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One mutation, several defects

Brazilian researchers have discovered the gene responsible for a rare syndrome that causes malformation of the jaw and larynx

Daniel BuenoThe genetic cause of Richieri-Costa Pereira syndrome, a rare disease that causes craniofacial abnormalities and defects in the formation of the hands and feet, has just been determined. An international group of doctors and geneticists, coordinated by Maria Rita Passos-Bueno of the University of São Paulo (USP), Human Genome and Stem Cell Research Center (CEGH-CEL), identified in 17 patients with the syndrome a type of mutation present in both alleles (copies) of the EIF4A3 gene, located on chromosome 17. The alteration is characterized by excessive repetitions of a section of the gene rich in the nucleotides C (cytosine) and G (guanine), two of the four nitrogenous bases that make up DNA.

The mutation was described in a scientific paper published in the January 2, 2014, issue of the American Journal of Human Genetics (AJHG). Also on that date and in the same journal, another USP team presented a second paper reporting the discovery of a defect in a gene on chromosome 3, which is responsible for triggering a rare form of dwarfism associated with vision problems (see box on page 38). CEGH-CEL is one of 17 Research, Innovation and Dissemination Centers (RIDC) funded by FAPESP.

Researchers still do not know how the mutation affects the gene’s behavior, which is linked to RNA metabolism and causes the syndrome to appear. But they believe that it may lead to reduced production of the protein associated with EIF4A3. This gene was partially disabled in colonies of a model fish of biological development—known as the zebrafish—and the descendants that inherited the modification developed problems with craniofacial bone formation comparable to the disease in humans, evidence that alterations in the EIF4A3 can trigger health problems.

Described in 1992 by the team led by Dr. Antonio da Costa Pereira, of USP’s Hospital for Rehabilitation of Craniofacial Anomalies, located in Bauru, better known as the “Centrinho,” the syndrome was discovered in the inhabitants of the Paraíba Valley, São Paulo State. The 20 patients diagnosed with the disease in Brazil—there is one reported case abroad—belong to 17 families of the region. Although unrelated in a formal sense, the families probably descended from a single ancestor.

The most characteristic feature of the syndrome, which has no cure, is a malformation of the jaw and larynx. The bones that usually fuse to form the jaw, which has a U-shape, do not do so in individuals with the disease. “In the most severe cases, patients cannot breathe properly, and we need to do a tracheotomy,” says Dr. Passos-Bueno. Patients have curved or smaller fingers, clubfeet and are short in stature. Half of those affected by the syndrome also have difficulties with verbal communication and learning disabilities.

Daniel BuenoPrior to identifying the new genetic alteration, the researchers knew only that it was an inherited autosomal recessive disease whose risk of transmission increases with inbreeding. To develop the syndrome, the patient has to carry mutations in both copies of the gene associated with the disease, one from the father and one from the mother. People with just one defective gene do not manifest clinical signs of the disease, but they can nevertheless pass the molecular alteration onto their descendants. Children of couples in which both the father and the mother are carriers of the mutation have a 25% risk of being affected by the syndrome.

Finding the mutation associated with the syndrome was a lengthy and complicated process. For years the Centrinho researchers tried to identify in which part of the genetic material the molecular change related to the disease resided. A Centrinho student spent some time in the United States looking for the gene, but with no success. “The mutation must be ancient in origin and most likely the region common to patients is very small,” says Dr. Passos-Bueno.

The circle began to close after researchers used a large number of markers along the entire genome, approximately 500,000 SNP (single nucleotide polymorphisms) markers. The term refers to the various forms a nucleotide can take. With the aid of computer programs, they compared the genetic material among patients and with healthy family members and arrived at a region of 122,000 bases of chromosome 17. This segment harbored four genes that could be related to the cause of the disease.

The clinical significance of these defects in three genes was already known: mutations in one gene were associated with psoriasis, in another a form of glycogenosis (glycogen storage disease) and in a third the respiratory disease known as primary ciliary dyskinesia. Since none of these clinical conditions resembled Richieri-Costa Pereira syndrome, their attention turned to the fourth gene, EIF4A3.

The resequencing of this gene in patients and comparison of the results with the version of EIF4A3 found in 520 Brazilians without the syndrome led to the location of the mutation. The alteration is in a small section of the sequence, comprising 18-20 nucleotides rich in cytosine and guanine bases, which regulate gene functioning; this is known as the promoter region in the jargon of molecular biology. People without the disease have 3-12 copies of this section of the gene. Patients with the disease have 14-16 repetitions of the segment.

An experiment coordinated by Nora Calcaterra, a researcher at Argentina’s National University of Rosario, confirmed that the mutation causes the disease. She is a co-author of the study and a collaborator of Dr. Passos-Bueno in a project financed through a Cooperation Agreement between FAPESP and the Argentine National Council for Scientific and Technical Research (CONICET). In her laboratory, she temporarily altered how the EIF4A3 gene functioned in colonies of zebrafish, a fish increasingly used as a biological model to study human diseases (see the cover story in Pesquisa FAPESP Issue No. 209). “We use an approach that leads to a lower expression of the gene,” says Dr. Calcaterra. “We mimicked the syndrome by reducing the amount of messenger RNA (needed to produce the protein associated with EIF4A3) transcribed by the gene.”

