Question
1: How does this finding implicate NOD2
in Crohn disease?
Could NOD2 be associated with Crohn disease
-- in the 17 people who did not inherit the 3020insC variant from
their parents?
-- in people from families without other affected members?
Your
answer:
To further investigate
the association of NOD2 3020insC with Crohn disease, the investigators
performed a case-control study. Cases were not related (only one
affected member was selected from each of the 416 families); controls
were drawn from 4 available groups of DNA samples obtained from
people without Crohn disease. The authors reported that all cases
and controls were Caucasian, and that allele frequencies were similar
in Jewish and non-Jewish cases. No further information was provided
about the cases or controls.
Question
2: At
minimum, what other information about cases and controls would be
important to know?
Your answer:
The distributions
of NOD2 3020insC in case and control groups were presented as allele
frequencies, which were significantly different by the large-sample
approximation to a two-sample binomial test (p = 0.0018).
Allele
frequency of 3020insC in unrelated Crohn disease patients
and controls*
|
|
Crohn
disease
|
|
Controls
|
Source
|
n
|
%
|
Source
|
n
|
%
|
Chicago
|
212
|
7.3
|
Chicago
|
65
|
3.8
|
Baltimore
|
88
|
6.8
|
Baltimore
|
46
|
3.2
|
Pittsburgh
|
116
|
10.8
|
San
Francisco
|
81
|
3.1
|
|
|
|
Germany
|
94
|
5.3
|
Total
|
416
|
8.2
|
Total
|
287
|
4.0
|
*Adapted
from Ogura et al.
Question
3: How
different were the distributions of the NOD2 3020insC variant in
cases and controls?
Your answer:
Genotype frequencies
among cases and controls were presented only in the text; they are
summarized in the following table.
NOD2
genotypes in unrelated Crohn disease patients and controls*
|
Genotype
|
Crohn
disease
|
Controls
|
OR
|
95%CI
|
+/+
|
359
|
264
|
ref
|
|
ins/+
|
46
|
23
|
1.5
|
(0.8-2.5)
|
ins/ins
|
11
|
0
|
17.6
|
(1.3-very
high)
|
Total
|
416
|
287
|
|
|
*Adapted
from Table 2, Ogura et al: + = wild type, ins = 3020insC, OR=odds
ratio
Expected number of controls=(0.04)2x287=0.4592, assuming Hardy-Weinberg
equilibrium and prevalence of Crohn disease 1 per 1000 people.
Because no NOD2
3020insC homozygotes were found among controls, the expected number
(assuming Hardy-Weinberg equilibrium) was used to estimate the corresponding
odds ratio (OR).
Question
4: How
strong was the association between NOD2 3020insC and Crohn disease?
Are heterozygotes at increased risk?
Your answer:
One way to quantify
the contribution of the NOD2 3020insC variant to the occurrence
of Crohn disease is to calculate the associated attributable fraction.
One way to do this is with the formula of Miettinen:
attributable fraction = fc (R - 1) / R
where fc is the fraction of cases with the risk factor
and R is the measure of relative risk (or odds ratio for rare diseases).
If we perform this calculation using the data in the chart,
AF(homozygotes) = (11/416)(17.6 - 1)/17.6 = 2.5%
AF(homozygotes+heterozygotes) = (57/416)(1.8-1)/1.8 = 6.0%
Question
5: What
assumptions are required to estimate an attributable fraction from
these data? If the result is correct, what does it suggest about
the role of NOD2 in Crohn disease at the population level?
Your answer:
Hugot et
al.,
who first described the IBD1 locus on chromosome 16, had already
examined and rejected two other candidate genes for Crohn disease
(CD19 and CD43) in this region. In this study, they used a positional
cloning strategy to identify three different single nucleotide polymorphisms
(SNPs) associated with Crohn disease. Their approach involved typing
26 microsatellite markers in the IBD1 region in 77 multiplex families,
followed by linkage analysis and linkage disequilibrium mapping
to hone in on the most promising region.
