Clinical features

Patient 1 was the first child of a consanguineous couple (first cousins) in Family 1 (Figs. 1a and 2a–d). There was no relevant family history. He was born at term after an uneventful pregnancy with a birth weight of 2000 g (−2.8 SD). Small head circumference (not recorded) and left microphthalmia were noted at birth. Ultrasound of the eyes showed normal right eye axial length (22 mm) while the axial length of the left eye was 15 mm. In addition, opaque lens, vitreous floaters, and membranes extending to the retina showing vitreoretinal adhesion and thickened choroid were documented. Further ophthalmological examination showed bilateral corneal opacity not affecting the vision on the right side, but the left side showed anterior segment dysgenesis. Visual evoked potential and electroretinography at the age of 1 year showed a normal functioning right eye. Echocardiography showed a small ventricular septal defect, an atrial septal defect, and tricuspid regurgitation. Abdominal ultrasound and electroencephalography showed normal results. A TORCH examination was negative.

Fig. 1: Genetic analysis of two families with homozygous GON4L variants.

Pedigree of Family 1 (a) and Family 2 (b). Wt wild-type, Var variant allele. Black dots indicate a GON4L variant carrier. Arrows indicate probands. c Human GON4L isoforms with the previously reported canonical splice variant (c.5517+1G>A) which is also identified in Family 2 and the frameshift variant identified in Family 1 [c.62_63del, p.(Gln21Argfs12*)] in NM_001282860.2 are indicated. The black bar in NM_001282858.2 indicates the lack of p.Ala1958. The short isoforms (NM_001282861.2 and NM_032292.6) have the same amino acid sequence from the first methionine to p.Gln1490 as the long isoforms, and the black boxes indicate the unique regions of the short isoforms. The gray boxes indicate paired amphipathic helix 1 (PAH1, 1624–1696 amino acids), PAH2, (1706–1777 amino acids), and Myb-like (2148–2201 amino acids) domains from N- to C-terminus, respectively, based on UniProtKB (Q3T8J9).

Fig. 2: Clinical features of the patients homozygous for GON4L variants.

Photographs of the face of Patient 1 at the age of 10 months (a) and 9 years and 6 months (b). He showed left microphthalmia. c Brain MRI of Patient 1 at the age of 5 months showed simplified gyral pattern. d Three-dimensional cranial CT images of Patient 1 at 10 months indicated metopic craniosynostosis. e Full picture of Patient 2 (left) and 3 (right). f, g Front and side of the face of Patient 2 at the age of 13 years old. She showed asymmetric face, a high forehead, thick eyebrows, upward slanting palpebral fissures, strabismus, broad nasal root, a broad nose with a beaked tip, deviated nasal septum, a prominent left cheek, short philtrum, thin upper lip, everted lower vermillion, broad chin, and low-set ears with folded helix. h, i Front and side of the face of Patient 3 at the age of 8 years. He shows dysmorphic features similar to Patient 2. Long fingers (j) and deviated feet with pes planus (k) of Patient 2. l, m Dorsum and palma of the hand with incomplete single transverse crease of Patient 3. Brain MRI of Patient 3 at the age of 6 [Axial T1- (n) and Sagittal T2-weighted (o)]. Deep Sylvian fissure is indicated by an arrow.

At the age of 10 months, his weight was 7 kg (−2 SD), length was 68 cm (−2 SD), and head circumference was 35 cm (−6.6 SD). His early developmental milestones showed mild delay (sitting at 9 months, standing with support at 14 months, and walking independently at 20 months). He had a history of repeated infection in the first years of life. At the age of 2 years, his parents noticed delayed speech, although a hearing test and auditory brain response were normal. Three-word sentences were not achieved until the age of 6 years, although the words were not pronounced clearly. He had a monotonous speech pattern (with a nasal tone and similar to that in some individuals with palatal anomalies). At 7 years of age, his weight was 18 kg (−1.6 SD), height was 120 cm (mean), and head circumference was 41 cm (−8.3 SD). At this age, the visual acuity of his right eye was normal. His muscle tone and reflexes were normal. No history of seizures or neurological regression was recognized. He showed hyperactivity with impulsivity but he had a very pleasant and friendly personality and was very cooperative with people. His intelligence quotient (IQ) was 65 at the age of 7 years and 55 at the age of 12 years, according to the Wechsler Preschool and Primary Scale of Intelligence. He was enrolled in regular school but performed poorly in reading, writing, and arithmetic tasks. He could read and write only a few words and counted with difficulty. At the age of 5 months, a brain MRI showed a simplified gyral pattern (Fig. 2c), hypogenesis of the corpus callosum, and a slightly small-sized pons. A cranial three-dimensional CT scan at the age of 10 months showed metopic craniosynostosis (Fig. 2d). Chromosomal G-banding showed a normal male karyotype (46,XY).

