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New genetic findings

Discovery of a mutation that mitigates muscular dystrophy in dogs and identification of human genes linked to rare diseases

Muscles affected by dystrophy and the dogs Ringo and Suflair: mutation in the Jagged1 gene mitigates symptoms of the disease

CDC / Dr. Edwin P. Ewing, Jr.Muscles affected by dystrophyCDC / Dr. Edwin P. Ewing, Jr.

Since 2004, researchers at the Human Genome and Stem Cell Research Center (CEGH-CEL), associated with the Biosciences Institute of the University of São Paulo (IB-USP), suspected that Ringo, the golden retriever, had some type of molecular mechanism that mitigated the appearance of the more severe symptoms of muscular dystrophy, such as trouble with walking and swallowing.  The dog, as what would later be the case with his pup Suflair, never produced dystrophin, an essential protein for maintaining muscle integrity, and should have succumbed to the disease’s progressive degeneration.  Both dogs, however, remained practically normal.  Ringo died in 2014 at age 11, after having lived a normal lifespan for dogs, and Suflair continues to be healthy at age nine and a half.  This month, the USP group, in partnership with colleagues from Harvard Medical School and the Broad Institute in the U.S., published an article in the journal Cell that explains the phenomenon:  the two golden retrievers present increased expression (activation) of the Jagged1 gene that protects them from developing the severe symptoms of muscular dystrophy.  The finding is promising because scientists believe that it signals the possibility of exploring a new therapeutic approach that could at least mitigate the symptoms of the disease in humans by controlling the function of this gene.

The newly discovered genetic alteration causes the Jagged1 levels to be two times higher in Ringo and Suflair than in dogs severely affected by the disease.  In other words, the gene is activated more intensively and produces twice the respective proteins in the USP dogs than in the sick animals.  According to the study, the anomaly in the Jagged1 gene compensates for the harmful effects of the absence of dystrophin, caused by mutations in the gene responsible for producing that protein.  This is because one of the functions of the Jagged1 gene is to act on an intracellular signaling pathway called Notch, which is involved in producing and repairing muscle cells.

The dogs Ringo and Suflair: mutation in the Jagged1 gene mitigates symptoms of the disease

Eduardo CesarThe dogs Ringo and Suflair: mutation in the Jagged1 gene mitigates symptoms of the diseaseEduardo Cesar

“We’ve been able to show for the first time that a large animal can have functional muscle without dystrophin,” says Mayana Zatz, coordinator of the recently-published study and of the CEGH-CEL, one of the 17 Research, Innovation and Dissemination Centers (RIDC) funded by FAPESP.  “The paper paves the way for us to test new treatments against the muscular dystrophies that are most commonly seen in humans, such as Duchenne and de Becker, through increased activation of the Jagged1 gene.”  Current therapeutic approaches to muscular dystrophy that for now show only modest results, focus on trying to restore normal function to the dystrophin gene, whose mutations interrupt the production of this protein and lead to the appearance of the disease.

Understanding the molecular bases of the diseases is an important step in developing more personalized treatments, a trend in 21st century medicine.  “In the near future, physicians will look at a person’s genetic information and metabolic functioning and prescribe the most appropriate medicine in an individualized dose,” Zatz says.  Cancer research is heading in this direction (see the report on page 64), and an initiative that involves five of the RIDCs, the Brazilian Initiative on Precision Medicine (BIPMed), has established a computer platform to integrate genetic and clinical data about diseases and thus generate treatments specifically designed for each patient.

Both forms of muscular dystrophy—the more severe, Duchenne, and the milder, de Becker—are recessive diseases that primarily attack males.  There is a simple explanation for this pattern of occurrence. The dystrophin gene is located on the X chromosome, of which males have one copy and females have two.  That is why males develop the disease by inheriting a single copy of the mutated gene, while females need to have two copies with the alteration in order for muscular dystrophy to manifest itself, which is something quite rare.  Women, when carrying a mutated copy of the gene, in most cases are asymptomatic but during pregnancy have a 50% chance of transmitting the gene to their offspring.  Duchenne muscular dystrophy affects one out of every 3,500 newborn males, a rate 10 times higher than that of de Becker.

