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Zika susceptibility linked to a baby's genetics
Three pairs of Brazilian twin infants with different birth outcomes have revealed their genetic clues of a distinct susceptibility to the Zika virus.
Neural progenitor cells (NPC) derived from congenital Zika syndrome (CZS) in affected and non-affected twins, reveals there are different rates of viral infection and different RNA expressions from genes associated with neural development. This imbalance existed even before infection by the Zika virus began. So, the research results confirm a hypothesis that a genetic or epigenetic influence increases susceptibility to CZS and microcephaly.
Findings are published in Nature Communications on Friday, 2 February 2018.
Research led by Mayana Zatz and Maria Rita Passos-Bueno - geneticists from the Human Genome and Stem Cell Research Center from the University of São Paulo (USP) - conclude that a single gene cannot explain the many cases of CZS development, nor why some brains resist Zika. But, about 6% to 12% of babies born from mothers infected with the Zika virus during pregnancy will have CZS. Why not all? This newest study sheds light on genetic components associated with the phenomenon.
"If the baby has these genetic susceptibility factors, we believe it will not have microcephaly unless it is infected by the Zika virus. Maybe we can identify these people and prioritize them in a future vaccine strategy."
Twins as a clue to genetic studies
Twins provide unique information to answer whether a condition has an environmental or a genetic cause. If genetic factors determine a congenital disease, there must be more agreement between identical (monozygotic) twins than between the non-identical or dizygotic twins. Identical siblings more often will both be affected, but not dizygotic twins who will show more "discordance" (one affected and one unaffected).
If environment leads to the condition, the pattern observed in two non-identically genetic siblings will be the same as observed in identical twins. Meaning either none of the siblings will be affected, or both, or just one of them, independently of being monozygotic or dizygotic twins.
The study began in 2016, during Zika epidemics in Brazil. Mayana Zatz and her team looked for twins in which one baby had microcephaly. They were able to get in touch with families from 9 pairs of twins, 6 sets from Brazilian states.
Two pairs were identical twins, with both showing microcephaly from ZIKA inffection; one pair was non-identical (dizygotic or discordant twins) but both also having ZIKA microcephaly; and 6 pairs were dizygotic and discordant - one affected twin and one unaffected twin. These results contribute to the hypothesis of genetic influence on Zika infection during fetal development.
Of the three pairs of non-identical, discordant, twins — blood samples collected were collected to generate human induced pluripotent stem cells (hiPSC). hiPSC cells that can induced to make almost any boy cell type. The hiPSC cells were transformed into neural progenitor cells (NPC) that form brain and other cells of the central nervous system.
These NPC cells were then infected with a Brazilian Zika virus strain. After four days, cells derived from affected babies had significanlty fewer cells than from non-affected ones. Analysis of neurospheres - cells grown in 3D structures - showed similar growth impairment from the affected twins' cells. Non-infected cultures from affected and non-affected derived NPCs used as a control, presented no differences after the same amount of time.
Using a fluorescence technique, researchers observed a larger amount of Zika virus in cells derived from affected babies compared to the cells from the healthy ones. More experiments pointed that the neural progenitor cells from babies with the syndrome produced significantly more viral RNA copies and more viral unities capable of infection. In short, they were more infected and did not proliferate as much as the cells from their protected siblings.
"In the laboratory, the cells mimicked what happened with the babies. Also, the results were the same for the three pairs of twins. We demonstrated that the infection is not random. It reinforces our hypothesis of a genetic component increasing the susceptibility to congenital Zika syndrome."
To identify this possible genetic component, the team analyzed all gene sequences from 8 pairs of twins and 10 non-twin babies who developed the Zika syndrome, and compared these results to normal controls. The exome analysis did not identify any one gene variation capable of determining susceptibility to ZIKA - instead, it excluded this possibility.
Later RNA sequencing allowed scientists to measure gene expression - meaning, which genes are silenced or which genes are active and sending messages to produce proteins in the cell. This was different from previous DNA sequencing which showed which genes are in existence, but not if or how they are expressed.
The RNA test from the 6 twins indicated a group of genes capable of distinguishing more susceptible cells from more resistant ones. The largest alteration occurred with the DDIT4L gene, 12.6 times less expressed in affected cells. Its related protein is an inhibitor of mTor signaling - a pathway involved in cell growth and cell death. Previous studies associated the mTor pathway and Zika virus replication.
The RNA sequencing also detected lower expressions of FOXG1 and LHX2 genes in the cells from babies with CZS. These genes participate in the brain regionalization process - characterized by development of cerebral areas during fetus growth. FOXG1 has also been linked to congenital brain disorders. The LHX2 gene, 9,6 times less present in affected cells, is responsible for attenuating the Wnt signaling, engaged in neural differentiation.
In conclusion, the results indicate that the development of Zika in babies is not random — it depends on the susceptibility of that particular infant's neural progenitor cells. It is possibly related to variants in several genes or mechanisms controlling gene expression and protein production.
Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.
Authors: Luiz Carlos Caires-Júnior, Ernesto Goulart, Uirá Souto Melo, Bruno Silva Henrique Araujo, Lucas Alvizi, Alessandra Soares-Schanoski, Danyllo Felipe de Oliveira, Gerson Shigeru Kobayashi, Karina Griesi-Oliveira, Camila Manso Musso, Murilo Sena Amaral, Lucas Ferreira daSilva, Renato Mancini Astray, Sandra Fernanda Suárez-Patiño, Daniella Cristina Ventini, Sérgio Gomes da Silva, Guilherme Lopes Yamamoto, Suzana Ezquina, Michel Satya Naslavsky, Kayque Alves Telles-Silva, Karina Weinmann, Vanessa van der Linden, Helio van der Linden, João Mendes Ricardo de Oliveira, Nivia Rodrigues Maria Arrais, Adriana Melo, Thalita Figueiredo, Silvana Santos, Joanna Castro Goes Meira, Saulo Duarte Passos, Roque Pacheco de Almeida, Ana Jovina Barreto Bispo, Esper Abrão Cavalheiro, Jorge Kalil, Edécio Cunha-Neto, Helder Nakaya, Robert Andreata-Santos, Luis Carlos de Souza Ferreira, Sergio Verjovski-Almeida, Paulo Lee Ho, Maria Rita Passos-Bueno and Mayana Zatz
This study was supported by grants from FAPESP (CEPID number 2013/08028-1 and INCT number 14/50931-3 to M.Z. and Thematic grant number 2014/03620-2 to S.V.-A.) and CNPq (INCT number 465355/2014-5 to M.Z.); Luiz Carlos de Caires Júnior is a fellow of FAPESP (2017/16283-2); Ernesto Goulart is a fellow of FAPESP (2015/14821-1); Bruno Araujo is a fellow of FAPESP (2014/08049-1); Vanessa van der Linden Mota is supported by AACD (Recife – PE); João Ricardo de Oliveira is supported by CNPq (440770/2016-5). We are extremely grateful to Dr. S. Raia, D. Bertola, M. Vibranoski, P. Cunha, E.L. Durigon, W. Cumpady, P. Zanotto, C.R.S. Maia, A.N. Melo, M.T.A.L. Bezerra, D.A.M. de Araújo, A. Tanuri, R.S. de Aguiar, and T.R. Gollop for scientific discussions and sample collections. L. Suzuki and E. Amaro-Júnior for helping with CT analysis. N. Lourenço, and M. Aguena for technical support in exome sequencing and MLPA assay.
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Computed tomography scans from two pairs of twins participant in the study show typical abnormalities (second and forth images) associated with congenital Zika syndrome and microcephaly.
Image credit: HUG-Cell