In Family 2, two affected siblings were born to first cousin healthy parents from Upper Egypt (IV-1 and IV-2 in Figs. 1b and 2e–o). History of a similarly affected male cousin with developmental delay and situs inversus totalis was recorded in the family pedigree, but we were not able to examine him (IV-4 in Fig. 1b). Patient 2 (IV-1 in Fig. 1b), a proband of family 2, is a 13-year-old female who was born after uneventful vaginal delivery. Her birth weight was 2.3 kg (−2.1 SD), length 45 cm (−2 SD), and head circumference 33 cm (−1.2 SD). Her Apgar score was 8/10 (1 min/5 min) and she did not require special neonatal care. Bilateral talipes equinovarus was noted. Several plaster casts were applied to the feet during the first year of life for talipes correction, which produced gradual improvement. At 4 months of age, she experienced febrile seizures (atonic and generalized tonic-clonic seizures) and a severe respiratory infection, and was admitted to an intensive care unit for 4 days. After this episode, the febrile seizures did not recur. Her developmental milestones were delayed: sitting at 18 months, standing without support at 2 years, and walking independently and speaking a meaningful word at 3 years. At the age of 7 years, she was continent. On examination, she was cooperative, understood and obeyed orders, reacted with her surroundings, talked in short sentences with dysarthric speech, and had an abnormal gait. At the age of 13 years her weight was 39 kg (−1 SD), height 117 cm (−5.7 SD), and head circumference 51 cm (−2 SD). Scoliosis was noted and the left lower limb was 5 cm longer than the right one. She had specific asymmetric facial features; a long face, a high forehead, thick eyebrows, upward slanting palpebral fissures, strabismus, broad nasal root, a broad nose with a beaked tip, deviated nasal septum, a prominent left cheek, short philtrum, thin upper lip, everted lower vermillion, broad chin, and low-set ears with folded helix (Fig. 2f, g). The extremities showed mild hyperextensibility of interphalangeal joints, long fingers (Fig. 2j), bilateral single transverse and increased palmar creases, and deviated feet with pes planus (Fig. 2k). In addition, echocardiography, abdominal ultrasonography, and chest and abdominal CT documented situs inversus totalis with normal heart structure. She showed hypotonia, normal reflexes, and good coordination. Her IQ was 62 on the Wechsler Intelligence Scale for Children at the age of 13 years. Brain MRI showed mild dilated lateral ventricles, prominent cortical sulci, a minimal high signal of white matter around the occipital horn, thin corpus callosum, and a relatively small vermis. Spine X-rays and chest CT documented scoliosis with Cobb’s angle of 35 degrees (moderate). Pubertal assessment showed breast B2, pubic hair P2, axillary hair A1 by Tanner stage, and at 13 years of age her menarche had not occurred.

Patient 3 (IV-2 in Fig. 1b) is the younger brother of Patient 2, and is now 8 years old. He was delivered by Caesarian section with no specific perinatal events. At birth, his weight was 2.5 kg (−1.7 SD), length 46 cm (−1.5 SD), and head circumference 33 cm (−1.2 SD). He also showed developmental delay, but with better achievement than his sister; sitting at 12 months, standing without support at 18 months, and walking independently and speaking a meaningful word at 2 years of age. He was continent at 5 years of age with infrequent nocturnal enuresis. On examination at 8 years, he was cooperative, understood orders, talked in short sentences with some unclear words, and had a normal gait. His weight was 26.8 kg (−0.5 SD), height 102 cm (−5 SD), and head circumference 49 cm (−2.7 SD). His dysmorphic features were similar to those of his sister (Fig. 2h, i). He also presented dextrocardia and situs inversus totalis with normal heart structure, and calcific hepatic foci with mild hepatomegaly. He showed mild hyperextensibility of interphalangeal joints, incomplete single transverse palmar crease on both hands (Fig. 2l, m), pes planus, and bilateral descended testicles. At the age of 8 years, neurological assessment showed hypotonia and normal reflexes and his IQ was 65 on the Wechsler Intelligence Scale for Children. No history of seizures was recorded. Brain MRI at the age of 6 years showed mild cortical simplified gyration, deep Sylvian fissure, thin corpus callosum, and small vermis (Fig. 2n, o). Blood examination including liver enzymes, metabolic screening, organic acid profile, electroencephalography, fundus, auditory brainstem response, and karyotype were normal in both Family 2 patients.

