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- 我是Laurent Abel
-I am Laurent Abel,

来自Necker校区Imagine研究所的
from the Laboratory of Human Genetics of Infectious Diseases

传染性疾病人类遗传学实验室
at the Imagine Institute at the Necker campus.

今天
Today,

我将探讨人类基因在结核病发展中的作用
I will speak about the role of human genes in the development of tuberculosis.

结核病的一个主要观察结果是
A major observation in tuberculosis

个体对于结核分枝杆菌的反应
is its very large variability observed between individuals

有非常大的差异性
in the response to M.tuberculosis.

这在感染过程的
This is true at the two main steps of the process,

两个主要步骤（接触和感染）中得以体现
going from exposure to infection,

因为一些密切接触
as some individuals who are heavily exposed to MTB

结核分枝杆菌（MTB）的人不会感染
do not get infected.

第二阶段
And for the second step,

从感染到发生临床疾病
going from infection to the development of the clinical disease,

估计只有5％的感染者
as it is estimated that only 5% of infected individuals

会在感染后的两年内发病
will develop primary TB,

即原发性结核
which is TB within two years of infection.

尤其是在高度流行地区的儿童
This is especially children in highly endemic areas.

另外5％会在之后一段时间发生结核病
And an additional 5% will develop TB later on,

有时在感染几十年后
sometimes decades after infection.

这是由于重新激活导致的结核病
This is classically pulmonary TB due to reactivation.

有几个因素可以解释这种差异性
So there are several factors that could explain this variability,

其中主要有两种类型
mainly two main kinds,

与分枝杆菌相关的
those related to the mycobacteria

与宿主相关的与分枝杆菌相关的
that I will not detail today

今天我不详细介绍
and those related to the host.

在宿主相关因素中
Among these host factors,

我们可以细分为非遗传的宿主因素
we could subdivide into non-genetic host factors.

这里的主要因素是获得性免疫缺陷
The major factor here is acquired immunodeficiency

当然
and of course,

获得性免疫缺陷的一个非常重要的影响因素
a very important factor presently of acquired immunodeficiency is HIV infection

是HIV感染
that strongly influences that process.

但是在大多数个体中
But in most individuals,

没有已知的获得性免疫缺陷
there is no known acquired immunodeficiency

居住在结核病流行地区的大量人群中
and it is likely that, in large populations,

遗传的宿主因素在很大程度上
which are living in endemic areas for TB,

可以解释这种差异性
host genetic factors could explain to a large extent this variability.

结核病人类遗传学的目标
The goal of human genetics of tuberculosis

是理解为什么一些感染者会患结核病
is to understand why some infected individuals develop TB and doing so,

确定在自然感染条件下
identifying the critical immunological pathways

关键的免疫学途径
that really matter in natural conditions of infection.

要做到这一点
To do this,

要找到能解释个体间差异
we are searching for genetic variants

并能影响对结核分枝杆菌的
that may explain these differences between individuals

免疫应答的遗传变异体
and could influence the immune response to M.tuberculosis.

为了找出这些遗传因素
To identify those genetic factors,

我们所说的基本假设
the basic hypothesis we are stating

是有主要倾向性的两个极端
is that there are two main poles of predisposition,

当然也有中间情况
and of course also intermediate situations,

但让我们把重点放在主要的两极上
but let us focus on these main poles.

一个是孟德尔遗传学（Mendelian genetics）
One is Mendelian genetics,

非常罕见的突变与强烈的个体效应
looking for very rare mutations with strong individual effects

意味着携带这些突变的个体很可能发病
meaning that individuals carrying these mutations are very likely to develop the disease.

这是传统的单基因疾病
These are classical monogenic disorders.

我将详细介绍一些严重的儿童结核病的例子
As I will detail, we have some examples in severe tuberculosis in children.

另一极是复杂的遗传学
The other pole is complex genetics,

更多的常见变异体和多态性
where we are looking for much more common variants, polymorphisms,

在个人层面上有相对温和的影响
which have a more modest effect at the individual level.

我们一直在寻找这种结核病的变异体
We have been looking for this kind of variants in pulmonary tuberculosis.

当然
Of course,

这项研究受益于
this research has benefited from the considerable progress

近期人类基因组学的可观进展
that has been made recently in human genomics,

特别是通过超高通量基因分型
especially by ultra-high throughput genotyping,

使得能够对数以百万计的
making it possible to genotype millions

SNP(单核苷酸多态性)进行分型最近
of polymorphisms or SNPs,

二代测序允许我们对全基因组
single nucleotide polymorphisms,

或至少被称为外显子的编码序列进行测序
and also more recently, next-generation sequencing allowed us

让我们从孟德尔那部分开始
to sequence whole genomes or at least their coding sequences which are called exomes.

