SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant

Abstract

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions.

Figure 1

Positions of the 8q24.3 Deletions and the Corresponding Photographs of Affected Individuals (A) Schematic representation of chromosome 8 showing the breakpoints of the 8q24.3 deletions. The minimal deletion (individual 1) includes SCRIB, PUF60, and NRBP2. (B) Photographs of individuals 1–5 capture the core facial gestalt, including microcephaly, bitemporal narrowing, wide nasal bridge, anteverted nares, long and flat philtrum, and thin upper lip.

Figure 2

Suppression of scrib and puf60 in Zebrafish Leads to Short Stature, Small Head Size, and Craniofacial Defects (A) Quantification of total body length was performed in embryo batches injected with sham (control), scrib MO, puf60a MO, and double MOs. Bars represent the mean length of 80 embryos at 3 dpf per condition, which were scored blind to injection cocktail. (B) Lateral and dorsal views of representative control embryos and embryos injected with scrib or puf60a MO at 5 dpf; yellow line indicates the distance across the convex tips of the eye cups. Right panel, ventral views of corresponding embryos stained with Alcian blue at 5 dpf to visualize cartilage structures; yellow line indicates distance between ceratohyal (CH) and Meckel’s cartilages (MK). (C and D) Quantification of head size (C) and craniofacial defects (D) was performed in control and embryo batches injected with scrib or puf60a MO by measuring distances as shown by the yellow lines in (B). Head size measurements are represented as a normal probability distribution curve in which the y axis represents the probability that the values of x fall within a certain interval. (C) Significant differences were observed for the microcephaly phenotype; p < 0.0001 between control and puf60a morphants and p < 0.0001 between control and scrib morphants (three independent experiments; two-tailed t test comparisons). (A and D) Data are shown as the standard error of the mean, SEM from three independent experiments, n = 80 embryos. The corresponding p values are denoted on the bar graphs (two-tailed t test comparisons).

Figure 3

Suppression of scrib or puf60 in Zebrafish Lead to Unique Phenotypes (A) Lateral views of representative control (sham-injected) embryos and those injected with scrib MO at 3 dpf. Coloboma was detected in scrib morphants (black arrow) but was absent in puf60a morphants. (A′) Corresponding quantification of the coloboma phenotype in control, scrib, and puf60a morphants. There is no statistical difference between scrib morphants and the double morphants; ∼50% of the scrib and double morphants have coloboma. (B) Lateral views of representative control embryos and those injected with scrib MO at 5 dpf. 50 kDa FITC-labeled dextran was injected in the cardiac venous sinus at 2 dpf followed by imaging. At 5 dpf Scrib morphants have pericardiac/yolk edema and absence of dextran in the intersegmental vessels compared to control. (B′) Qualitative scoring of the glomerular filtration was performed in control and embryo batches injected with scrib MO or puf60a MO (n = 25, Dextran-injected embryos at 2 dpf followed by scoring at 5 dpf). (C) Lateral views of representative control embryos and those injected with puf60a MO at 3 dpf. (C′) Qualitative scoring of heart edema was performed in embryo batches injected with scrib, puf60a, and double MOs (80 embryos per injection cocktail).

Figure 4

Puf60 p.His169Tyr Is Nonfunctional in Splicing (A) Immunoblot analysis of PUF60 in lysates from cells transfected with a nonspecific control siRNA (siC) or a siRNA specific for PUF60. Cells were cotransfected with an empty expression plasmid (C) or a plasmid expressing wild-type PUF60 (PUF) or PUF60 p.His169Tyr mutant (PUFm). Both PUF60 cDNA expression plasmids have silent mutations introduced into the siPUF target region to render them insensitive to siRNA silencing. Blots were probed with an Alexa 594-tagged secondary antibody and quantitated with a Typhoon phosphorimager (right). Error bars represent SEM (n = 4); ^∗p < 0.05 (two-tailed, paired Student’s t test). Blots were subsequently probed with an HRP-labeled secondary antibody (left). β-actin is included as a loading control. Asterisk indicates an SDS-resistant PUF60 dimer. (B) Representative images of radiolabelled RT-PCR analysis of SMN2, BIN1, and APP alternatively spliced RNA isolated from cells treated as described in (A). (C) Quantitation of alternative splicing shown in (B) by a Typhoon phosphorimager. Error bars represent standard error (n = 6). Asterisk (^∗) indicates statistically different from siPUF rescued with PUF60, and hatch sign (#) indicates statistically different from equivalently treated siC (ANOVA with Tukey post-test).

Figure 5

In Vivo Data: Individuals’ RNA and Zebrafish (A) Deregulation of PUF60-dependent alternative splicing in affected individuals. Quantitation of RT-PCR analysis of alternatively spliced mRNA isoforms from SMN2, PUF60, BIN1, and APP from relatives (n = 6) or proband (n = 4) with a mutation or deletion of PUF60. (B) Quantification of the total body length was performed in embryo batches injected with puf60a MO alone or puf60a MO along with either human wild-type (WT) or mutant PUF60 (p.His169Tyr) messages. The p.His169Tyr mutation was introduced in the long and short isoforms. Bars represent the average length of 80 embryos at 3 dpf, which were scored blind to injection cocktail. Injection of long WT and mutant mRNAs rescued the body length phenotype observed in puf60a morphants (p < 0.0001; two-tailed t test comparisons). The short mutant PUF60 mRNA failed to rescue the phenotype and showed no significant difference with the embryos injected with the puf60a MO alone. Data are shown as the mean ± SEM (three independent experiments). Hatch sign (#) indicates nonsignificant.