Biologist Natássia Vieira, who received her doctorate from IB-USP and has now spent nearly four years in the United States looking for the protective mutation in Ringo and Suflair, was one of the key individuals responsible for the discovery.  She spent time at the Harvard Medical School laboratory of Louis M. Kunkel, who in the late 1980s identified the mutation of the dystrophin gene associated with the disease and convinced Swedish geneticist Kerstin Lindblad-Toh to take part in the project. Lindblad-Toh is the scientific director of the Vertebrate Genome Biology Group at the Broad Institute, maintained by Harvard and the Massachusetts Institute of Technology (MIT).  For this reason, Kunkel and Lindblad-Toh, along with Zatz, are senior co-authors of the study in Cell.

As an early clue for where to look for the hypothetical protective mutation, Vieira used data from an experiment conducted five years ago in collaboration with the laboratory of Sergio Verjovski-Almeida, at the USP Chemistry Institute (IQ).  “By using a DNA chip, we saw that 66 genes had an altered expression profile in the asymptomatic dogs compared with the affected animals,” says Verjovski-Almeida, who also contributed to the new study.  In Lindblad-Toh’s laboratory, Vieira performed whole-genome sequencing on Ringo, Suflair and a third golden retriever with a severe case of muscular dystrophy.  She discovered that there was a small mutation in one of the 66 genes that had caught their attention in the IQ-USP study: a change in a single nitrogenous base (the chemical unit of which all DNA is composed) in the region that regulates the Jagged1 gene function, located on chromosome 24 of the dog, was the mutation they had sought for years.

The biologist then returned to Kunkel’s laboratory and ran experiments with messenger RNA in zebrafish, one of the biological models most often used to simulate human diseases in animals.  The idea was to see, by stimulating activation of the Jagged1 gene as it occurs in the dogs, whether the fish would present a lower prevalence of a condition equivalent to muscular dystrophy in humans.  It worked.  “The incidence of the disease fell from the expected 25% to 6%,” says Vieira.  Thus, the researchers collected strong evidence that the mutation in the Jagged1 gene, which is found on chromosome 20 in humans, is capable of attenuating the clinical manifestation of muscular dystrophy.

014-021_Capa_doenças genéticas_237-01Advances in rare diseases
Identification of a likely molecular mechanism mitigating the development of dystrophies was not the only recent achievement by CEGH-CEL to produce significant findings towards understanding genetic diseases.  In an online article published September 29, 2015 in the journal Human Molecular Genetics, another group of researchers associated with the USP center showed that patients with a rare hereditary neurodegenerative syndrome do not present a 216 base pair sequence in the region that controls the function of the KLC2 gene on chromosome 11.  It took 10 years to identify the genetic alteration.  In 2005, while doing a post-doc fellowship at the CEGH-CEL, São Paulo biologist Silvana Santos was the key player in discovery of the disease in Serrinha dos Pintos, a town of 5,000 inhabitants in interior Rio Grande do Norte State, where one-third of all marriages were consanguineous, meaning between two individuals who are related as second cousins or closer.  “It was very hard to find the mutation.  We were only successful because we were able to use new sequencing techniques,” Mayana Zatz says.

The disease known as SPOAN syndrome, which stands for Spastic Paraplegia, Optic Atrophy and Neuropathy, causes a series of clinical symptoms that result in patients becoming wheelchair-bound by their teenage years: continuous stiffness and weakening of the arms and legs, progressive lesions in the motor and sensory neurons and a decrease in the field of vision as a result of the congenital atrophy of the optic nerve (see Pesquisa FAPESP Issue nº 113).  This decade-long effort mobilized Santos who, since 2008, has been a professor at the State University of Paraíba (UEPB), in Campina Grande, as well as researchers from the CEGH-CEL at USP, along with other Brazilian and even foreign universities.  The work has included several field visits to the interior of the states of Paraíba and Rio Grande do Norte.  During those trips, researchers discovered, in addition to information about SPOAN, two new neurological disorders as well as the genetic mechanism that causes them in relatively isolated Northeastern communities.  These new diseases, equally as rare, were presented to the scientific community in two recently published studies.  “Just as with SPOAN, these two diseases are found in families that have numerous consanguineous unions,” Santos says.

As initial studies had indicated a decade ago, the genetic alteration responsible for causing SPOAN is found in a region on chromosome 11 that has more than 140 genes.  This trail was always correct.  What the researchers had no way of knowing was that the mutation was on a segment of DNA that had been the subject of little study up to just a few years ago.  The mutation associated with the syndrome is not in what is known as the coding region, the part of the sequence that contains specific instructions for producing the protein associated with each individual gene.  Such region is the most common location for mutations associated with the appearance of diseases and is therefore traditionally the prime target when searching for genetic alterations with clinical repercussions.  The mutation linked to the Serrinha dos Pintos syndrome, however, is found in a regulatory area of the KLC2 gene.

