SPLIT HAND/FOOT VARIANTS FAIL TO RESCUE PRDM1A MUTANT CRANIOFACIAL DEFECTS
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Abstract
Background
Split Hand/Foot Malformation (SHFM) is a congenital limb disorder presenting with limb anomalies, such as missing, hypoplastic, or fused digits, and often craniofacial defects, including a cleft lip/palate, microdontia, micrognathia, or maxillary hypoplasia. We previously identified three novel variants in the transcription factor, PRDM1 , that are associated with SHFM phenotypes. One individual also presented with a high arch palate. Studies in vertebrates indicate that PRDM1 is important for development of the skull; however, prior to our study, human variants in PRDM1 had not been associated with craniofacial anomalies.
Methods
Using transient mRNA overexpression assays in prdm1a -/- mutant zebrafish, we tested whether the PRDM1 SHFM variants were functional and could lead to a rescue of the craniofacial defects observed in prdm1a -/- mutants. We also mined a CUT&RUN and RNA-seq dataset to examine Prdm1a binding and the effect of Prdm1a loss on craniofacial genes.
Results
prdm1a -/- mutants exhibit craniofacial defects including a hypoplastic neurocranium, a loss of posterior ceratobranchial arches, a shorter palatoquadrate, and an inverted ceratohyal. Injection of wildtype hPRDM1 in prdm1a -/- mutants partially rescues these structures. However, injection of each of the three SHFM variants fails to rescue the skeletal defects. Loss of prdm1a leads to a decreased expression of important craniofacial genes, such as dlx5a/dlx6a, hand2, sox9b, col2a1a , and hoxb genes.
Conclusion
These data suggest that the three SHFM variants are not functional and may have led to the craniofacial defects observed in the humans. Finally, they demonstrate how Prdm1a can directly bind and regulate craniofacial gene expression.
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Of the three variants we tested, two are conserved and one was not between zebrafish and humans. The one what was not was in a conserved domain however. Yes we tried to generate mutants but we could not recover the precise single nucleotide mutation in the germ line. It is possible that we did not screen enough lines or that crispr method we used was not the efficient enough. We hope to make these in the future.
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Thank you for your comments. This approach is used a lot in zebrafish but I not as much in other model systems. Yes it could be used as a diagnostic in the future.
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This study uses prdm1a -/- mutant zebrafish to evaluate the function of human PRDM1 variants within patients presenting craniofacial defects. By transiently expressing hPRDM1 variants and comparing their rescue potential to wildtype hPRDM1, the authors demonstrate that the variants have reduced capacity for rescue of craniofacial defects.
It would be really interesting to see how this kind of rare disease allele functional assessment in model organisms could be scaled to improve diagnosis of rare diseases. It's great that the authors performed these experiments and reported this result, which seems to stem from a previous study on related alleles.
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Together, these data suggest that the SHFM PRDM1 variants are not functional and may lead to craniofacial defects.
This is a really cool outcome suggesting that you could determine how human variants from individual patients contribute to disease etiology.
How common is it to explore clinical variants using mutant rescue experiments in model organisms? Would it be possible to use this kind of strategy as a diagnostic approach for rare diseases?
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This was expected given that overexpression of zebrafish prdm1a mRNA sufficiently rescues NCCs in mutants (Hernandez-Lagunas et al., 2005)
How different in sequence are the zebrafish and human versions of prdm1a?
Would it be possible to generate mutant versions of the zebrafish gene based on specific mutations found in the human variants and examine their capacity for rescue?
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