这项研究来自对相关疾病的观察和研究
Let us start with the Mendelian part.

称为孟德尔遗传易感性的分枝杆菌病
This research came from the observations and the studies made on a related disorder

或MSMD
called Mendelian Susceptibility to Mycobacterial Diseases or MSMD.

这是一种非常罕见的疾病
This is a very rare disorder

其特征在于弱毒性分枝杆菌
characterized by very severe and disseminated infection

制造严重的大面积感染
by weakly virulent mycobacteria

如活卡介苗
such as live BCG vaccines.

特别是我的同事Jean-Laurent Casanova
In particular my colleague, Jean-Laurent Casanova,

在这个症状中发现了
identified in this syndrome

大量与白细胞介素-12-干扰素-γ通路
a large number of mutations in genes

有关的基因突变
which are all involved in the interleukin-12-interferon-gamma pathway.

这些突变分子的编码基因中
These mutations are in genes coding for the molecules

在图中以红色表示
indicated in red on that figure.

不同位置的突变
These mutations lead to defects either in the production of interferon-gamma

会导致相应的γ干扰素生成或应答障碍
which are mutations in that part, or in defects in the response to interferon-gamma

因此这些发现使我们首先确定了
which are mutations in that part roughly.

首例孟德尔结核病例
So these findings led us to the identification of the first cases of Mendelian TB,

特别是通过观察经典MSMD病人的家属
especially by looking at families of patients who have classical MSMD,

更准确地说是整个家庭
I mean this family.

我们发现其他兄弟姐妹的遗传缺陷
We identified additional siblings

和这个病人完全一样
who had exactly the same genetic defect as this patient here.

这是他的兄弟姐妹之一 
This is a sibling

实际患有腹部结核病
and this sibling had in fact abdominal tuberculosis in that case.

因此 在这种情况下
So the same genetic defect,

同样的遗传缺陷
which was in that case a complete defect

是白细胞介素-12受体β-1的
in interleukin-12 receptor beta-1 deficiency,

完全缺陷当接触结核分枝杆菌时
could lead to MSMD or TB,

可能导致MSMD或结核病
of course, upon exposure to Mycobacterium tuberculosis.

当我们更系统地调查
When we investigated more systematically the role of this gene,

这个基因-白细胞介素-12受体β1
interleukin-12 receptor beta 1

在50例严重结核患儿样本中的作用时
in a sample of fifty children with severe TB,

我们发现这些孩子中
we found that two of these children were carrying complete deficiency

有两个完全缺乏该基因和功能丧失性突变
in that gene and loss-of-function mutation,

一个来自摩洛哥
one from Morocco,

一个来自伊朗
one from Iran,

这意味着孟德尔结核的比例
meaning that the proportion of Mendelian TB

绝不可以忽略不计
could be far from negligible

因为在这个小样本只是测试单一基因
because we found 4% in that small sample

就发现有4％的比例
and by testing a single gene.

如今我们正在使用二代测序方法
We are now looking for the role of mutations in other genes,

寻找其它基因突变的作用
especially using these next-generation sequencing approaches.

我们已经确定了
And we already identified

另外几个引起孟德尔结核的基因
several additional genes causing Mendelian TB,

验证了一般假设
validating the general hypothesis

即严重的儿童结核病
that severe TB of children

可能在很大程度上归因于孟德尔易感性
could be due to a large extent to a Mendelian predisposition.

现在让我们切换到结核病
So let us switch to pulmonary tuberculosis.

在结核病中
In that pulmonary TB,

常见变异体的作用
the role of common variants

首先在候选基因中进行了研究
was first studied in candidate-gene studies

最近在全基因组关联研究中被研究
but more recently by genome-wide association studies,

通过对数百万个
allowing to search

单核苷酸多态性（Single Nucleotide Polymorphism,SNP）进行基因分型
for the role of these common variants

寻找这些常见变异体在整个基因组中的作用
across the whole genome by genotyping millions of SNPs.

有两个主要的研究报
There were two main studies that were reported:

道 一个来自非洲
one from Africa

一个来自俄罗斯样本量非常大
and one from Russia with very large samples.

事实上
In fact,

它们相当令人失望
they were rather disappointing.

只发现了三个变异的作用
They only found the role of three variants

在比值比（odds ratio, OR）
with very weak individual effects

非常接近1的情况下
in terms of odds ratio,

个体效应非常弱
very close to 1,

这意味着至少在这些样本中
meaning that, at least in those samples,

由于常见变异
there were no strong signals in pulmonary TB

肺结核中没有强信号
due to common variants.

而且
By the way,

他们没有发现
they did not find

位于先前测试的候选基因中多态性的作用
the role of polymorphisms located in candidate genes that were previously tested.

