Mapping of the Breakpoints in Two Balanced Translocations
(A and B) Disruption of PHF21A by two translocations. A schematic diagram of the DGAP012 breakpoints (not to scale) shows, in blue, PHF21A on chromosome 11 (exons 1–18) and, in white, either (A) ELAVL1 on chromosome 19 (exons 1–6) or (B) chromosome 1. All exons are depicted as vertical black boxes, and the start and direction of transcription are indicated by an arrow above the corresponding exons. The translocation occurred at the site of the red vertical line. In DGAP012, the translocation produced der(11) and der(19) chromosomes that encode potential fusion transcripts (A). Note that the stop codon of the potential PHF21AΔex15-18/ELAVL1Δex1-5 fusion gene is at the same location as wild-type ELAVL1 because there is no frameshift, whereas ELAVL1Δex6/ PHF21AΔex1-14 has a frameshift with a premature stop codon in the new exon 6 (equivalent to exon 15 of PHF21A). The translocation in MCN1762 does not predict any potential fusion product because the breakpoint on chromosome 1 is located in a gene desert.
(C and D) Genomic DNA sequence from the normal chromosomes and at the breakpoints on derivative chromosomes. In DGAP012, at the der(19) breakpoint, there was a 5 nt CTCCT deletion of chromosome 11 sequence and a 5 nt TTCAG deletion of chromosome 19 sequence, whereas at the der(11) breakpoint, there was no loss or gain of sequence (C). In MCN1762, the junction sequences revealed a 3 bp TTA deletion of chromosome 1 sequence on der(11) and a 2 bp TT deletion of chromosome 11 sequence and an 8 bp CTCCAAAT insertion on der(1). Details of the mapping of both breakpoints in DGAP012 and MCN1762 are described in Figure S1 as well as in the Subjects and Methods section.
(E) Immunoblot analysis of PHF21A levels in DGAP012 and MCN1762 and in controls. The PHF21A antibody against an N-terminal 93 residue polypeptide 28 recognizes the ∼92 kDa PHF21A in both female (“F”) and male (“M”) controls, as well as in DGAP012 and MCN1762 lymphoblastoid cell line extracts. It shows notably reduced protein levels due to disruption of PHF21A (arrow) in both DGAP012 and MCN1762. A 73 kDa protein (arrowhead) was noted in DGAP012 and is likely to be a product of the PHF21AΔex15-18/ELAVL1Δex1-5 fusion gene, which deletes the critical plant homeodomain (PHD) finger domain. α-tubulin was used as an internal loading control. The bar graph shows the mean and ± standard deviations from three independent experiments (^∗p < 0.001, ^∗∗p < 0.0001). The subcellular localization of PHF21A with the same antibody is described in Figure S3.
(F) PHF21A functional domains in wild-type and theoretical truncated proteins in two balanced translocation subjects. PHF21A contains two leucine zipper domains (LZD1 and LZD2), one AT-hook domain, and one PHD zinc finger domain. The amino acid positions of all domains are indicated as numbers below the domain structures. Note that if any protein were produced from the truncated PHF21A of DGAP012 or MCN1762, it would lack the PHD finger domain essential for binding H3K4me0. 14