Genetic analysis

To identify the genetic cause of the patients’ conditions, we performed whole exome sequencing. For Patient 1, we did not identify any strong candidate variants in genes known to be associated with human diseases. However, we identified a homozygous variant in GON4L [NM_001282860.2:　c.62_63del, p.(Gln21Argfs*12), dbSNP ID: rs755827429], which was confirmed by Sanger sequencing (Supplementary Fig. 1). His parents and sibling were heterozygous for this variant, but presented no obvious phenotype (Fig. 1a). At least five GON4L isoforms are archived in the RefSeq database. Two long isoforms are 2241 amino acids (NM_001282860.2 and NM_001282856.2) and a third long isoform lacks p. Ala1958 of the other two long forms and is 2240 amino acids (NM001282858.2). Two short isoforms have the same amino acid sequence from the first methionine to p.Gln1490 as the long isoforms and have unique C-terminal regions of 39 amino acids (Fig. 1c and Supplementary Fig. 2). The frameshift variant, c.62_63del, p.(Gln21Argfs*12), identified in Patient 1 is located in a region common to all five isoforms (in exon 2 out of 32 coding exons in NM_001282860.2) (Fig. 1c). All isoforms with this variant are predicted to be subject to nonsense-mediated mRNA decay, which would result in complete loss of GON4L function. In addition, this variant was rare in gnomAD, with a minor allele frequency (MAF) = 0.00004377, and no record of homozygosity. We also identified a homozygous GON4L canonical splice site variant (c.5517+1G>A) in two affected individuals in an unrelated pedigree (Family 2, Fig. 1b). Sanger sequencing confirmed the familial segregation of the variant (Supplementary Fig. 3). This variant is located in a region specific for longer isoforms (NM_001282860.2, NM_001282856.2, NM_012812858.2) (Fig. 1c), and c.5517 is located in exon 27 of 32 coding exons (NM_001282860.2). If this variant causes a frameshift, the mRNA would be subject to nonsense-mediated mRNA decay resulting in no protein production. The clinical similarities among these three patients (Table 1 and Supplementary Table 1) indicate that the longer isoforms are important in human development. Both variants were located within the absence of heterozygosity regions: Chr1: 154,112,167–155,934,683 (1.8 Mb) in Family 1 and Chr1: 52,281,228–171,076,768 (18.8 Mb) in Family 2.

Table 1 Comparison of the clinical characteristics of diseases caused by pathogenic variants in GON4L, YY1, and SIN3AHuman GON4L expression

To further explore GON4L expression in humans, we analyzed multiple human tissues at fetal and adult stages using TaqMan assays with two probes common for short and long isoforms, and found GON4L to be ubiquitously expressed, including in the fetal brain (Supplementary Fig. 4a, b). Additionally, the long isoforms specific GON4L expressed ubiquitously including the fetal brain (Supplementary Fig. 4c), supporting that the longer isoforms are crucial in human brain development.

Neurite outgrowth in PC12 cells

To analyze the impact of GON4L on nerve development, we generated Gon4l-knockdown PC12 cells. PC12 cells treated with nerve growth factor (NGF) have been widely used as a neuronal differentiation model28,29. We designed three short hairpin RNA (shRNA) sequences for Gon4l knockdown: shGon4l_2915/5252/4283, two of which, shGon4l_5252 and shGon4l_4283, lowered Gon4l mRNA levels and GON4L protein levels efficiently (Fig. 3a–c). Gon4l knockdown did not influence PC12 cell growth (Supplementary Fig. 5). To analyze the effects of Gon4l knockdown on neural development, neurite length after NGF treatment was measured. Neurites of PC12 cells were elongated by NGF treatment, whereas neurite lengths of Gon4l-knockdown cells were shorter than those of control cells (Fig. 3d, e).

Fig. 3: Gon4l knockdown in PC12 cells and neurite outgrowth.

