Transcript Classification of leprosy

Prepared by:
- Ilham Abd Allah EL-bushra
- Safaa Othman Andarawi
CONTENT
• Introduction to leprosy
• Genome wide scan
• Candidate genes:– HLA genes
– Non-HLA genes
•
Leprosy is a chronic infectious disease caused by
the organism Mycobacterium leprae.
• Leprosy is primarily a disease of the peripheral
nerves and mucosa of the upper respiratory tract,
skin lesions are the primary external symptom.
• it has long been recognized that leprosy clusters in
families and it is often regarded as genetic disease
• Evidence suggests that the incidence of leprosy
infection is much higher than the incidence of clinical
leprosy.
• Only a small proportion (about 5%) of those infected
are thought to develop clinical symptoms; This may
be due in part to environmental factors but there is
also a wealth of evidence for a genetic basis for host
susceptibility to both disease per se and diseases subtype. (M-A Shaw1,2,)
Who is at risk of leprosy?
• Leprosy can affect people of all races all around
the world. However, it is most common in warm,
wet areas in the tropics and subtropics.
• Worldwide prevalence is reported to be around
5.5 million, with 80% of these cases found in 5
countries: India, Indonesia, Myanmar, Brazil and
Nigeria.
• Leprosy presents most often during two different
periods of life, between the ages of 10 and 14 and
in those aged 35-44 years old. It is rarely seen in
infants.( Vanessa Ngan2009)
Classification of leprosy:
• classic classification system for leprosy clinical forms was
• proposed in 1966 by Ridley and Jopling . Five groups of
leprosy cases spanning the entire disease spectrum :
• tuberculoid (TT), borderline tuberculoid (BT),
midborderline(BB), borderline lepromatous (BL) and
lepromatous(LL) leprosy.
• More recently, a treatment-oriented system created by WHO
classifies leprosy patients into three groups.:
• Patients presenting a single skin lesion are classified as
• single-lesion paucibacillary cases, paucibacillary leprosy (PB)
includes all cases presenting five or fewer skin lesions
Classification of leprosy:
(S. L. Walker.et al.2006)
Classification of leprosy:
• patients presenting six or more skin lesions plus detectable
M. leprae are classified as multibacillary (MB).
• When comparing the two systems:
• the single and multi-lesion PB patients are generally
equivalent to the TT , BT.
• while MB patients represent BB , BL , LL disease of the Ridley
and Jopling system.
Genetics of susceptibility
• Epidemiological and clinical observations suggested that the disease does
not occur in most of the exposed individuals, Thus, the vast majority of
people have some sort of innate resistance that can be, explained by their
genetic background. There is a large evidence indicating genetic influence
in leprosy outcome, suchas
• (i) familial clustering;
• (ii) distinct prevalence rates associated with distinct ethnic groups in an
endemic area with similar exposure to the infectious agent;
• (iii) high-throughput sequencing data from M. leprae genomes suggest
that this pathogen has a very low genetic diversity,indicating a crucial role
for the host genetics in disease outcome.
• (Kerrie Tosh,et al .2002)
Genetics of susceptibility
Genetics of susceptibility
• The suggestion that leprosy is a hereditary disease dates back to the
Middle Ages.
• Modern scientific investigation of the hypothesis of a genetic component
for human susceptibility to leprosy started at the turn of the nineteenth
century with the first reports of leprosy occurrence among twins. Twin
studies in India demonstrated higher concordance rates for leprosy in
monozygotic twins (60%–85%) than in dizygotic twins (5%–20%)
(M.R.Chakravartti,1937) .
• Complex segregation analyses have found models consistent with a single
major codominant or recessive gene and perhaps several modifying genes
controlling susceptibility to leprosy in some populations
Review of segregation analysis
Leprosy in Sudan
• Socio-cultural aspects of leprosy among the Masalit and Hawsa tribes in
the Sudan.
• The target populations were Masalit and Hawsa, the two main tribes in
the area. Knowledge about the pathological cause of leprosy was lacking
but the clinical manifestations were well recognized, particularly among
the Masalit, in whom the disease is more common than the Hawsa.
