Unlocking the secrets of genetics

The Human Genome and Stem-Cell Research Center has identified more than a dozen genes responsible for diseases, have been developed tests for 45 of them, and through the use of genetic counseling, families can plan their child-bearing.

By Claudia Izique

Genetic diseases affect 2-3% of children born to healthy parents. An “error” in a single gene, unexpected mutations, or the presence of genes that increase our susceptibility to complex diseases (depending on the environment) can result in more than 20,000 conditions, including muscular dystrophies, Parkinson’s disease, Alzheimer’s disease, cardiopathies, autism, and nanism.

The Human Genome and Stem-Cell Research Center (HUG-CELL), one of the Research, Innovation, and Dissemination Centers (RIDC) funded by FAPESP, has already identified more than a dozen genes responsible for some of these diseases. Tests have been developed for 45 of the diseases, and through the use of genetic counseling, thousands of families can plan their child-bearing.

This path of study and patient care began a little more than ten years ago, when the investigation of the genetic mechanisms that trigger disease required, in addition to research, a certain bit of luck. When at least six members of a family were affected by a given disease, researchers extracted DNA – a molecule nearly 2 meters long and 2 nanometers thick – from the cells of each person. This DNA had to then be separated into fragments through electrophoresis, transferred to photographic films, and printed in acetate before the researchers could begin, half blindly, to investigate the millions of base pairs.

“It was like arriving in a city you knew nothing about and trying to find a specific house number,” explains Mayana Zatz, coordinator of the Center, based at the Biosciences Institute (IB) of the University of São Paulo (USP).

Between 1995 and 1997, science and luck helped the IB team, led by Zatz, to identify two genes linked to a severe form of Limb-Girdle Muscular Dystrophy. The team also identified a genetic mutation on chromosome 21 responsible for Knobloch Syndrome, a condition that causes progressive blindness and affects members of a family from the municipality of Euclides da Cunha in the state of Bahia.

The discovery, which was made within the scope of the FAPESP-funded thematic project Genotype-phenotype correlations: our contribution to the Human Genome Project, was one of Brazil’s first contributions to the Human Genome Project (HGP), an international initiative that sequenced nearly 21,000 protein-coding genes between 1990 and 2003. Once collected into a database, this information was made available to researchers all over the world as a type of “address book” of genetic research.

Shortly before the conclusion of the HGP, and already anticipating the possibilities for future molecular biological research projects, IB researchers proposed the establishment of a RIDC to study the human genome. This proposal was approved during the first round of the program launched by FAPESP. The goal was to identify and map new genes related to genetic diseases and understand the variability of these diseases. “We discovered more than a dozen genes,” Zatz recalls.

Gene function

The discovery of a gene represents a significant advance in understanding the body’s internal mechanisms; however, such a discovery does not immediately translate into a cure for a given disease. Once the gene is identified, the next step is discovering what it does, which mutations or errors are responsible for a genetic disease or malformation, and what its clinical impact will be. “The absence of the dystrophin protein in muscle, for example, leads to muscular dystrophy. Once you discover the gene that causes the dystrophy, you know that that gene is important in normal muscle function. In other words, starting from the disease, we end up finding out how a gene works.”

The researchers discovered that the VAPB gene, which encodes a protein involved in the transfer of substances inside the cell and is located on chromosome 20, is related to three different types of degenerative diseases in motor neurons: late-onset spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), and ALS8, which is an atypical variant of ALS. The researchers found alterations in the VAPB gene in dozens of individuals from seven families, all of whom were carriers of one of the three diseases, and they now suspect that a deficiency in the amount of this protein causes the death of motor neurons.

Findings from the research study were published in the November 2004 issue of the American Journal of Human Genetics, and the article has been cited more than 300 times. The study continues with the use of stem-cells in model animals.

Often, the gene related to the disease may remain “hidden” in a particular family over several generations, and the disease will only manifest itself when an individual receives one defective copy of the gene from the mother and another defective copy from the father. Such a situation occurred in a RIDC research project conducted to examine a set of symptoms that confined 26 people to wheelchairs. All subjects were related and were residents of Serrinha dos Pintos, a rural municipality with just over 4,000 inhabitants in the state of Rio Grande do Norte.

The researchers determined that the subjects were affected by a new neurodegenerative disease, which they named SPOAN Syndrome (using the initials for Spastic paraplegia, optic atrophy, and neuropathy), and they described it in a 2005 article in the journal Annals of Neurology.

The team recently discovered that the gene responsible for the disease is located in a region of chromosome 11 that contains at least 143 genes – 96 of which were strong candidates because they are activated in nerve tissue. “Determining which of these genes was behind SPOAN Syndrome was not an easy task,” says Zatz. Now, the challenge is to learn how the gene works.

The discovery of the gene responsible for SPOAN may enable the development of a diagnostic test to distinguish asymptomatic carriers of the disease (i.e., those who have an abnormal copy of the gene and thus may transmit the mutation) from those who are free from the disease.

