Family 1. The proband (Fig. 1A, B, online supplemental Fig. S2 and S3) was first seen at 17 months of age. He was born early at 35 week gestation because of pre-eclampsia. An abnormal skull shape was previously identified on fetal ultrasound and craniofacial dysmorphisms were noted at birth. In addition, he had a small and narrow oropharynx and ear canals, small nasal airways, and a right diaphragm paralysis. An ophthalmic examination revealed normal optic nerves but no other deformities except for epiphora and hyperopia. He subsequently developed chronic rhinitis, severe conductive hearing loss (requiring hearing aids), eczema, chronic constipation, and joint stiffness affecting the fingers/toes, elbows, and ankles. He suffers episodic headaches and has experienced several seizure-like episodes. He has mild developmental delay and learning disabilities requiring an individualized education plan, especially for reading and math.

Clinical features of siblings from family 1. A, B Facial characteristics of the proband (11 years) and his more severely affected sister (C, D 15 years). Note turribrachycephaly with a very high forehead and triangular face, interrupted eyebrows, shallow orbits, and small-looking palpebral fissures

His older sister (Fig. 1C, D, online supplemental Figs. S2 and S3) has a similar phenotype. Her antenatal period was unremarkable except for the identification of an abnormal cranial shape on fetal ultrasound. Despite third-trimester pre-eclampsia, the pregnancy continued to term and she was born via a normal vaginal delivery. Her medical history was significant for recurrent otitis media due to narrow external ear canals and conductive hearing loss requiring hearing aids. She also had nasal polyps, a nasal septal deviation and a nasolacrimal duct obstruction that improved with time. She complained of recurrent/voluntary shoulder dislocation. Her ophthalmologic examination was normal except for mild peripapillary atrophy which was not clinically relevant. She suffered from mild asthma, eczema, and migraine-like episodic headaches. She was also diagnosed with mild developmental delays and learning disabilities, especially in reading/comprehension and math. Craniofacial reconstructive surgery was undertaken at age 10.

The siblings' physical examination revealed strikingly similar features summarized in supplemental Table S2. Their head circumference and upper-to-lower body ratio were normal. Other common features included turribrachycephaly with a very high forehead and triangular face, shallow orbits and small-looking palpebral fissures, a prominent nose with a high bridge and bulbous tip, a small philtrum and mouth, a high palatal arch and crowded teeth, a flat facial profile due to malar hypoplasia, pointy chins and prominent low set and posteriorly rotated ears. Only the female sibling presented ocular hypertelorism. The proband had pectus excavatum, a short fourth metatarsal, and overriding of his fourth left toe. Both siblings displayed a Marfanoid habitus with slim limbs, arachnodactyly, and a drumstick appearance to the distal phalanges and long toes. Bony protrusion at the ulnar area of the wrist (palmar side) and restricted pronation/supination movements were identified in both. Their parents were non-consanguineous, of Caucasian descent and had another healthy child together. In addition, each parent also had a healthy child from a previous relationship.

Head computerized tomography (CT) in the proband was performed a few months after birth and ruled out craniosynostosis but showed wide orbital fissures, thought to be related to the partial absence of the medial portions of the greater sphenoid wing, and maxillary hypoplasia. There was prominence of the extracerebral space and mild ventriculomegaly. A brain MRI at age 11 years showed nonspecific multiple foci of white matter hyperintensities distributed in the left parietal and bifrontal areas and left periventricular gliosis. His skeletal survey at 13 years showed a flattened appearance of the frontal and midface bones and a pointy mandibula. A short left fourth metatarsal bone and fusion of the left second metatarsal and medial cuneiform were identified. Both piriformes appear elongated, extending peripherally and projecting over the hamate. His bone age was consistent with his chronological age.

His sister’s head CT excluded craniosynostosis but identified enlarged subarachnoid spaces with enlarged ventricles. She also had a partial absence of the greater sphenoid wings bilaterally resulting in an enlarged appearance of the orbital fissures and maxillary hypoplasia. A skeletal survey done at 15 years of age revealed subtle mid-thoracic scoliosis and a slightly exaggerated lumbar lordosis. There was no evidence of elbow joint fusions, although there was a suspicion of a bony bar between the right radial head and proximal ulna. She had elongated metatarsals, phalanges, and metacarpals. Bone mineralization was preserved with a bone age of 17y (chronological age 15 y 5 m), still within 2SD and considered appropriate skeletal maturation.

