptibility to
leprosy per se. The peak for leprosy in Brazil is distinct
from the peak for tuberculosis at 20p12.1 (Figure 1).
There are no obvious functional candidate genes under
either of these regions of linkage.
For tuberculosis, the regions under the linkage curves
significant at the P¼0.05 level spanned intervals of
B26 cM on chromosome 10, B54 cM on chromosome
Table 1 Details of family structure for families used in 2-stage genome scan of tuberculosis and leprosy in Brazil
Numbers observed
Family structure TB
Scan 1
TB
Scan 2
Leprosy per
se Scan 1
Leprosy per
se Scan 2
Leprosy LL
Scan 1
Leprosy LL
Scan 2
Leprosy TT
Scan 1
Leprosy TT
Scan 2
No. families 16 22 21 50 15 29 17 22
No. nuclear families 24 31 32 54 16 31 18 23
Nuclear families with 1 affected sib 8 14 10 7 9 9 7 8
Nuclear families with 2 affected sibs 11 10 12 39 6 19 5 12
Nuclear families with 3 affected sibs 4 5 4 3 1 2 5 2
Nuclear families with 4 affected sibs 1 2 3 3 1 1
Nuclear families with 5 affected sibs 3 1
Nuclear families with 6 affected sibs 1
Nuclear families with 7 affected sibs 1
No. affected offspring 48 57 73 118 24 57 36 44
No. of affected parents 9 19 16 23 6 12 6 4
Total No. of affected individuals 58 73 86 140 30 67 42 48
Total No. individuals 178 176 173 192 122 124 146 100
Leprosy and tuberculosis families were ascertained through medical records at local Ministerio de Sanidad health centres in Belem, Para,
Brazil. All health centres were staffed by clinicians highly experienced in the diagnosis and treatment of leprosy and tuberculosis. The study
was performed with approval of the ethical review committee of the Institute Evandro Chagas, Belem, Para, Brazil. For tuberculosis families,
diagnosis was made on the basis of a sputum test positive for acid-fast bacilli and/or chest X-ray. Sputum tests were carried out at the centres,
stained (Ziehl-Nielson) and read for acid-fast bacilli by qualified laboratory technicians. If tuberculosis symptoms persisted following two
negative sputum results, patients were referred to the central tuberculosis hospital in Belem, where all X-rays were read by experienced
specialist clinicians. Families came from an area of high tuberculosis prevalence. The incidence rate for TB in Belem City at the beginning
(1991) of the study was 62 per 100 000 habitants. The prevalence rate for TB in the whole of Brazil is 68 cases per 100 000 habitants. For leprosy
families, diagnosis was made following clinical examination for anaesthetic skin lesions, results from slit skin smear testing for acid fast
bacilli, and, in some health centres, histological analysis. Patients were categorised into disease sub-type groups according to the Ridley-
Jopling
19
histological scale and/or bacterial load. Subtypes recorded were lepromatous (LL), borderline lepromatous (BL), borderline (BB) or
borderline tuberculoid/tuberculoid (BT/TT). For genetic analyses in the present study, lepromatous patients comprised LL plus BL subtypes
(referred to hereafter as LL) and tuberculoid patients comprised BB plus BT/TT subtypes (referred to hereafter as TT). The incidence rate for
leprosy in Belem City at the beginning of the study was 45 per 100 000 population. The prevalence rate for leprosy in the whole of Brazil is 45
cases per 100 000 habitants.