Among the Masalit there was a widely held belief that leprosy was caused
by eating meat of the wild pig and a certain type of fish. The Hawsa, who
are more devout Muslims, do not eat pig and associate leprosy with
consumption of two types of fish. Between both tribes, the stigma of
leprosy was not strong and the degree of rejection was more towards
those with severe disease, particularly patients with ulcerated lesions and
severe deformities. Patients were cared for by the family and lived in a
separate hut within the families' housing compounds
Chromosome 10p13
Family study was done in south India ,all patients had
been diagnosed as TT leprosy form.(M. Ruby
Siddiqui1)
.
stages
Affected sib No of
pair no
markers
result
Stage 1
103
388
stage2
245
37
stage3
245
8
Suggestive
linkage
analysis
region
D10s548
MLS >3.0
D10s1661
MLS =4.0
Chromosome 6 p25
• In 197 Vietnamese families they found a significant
association between leprosy and 17 markers located
in the 5' regulatory region shared by PARK2 gene and
the co-regulated gene PACRG.(mira mt 2004)
• This was confirmed in a sample of 975 unrelated
leprosy cases and controls from Brazil in whom the
same alleles were strongly associated with leprosy.
Variants in the regulatory region shared by PARK2 and
PACRG therefore act as common risk factors for
leprosy.
Chromosome 20
•In 2002 asusceptibility locus in chromosome 20 was found in
a study done in nourth india in two ethnic group where the
families show cluster of leprosy
•It was a non parametric linkage study where :
•175 families (which included 185 independent affected sib
pairs) were found in Sakthi Nagar and Kumbakonam in Tamil
Nadu.58 other families (including 71 independent sib pairs)
were found in Vizag in Andhra Pradesh. Both parents were
available for study participation.
Study design
The first stage
93 families(103 independent sib pairs), all of which
were from Tamil Nadu, India.
No members from the Andhra Pradesh region of India.
involved in this stage
markers attained a significance level of P < 0.1
11 markers (D1S2800, D2S347, D3S2338, D3S3681, D3S1278, D3S1292, D6S446, D7S661, D7S2465, D18S478,
and D20S115) that met thIs criteria in the first round were typed in the second-stage families. Eight more markers
on chromosome 20p12 were typed in all families to increase the marker density .
The second stage
(153 independent sib pairs), 82 families (82
independent sib pairs) of which were from Tamil Nadu
58 families (71 independent sib pairs) of which were
from the Andhra Pradesh region of India.
Chromosome 20 linkage map for families from Tamil Nadu, India. Singlepoint (solid line) and multipoint (broken line) maximum
logarithm of odds score (MLS) values are shown.
Result
1est,2end
• Significant evidence for linkage was detected at markerD20S115
• additional marker were added and the peak of linkage on chromosome 20 was still
found to be at marker D20S115
• No evidence for linkage on chromosome 20 among the 58 families(71 independent sib
pairs) from Andhra Pradesh
• the linkage became more significant when only the 175 families (185 independent sib
Separate region
pairs) from Tamil Nadu were included
analyzation
Strong linkage
• there were no significant differences in either the location or the strength of linkage
when siblings with multibacillary leprosy were excluded.
• These data suggest that the region of linkage on chromosome 20 may be linked more
strongly to paucibacillary leprosy susceptibility in the Tamil Nadu population than in the
Andhra Pradesh population.
ASSOCIATION ANALYSIS
ASSOCIATION
ANALYSIS
• Because linkage in the region was only observed in the
families from Tamil Nadu, only this group was included in
testing for association
TDT
• Transmission disequilibrium testing of D20S115 and its 8
flanking markers was carried out
• An allele of the microsatellite marker D20S835 was found
to be associated with protection against leprosy (Pp.021).
Mitsuda Reaction and Chromosomal
Regions 2q35 and 17q21
• These region found to involved in the clinical out come of the
infection with M.leprae, by controlling the formation of active
granulomatus reaction
• This was tested in a study done in Vietnam by doing the mitsuda
reaction
Mitsuda reaction
• The Mitsuda reaction is a granulomatous skin reaction, measured
21–30 days after the intradermal injection of 0.1 mL of lepromin
(heat-killed M. leprae). The results of the Mitsuda test are classically
interpreted as a binary outcome, either negative (<3 mm) or
positive (>3 mm).
positive
(>3 mm).
negative
(<3 mm)
WHY??