Knockdown efficiencies of Gon4l mRNA (a) and GON4L protein (b, c) levels in PC12 cells. mRNA levels were normalized against Gusb, and protein levels were normalized against ACTB. Data are shown as the mean ± standard error of the mean (SEM) from three independent experiments. d, e Neurite outgrowth assay in Gon4l-knockdown PC12 cells in response to NGF treatment. Representative images (d) and total neurite length in one cell (e) are shown. Dots indicate means of each independent experiment (n = 3), and the bars represent means and SEM of three independent experiments. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001 using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

Zebrafish gon4lb-knockdown phenotype and rescue by human GON4L mRNA

We next examined the morphological effects of GON4L knockdown in zebrafish. In zebrafish, two orthologous genes of GON4L are known: gon4la (XM_003200603.5, XP_003200651.2) on chromosome 19 and gon4lb (NM_001201535.1, NP_001188464.1) on chromosome 16 (in the NCBI database). Neither encoded protein shows high homology with human GON4L, but only gon4lb is “validated” in RefSeq (the status of gonl4a is only “model”) at present (accessed March 5, 2024) and shows a more similar protein sequence alignment using Clustal W (https://www.genome.jp/tools-bin/clustalw) (Supplementary Fig. 6). In addition, zebrafish phenotypes have been well studied in gon4lb previously1,3,4,8, so we chose gon4lb as a GON4L ortholog. We generated gon4lb-knockdown zebrafish using an MO, and reconfirmed the decreased eye size, head size, and body length at 50 hpf, and increased apoptosis in the central nervous system as previously reported1,3,4,8 (Supplementary Figs. 7 and 8).

gon4lb-knockout zebrafish phenotype and rescue by human GON4L mRNA

We created gon4lb-knockout zebrafish using the CRISPR/Cas9 system and successfully obtained a knockout germline allele, a 13 bp deletion in exon 2, that leads to a premature stop codon (Fig. 4a and Supplementary Fig. 9). A lateral view of gon4lb-knockout embryo morphology is shown in Fig. 4b. Consistent with our MO knockdown experiments and previous reports1,3,4,8, functional impairment of gon4lb resulted in significant reduction of three measured parameters: eye size (19%), head size (22%), and body length (19%) compared with those of gon4lb+/+ embryos (Fig. 4c–g). Moreover, in comparison to gon4lb+/+ embryos, gon4lb+/− embryos did not exhibit notable alterations in the size of eye, head, or body length. Rescue experiments conducted on gon4lb-knockout embryos demonstrated that injection of human GON4L mRNA was capable of rescuing phenotypic abnormalities, recovering eye size (Fig. 4c, d), head size (Fig. 4c, e), and body length (Fig. 4f, g), thereby reconfirming the essential role of gon4lb in head, eye, and body axis development as reported previuosly1,3,4,8. Similarly, akin to the results of MO knockdown, Acridine Orange staining revealed that impaired gon4lb function also caused a significant increase in the number of positive cells in the head region corresponding to dead cells, whereas gon4lb+/− embryos did not exhibit a significant change in the number of positive cells in the head region compared to gon4lb+/+ embryos (Supplementary Fig. 10). Rescuing gon4lb-knockout zebrafish showed that the number of positive cells in the head region was recovered (Supplementary Fig. 10). Therefore, the loss-of-function gon4lb induced cell death in developing brain of zebrafish, as previously reported1,3,4,8.

Fig. 4: Establishment of gon4lb-null zebrafish and rescue using human GON4L mRNA.

a Target design to knockout gon4lb using CRISPR/Cas9. A crRNA targeted exon 2 of gon4lb and resulted in a 13 bp deletion that generated a premature termination codon to knockout. b Representative images of 50-hpf zebrafish embryos from the gon4lb-knockout line: gon4lb+/+, gon4lb+/−, gon4lb−/−, and gon4lb−/− injected with human GON4L mRNA (rescue). All images are lateral views, with the anterior surface to the left. Scale bar: 200 µm. c Representative images of eye size and head size of 50-hpf zebrafish embryos from the gon4lb-knockout line. All images are dorsal views, with the anterior aspect at the top. The blue line delineates the contour of the head, and the pink line delineates the contour of the eye. Scale bar: 50 µm. Quantitative data showing eye (d) and head size (e) of 50-hpf zebrafish embryos: gon4lb+/+ (n = 12), gon4lb+/− (n = 12), gon4lb−/− (n = 10), and gon4lb−/− with rescue (n = 15). f Representative images of the body length of 50-hpf zebrafish embryos. All images are dorsal views, with the anterior aspect at the top. The red line indicates the position for measuring the body axis. Scale bar: 200 µm. g Quantitative data showing the body length of 50-hpf zebrafish embryos. Sample numbers are as follows: gon4lb+/+ (n = 30), gon4lb+/− (n = 30), gon4lb−/− (n = 31), and gon4lb−/− embryos with rescue (n = 17). Data are shown as the mean ± SEM; ****p ≤ 0.0001, §§§§p ≤ 0.0001 using one-way ANOVA with post hoc Tukey’s test.