Human Craniofacial Anomalies and Murine Phf21a Expression in Craniofacial and Brain Regions
(A–E) Human craniofacial anomalies with PHF21A truncation and PHF21A deletion.
(A–C) Female subject MCN1762 with balanced translocation t(1;11) exhibits ID and CFAs that include brachycephaly, microcephaly, a long narrow nose, mild midfacial hypoplasia, thin lips, and prominent ear lobes.
(D) The PSS male proband (PSS-Romeike) with a deletion including PHF21A is shown at the age of 31 years. He shows microcephaly, brachycephaly, a broad forehead, a long narrow nose, a hypoplastic mandible, very thin lips, hypotelorism, and dysplastic low set ears, in addition to ID and no speech, among other phenotypes.
(E) A topogram of a cerebral CT of individual PSS-Romeike at the age of 33 years, shortly before he died, shows a hypoplastic mandible and the parietal foramen at the back of the head.
(F–J) Phf21a expression during murine craniofacial development.
(F and G) Expression of Phf21a at embryonic days (E) 13.5 and 14.5. Highest transcript abundance is seen in the developing CNS with [³⁵S]-UTP labeled in situ probes. The following abbreviations are used: rom, roof of midbrain; rnc, roof of neopallial cortex; sc, spinal cord; and cp, intraventricular portion of cerebellar primordiu.
(H and I) At E17.5, prominent signals were detected with Phf21a antisense probes in bones of the facial skeleton. The palatine bone is marked by arrows, and the orbitosphenoidal bone is marked by a black arrowhead. Signals in the intestine were also detected with the sense probes and are most likely not specific (these are marked by asterisks). The following abbreviation is used: cal, calvaria.
(J) Sagittal sections of the adult mouse brain showed expression of Phf21a within the hippocampal formation, the cerebellum, and the olfactory bulb. Three different Phf21a antisense probes gave consistent results. The following abbreviations are used: mob, main olfactory bulb; hp, hippocampus; and gcl, granule cell layer of the cerebellum.

SCN3A Transcriptional Derepression and Reduced LSD1 Promoter Occupancy in DGAP012 and MCN1762 Lymphoblastoid Cells
SCN3A transcription is derepressed in affected individuals DGAP012 and MCN1762. Semiquantitative RT-PCR reactions were carried out in separate experiments for comparing the levels of SCN3A transcript (top box) in normal male and female controls with those of DGAP012 (A) and MCN1762 (B). Beta-actin (lower box) was used as a control in both experiments.
(C) LSD1 occupancy at the SCN3A promoter was compared in separate experiments between a normal male (#1 in left panel) and DGAP012 and between a normal female (#1 in right panel) and MCN1762 with the use of a ChIP assay. Five sets of PCR primers spanning the SCN3A promoter were used. These assays revealed reduced LSD1 occupancy at the SCN3A promoter in DGAP012 and MCN1762. The two panels cannot be directly compared because the two sets of assays were carried out at different times with different reagents (Abcam ab-17721 in left panel and Cell Signaling 2184 in right panel) and detection methods (radioactive PCR in left panel and SYBR Green real-time PCR in right panel). Error bars represent the standard error of the mean calculated on the basis of three independent experiments. An intragenic region upstream of the ACTG promoter not bound by LSD1 was used as a reference region in the ChIP assay for normalization.

Ideograms of Balanced Chromosome Translocations and FISH Mapping of the Chromosomal Breakpoints
(A and B) Ideograms illustrating the t(11;19)(p11.2;p13.2)dn karyotype in DGAP012 and the t(1;11)(p21.1;p11.2)dn karyotype in MCN1762; this latter karyotype was revised from the original karyotypic assessment, t(1;11)(p13;p11)dn.
(C) FISH analysis with a 30 kb SnaBI restriction fragment (signal shown in green) from BAC CTD-3193O13, which has previously been reported as a breakpoint-crossing clone. 24 Probe hybridization is seen on the normal 19, der(19), and der(11) chromosomes, indicating that this 30 kb restriction fragment spans the chromosome 19 breakpoint region (Figure S1B).
(D) FISH analysis with 37 kb fosmid G248P88722D5 spanning the breakpoints on the normal 11, der(1), and der(11) chromosomes, indicating that the translocation breakpoint of the chromosome 11 is located within the sequence of this genomic clone.