Family 2. The proposita in family 2 (Fig. 2B–D) was the first pregnancy of a healthy and consanguineous Brazilian couple (F = 1/32, Fig. 2A). There was no family history of congenital defects. The parents are of a Caucasian background and originate from a southern Brazilian town with a Polish settlement. A prenatal ultrasound at 31 + 5 weeks identified a single fetus with associated polyhydramnios, voluminous hydrocephalus (cephalic circumference = 35.1 cm [z-score + 4.92]), an elliptical skull shape, agenesis of the corpus callosum and absence of posterior fossa elements. The abdominal circumference was 21.9 cm (z-score – 3.47), and the long bones were short (below p 2.5). Severe kyphosis, a narrow thorax, and pulmonary hypoplasia were also observed. Mineralization was decreased, especially in the long bones. The following skeletal defects were observed: bowing of humerus, radius, and ulnae, absence of the femora, shortening of the tibiae, and abnormally shaped hands and feet with a lack of some fingers and toes. The phenotype was consistent with a lethal skeletal disorder; the sonographer raised two hypotheses—hypophosphatasia (OMIM 241500) and osteogenesis imperfecta (OMIM 166210). The fetus, a stillborn female, was born at 33 weeks by cesarian section. Growth birth parameters were: birth weight 539 g (z-score – 0.92); birth length 41.3 cm (z-score – 0.51); and occipital frontal circumference 41.0 cm (z-score + 7.57). A review of clinical photographs showed a large head with dolichocephaly, a very small and low set right ear, mild ocular protrusion, lingual protrusion, a short neck, thoracic kyphosis, short limbs and four finger oligodactyly, including the right thumb, and feet with a single median toe.

Clinical features and genetic investigation of proposita from family 2. A Family pedigree. B Lateral view of the fetus. Note dolichocephaly, very small ears, lingual protrusion, short limbs with oligodactyly—the left foot presents an absence of hallux and a single middle toe with an appearance of arachnodactyly. C, D Babygram presents a skeleton poorly mineralized, the vault not calcified, absence of clavicles, generalized spina bifida occulta, severe thoracic kyphosis with tall vertebrae (better seen in the lateral view), short and thin ribs, hypoplasia of scapulae, short humerus with shaft fracture at right, synostosis of the elbow joint, carpal and tarsal ossification nucleus, four metacarpal bones, thumb with one phalanx, the next two fingers with two phalanges and the last with only one phalanx, hypoplastic femur, bowing of tibiae and fibulae are bowing, presence of three metatarsals and only a single toe with a single phalanx. E Growth plate of the fetus showing the resting zone (RZ) with slight hypercellularity of the chondrocytes, a short proliferative zone (PZ) with small cells with poor cytoplasm, and hypertrophic zone (HZ), also short, and few cells more widely spaced than expected. F Electropherogram of exon 5 displaying the variant c.1083C > A in homozygosity in the proposita (red arrow) and in heterozygosity in both parents. G RT-PCR analysis of the minigene assay product comparing the wild-type (wt) and the mutant (mut) plasmid. Note the difference in sizes, showing the deletion of 66 amino acids caused by the splicing defect. H Electropherogram of the RT-PCR product showing the exact breakpoint of the splicing. bp base pair; L 100 bp ladder

Radiographs revealed generalized osteopenia and absent skull vault ossification. The following findings were also observed: absent clavicles, hypoplastic scapulae, abnormal vertebral pedicle morphology in the anterior–posterior view compatible with generalized spina bifida occulta, severe thoracic kyphosis and short and thin ribs. Lateral spinal views indicated the appearance of tall vertebrae. Both humeri were short and there was a right midshaft humeral fracture. In addition, elbow joint synostosis and a large carpal ossification nucleus (probably synostosis of capitate-hamate) were also observed. The right hand presented only four metacarpal bones and the left hand was not evaluated; the thumb showed one phalanx, the next two fingers had two phalanges, and the last one had only one phalanx. Both femora were severely hypoplastic, the tibiae and fibulae were bowed, two tarsal ossification centres were noted, and there were three metatarsals and a single phalanx in the middle toe. The pelvis could not be seen in the available radiographs (Fig. 2C, D).

The autopsy revealed hydrocephalus and cerebral atrophy due to lateral ventricle dilatation and compression of the parenchyma, deformity of the cranial base bones; an apparently normal spinal cord despite complete spina bifida occulta (absence of the posterior process of the vertebrae from C1 to S1); and hypoplasia of the lower pulmonary lobes, without other internal anomalies. Detailed clinical findings are described in the online supplemental Table S3. Four years after the proposita’s birth, the parents had a healthy boy.

