January 2015

Exploring HLA Diversity in Brazil
Drawing upon the great genetic diversity of the people of Brazil, Erick C. Castelli, PhD, isdeveloping a database for the exploration of human HLA variation.

Introduction
The sixth chromosome of the human genome is home tothe human leukocyte antigen (HLA) system, a unique groupof genes essential for human health and immunity. A majorrole of HLA is enabling the immune system to differentiate
between healthy and diseased cells. The immune system
can detect certain threats, such as the presence in a cellof a viral protein or the product of a mutated gene. When
this happens, HLA brings evidence of the threat, in theform of a small piece of protein, to the surface of the cell.
Cells bearing such markers are then rapidly recognized anddestroyed by other components of the immune system.
Among other wonders, the immune system makes a
special exception in the case of pregnancy, sparing thefetus from annihilation by the mother’s immune system.
The basis of this “immune tolerance” mechanism appears
to be associated with HLA-G, one of several genes in theHLA system.
As a biologist and a computer programmer, Erick C.
Castelli, PhD, has been working with HLA for more than
10 years. His graduate research, carried out in the lab ofEduardo Donadi, MD, PhD, at Ribeirão Preto University ofSão Paulo, was focused on the relationship between HLA-G
gene polymorphism and cancer susceptibility.
Now, as a member of the Department of Pathology atSão Paulo State University (UNESP – Botucatu) in Brazil,
Dr. Castelli is taking advantage of the country’s vast
population diversity to establish a database of human HLA
gene variation. This database is becoming an essential
resource for Dr. Castelli and other biologists who wish
to understand how HLA genes are regulated and theassociations between HLA region polymorphisms andhuman phenotypes.
iCommunity spoke with Dr. Castelli to learn how nextgeneration sequencing (NGS) is contributing to theadvancement of knowledge about this complex andessential region of the human genome.
Q: What sparked your interest in conducting HLA research?
Erick Castelli (EC): I began by studying the HLA-G gene in Dr.
Donadi’s laboratory. He’s an expert in the field and has been
conducting HLA research for more than 30 years. The HLA-GErick C. Castelli, PhD is a Principal Investigator in the Department of Pathology
at the São Paulo State University (UNESP – Botucatu) School of Medicine.

gene produces an immune tolerance protein that is active at
the fetal/maternal interface. We were trying to understandthe genetics of HLA-G. It is quite different from the other HLA
genes because it is highly conserved. Our main focus was on
the population genetics around this gene. As we learned more
about its structure, we were able to start looking at how theexpression of the gene is managed.
Now that I am here in Botucatu, my research is expanding
to include the other genes in the HLA region, and theassociations between sequence variants and disease states. Itis helpful that I am situated here in Brazil, which is a very goodplace to do HLA research.
Q: Why is Brazil a good place for HLA research?
EC: We have a very mixed population here in Brazil. In fact, wehave a saying among scientists that “Brazil is a good variation
repository.” This country has been influenced by immigrationfrom Europe, Africa, and Asia. The genetic background of
the Brazilian population is highly diverse as a consequenceof five centuries of interethnic crosses of people from threecontinents, including Portuguese, Africans, and autochthonous
Amerindians. Later, other immigrants from Europe (mainly Italyand the Netherlands) and Asia (mainly Japan and China) cameto Brazil in the past century.
I saw that variability when I started working on HLA-G eight
years ago. Even though HLA-G itself is very highly conserved,
we found divergent sequences when evaluating its regulatory
regions. Any time I look at HLA in the Brazilian population, IJanuary 2015
can find something new. We have genetic variation here thatyou do not find elsewhere, but most of the frequent variations
found worldwide are also found in Brazil.
Q: What is the focus of your current research studies?
EC: We’re conducting two different research studies. The
first study is an evaluation of HLA genetics from a population
point of view. For this study, we are using normal, healthy
individuals. No one has performed deep sequencing of theHLA region on such a mixed population, and the available
databases describing the polymorphisms in the HLA genes
usually does not consider their regulatory regions. So, we aredetermining the genetic structure of this region and creating
a database that can be used for future comparative studies.
We’re looking at the sequence of the entire region, not just theexons, because as we saw with HLA-G that there is a lot ofinteresting variation outside the exons.
The second study is focused on the relationship between
genetic structure in the HLA region and various disease states.
Specifically, we are looking at the genetic variation in theHLA region between several tumor types and autoimmune
diseases (such as diabetes), and normal individuals, andcharacterizing the correlations between HLA polymorphisms
and such diseases.