Genome-wide scans for leprosy and tuberculosis
EN Miller et al
64
Genes and Immunity

11, and B15 cM on chromosome 20 (data not shown). To
try to pinpoint the location of the putative susceptibility
loci within these regions more precisely, we used the
transmission disequilibium test (TDT),
15
using a robust
sandwich estimator for the variance and a Wald chi-
squared test to control for pedigree clustering, to look for
association between specific markers and tuberculosis
susceptibility. Robust TDT statistical tests developed by
Table 2 Results from 2-stage genome scan of tuberculosis and leprosy in Brazil
Scan 1 Scan 2 Combined analysis
Marker Location (cM) LOD Z
lr
P LOD Z
lr
P LOD Z
lr
P
Tuberculosis
D10S587 156.6 0.63 1.70 0.044 0.91 2.05 0.020 1.07 2.23 0.013
D10S1656 158.3 — — — 1.22 2.37 0.009 1.31 2.45 0.007
D10S575 162.9 — — — 1.11 2.26 0.012 1.16 2.31 0.010
D10S217 167.2 0.62 1.69 0.046 0.51 1.53 NS 1.05 2.20 0.014
D11S4191 63.4 1.31 2.46 0.007 0.29 1.15 NS 1.59 2.70 0.003
D11S4205 67.4 — — — 0.28 1.13 NS 1.85 2.92 0.002
D11S987 —
a
1.58 2.70 0.004 0.29 1.16 NS 1.70 2.79 0.003
D11S1314 77.5 1.47 2.61 0.005 0.19 0.93 NS 1.45 2.59 0.005
D20S186 33.2 0.39 1.34 NS 0 0.07 NS 0.15 0.82 NS
D20S898 35.8 — — — 1.69 2.79 0.003 1.78 2.86 0.002
D20S112 39.3 0.99 2.14 0.016 0.12 0.73 NS 0.95 2.09 0.018
Leprosy per se
D6S289 29.6 2.41 3.33 0.0004 0.42 1.39 NS 2.41 3.33 0.0004
HLADRB —
b
1.97 3.01 0.003 1.09 2.25 0.013 3.00 3.72 0.0001
HLADQA —
b
1.97 3.01 0.003 1.30 2.44 0.007 3.23 3.85 5.8  10
5
HLADQB —
b
1.97 3.01 0.003 1.11 2.25 0.012 3.01 3.72 0.0001
D17S1868 65.1 1.97 3.01 0.003 0.60 1.67 0.048 2.38 3.31 0.0005
D17S787 75.7 1.58 2.70 0.004 0.52 1.55 0.060 2.04 3.10 0.001
D17S944 84.2 2.01 3.04 0.001 0.01 0.08 NS 0.80 1.91 0.027
D20S117 2.9 0.34 1.26 NS 0.28 1.13 NS 0.87 2.00 0.023
D20S889 11 1.64 2.75 0.003 0.20 0.96 NS 1.51 2.64 0.004
D20S835 14.8 — — — 0.05 0.49 NS 0.96 2.10 0.018
D20S115 20.9 0.76 1.88 0.031 — 1.12 NS 0 0.06 NS
LL Leprosy
D20S117 2.9 0.70 1.80 0.036 0.44 1.43 NS 1.06 2.21 0.014
D20S889 11 1.16 2.31 0.010 0.55 1.58 0.056 1.36 2.50 0.006
D20S835 14.8 — — — 0.43 1.40 NS 1.22 2.37 0.009
D20S115 20.9 1.71 2.81 0.003 — 0.28 NS 0.23 1.02 NS
TT Leprosy
D20S117 2.9 0 0.03 NS 0.05 0.48 NS 0.11 0.73 NS
D20S889 11 0.40 1.36 NS 0.06 0.53 NS 0.62 1.69 0.046
D20S835 14.8 — — — 0.03 0.34 NS 0.74 1.84 0.033
D20S115 20.9 0.77 1.88 0.030 — 0.57 NS 0.06 0.53 NS
Methods: For the genome scan, a 2-stage strategy was employed. At stage 1, 178 DNAs from 16 extended (24 nuclear) multicase tuberculosis
families and 173 DNAs from 21 extended (32 nuclear) multicase leprosy families were genotyped for the 400 markers that make up the
Applied Biosystems ABI Prism Linkage Mapping Set version 2 with markers at B10 cM intervals (www.appliedbiosystems.com/products/
linkmapping.cfm) across the genome, with 5 additional markers typed for HLA at 6p21. At stage 2, 58 markers from the positive regions were
typed in 176 DNAs from a second set of 22 extended (31 nuclear) multicase tuberculosis families, with 22 additional markers typed in both
family sets. For leprosy, 42 markers from positive regions were typed in a second set of 192 DNAs from 50 extended (54 nuclear) multicase
leprosy families, with 30 additional markers typed in both family sets. Although the additional markers were typed in the scan 1 families as
part of the grid tightening strategy that contributes to the combined analysis, we have not given the scan 1 only data for these markers. The
scan 1 data provided is to indicate the level of evidence that we had for a gene in the region after the initial scan with 405 markers.
Nonparametric multipoint linkage analyses were performed in ALLEGRO,
12
with results reported as maximum Z scores for the likelihood
ratio (Z
lr
) or allele sharing LOD scores.
