Biology subjects

Louzada, S.

Publications and source records attributed to Louzada, S..

3 recordsLinked to original sources

bioRxiv2018

The malaria-protective human glycophorin structural variant DUP4 shows somatic mosaicism and association with hemoglobin levels

Glycophorin A and glycophorin B are red blood cell surface proteins that are both receptors for the parasite Plasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A - glycophorin B fusion gene, and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and reveal somatic variation in copy number of the glycophorin A-glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation, and show that it is associated with a malarial-related phenotype in a non-hospitalized population.\n\nSignificance statementPrevious work has identified a human complex genomic structural variant called DUP4, which includes two novel glycophorin A-glycophorin B fusion genes, is associated with a profound protection against severe malaria. In this study, we present data showing the molecular basis of this complex variant. We also show evidence of somatic variation in the copy number of the fusion genes. We develop a simple robust assay for this variant and demonstrate that DUP4 is at an appreciable population frequency in Tanzania and that it is associated with higher hemoglobin levels in a malaria-endemic village. We suggest that DUP4 is therefore protective against malarial anemia.

genetics↗

bioRxiv2018

Tandem duplications lead to loss of fitness effects in CRISPR-Cas9 data

CRISPR-Cas9 gene-editing is widely used to study gene function and is being advanced for therapeutic applications. Structural rearrangements are a ubiquitous feature of cancers and their impact on CRISPR-Cas9 gene-editing has not yet been systematically assessed. Utilising CRISPR-Cas9 knockout screens for 163 cancer cell lines, we demonstrate that targeting tandem amplified regions is highly detrimental to cellular fitness, in contrast to amplifications caused by chromosomal duplications which have little to no effect. Genomically clustered Cas9 double-strand DNA breaks are associated with a strong gene-independent decrease in cell fitness. We systematically identified collateral vulnerabilities in 25% of cancer cells, introduced by tandem amplifications of tissue non-expressed genes. Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing technology, and how resulting collateral vulnerabilities are a generalisable strategy to target cancer cells.

genomics↗

bioRxiv2017

Targeted gene correction of FKRP by CRISPR/Cas9 restores functional glycosylation of α-dystroglycan in cortical neurons derived from human induced pluripotent stem cells

Mutations in genes required for functional glycosylation of -dystroglycan cause a group of congenital muscular dystrophies associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic, physiology-relevant human cellular models has limited our understanding of the cortical abnormalities in dystroglycanopathies. Here we generate induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous mutations in the ribitol-5-phosphate transferase gene, FKRP. We carry out targeted gene correction in FKRP-iPSCs using CRISPR/Cas9-mediated genome editing. We characterise the directed differentiation of FKRP- and corrected-iPSCs to neural stem cells, cortical progenitors and cortical neurons. Importantly, we show that targeted gene correction of FKRP restores functional glycosylation of -dystroglycan in iPSC-derived cortical neurons. We independently validate this result by showing targeted gene mutation of FKRP disrupts functional glycosylation of -dystroglycan. This work demonstrates the feasibility of using CRISPR/Cas9-engineered human iPSCs for modelling dystroglycanopathies and provides a foundation for therapeutic development.\n\nHighlightsO_LIGeneration of FKRP-iPSCs for modelling cortical abnormalities in dystroglycanopathies\nC_LIO_LIPrecise gene correction by CRISPR/Cas9-mediated genome editing\nC_LIO_LIDirected differentiation of isogenic control and FKRP-iPSC to cortical neurons\nC_LIO_LIFunctional glycosylation of -dystroglycan is restored in cortical neurons derived from CRISPR/Cas9-corrected iPSCs\nC_LIO_LITargeted gene mutation of FKRP disrupts functional glycosylation of -dystroglycan in cortical neurons\nC_LI

molecular biology↗