• Mitsuda reaction corresponds to the formation of a nodular
granuloma composed of epithelioid cells with giant multinucleated
cells and associated with a lymphocytic infiltrate.
• Tuberculoid form
• patients with a negative Mitsuda reaction are likely to develop the
more disseminated and contagious,lepromatous form of leprosy
• WHY DO SOME PEOPLE DEVELOP TUBERCULIOD FORM AND THE OTHER
DEVELOP THE LEPROMATOUS FORM ALTHUOGHT THEY SHARE THE SAME
ENVIRONMENTAL FACTORS AND THEY INFECTED BY THE SAME STREIN?
Mitsuda Reaction and Chromosomal
Regions 2q35 and 17q21
• Many studies strongly suggested the presence of
genetic
components influencing Mitsuda-reaction values.
• The first study was a candidate-gene study that showed evidence
for linkage between Mitsuda reactivity and the SLC11A1 gene (also
known as“NRAMP1”) in a population from Vietnam (Alcaı¨s A,ET AL
2000)
• The latest one was a genome wide linkage scan in a sample of
families from Vietnam.( Brigitte R.et al,2007)
Mitsuda Reaction and Chromosomal
Regions 2q35 and 17q21
Criteria
Mitsuda
• families with at least 2 affected siblings were involved
• thus minimizing the Mitsuda-reaction variability attributable to
differences in the antigenic trigger
• performed both in patients with leprosy(at the time of diagnosis, prior to
multidrug therapy) and in healthy individuals, by intradermal injection of 0.1 mL
of lepromin
• The reaction was measured 28–30 days later, and the diameter of induration was
measured, in ml, by 2 independent leprologists
• Mitsuda reaction were adjusted for sex and ethnicity , because these variables
had previously been shown to influencethe extent of Mitsuda reactivity
Adjustment
Mitsuda Reaction and Chromosomal
Regions 2q35 and 17q21
• A panel of 388 highly informative microsatellite markers spanning the
genome at ∼10-cM intervals
Linkage
Fine mapping
• highly informative extended haplotype of ∼4 Mb, including 4
microsatellites , 5 single-nucleotide polymorphismsand 1 tandem repeat
all within the SLC11A1 gene,
• additional set of 12 microsatellite markers, under the same conditions, in
the 2 genomic regions that we selected for fine mapping
Mitsuda Reaction and Chromosomal
Regions 2q35 and 17q21
Mitsuda Reaction and Chromosomal
Regions 2q35 and 17q21
MHC region susceptibility to leprosy
• Association and Linkage analysis of leprosy phenotypes with
HLA class II and TNF genes performed in brazilian population
found the significant linkage between HLA class II region
(HLA-DQB1 ,HLA-DQA1) and HLA-DRB1)
• Linkage was also found to the TNF locus(LOD = 4.000, P =
0.00002).
MHC Region Study In India
South Indian families, consisting of sib pairs with
mainly(TT) form of disease. were genotyped at the
DRB1 and DQB1 loci of HLA, the TNF-308 promoter
polymorphism ,(LTA) gene, MICA and
MICB gene (Muthuswamy Ravikumar 2006).
Continue …..
• in numerous case control studies DQ alleles especially
DQw1 have been shown to be associated with tuberculoid
leprosy in India, Korea, Thailand and Japan, and with
lepromatous leprosy in India and Japan. (Wellcome Trus
2002)
• (Rani et al.17 2007) confirmed by molecular typing, and
reported association of DRB1*1501 with leprosy
susceptibility, whereas DRB1*04 and DRB1*07 were
associated with resistance to leprosy. In addition, HLA-DR3
has been found associated with leprosy susceptibility in
Surinam and Mexico.
Continue …..
• A small study of leprosy cases and controls in Southern China
suggested that an exon 5 variant of the MICA gene may be
associated with resistance to multibacillary form of the
disease.