Deletion of a 216 base pair segment in the region that controls the gene causes it to be overexpressed.  The KLC2 gene is more activated than normal in victims of the disease and produces additional amounts of kinesin, which belongs to a class of motor proteins that transports cellular organelles to the axon, the part of the neuron that conducts electrical impulses.  In other words, the mutation in the regulatory region does not lead to production of an altered form of the protein nor does it impede its production, as is the case in most genetic diseases.  Instead, it deregulates the amount of protein that is produced.  “This is the first recessive autosomal disease described as a result of a mutation that causes a gene to gain rather than lose a function,” Santos says.

Patients who develop SPOAN inherited two copies of the KLC2 gene; one from the father and one from the mother, with the alteration that causes the disease.  That is why the syndrome is called recessive.  People with just one altered copy carry the mutation that can be transmitted to their children but they themselves are clinically normal.  If the disease were dominant, a single copy of the mutated gene would be enough to result in development of the health problem.  “The defect that causes SPOAN was in a gene we had studied in 2006,” says biologist Lúcia Inês Macedo de Souza, who dedicated herself to finding the genetic alteration responsible for the syndrome during her doctoral and post-doc studies at the IB-USP from 2005 to 2013.  “But at that time, we didn’t have the affordable technology to sequence the whole genome.  We were only able to do that in December 2012.”

Reproduction of the cover of the Book Brazil and the Dutch – 1630-1654Rua dos Judeus, in 17th century Recife: the mutation that causes SPOAN syndrome likely came into the Northeast at the time of Dutch Brazil with the arrival of Sephardic JewsReproduction of the cover of the Book Brazil and the Dutch – 1630-1654

From the tail of the zebrafish
A native of Campina Grande, the biologist Uirá Souto Melo, who is pursuing his doctorate on SPOAN under the supervision of researchers from the CEGH-CEL, conducted an experiment that reinforced the link between the KLC2 gene and the syndrome.  He spent three months on the team led by Nora Calcaterra of the National University of Rosário (Argentina), former partner of the center at USP, where he used zebrafish in studies about gene function.  Melo injected zebrafish embryos with additional doses of messenger RNA (the chemical recipe for producing a protein) extracted from the KLC2.  As the RNA dose was increased, the tails of the fish became more twisted, a phenotype that can be interpreted as manifestation of a neurodegenerative disease.  “Right now I’m trying to develop a transgenic mouse with the SPOAN mutation,” says Melo, who is spending time at the University of California in Berkeley.  “That way it will be easier to make a biological model of the disease in an animal more genetically similar to humans.”

Last decade, due to cost issues and technological limitations, the sequencings focused on the coding region of the human genome, which represents 2% to 3% of the total sequence.  The rest of the genome, nearly 97% of the sequence, even came to be known as “garbage DNA” during the first few years after completion of the human genome sequencing project in 2003.  It was thought that it served no purpose.  But this view changed drastically as time passed and new sequencing techniques were created and publicized.  Today, understanding the chemical composition of all human genetic material, including that from non-coding areas, has become a much quicker, cheaper, and, as shown by the SPOAN study, important task.  According to a survey conducted by the National Human Genome Research Institute (NHGRI) of the U.S., sequencing a person’s entire genome cost $95 million in September 2001.  By July 2015, the same endeavor cost only $1,300.

The discovery of the gene mutation does not immediately pave the way to a cure for SPOAN, the victims of which have neither cognitive loss nor pain, but who do see their quality of life quickly deteriorate.  It does, however, facilitate the development of a test that can predict the occurrence of the disease in children of parents who carry the mutation.  Ten years ago, when first discovered, SPOAN affected 26 residents (17 women and nine men) from Serrinha dos Pintos, all descendants of 19 consanguineous unions.  Now, there are 61 victims in Rio Grande do Norte, spread out among eight cities (two-thirds of them in Serrinha and São Miguel), and 14 in four other states (5 in São Paulo, 5 in Paraíba, 2 in Ceará and 2 in Rio Grande do Sul).  We have also been in contact with two cases in Egypt for which we have confirmed the mutation in the regulatory region of the KLC2 gene,” Santos says.

