<i>EFTUD2</i>deficiency in vertebrates: Identification of a novel human mutation and generation of a zebrafish model

BACKGROUND: Congenital microphthalmia and coloboma are severe developmental defects that are frequently associated with additional systemic anomalies and display a high level of genetic heterogeneity. METHODS: To identify the pathogenic variant in a patient with microphthalmia, coloboma, retinal dystrophy, microcephaly and other features, whole exome sequencing (WES) analysis of the patient and parental samples was undertaken. To further explore the identified variant/gene, expression and functional studies in zebrafish were performed. RESULTS: WES revealed a de novo variant, c.473_474delGA, p.(Arg158Lysfs*4), in EFTUD2 which encodes a component of the spliceosome complex. Dominant mutations in EFTUD2 cause Mandibulofacial Dysostosis, Guion-Almeida type (MFDGA) which does not involve microphthalmia, coloboma or retinal dystrophy; analysis of genes known to cause these ocular phenotypes identified several variants of unknown significance but no causal alleles in the affected patient. Zebrafish eftud2 demonstrated high sequence conservation with the human gene and broad embryonic expression. TALEN-mediated disruption was employed to generate a c.378_385 del, p.(Ser127Aspfs*23) truncation mutation in eftud2. Homozygous mutants displayed a reduced head size, small eye, curved body, and early embryonic lethality. Apoptosis assays demonstrated a striking increase in TUNEL-positive cells in the developing brain, eye, spinal cord and other tissues starting at 30 hours post fertilization. CONCLUSION: This study reports a novel mutation in EFTUD2 in an MFDGA patient with unusual ocular features and the generation of a first animal model of eftud2 deficiency. The severe embryonic phenotype observed in eftud2 mutants indicates an important conserved role during development of diverse tissues in vertebrates

Mutations in <i>MAB21L2</i> Result in Ocular Coloboma, Microcornea and Cataracts

<div><p>Ocular coloboma results from abnormal embryonic development and is often associated with additional ocular and systemic features. Coloboma is a highly heterogeneous disorder with many cases remaining unexplained. Whole exome sequencing from two cousins affected with dominant coloboma with microcornea, cataracts, and skeletal dysplasia identified a novel heterozygous allele in <i>MAB21L2</i>, c.151 C>G, p.(Arg51Gly); the mutation was present in all five family members with the disease and appeared de novo in the first affected generation of the three-generational pedigree. <i>MAB21L2</i> encodes a protein similar to <i>C. elegans</i> mab-21 cell fate-determining factor; the molecular function of MAB21L2 is largely unknown. To further evaluate the role of <i>MAB21L2</i>, zebrafish mutants carrying a p.(Gln48Serfs*5) frameshift truncation (<i>mab21l2^Q48Sfs*5</i>) and a p.(Arg51_Phe52del) in-frame deletion (<i>mab21l2^R51_F52del</i>) were developed with TALEN technology. Homozygous zebrafish embryos from both lines developed variable lens and coloboma phenotypes: <i>mab21l2^Q48Sfs*5</i> embryos demonstrated severe lens and retinal defects with complete lethality while <i>mab21l2^R51_F52del</i> mutants displayed a milder lens phenotype and severe coloboma with a small number of fish surviving to adulthood. Protein studies showed decreased stability for the human p.(Arg51Gly) and zebrafish p.(Arg51_Phe52del) mutant proteins and predicted a complete loss-of-function for the zebrafish p.(Gln48Serfs*5) frameshift truncation. Additionally, in contrast to wild-type human <i>MAB21L2</i> transcript, mutant p.(Arg51Gly) mRNA failed to efficiently rescue the ocular phenotype when injected into <i>mab21l2^Q48Sfs*5</i> embryos, suggesting this allele is functionally deficient. Histology, immunohistochemistry, and in situ hybridization experiments identified retinal invagination defects, an increase in cell death, abnormal proliferation patterns, and altered expression of several ocular markers in the <i>mab21l2</i> mutants. These findings support the identification of <i>MAB21L2</i> as a novel factor involved in human coloboma and highlight the power of genome editing manipulation in model organisms for analysis of the effects of whole exome variation in humans.</p></div

Summary of TUNEL assays in zebrafish wild-type and <i>mab21l2</i> mutant embryos.