“We have a saying amongscientists that ‘Brazil is a goodvariation repository.”
Q: What’s unique about your approach to studying theHLA region?
EC: First, we are sequencing the entirety of every gene — all
the promoter segments and the 3’ untranslated regions, inaddition to the coding regions. Second, we’re performing deep
sequencing on many individuals. And third, we are creating
new tools that are allowing us to complete this work. These
include our own software analysis program and strategies todevelop a good sequence map of the HLA region.
Q: What are some of the challenges you’ve encountered inyour HLA research?
EC: We are starting from the beginning, in the sense that
there is no established methodology to evaluate all HLA genesegments and regions. Much of the genetic analysis of HLA
has been focused on answering specific clinical questions,mainly detecting the presence of known sequences to make apatient/donor comparison. We need to look at much more of
the data so we can ask many different questions.

Also, working with HLA is not the same as working withanother genome region. The HLA genes are very similar toeach other. You cannot mix several genes at the same time,
unless specific bioinformatics approaches are developed tocorrectly separate data from each gene.
Today our main problem is the presence of repeated sequences
within the region and the fact that the HLA genes are quite similar.
Sometimes, for a short nucleotide segment (~ 40 nucleotides), it is
not possible to define the origin of this segment. We have written
new software and created data flow pipelines using open-source
tools to help us address this challenge, but we are still developing
strategies to work with the data.

“We are starting from thebeginning, in the sense that there
is no established methodology toevaluate all HLA gene segments
and regions.”
Q: How long have you been working with theMiSeq System?
EC: We have three MiSeq systems here in the University
campus, and I have been working with the MiSeq System forthe past two years. We are performing sequencing with NGS
systems to determine HLA variability.
Using Sanger sequencing, we were limited to working with justone gene at a time. It took me a whole year to evaluate justone or two genes. We moved to NGS because it enables us
to work with several genes at the same time. With the MiSeq
system, I can work with several thousand nucleotides in one
experiment and process all of my genes at the same time.
Q: What have you discovered in your research?
EC: We are finding a lot of polymorphism in HLA regions outsideof the ones that have been investigated in clinical studies.
Over the entire region, we find a great amount of variability thathas not been seen before because the Brazilian population isso much more diverse than other countries. This is not reallysurprising to us. What’s exciting is that it’s all new information.
We’re creating a database covering the genetic diversity of theHLA region in Brazil. It will be a comprehensive HLA databasesince it also considers the regulatory regions and introns.
Q: Were you surprised by the extent of polymorphism in theHLA regions?
EC: I was expecting some extent of variation. We weren’t
sure how much there would be. It was very exciting for us todiscover such a high degree of variability in the HLA region inthe segments that are usually not evaluated.

January 2015

“With the MiSeq System, I canwork with several thousand
nucleotides in one experiment andprocess all of my genes at thesame time.”
Q: Is there any potential clinical relevance in thepolymorphisms you are finding in the HLA region?
EC: A large part of our curiosity about HLA genes and theirsurrounding sequences is that we believe that most of thesepolymorphisms have some effect on gene expression. After
we have established our initial database and methodology, wewant to investigate that more closely. We want to establish
specific associations between polymorphisms and theexpression levels of HLA genes.
We already know that some of HLA genes, like HLA-G, areassociated with immune tolerance, and several groups in theworld are trying to figure out how they work. There are several
situations where being able to control gene expression in thisarea would be useful. For instance, it would be beneficial if
we could treat autoimmune disorders by enhancing immune
tolerance by increasing HLA-G expression.

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Pub. No. 070-2014-005 Current as of 06 January 2015