TLR2
TLR2:
• 86 Korean leprosy patients were screened for polymorphisms
in a highly conserved part of the TLR2 intracellular domain. C
to T substitution that results in an arginine to tryptophan
change at the highly conserved amino acid 677 was found in
22% of the 45 lepromatous leprosy patients (J Fitness 2002)
TLR2 polymorphisms are
Continue TLR-2
•
Three polymorphisms in TLR2 (597C3T, 1350T3C,
and a microsatellite marker) were analyzed in 431
Ethiopian patients withleprosy and 187 control
subjects.result in , The microsatellite and the 597 T
polymorphisms both influenced susceptibility to
reversal reaction. (Marta Janer2008)
Vitamin D receptor (VDR)
Vitamin D receptor (VDR):
The evidence for association of the VDR gene in leprosy was
firstly reported in a study in an Indian population (Bengalis),
testing for a T-C substitution at codon 352.(OR 1.67; P value
0.03. This work detected susceptibility to lepromatous and
tuberculoid leprosy in the presence of genotypes TT and CC
respectively.
a larger case-control study conducted in Malawians reported
susceptibility to leprosy per se in the presence of the CC
genotype. (M. O. 198 Moraes et al).
IL-10
interleukin-10 has many effects upon the functions of cells such as
lymphocytes, monocytes, natural killer cells, and dendritic
cells. Specifically, IL-10 is a cytokine that regulates immunemediated inflammation. It appears to have two major functions: (1)
to inhibit cytokine (i.e., TNF, IL-1, chemokine, and IL-12) production
by macrophages and (2) to inhibit the accessory functions of
macrophages in T cell activation. IL-10 accomplishes the latter
function through the reduced expression of MHC class II molecules
and certain co-stimulators (e.g., B7).
IL-10 down regulates T-helper 1 cytokines and
costimulatory molecules on macrophages,
contributing to the persistence of the
pathogens.
(AC Pereira ,et al.2009)
IL-10
two different studies demonstrated the
association of IL10 loci with leprosy.
showed that -819T allele was associated with
leprosy susceptibility (Santos AR,et al.2002)
implicated a different promoter haplotype
associated with leprosy susceptibility (3575T/
2849G/ 2763C/1082A/-819T/592A),but again
819T was associated with the disease
outcome.(Malhotra D,ETAL.2005)
in a study among a Malawi population, proximal IL10
SNPs were not associated with leprosy.(Fitness
J,ETAL.2004)
Association analysis
IL-10
genotyping
IL-10
genotyping
Result
• linkage disequilibrium analysis using genotypes obtained from the control group.
• the r2 values showed a low correlation between the markers and according to
the r>0.8 threshold defined by the HapMap project,THEY could not define a tag
to predict haplotype bins and all SNPs were compared between cases and
controls
• They found that -- 819CT and --819TT genotypes as well as T-allele carriers were
associated with leprosy susceptibility,
(AC Pereira ,et al.2009)
Genotypic , allelic and carrier frequency of promoter IL-10
(AC Pereira ,et
al.2009)
IL-10
Meta
analysis
Meta
analysis
Meta
analysis
• A total of four case–control studies were searched in the
PubMed, combined with the present data to generate(819C/T) meta-analysis
• There was no significant evidence for publication bias .
• They found significant associations of the -819 SNP with
leprosy susceptibility
(AC Pereira ,et al.2009)
Summery of meta analysis of case-control studies of
IL-10 -819
(AC Pereira ,et al.2009)
IL-10 measurement in PBMC culture supernatants
(PBMC)
cultures
(PBMC)
result
Conclusion
• To evaluate the biological significance of _819C/T SNP
genotypes and IL-10 production, peripheral blood mononuclear
cell (PBMC) cultures were stimulated with different antigens .
• Individuals with the _819CC genotype produced higher amounts
of IL-10 under any stimulus than _819T allele carriers
• (_819TT/TC genotypes)
• carriers as( low IL-10 producers) bring up a new understanding
for the disease, these sustained low levels of IL-10 are necessary
to lead leprosy to a chronic and unresponsive state
(AC Pereira ,et al.2009)
IL-10 measurement in PBMC culture supernatants
(AC Pereira ,et al.200
conclusion
• Leprosy is quantative trait(polygenic).
• The most genes that associated/protactive to leprosy are
genes that responsible for immune response .
• The genetic factors play big role in disease in contrast with
environmental factors
• Some genes are sucseptible to clinical disease such as TLR2
and TNFa.