PHF21A Is Disrupted in DGAP012 and MCN1762 and Maps within the Refined Interval Associated with ID and CFAs
The minimal deletion region associated with the full spectrum of PSS phenotypes, compiled from a consensus of ten PSS subjects, is shown as a gray line encompassing deleted markers and flanked by small white boxes leading to markers D11S1393 and D11S1319, which are at the end of each box and are definitely not deleted. 15 The PSS-associated genes EXT2 and ALX4, together with PHF21A, all map to this deletion region. Black lines show regions of deletion in three subjects showing neither ID nor CFAs. 34–36 These deletion regions, subtracted from the above gray line, predict the ID and CFA candidate region (shown as a red line), a segment encompassing ∼1.1 Mb and containing 12 annotated genes between D11S554 and D11S1319. The proximal end of the ID and CFA candidate region overlaps with the terminal 98 kb of PHF21A. The blue lines of PSS subjects with ID and CFAs show unanimous deletion of PHF21A. At the bottom right, the breakpoints of DGAP012 and MCN1762 are shown. PHF21A is disrupted in both of these balanced translocation subjects with ID and CFAs but without multiple exostoses and parietal foramina. All together, the above genotype-phenotype analyses, combined with these two translocation cases, support a causative role for PHF21A in ID and CFAs. In the lower left corner is FISH mapping showing deletion of PHF21A in the five 11p11.2-deletion ID and CFA subjects shown above. FISH was carried out with BAC RP11-618K13 (red), which encompasses the 3′ portion of PHF21A, for three PSS subjects (PSS02, PSS08, and PSS10) whose centromeric PSS deletion boundaries had not been fully delineated 15 and for two additional 11p11.2-deletion subjects (GC14361 and GM03316). Both chromosomes 11 from the same metaphase spread are shown, indicating the absence of PHF21A from one chromosome in each subject. Clone GS-770G7 from 11q25 (green) was used as a positive chromosome 11q control.

PHF21A Regulates Zebrafish Neuronal Cell Survival and Craniofacial Development
(A–C) A noninjected control embryo (A), an embryo injected with a control MO (B), and an embryo injected with a phf21a MO (C). Knockdown of phf21a causes a reduction in head size, a change of head and face shape (arrowhead), and a major reduction of the jaw (arrow) at 3 dpf; these features are reminiscent of the microcephaly and craniofacial dysmorphism seen in the translocation subjects. The scale bar in (A) represents 300 μm. The following abbreviations are used: ov, otic vesicle; and h, heart.
(D–G) Cartilage staining of embryos injected with the control MO (D and F) or phf21a MO (E and G). Compared with that of the control embryo, Meckel's and palatoquadrate cartilage in the phf21a-MO-injected embryos are severely distorted in their size and shape. Five-day-old embryos are shown in ventral (D and E) or lateral (F and G) views. The following abbreviations are used: ch, ceratohyal; bh, basihyal cartilage; m, Meckel's cartilage; pq, palatoquadrate cartilage; and cb 1–5, ceratobranchial cartilage 1–5.
(H and I) dlx2a expression in the control (H) and the phf21a morphant (I) at 2 dpf. phf21a morphants fail to expand dlx2a-expressing pharyngeal-arch progenitor cells (arrow). The scale bar in (H) represents 300 μm, and the scale bar in (I) represents 230 μm. The following abbreviation is used: PA 1–7, pharyngeal arches 1–7.
(J and K) Formation and patterning of arch-associated blood vessels in the control (J) and the phf21a morphant (K). A lateral view of the flk1:GFP transgenic line at 4 dpf is shown. In phf21a-MO-injected embryos, aortic arches (arrowheads) form normally but fail to swing to a more anterior position (compare the position of arrows in J and K). Also, the vessels associated with gill filaments develop poorly in morphants.
(L–O) Effects on neuronal cell survival. Compared with the control MO (L) and PHF21A-mRNA-injected embryos (M), phf21a-MO-injected embryos (N) show a dramatically increased number of acridine-orange-positive apoptotic cells. The apoptotic phenotype of the phf21a morphant is rescued by coinjection of PHF21A mRNA (O).
(P–S) The phf21a-MO causes craniofacial, morphological, and growth defects in the developing zebrafish embryo. Reintroduction of wild-type phf21a/PHF21A rescues the zebrafish phenotype. A control-MO-injected embryo is shown in (P). Knockdown of phf21a causes ventral curvature of the body and a small-head phenotype (Q). These phenotypes of the phf21a MO are rescued by coinjection of either zebrafish phf21a mRNA (R) or human PHF21A mRNA (S). Expression of phf21a during zebrafish early development is described in Figure S4.