Serrinha dos Pintos: SPOAN disease was discovered in 2005 in a town in rural Rio Grande do Norte State

Eduardo CesarSerrinha dos Pintos: SPOAN disease was discovered in 2005 in a town in rural Rio Grande do Norte StateEduardo Cesar

Mutation and Sephardic Jews
One detail always called the attention of researchers when they identified the initial cases of those affected by SPOAN in Serrinha dos Pintos.  Every patient had been born in that region, was Caucasian and was a distant descendant of the Portuguese or Dutch who had dominated part of Brazil’s northeast in the 17th century.  This situation led them to formulate the hypothesis that the Europeans could have introduced the mutation to the region back in the time of Dutch Brazil.  The DNA of 68 victims affected by the syndrome and of 85 family members who carry the mutation (but do not have the disease) is being analyzed in an attempt to determine when the genetic alteration first appeared.  With any luck, scientists will also be able to determine whether the mutation first appeared in Brazil or was brought there by immigrants.  “Preliminary data suggest that the mutation may have arrived in the Northeast with Sephardic Jews, natives of the Jewish community in Portugal and Spain during the time of Dutch Brazil,” Santos says.

The studies that led to the discovery of SPOAN began during the first half of the 2000s.  Santos still lived in São Paulo and had become intrigued by the story of a neighbor who had a type of paraplegia that no one could explain.  The girl was said to be from a Serrinha dos Pintos family that had several members affected by the same health problem.  Santos went to the small town in the hinterlands of Rio Grande do Norte and saw that the incidence of the disease and of other neuromotor disturbances was significant throughout the entire region.  SPOAN, described in the scientific literature in 2005, was her first discovery while still part of the CEGH-CEL team at USP.  In 2008, she was hired by the UEPB and continued to pursue her studies of diseases linked to consanguinity.  “Since then, she’s been a wonderful partner to our center, sending students here and doing joint studies,” says Mayana Zatz.

In 2009, Santos and her students at UEPB established a partnership with 39 towns in the hinterlands of Paraíba State, and with the support of community health officials, interviewed 20,462 couples to try to establish the frequency of consanguineous unions in various localities.  The results of the study indicated high numbers at a rate that ranged from 6% to 41% depending on the city.  In 2012, the UEPB group along with a team of neurologists from USP examined 109 people with some type of physical or mental disability from six municipalities in Paraíba with high rates of consanguinity (Bom Sucesso, Brejo dos Santos, Catolé do Rocha, Belém do Brejo do Cruz, São José do Brejo do Cruz and Brejo do Cruz).  These incursions to places where there were many marriages between relatives led to the discovery of additional cases of SPOAN as well as other rare diseases such as the two neurological problems now reported in scientific journals.  “We did up a free electronic book entitled Does your family have anyone with disabilities? and made it available on the UEPB site,” Santos says.   

One of the new diseases is a type of intellectual disability (once called mental retardation) that causes exacerbated sexuality and anatomical alterations of the face (prognathism, prominent chin and very large nose), and it is found in seven members of a single family from the town of Catolé do Rocha, Paraíba.  Today, three of those affected live in Mossoró, in Rio Grande do Norte, but the original nucleus of the clan is the town in Paraíba.  In an article published in December 2014 in the Journal of Medical Genetics, scientists described the disease caused by a mutation of the MED25 gene on chromosome 19.  “We were lucky,” says biologist Thalita Figueiredo, who completed her doctorate at UEPB under the advisorship of Santos and is first author of the study.  “Three months after our study, an international group described families with another mutation in this same gene.”  The MED25 is part of a large family of genes associated with several forms of intellectual disability.

Zebrafish embryo: (from left to right) normal, slightly affected and highly altered by the SPOAN mutation

UIRÁ MELO Zebrafish embryo: (from left to right) normal, slightly affected and highly altered by the SPOAN mutationUIRÁ MELO

The second neurological disorder, yet to be named, occurs as a result of a mutation identified in the Inositol monophosphatase 1 (Impa1) gene located on chromosome 8.  It is a disease that causes intellectual disability and behavioral changes such as agitation and aggressiveness.  It was discovered in nine members of a family from Brejo dos Santos.  “This work offers an interesting perspective to our studies,” says neurologist Fernando Kok, a CEGH-CEL researcher and medical director of Mendelics, a private genomic analysis laboratory that took part in the sequencing of the three diseases.  “The lithium that is administered to patients with bipolar disorder inhibits the action of the gene.”  However, before the Brazilian researchers’ study was published on October 2, 2015 in the journal Molecular Psychiatry, no mental problem had been linked to Impa1.