<p>TUNEL results in 24-hpf wild-type (A-B), <i>mab21l2</i>^{<i>Q48Sfs*5</i>} embryos (C-D) and <i>mab21l2</i>^{<i>R51_F52del</i>} mutants (E-F) are shown. An increase in TUNEL staining was observed in both <i>mab21l2</i> mutants with remarkably high levels in the <i>mab21l2</i>^{<i>Q48Sfs*5</i>} embryos, particularly in the lens and ventral retina (C-D), and moderately increased levels in the <i>mab21l2</i>^{<i>R51_F52del</i>} embryos (E-F); arrowheads indicate sites of increased TUNEL staining in the eye and brain; le, lens; r, retina.</p

Expression and mutations of zebrafish <i>mab21l2</i>.

<p><b>A-K.</b> Expression pattern of <i>mab21l2</i> in zebrafish 18–72-hpf embryos. Whole mount images (A-H) and sections (I-K) are shown. <b>A, B.</b> At 18-hpf, expression in the presumptive eye field (e) and midbrain (m) is observed. <b>C, D, I.</b> At 24-hpf, <i>mab21l2</i> expression is seen in the periphery of the retina (r), lens (le), spinal cord (sc), midbrain (m) and pharyngeal arch region (pa). <b>E-H, J, K.</b> At 48–72-hpf, expression in the ciliary marginal zone (cmz), inner nuclear layer (inl) and ganglion cell layer (gcl) of the retina, and the region of the optic fissure (of) in the eye as well as the midbrain (m), hindbrain (h), developing fins (f), and branchial arches (ba) is shown with arrows. <b>L.</b> Distribution and expression of <i>mab21l2</i> mutations induced by TALENs. On the left, a schematic of the zebrafish mab21l2 protein is shown as a light grey box with the mab-21 domain (amino acids 62–346) indicated in dark grey color; the positions of the zebrafish mutations identified in the progeny of TALEN-injected fish are shown at the top of the box and the position of the human mutation identified in Patient 1 is indicated at the bottom; the positions of the p.(Gln48Serfs*5) and p.(Arg51_Phe52del) mutations are shown with red arrows. On the right, a graph summarizing results of semi-quantitative RT-PCR analysis of wild-type and mutant <i>mab21l2</i> transcript levels in 48-hpf homozygous embryos is shown.</p

Analysis of <i>pax6b</i>, <i>pax2</i>.<i>1</i> and <i>foxe3</i> expression in wild-type, <i>mab21l2</i>^{<i>Q48Sfs*5</i>} and <i>mab21l2</i>^{<i>R51_F52del</i>} embryos.

<p>Wild-type (A-E) and mutant (F-O) zebrafish embryos at 24–48-hpf were analyzed as indicated in the right bottom corner of each image. Please note a change in <i>pax6b</i> transcript distribution at 24-hpf and 48-hpf in <i>mab21l2</i>^{<i>Q48Sfs*5</i>} (arrowheads in F-H), retinal folding defect in <i>mab21l2</i>^{<i>R51_F52del</i>} embryos at 24-hpf (arrowheads in K, L) and visibly abnormal <i>pax6b</i> pattern at 48-hpf in some (arrowheads in M) but not all (N) <i>mab21l2</i>^{<i>R51_F52del</i>} embryos. <i>pax2</i>.<i>1</i> expression seems to be unaffected in 24-hpf frameshift mutant embryos (I) but shows an abnormal pattern in both mutants at 48-hpf (J,O). At 48-hpf, in addition to more broad and intense <i>pax2</i>.<i>1</i> expression in the region of optic fissure, abnormal <i>pax2</i>.<i>1</i> staining was detected in central retina in <i>mab21l2</i>^{<i>Q48Sfs*5</i>} embryos (arrowheads in J). le, lens; of, optic fissure; retina.</p

<i>MAB21L2</i> mutations and protein sequence conservation.

<p><b>A.</b> Three-generation pedigree of Patient 1 with <i>MAB21L2</i> genotype information. DNA chromatograms for all tested family members are shown with c.151C position indicated with black (WT allele) or red (heterozygous mutant allele) arrows. The proband (Patient 1) is indicated with a black arrowhead. Please note the presence of the mutant allele in all affected individuals, its absence in unaffected family members, and the presence of a low ‘G’ peak in addition to the normal ‘C’ nucleotide at the mutant position in the proband’s unaffected mother. <b>B.</b> Amino acid alignment of the MAB21L1-3 and mab-21 regions surrounding the arginine at position 51; amino acids identical between different homologs are highlighted with a light grey color, three invariant residues are shown in dark grey; the glycine (G) predicted to replace arginine 51 in Patient 1 is shown in red font; the positions and predicted effects of the zebrafish <i>mab21l2</i> mutations involving the same region are also shown in red font. Accession numbers for sequences utilized in the alignment are provided in Methods.</p