Microcephaly, brain calcification, skeletal alterations, auditory and visual deficits – these are examples of the damage that can be caused by Zika virus in babies born to mothers infected during pregnancy.

However, these problems are observed in only 6%-12% of cases, and scientists suspect other factors besides viral infection are associated with the development of congenital Zika syndrome.

A new study published on February 2 in the journal Nature Communications offers strong evidence that the higher susceptibility of some babies to the virus has a genetic origin.

“We studied pairs of discordant twins – cases in which only one twin was affected by the virus, although both had been equally exposed during pregnancy. In the laboratory, we found some 60 genes with differentiated expression in the neural progenitor cells of the affected babies,” said Mayana Zatz, a professor at the University of São Paulo’s Bioscience Institute (IB-USP) and a principal investigator for the research project.

Most of the investigation was conducted at the Human Genome & Stem Cell Research Centre (HUG-CELL), one of the Research, Innovation and Dissemination Centres (RIDCs) supported by São Paulo Research Foundation – FAPESP. HUG-CELL is hosted by the University of São Paulo (USP). Other participants included scientists affiliated with the National Energy & Materials Research Centre (CNPEM), the Federal University of São Paulo (UNIFESP), Butantan Institute, the Albert Einstein Jewish Hospital (HIAE) in São Paulo, USP’s Chemistry Institute, and the Federal Universities of Paraíba (UFPB), Rio Grande do Norte (UFRN) and Pernambuco (UFPE). Data collection was performed in 2016, mainly in states of the Northeast affected by the Zika epidemic in the previous year.

The researchers evaluated data for 91 babies born to infected mothers, including two pairs of monozygotic twins (identical twins that split from one egg and one sperm after fertilization) and seven pairs of dizygotic twins (fraternal twins from separate eggs and sperm).

The first piece of evidence in favour of the hypothesis that genetic background determines the development of congenital Zika syndrome was the finding that all the monozygotic twins were concordant: all the identical twins were equally affected by the virus, whereas six of the seven pairs of dizygotic twins were discordant – only one of each pair was affected.

From three pairs of dizygotic twins, the researchers obtained blood samples in sufficient quantities for more complex laboratory studies. Inspired by the induced pluripotent stem cell (iPSC) technique for which Shinya Yamanaka, a scientist affiliated with the University of Kyoto (Japan), was awarded the 2012 Nobel Prize in Medicine, the HUG-CELL team developed a method to convert blood cells into iPSCs, which like embryonic stem cells can differentiate into almost any type of human cell or tissue.

The next step was to convert the iPSCs into neural progenitor cells (NPCs), the type most affected by Zika in the developing brain. As soon as the NPCs from the affected babies and their unaffected siblings were established in vitro, all cultures were infected with a Brazilian strain of Zika virus.

“From the start, we were struck by the fact that the virus replicated much faster in the NPCs from the affected babies,” Zatz said. “In addition, these cells proliferated less and died more than cells from unaffected babies. We appear to have succeeded in reproducing in vitro what happens in vivo.”

Next, in collaboration with a team of researchers led by Sergio Verjovski-Almeida at IQ-USP, the scientists used sequencing techniques to analyze all the messenger RNA molecules expressed by the cultured NPCs, with the aim of discovering which genes were more active or less active in each case.

Differences were observed in two cell signaling pathways of key significance for brain development during the embryonic period – one mediated by the protein mTOR and the other by Wnt.

“These pathways regulate the proliferation and migration of central nervous system cells, among other things,” Zatz said. “The expression of several genes linked to these pathways was reduced in the NPCs from affected babies. One was 12.6 times less expressed in affected cells than the same gene in cells from the unaffected twin.”

The results were similar for all three pairs of twins studied, reinforcing the hypothesis that congenital Zika syndrome is not a random event but is favoured by genetic factors.

Complex inheritance

Prior to their work on NPCs, the researchers conducted a study of genetic material from 18 babies affected by congenital Zika syndrome (including five of the dizygotic twins evaluated in the later study) to investigate whether a single gene was involved in the increase in susceptibility. They also analyzed DNA from the five unaffected twins and from 609 control individuals not infected by Zika and without neurological alterations.

In this study, the researchers performed whole exome sequencing (WES) to look for disease-causing DNA variants in the genome’s exons, the pieces of genes that code for proteins.

“We wanted to see if any genetic variants were present only in the affected babies, but we found none. This rules out the hypothesis of a Mendelian inheritance [linked to a single gene] and suggests that we’re dealing with a complex inheritance [a combination of differentially expressed genes], like susceptibility to diabetes, for example,” Zatz said.

In individuals with a genetic profile that predisposes them to develop diabetes, the disease may manifest itself only if certain environmental factors are also present, such as an unbalanced diet and excess weight gain. In the case of congenital Zika syndrome, viral infection may be the environmental factor that triggers the development of the disease.

According to Zatz, in time the new discoveries could enable the identification of parents at risk of having children with this more vulnerable genetic profile. “If and when a vaccine against Zika is developed, these parents could be prioritized in immunization strategies,” she said.

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