X-rays of the spine, pelvis, legs, arm, forearm and hands: shortened and bent bones and prominent joints

Yamamoto et al. ajhg, 2014 X-rays of the spine, pelvis, legs, arm, forearm and hands: shortened and bent bones and prominent jointsYamamoto et al. ajhg, 2014

She then followed the development of the genetically engineered fish strains by using light microscopy and recording their general morphology. The altered zebrafish showed malformations in their craniofacial cartilage comparable to the syndrome reported in humans. To prove that the changes were indeed caused by the deficiency, Dr. Calcaterra injected the RNA of the EIF4A3 into the fish. The procedure is the equivalent of restoring the gene’s default behavior and allowing normal development of the zebrafish colonies.

Thus, the mutation identified by the USP center in the promoter region of gene EIF4A3 was proven to be the main culprit in causing the syndrome. The main culprit, but not the only one. Dr. Passos-Bueno’s team identified another type of mutation in the same gene in a patient with a clinically milder version of the syndrome (the jaw was formed normally, but the patient has some minor anatomical problems in the limbs and larynx). This second genetic alteration, also described in the scientific article, is different in nature from the previous, but seems to be sufficient to cause milder forms of the disease. Her finding supports the idea that how the EIF4A3 gene functions is key to the development of the rare syndrome.

Small and with limited vision
A genetic defect causes a form of dwarfism associated with progressive retinal problems

The gene responsible for a very rare form of dwarfism associated with progressive loss of vision, known as spondymetaphyseal dysplasia with cone-rod dystrophy, has been discovered by USP researchers. After sequencing all segments of the genome responsible for encoding the proteins of four Brazilian patients from two different families, the team led by Dr. Deborah Bertola, a geneticist at CEGH-CEL and also a physician at Hospital das Clinicas Children’s Institute, found two mutations in the PCYT1A gene, located on chromosome 3. The study’s findings were published in the January 2, 2014 issue of the American Journal of Human Genetics (AJHG).

In that same issue, a group at the prestigious Johns Hopkins University in the US authored another article, which also reported the identification of additional gene PCYT1A mutations, also capable of causing this type of dysplasia. US researchers used the same sequencing technique employed by Brazilian researchers and analyzed genetic material from three patients from different countries. “The two studies were done independently and concurrently. They are equivalent,” says Dr. Bertola. “We are proud. Our group used the same cutting-edge technology and achieved the same results as quickly as one of the most prestigious centers for the study of genetic diseases in the United States.”

Until now there had been less than 20 cases of the disease in the entire world described in the scientific literature. For this dysplasia to manifest itself clinically, the individual must carry mutations in both alleles (copies) of the PCYT1A gene. Some of those affected do not reach a height of more than one meter in adulthood. Dwarfism results from bone changes in the spine and lower limbs, which are very curved. Patients also have altered retinal cells (rods and cones that are part of the name of the disease) which progressively undermine their vision. There is no effective treatment to prevent the disease’s progression. Corrective orthopedic surgery can only be done in a palliative manner.

It was a surprise to discover the PCYT1A gene as the source of the mutations involved in this dysplasia, since it had never been associated with any genetic disease. “At first it was difficult to tie it directly to bone and retinal problems, since there had been no previous description of their involvement in human diseases of bone metabolism or retinal formation,” explains Guilherme Yamamoto, first author of the Brazilian study. The gene encodes an enzyme that acts metabolically in the formation of phosphatidylcholine, an important phospholipid for cellular membrane development. “Our work shows only that the gene is responsible for the disease. It remains to be seen how this happens,” says Dr. Bertola. “For us to know the actual mechanism of causality, studies of how this protein works should be conducted.”

1. Investigation of the role of oxidative stress ND the CNBP protein in treacle-deficient mesenchymal stem-cells and in zebrafish models (FAPESP-CONICET) (No. 2010/52446-4); Grant Mechanism Regular Line of Research Project Award; Principal investigator Maria Rita Passos-Bueno-USP; Investment R$12,708.67 (FAPESP).
2. Human Genome and Stem Cell Research Center (CEGH-CEL) (No. 13/08028-1); Grant Mechanism Research, Innovation and Dissemination Centers-RIDC; Principal investigator Mayana Zatz-USP; Investment R$2,266,005.51 and $940,000 per year for all RIDC (FAPESP).

Scientific article
FAVARO, F.P. et al. A noncoding expansion in EIF4A3 causes Richieri-Costa Pereira syndrome, a craniofacial disorder associated with limb defects. American Journal of Human Genetics. V. 94, No. 1, p. 120-8.
YAMAMOTO, G.L. et al. Mutations in PCYT1A Cause spondylometaphyseal dysplasia with cone-rod dystrophy. American Journal of Human Genetics. V. 94, No. 1, p. 113-9. January 2, 2014.