13
For tuberculosis, where only complex multi-generation pedigrees were used, the S
all
scoring function
with equal weighting was used. For leprosy, where a mixture of pedigrees of different sizes and complexity were used, the S
pairs
scoring
function with 0.5 weighting was used to take account of differences in family size. Simulations (100) performed within ALLEGRO using data
for a typical set of six linked polymorphic microsatellite markers (7–10 alleles; heterozygosity 0.73) showed that the scan 1 family set and the
scan 2 family set for tuberculosis each had 100% power to detect Z
lr
scores of 1.64 (P¼0.05), and 489% power to detect Z
lr
scores of 2.33
(P¼0.01). The combined family set had 495% power to detect Z
lr
scores of 3.09 (P¼0.001). For leprosy per se, the scan 1 family set had 99%
power to detect Z
lr
scores of 3.71 (P¼1x10
4
), and 95% power to detect Z
lr
scores of 4.26 (P¼1  10
5
). The scan 2 family set had 90% power to
detect Z
lr
scores of 3.08 (P¼0.001). The combined family set had 99% power to detect Z
lr
scores of 5.20 (P¼1  10
7
). Not all data for all of the
additional markers genotyped are shown in the table, the purpose of which is to demonstrate evidence for linkage (scan 1 alone) supported
by replication (scan 2 alone) and/or grid tightening (scan 1+2) in and around the peak of linkage.
a
Not in Genethon map. Position based on Marshfield map.
b
Not in Genethon or Marshfield map. Position based on physical location.
Genome-wide scans for leprosy and tuberculosis
EN Miller et al
65
Genes and Immunity

Heather Cordell and David Clayton at the Cambridge
Institute for Medical Research were implemented within
Stata and are available at http://www-gene.cimr.cam.
ac.uk/clayton/software/. For chromosome 10, a signifi-
cant (w
2
¼11.52, 4 df, P¼0.021) global association was
observed at D10S1656, with significant protection asso-
ciated with allele 6 (w
2
¼9; 1 df; 0.002) that was robust to
pedigree clustering (Wald w
2
¼4.76; 1 df; P¼0.029). No
other markers in the region showed significant global
associations, effectively narrowing down the interval on
this chromosome to 6 cM between D10S1656 and the two
nearest proximal (D10S587) and distal (D10S575) mar-
kers typed. For chromosome 11, a significant global
association (w
2
¼16.9; 7 df; P¼0.018) was observed at
marker D11S987, with allele 5 associated with protection
(w
2
¼4.57; 1 df; P¼0.033; Wald w
2
¼4.57; 1 df; P¼0.033) and
allele 7 associated with disease (w
2
¼8.00; 1 df; P¼0.005;
Wald w
2
¼4.23; 1 df; P¼0.039). This marker lies 1.63 Mb
distal to D11S4205 which lies at the peak of a broad
multipoint linkage curve on 11q13. A significant global
association (w
2
¼11.7; 3 df; P¼0.008), with allele 4 (w
2
¼11;
1 df; P¼0.0009; Wald w
2
¼6.37; 1 df; P¼0.012) disease
associated, was also observed for marker D11S4175
located at 11q14.3 B30 Mb distal to D11S987, indicating
that there may be more than one susceptibility gene
contributing to the broad region of linkage on this
chromosome. No other markers in the region showed
significant global associations. No markers on chromo-
some 20 showed significant global TDT associations.
There are no obvious candidate genes likely to be in
linkage disequilibrium with any of the markers at the
peaks of linkage or showing significant associations on
any of these chromosomes. The leprosy families have an
insufficient number of parent child trios to perform
allelic association testing.
None of the regions we identified as carrying putative
susceptibility loci for tuberculosis in Brazil match those
(15q11-q13, Xq) identified for tuberculosis in the African
5
study. There was only one possible region of overlap
(20p13) between Brazil and data reported earlier (6q25,
10p13, 20p12) for leprosy in Indian
7,9
or Vietnamese
8
populations. Interestingly, the HLA complex was not
significant for linkage to tuberculosis in our Brazilian
population, although it does make a major contribution
(peak parametric LOD score¼5.78; P¼2.5  10
7
)to
susceptibility to leprosy in the same population.
10
HLA
was identified as a positive region of linkage in the
Vietnamese
8
but not the Indian
7
leprosy study. Further
investigation of the Vietnamese population
16
indicates
linkage to clinical sub-type, as many earlier studies
(e.g.
17,18
) had suggested. Overall, a complex picture of
geographic heterogeneity in genetic effects on these
different mycobacterial infections is emerging. Our
ability to dissect out these genetic pathways in the
future will require much larger sample sizes and a better
definition of pathology and immune response pheno-
types associated with clinical disease, and with resis-
tance in those who are exposed to infection but do not
succumb to clinical disease. For the moment, our study
here provides additional leads that may be worth
examining in other populations, and may have some
bearing on our further analysis of genes involved in
susceptibility to the complex phenotypes of pulmonary
tuberculosis and leprosy in Brazil.
Acknowledgements
This work was funded by grants from the Wellcome
Trust. We would also like to thank the people of Belem
for their contribution to this study.
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