Family planning

Between 2000 and 2012, the Human Genome Research Center developed new tests for 45 genetic diseases in addition to a diagnostic kit for autism, which is already patent-protected, according to Maria Rita Passos-Bueno. “Rather than using imported tests that are compatible with other ethnic groups but not always suitable for Brazilians, we discovered the most common mutations among our population and created our own specific tests.” These tests allow the identification of clinically normal couples who are at risk for having children with genetic diseases. “We made it possible for these couples to plan their child-bearing,” Zatz notes.

The Center has used FAPESP funds to purchase equipment that will allow thousands of mutations to be tested at the same time, facilitating the diagnosis of diseases such as cystic fibrosis, which involves more than 1,500 mutations in a single gene. All of these tests can be conducted at the RIDC, and the team is “fighting” (to use Zatz’s term) to have the tests covered by Brazil’s National Healthcare System (SUS).

Genetic counseling has been one of the RIDC missions since its inception in 2000. “When a disease is identified in a family, we need to determine if there is a risk of repetition,” explains Zatz. In the case of SPOAN, which is caused by a recessive gene, men and women have the same likelihood of presenting the pathology. However, the disease only develops in individuals born with two mutated copies of the gene associated with the syndrome: one inherited from the father and one inherited from the mother.

The parents do not manifest the syndrome because each parent has only one copy of the mutated gene; however, they are able to transmit it. This phenomenon can be thought of as a biological Russian roulette, the risks of which increase according to the number of marriages within the same family. In the case of the disease identified in Serrinha, 1 of every 250 inhabitants of the municipality had SPOAN, and one of every nine inhabitants was a potential transmitter of the mutation to his or her offspring at the time of the study.

Genetic counseling involves clinical and genetic tests, calculation of the risk of occurrence or recurrence, and prenatal guidance for couples or pregnant women. “We see approximately 3,500 people a year,” Zatz states.

Stem cell research

In 2005, researchers realized that they would have to include stem cell studies in the portfolio of research at the Human Genome Research Center to compete with international studies.

The first task was to “fight,” as Zatz says, for the approval of the Biosecurity Law. Enacted in March 2005, this law authorized research using genetically modified organisms as well as embryonic stem cells. The chapter of the law that refers to the use of stem cells for research was challenged under the argument that it would violate article 5 of the Brazilian Constitution that guarantees the right to life. In 2008, the Federal Supreme Court ruled in favor of the research after hearing testimony from scientists in the Court’s first-ever public session. “From that point on, we began to have more research funds available to study various sources, and we have been able to offer some hope for the families of patients afflicted by incurable diseases,” says Zatz.

Research is conducted using stem cells from patients and animal models. “Starting with the patients’ stem cells, we derive cell lines in the laboratory that represent various tissues such as bone, cartilage, fat, muscle, or nerve cells. These lines are our patients in vitro,” she says. Using this methodology, it is possible to study how a mutation in a particular gene is expressed in the cells of a patient, why one tissue type is more severely affected than another, and why two people who have the same mutation can have such different medical conditions.

Furthermore, use of this methodology allows the testing of numerous drugs and gene therapies to attempt to correct that defect. “Since research began, we have collected stem cell lines from over 500 individuals in affected families,” states the RIDC coordinator. Seven of these lines were obtained from four carriers of amyotrophic lateral sclerosis (ALS), which is caused by an alteration in the VAPB gene identified by the Center’s team, and from their healthy family members.

Starting from the patients’ stem cells, a methodology was established to develop induced pluripotent stem (IPS) cells that enable adult cells to be converted to pluripotent cells, permitting their use as embryonic stem cells. “The technology earned Japanese physician Shinya Yamanaka the Nobel Prize in Medicine in 2012,” recalls Zatz. Yamanaka shared the award with Englishman John Bertrand Gurdon. “We already have 40 lines of IPS cells and are offering this as a service to the community,” adds Zatz.

Another research strategy involves evaluating the clinical effects of injecting stem cells into animal models. “We have several mouse models that we use to study muscle regeneration, rat models for bone research, and dog models for the study of muscular dystrophy,” Zatz explains. Two of the golden retrievers carry a genetic mutation that prevents the production of dystrophin, a protein essential for maintaining muscle integrity, and are asymptomatic. The researchers work with the hypothesis that the dogs have protective genes or mechanisms that neutralize the negative effects of the mutation, and they want to discover what these genes or mechanisms are.

Zatz goes on to say that the Center also conducts research with stem cells that may be used in cell therapy. “Nine new sources of mesenchymal stem cells – which have the potential to form several tissues such as fat, bone, cartilage and muscle – have been identified in the fallopian tubes and in the orbital muscles of the lips and have led to another patent.” The studies further show that the umbilical cord tissue – but not the cord blood – is rich in mesenchymal stem cells. “The publication describing this work has already been cited over 100 times, and this finding has an important practical application because umbilical cord banks typically save the blood and discard the tissue.”

In preclinical studies, mesenchymal stem cells from various sources (e.g., tissue from the umbilical cord, fallopian tube, fat, and dental pulp) were compared to determine their capacity for muscle and bone regeneration in various animal models. “The findings from these studies have resulted in 41 international publications and two patents,” Zatz says.

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