gon4lb-knockout and knockdown zebrafish exhibits craniofacial abnormalities

We performed Alcian blue/Alizarin Red bone staining on gon4lb-knockdown and knockout zebrafish larvae at 5 dpf. In knockout model (Fig. 5), we found that in 78.6% of gon4lb−/− larvae, although ventral cartilage was formed, craniofacial cartilage abnormalities were observed. These included a smaller and narrower Meckel’s cartilage, ectopic palatoquadrate cartilage, and a lack of ceratobranchial cartilage in gon4lb−/− deformities, similar to gon4lb MO knockdown zebrafish larvae, and the ceratohyal angle expanded to nearly 180° (Fig. 5d). Compared with gon4lb+/+ larvae, three out of four craniofacial cartilage angle parameters significantly increased in gon4lb−/− larvae (Fig. 5e), and four out of six craniofacial cartilage length parameters were significantly decreased (Fig. 5f). Subsequent rescue experiments were conducted on gon4lb-knockout embryos. In 5 dpf gon4lb−/− larvae injected with human GON4L mRNA, there was no lack of ceratobranchial cartilage, no morphological abnormalities of Meckel’s cartilage and palatoquadrate cartilage, and no abnormalities of the ceratohyal angle (Fig. 5d). Compared with gon4lb+/+ larvae, all four significantly decreased craniofacial cartilage length parameters in gon4lb−/− larvae were rescued, and two out of three significantly increased craniofacial cartilage angle parameters were also rescued (Fig. 5e, f). These results also indicate that the craniofacial cartilage developmental abnormalities in 5-dpf larvae are caused by the loss of gon4lb function. The similar phenotypes were observed in knockdown zebrafish (Supplementary Fig. 11).

Fig. 5: gon4lb knockout causes abnormal craniofacial development.

a Craniofacial bone structure of zebrafish larvae at 5 dpf. b Measurement parameters of craniofacial bone length in wild-type zebrafish larvae: (1) Total length of the head, (2) length from the ceratohyal cartilage to the anterior end of the head, (3) length from the ceratohyal cartilage to the posterior end of the head, (4) width of the Meckel’s cartilage and palatoquadrate joint, (5) width of the ceratohyal cartilage and palatoquadrate joint, and (6) length of the ceratohyal cartilage. c Four angle parameters for craniofacial cartilage: (1) the angle between ceratohyal (CH) cartilages (CH Angle), (2) the angle between palatoquadrate (PQ) cartilage and ceratohyal cartilage (PQ/CH Angle), (3) the angle between Meckel’s cartilages (Meckel’s Angle), and (4) the angle between palatoquadrate cartilage and Meckel’s cartilage (PQ/Meckel’s Angle). All images are ventral views with the anterior at the top. Scale bars: 100 µm. d Representative images of the craniofacial cartilage of 5 dpf zebrafish larvae stained with Alcian blue and Alizarin Red (10 embryos each). The magenta-colored arrows in the panel of gon4lb−/− represent the abnormal craniofacial cartilages. All images are ventral views with the anterior at the top. Scale bar: 100 µm. e Quantitative data showing the angles of four different mineralized craniofacial cartilage elements 5 dpf zebrafish larvae. f Quantitative data showing the lengths of six different mineralized craniofacial cartilage elements in 5 dpf zebrafish larvae. Data are represented as mean ± SEM; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 using two-way ANOVA with Tukey’s multiple comparisons test (α = 0.05). CH ceratohyal, PQ palatoquadrate.

Situs inversus in gon4lb-knockout zebrafish

Since two of three patients showed situs inversus totalis, we examined the heart positioning in 48 hpf gon4lb mutant zebrafish embryos. Ectopic heart positioning on the right side was more frequently observed in gon4lb−/− (82%) compared to gon4lb+/+ (15%) with the statistical significance of p < 0.01 (Supplementary Fig. 12). We also examined the liver positioning in 5 dpf gon4lb mutant embryos. Normally, the left lobe of the liver (as observed from the left lateral view) was significantly larger than the right lobe (from the right lateral view) (Supplementary Fig. 13). Abnormal liver positioning was more frequently observed in gon4lb−/− embryos (37%) compared to gon4lb+/+ (5%) with the statistical significance of <0.001.