1. De Novo Mutations of CCNK Cause a Syndromic Neurodevelopmental Disorder with Distinctive Facial Dysmorphism 
Yanjie Fan, Wu Yin, Bing Hu, Antonie D. Kline, Victor Wei Zhang, Desheng Liang, Yu Sun, Lili Wang, Sha Tang, Zöe Powis, Lei Li, Huifang Yan, Zhen Shi, Xiaoping Yang, Yinyin Chen, Jingmin Wang, Yuwu Jiang, Hu Tan, Xuefan Gu, Lingqian Wu, Yongguo Yu 
The American Journal of Human Genetics 
Volume 103, Issue 3, Pages (September 2018)
DOI: /j.ajhg
Copyright © 2018 American Society of Human Genetics Terms and Conditions

2. Figure 1 Facial Dysmorphism of Four Subjects
Front and side views of subject 1 (at age 4 years), subject 2 (at age 9 years), subject 3 (at age 8 years), and subject 4 (at age 4 years). Common facial dysmporphism includes high hairline, hypertelorism, thin eyebrows, abnormal ears, broad nasal bridge and tip, thin upper vermilion, and narrow jaw.
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2018 American Society of Human Genetics Terms and Conditions

3. Figure 2 De Novo CCNK Variants Identified in Four Subjects
(A) Chromosomal positions and gene contents of deletions identified in subject 1, 2, and 3 and Decipher cases (#291402, #260834, #296462, #254518, #296477) with the minimal overlapping regions highlighted.
(B) Conservation of amino acid sequence flanking the p.Lys111Glu variant detected in subject 4.
(C) Protein structural modeling of CDK12/CCNK and p.Lys111Glu variant by pyMol (using a 2.2 Å resolution crystal structure, PDB: 4un0).
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2018 American Society of Human Genetics Terms and Conditions

4. Figure 3
CCNK mRNA Level in Lymphocytes of Affected Subjects and the Morpholino Knockdown Approach of Zebrafish Ccnk
(A and B) The relative quantification of CCNK RNA level based on quantitative PCR of reverse-transcribed mRNA (using PrimeScript RT reagent Kit from Takara and SYBR green master PCR mix from Applied Biosystems; primers were described in Table S6). As the available subject samples were in different forms, control samples from age-matched individuals were prepared accordingly. The mRNA sample of subject 1 was extracted from uncultured lymphocytes (n = 3, control n = 12); mRNA of subject 2 was extracted from cultured lymphocytes (n = 3). Error bars: SEM, ∗p < 0.001, t test.
(C) The predicted human CCNK and zebrafish Ccnk protein structure. Pink horizontal bars: the low-complexity motifs (proline-rich domains); triangles: cyclin box; dashed lines: cyclin_C domains (the regulatory region to activate the CDK).
(D and E) The knockdown specificity and efficiency of the ccnk MO1 and MO2 were analyzed by western blot. The band density was quantified with ImageJ software and Gapdh protein was used for normalization. The relative level of wild-type (WT) was normalized to 1. Embryos that received injections of the ccnk MO1 or MO2 (4 ng per embryo) showed reduction of Ccnk expression (WT: ± 0.007; MO1: ± 0.049, at ∗∗∗p < 0.001; MO2: ± 0.021, at ∗∗∗p < 0.001; 5MIS: ± 0.001; n = 3 batches, with 30 embryos per batch). Antibodies used: rabbit anti-CCNK, 1:1,000, ab130475, Abcam; mouse anti- GAPDH, 1:1,000, Proteintech.
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2018 American Society of Human Genetics Terms and Conditions

5. Figure 4 Knockdown of Ccnk Caused Developmental Defects in Zebrafish
(A) The gross morphology of the 3 dpf morphants. The morphology of the uninjected (WT) and control MO-injected embryos (5MIS group, 4 ng per embryo) appeared largely normal, whereas the embryos injected with ccnk MO1 or ccnk MO2 showed growth defects, including unconsumed yolk sac, small eyes, deformed head, and curly spinal cord. The abnormal phenotypes were partially rescued by co-injections of zebrafish ccnk mRNA (RES) or human CCNK mRNA (hRES) (200 pg per embryo) but were not rescued by human CCNK mutant mRNA with c.331A>G (hMUT).
(B and C) The phenotypic classification of morphants, with 4 ng (B) and 2 ng (C) dosage of MO injection. The 3-dpf injected embryos were classified by morphological criteria into five categories (normal, mild, moderate, severe, and dead, as indicated below the chart), and their proportions were shown in the stacked-bar graphs (the number of embryos used was labeled under each group name, pooled from three different batches). ∗∗∗p < 0.001, ns: p > 0.05, Chi-square test.
(D) Ventral views of 5 dpf whole-mount Alcian-blue staining embryos at two different MO doses, 4 ng and 2 ng. The cartilage staining method was as previously described.11 The cartilage structures were visualized and the distance between ceratohyal cartilage (ch) and Meckel’s cartilage (Mk) were measured by ImageJ software (red lines).
(E) The distance between ch and Mk cartilages with 4 ng and 2 ng MO injection. While the mean distance in wild-type was 258 μm (SEM: 1.8 μm), the MO1 4 ng group was significantly shorter, 146 μm (SEM: 4.4 μm). If compared with the 2 ng MO1 group (mean distance 173 μm, SEM: 4.2 μm), the 4 ng MO1 group was approximately 27 μm shorter with statistical difference (n = 30 embryos for each group, ∗∗∗p < 0.001, ∗p < 0.05, ns: p > 0.05, t test).
(F and G) Knockdown of Ccnk affects primary motor neuronal development. The primary motor neuronal large-caliber Mauthner axons were analyzed in Tg(tol-056:GFP) zebrafish line. The branches of the Mauthner axons disappeared after Ccnk knockdown (n = 6 embryos, ∗∗∗p < 0.001, t test). The dashed line labels spinal cord, and the anal pore site was marked by an asterisk (*).
(H and I) Knockdown of Ccnk induced neural cell death. The intense fluorescent apoptotic foci in the head were detected by TUNEL assay of 3 dpf embryos (n = 10 embryos per group, ∗∗∗p < 0.001). Dashed lines: eye region.
Scale bar: 200 μm (A, B, and D), 50 μm (F and H).
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2018 American Society of Human Genetics Terms and Conditions