这意味着常见变异体
That probably means

在结核病中的作用可能很有限
that common variants have a limited role

并产生了通过二代测序方法
in pulmonary tuberculosis and leads to the idea of testing rarer variants,

检测罕见突变的想法
especially by next-generation sequencing approaches,

并且还应关注更具体的结核病表型
and also of focusing on more specific pulmonary tuberculosis phenotypes,

如家族性病例或早发性肺结核这些病例
such as familial cases or early onset pulmonary TB

更可能携带者具有更强个体效应的变异
which may be more likely to carry variants with stronger individual effects

可以通过二代测序方法检测出来
that could be detected by next-generation sequencing approaches.

为了说明这一点
To illustrate that,

我提到我们在摩洛哥进行的这项研究
I mention this study that we performed in Morocco,

我们关注的是多发家庭
where we focused on multiplex families.

我们收集了至少有两个受影响的兄弟姐妹的
We collected about 100 families

100个家庭
with at least two affected siblings,

并且通过连锁研究
and by linkage studies,

发现了8号染色体上
we found the role of a major locus

一个主要基因位点的作用
on chromosome 8.

为了进一步研究这个位点
To further investigate that locus,

我们把重点放在这个连接区域上
we focused on this linked region

并且在该区域内进行
and performed an association study

了3000个SNP位点的关联研究
with 3 000 SNPs within that region.

通过在摩洛哥的第一个研究
By a first study and a replication study

和一个复制研究
in Morocco,

我们确定了位于一个名为TOX的基因中
we identified the role of two SNPs located

的两个SNP的作用
in a gene called TOX

我将在下一张幻灯片中详细说明
that I will detail in the next slide.

最初的研究结果发现
The initial findings found

OR值在2左右但有趣的是
an odds ratio around 2 but very interestingly,

当我们根据患者的发病年龄将样本分层时
when we split the sample according to the age of onset of the patients,

发现在25岁以前
we found a much stronger effect in patients

发生结核病的患者的效果更强
who developed TB before the age of 25,

OR值是2或3
with an odds ratio of about 2 or 3.

这种效应
This effect

也在马达加斯加的一个样本中得到验证
was also validated in an additional sample from another country,

有趣的是
in Madagascar.

TOX基因是参与几种免疫细胞发育的核因子
What is interesting is that the TOX gene is a nuclear factor

特别是在结核病中非常重要的CD4T细胞
which is involved in the development of several immune cells,

这点可以从HIV感染个体
in particular CD4 T cells which are very important in TB,

具有非常低CD4T细胞计数
as shown by HIV-infected individuals who have very low CD4 T cell counts

但对结核病高度敏感看出来
and who are highly susceptible to TB.

我们目前正在研究
We are presently investigating

一个位于调节区域的SNP的作用
the role of one of these SNPs,

采用的方法是根据该SNP的基因分型结果
which is located in a regulatory region by performing functional studies in T cells

在T细胞中进行功能研究
according to the genotype of this SNP.

总而言之
Just to summarize,

白细胞介素-12-γ干扰素
there are therefore rare mutations,

通路基因中的
especially in genes of the interleukin-12-interferon-gamma pathway

一些罕见突变与儿童原发性严重结核病有关
which are involved in the development of primary severe TB in children.

而对于肺结核的发展
While, for the development of pulmonary TB,

常见变异的作用可能是有限的
the role of common variants is probably limited

TOX变异体
and TOX variants may play a role in the development

可能在早发型结核病的发展中起作用
of early onset pulmonary tuberculosis.

我们也初步发现了
Just to mention that we have also preliminary findings showing

遗传因素在抵抗感染中的作用
the role of genetic factors in the resistance to infection.

因此 这项研究的目标
So the goal of this research is to identify the critical pathways

是确定感染的关键途径和自然条件
and natural conditions of infection to understand the pathogenesis of TB,

以了解结核病的发病机制
which is largely unknown.

这在很大程度上是未知的
This will have major implications,

这对于预防（如开发新的疫苗）
both for prevention, such as the development of new vaccines,

或者确定高危目标人群将具有重大影响
or the identification of target populations specifically at risk of developing TB.

而且能用于开发恢复免疫缺陷的新疗法
And also for the development of new treatments aimed at restoring deficient immunity.

这里 一个非常好的例子
Here, a very good example

是白介素-12β1受体突变的受试者
are the subjects who have mutations in interleukin-12 receptor beta 1

因为他们在产生γ干扰素方面有缺陷
as they have a defect in the production of interferon-gamma

除了传统的抗生素外
and could be treated by recombinant interferon-gamma

还可以用重组γ干扰素治疗
in addition to classical antibiotics.

非常感谢您的关注
Thank you very much for your attention.