This region
did not include any obvious candidate genes but contained a number
of transcribed regions that could be identified by expressed sequence
tags. Eventually, 13 SNPs were examined for linkage disequilibrium,
and three SNPs (8, 12 and 13) were independently associated with
Crohn disease in 235 families. These three SNPs never occurred on
the same haplotype. The strongest association was found for SNP
13, which codes for a one base-pair insertion identical to that
found by Ogura et al. All three SNPs proved to be variants of the
NOD2 gene.
The investigators
presented allele and genotype frequencies for 468 unrelated people
with Crohn disease, 159 people with ulcerative colitis, and 103
unaffected people (78 unaffected spouses of people with Crohn disease
and 25 relatives of the French investigators). No additional information
was provided about the study subjects.
Allele
frequencies of three NOD2 variants associated with Crohn disease*
|
Condition
|
#
chromosomes
|
SNP8
|
SNP12
|
SNP13
|
Total
|
Unaffected
|
206
|
0.04
|
0.01
|
0.02
|
0.07
|
Ulcerative
colitis
|
318
|
0.03
|
0.00
|
0.01
|
0.05
|
Crohn
disease
|
936
|
0.11
|
0.06
|
0.12
|
0.29
|
* Adapted from
Hugot et al.
Identical to the NOD2 3020insC allele described by Ogura et
al.
Question
6: How
did frequencies of these variants compare in the Crohn disease and
unaffected control groups? How did frequencies of SNP 13 compare
with the results of Ogura et al.?
Your answer:
Question
7:
Were any of the three variants associated with ulcerative colitis?
Why is this finding important?
Your answer:
Hugot et al.
also presented genotype frequencies, summarizing data for the 16
possible genotypes to calculate odds ratios.
NOD2
genotypes in Crohn disease patients and controls*
|
Genotype
|
Crohn
disease
|
Controls
|
OR
|
95%
CI
|
Wild
type
|
267
|
88
|
ref
|
|
Simple
heterozygous
|
133
|
15
|
2.9
|
(1.6-5.5)
|
Homozygous |
28
|
0a
|
42
|
(1.7-very
high)
|
Compound
heterozygous |
40
|
0b
|
45
|
(2.4-very
high)
|
Total |
468
|
103
|
|
|
*Adapted from
Hugot et al.; OR=odds ratio.
Assuming Hardy-Weinberg equilibrium and prevalence of Crohn
disease 1 per 1000, expected values are a=0.22, b=0.29 .
Differ slightly from those reported (which may contain rounding
error).
Question
8:
How were these NOD2 variants associated with Crohn disease?
Your answer:
If we make similar
assumptions and calculate attributable fractions as for the other
study,
AF(homozygotes+compound heterozygotes) = (68/468)(44.4-1)/44.4 =
14%
AF(homozygotes+all heterozygotes) = (201/468)(4.3-1)/4.3 = 33%
Question
9:
How important are these NOD2 variants as risk factors for Crohn
disease at the population level?
Your answer:
Hugot et al.
also presented estimates of the absolute risk of Crohn disease by
genotype, using the estimated relative risks described above and
assuming that the total population risk was 1 per 1000 people.
Question
10:
What is the estimated absolute risk (incidence) of Crohn disease
by genotype?
Your answer:
In June 2001,
a third study by Hampe et al. reported an association with Crohn
disease for the insertion mutation of NOD2 reported by both Ogura
and Hugot. These investigators also found significantly increased
odds ratios for insertion variant heterozygotes (OR=2.6) and homozygotes
(OR=42.1, assuming Hardy-Weinberg equilibrium and population prevalence
of Crohn disease of 4 per 10,000 people).
All three authors
highlighted the importance of this finding for improving understanding
of the pathogenesis of Crohn disease. The NOD2 gene product is thought
to be important in the innate human immune response, functioning
as an intracellular receptor for bacterial lipopolysaccharides and
activating NF-kB. The frameshift mutation NOD2 3020insC encodes
a truncated protein, which Ogura et al. showed in vitro to have
diminished ability to confer responsiveness. This observation suggests
several ways that a faulty immune response to bacteria could contribute
to the Crohn disease process.
Question
11:
Considered together, what do the findings of these three studies
mean for researchers investigating the genetic basis of Crohn disease?
For people with one or more of these NOD2 variants? For people with
Crohn disease?
Your answer:
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