The studies of rare genetic diseases both old and new in Brazil’s Northeast are far from over.  In the case of SPOAN, the challenge now is to understand the physiological mechanism, the chain of events, caused by the mutation in those affected by the syndrome, which progressively forces them to life in a wheelchair.  The high prevalence of disturbances and syndromes caused by marriages between relatives is one indication that there is still much to be studied.  A national survey published in 2014 points to the fact that there are nearly 4,000 people from more than 80 towns, mostly in Brazil’s Northeast, who present low-incidence genetic diseases in communities that are relatively isolated from the rest of the country.  “Several countries pay a lot of attention to highly inbred populations or those with low genetic diversity, such as the Amish in the United States, the Franco-Canadians in Canada, the Arab-Israelis in Israel and the Finnish,” notes Kok.  “We still know very little about the genetic diseases in Brazil.”  Advances in genetic studies, driven by new technologies and by a better ability to analyze vast amounts of data, are leading to a better understanding of the roots of rare diseases like SPOAN, as well as much more common conditions such as muscular dystrophy.

Mutations in two new genes cause Noonan’s syndrome 

Study expands the molecular basis of the disease and raises to 11 the number of genes involved in the clinical condition 

014-021_Capa_doenças genéticas_237-02Researchers at the CEGH-CEL discovered two new genes associated with Noonan’s syndrome, a disease nearly as prevalent as Down’s syndrome.  According to an article published in the Journal of Medical Genetics in June 2015, mutations in the S0S2 and LZTR1 genes are responsible for 3% of all cases of the disease.  The first gene is on chromosome 2 and the second on chromosome 22.

According to some estimates, the syndrome that can affect different areas of the body (see drawing) strikes one out of every 2,500 newborns.  Other estimates place the number at one out of every 1,000 newborns.  “The most important anomalies that lead parents to seek medical care are cardiac issues and short stature,” says CEGH-CEL geneticist Débora Bertola, a physician at the Children’s Institute of the Hospital das Clínicas (HC) at USP, principal author of the study.  “The clinical characteristics of the syndrome vary greatly.  There are probably many affected individuals who do not even know they have it.”  The disease does not generally affect an individual’s life expectancy.

Noonan’s syndrome is classified as a monogenic autosomal dominant disorder.  One need only inherit a single copy of one gene with the pathogenic mutation to develop the disease.  Before the CEGH-CEL study, mutations in nine genes were associated with 80% of all cases of the disease.  In general, those affected have some harmful mutation in just one of the genes that can cause the syndrome.

With the Brazilian study, the number of genes associated with the syndrome increased to 11.  “The trouble today is finding genes that are responsible for a very small percentage of cases of the disease,” Bertola explains.  “The discovery of the mutations in these two genes improves the effectiveness of the molecular diagnosis of the syndrome,” says Maria Rita Passos-Bueno, coordinator of the Technology Transfer Department of the CEGH-CEL.  With the exception of the LZTR1, all the other genes responsible for  development of the syndrome are associated with the RAS/MAPK signaling pathway, whose deregulation seems to be crucial in the emergence of the disease.  In order to discover what links the two genes to the syndrome, scientists sequenced the coding portion of the genome of 50 patents from the HC who had the disease but presented no mutations on the nine genes known to be associated with the disease up to that time.

1. CEGH-CEL – Human Genome and Stem-Cell Research Center (nº 2013/08028-1); Grant Mechanism Research, Innovation and Dissemination Centers (RIDC); Principal Investigator Mayana Zatz (IB-USP); Investment R$9,609,746.03 and US$4,676,005.00 for the entire project.
2. Program of the Center for Genetic Studies and Education at the State University of Paraíba (NEGE-UEPB); Principal Investigator Silvana Santos (UEPB); Investment R$ 200.000,00 (PROPESQ/UEPB, FAPESQ/CNPq, Biomarin).

Scientific articles
VIEIRA, N. M. et al. Jagged1 mitigates the Duchenne muscular dystrophy phenotypeCell.  Nov. 12 2015.
MELO, U. S. et al. Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndromeHuman Molecular Genetics.  Sept. 18 2015.
FIGUEIREDO, T. et al. A homozygous loss-of-function mutation in inositol monophosphatase 1 (Impa1) causes severe intellectual disability. Molecular Psychiatry. Sept. 29 2015.
FIGUEIREDO, T. et al. Homozygous missense mutation in MED25 segregates with syndromic intellectual disability in a large consanguineous family. Journal of Medical Genetics. 19 Dec. 2014
YAMAMOTO, G. L. et al. Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome. Journal of Medical Genetics. V. 52, No. 6, p. 413-21. June 2015.