Femoral growth plate histology revealed disorganization of the resting zone and hypercellularity; the proliferative and hypertrophic zones were short with small cells and reduced cytoplasm in the proliferative zone and fewer more widely spaced cells in the hypertrophic zone. Osteocytes and osteoblasts were observed in the trabecular zone (Fig. 2E).

Family 1. Chromosomal microarray analysis and targeted single gene variant testing (FGFR1 (p.P252R), FGFR2 (IIIa and IIIc), FGFR3 and TWIST1) was uninformative. Later, quadruplet (parents and affected siblings) whole exome sequencing (WES) was undertaken and revealed two compound heterozygous variants of unknown significance (VUS) in trans in the CYP26B1 gene (c.353C > T; p.(Pro118Leu) and c.701G > A; p.(Arg234Gln)) in both siblings. The first variant, c.353C > T in exon 2 was paternally inherited and absent in large population cohorts. The second variant c.701G > A in exon 3, was maternally inherited and has been observed with an allele frequency of 0.00004631 (Popmax filtering allele frequency = 0.0003353, gnomAD—https://gnomad.broadinstitute.org/).

Family 2. Initial SS analysis revealed that the parents were heterozygous for the c.1083C > A variant (Fig. 2F) in exon 5. Later, SS of exon 5 of the proposita’s DNA confirmed the c.1083C > A variant in homozygosity. This variant is absent in gnomAD, EVS, ABraOM, HGMD, and ClinVar databases. The variant encoded a synonymous change (p.(Val361 =)) but further in-silico analysis suggested the creation of a new cryptic donor splice site, prompting the hypothesis that this variant led to a partial deletion of exon 5.

Minigene analysis revealed a near-exclusive expression of an abnormally spliced transcript (226 bp) for the c.1083C > A variant (Fig. 2G). Sequencing of the amplicon confirmed a 66-nucleotide exon 5 deletion. (Fig. 2H). In comparison, the WT minigene only expressed the normally spliced exon 5 isoform (292 bp, Fig. 2G). Therefore, the variant was re-classified as “pathogenic” (PVS1_strong, PS3, PM2, and PP4) according to ACMG guidelines and Clingen recommendations (Richards et al. 2015; Abou Tayoun et al. 2018).

Ectopic expression of the p.Val361_Asp382del protein caused an accumulation of intracellular RA (~ 3.5-fold increase in luciferase activity), confirming a notable loss in the ability to metabolize RA. The difference in the enzymatic function was significant between p.Val361_Asp382del and the other tested variants. The other two variants, p.(Pro118Leu) and p.(Arg234Gln), also caused a partial loss of CYP26B1 enzymatic activity, with 2.3- and 1.7-fold increases in luciferase activity, respectively (Fig. 3E). The enzymatic function did not differ significantly between the p.Arg234Gln variant and the other constructs. Finally, the p.Arg363Leu variant showed a 2.1-fold increase compared to the WT. Western blotting of transiently transfected HEK293T/17 and NIH/3T3 cells confirmed similar expression levels for the various CYP26B1 constructs (online supplemental Fig. S4).

A Described variants along CYP26B1. The variants on the top are found in patients presenting mild phenotypes and those on the bottom represent variants in patients with a severe or lethal phenotype. Individuals are referenced on the right and variants are colour-coded by individual (two variants with the same colour represent compound heterozygosity). The figure was adapted from the ProteinPaint website (https://proteinpaint.stjude.org/). B Structural consequences of p.Pro118Leu and p.Arg234Gln substitutions. Missense variant substitutions are labelled in pink with the wild-type (WT) in blue, retinoic acid and heme are present in the interior cavity coloured lime and orange, respectively. C Predicted p.Val361_Asp382del structure superimposed on WT CYP26B1. The pink region represents the WT peptide sequence that is deleted resulting in the chromatic predicted structure; this region extends from the ExxR motif through β 2–2. D CYP26B1 as a topological schematic. All secondary structures are annotated chromatically from N to C termini, alpha helices are represented as tubes and beta strands as directional arrows. General locations of the cytochrome p450 conserved substrate recognition sites and ExxR motif are labelled with gold disks. The meander loop is defined as the region between αK’ and the heme binding site shown by a white octagon. Adapted from (36). E Luciferase assay results showed the normalized fold change in activity (constructs/WT) when cells were treated with RA. N = 5 independent experiments. Statistical analysis was performed using ANOVA followed by a corrected Bonferroni post hoc test. WT wild-type plasmid, NTC non-transfected cells, # Individual also has pathogenic variants in the gene NAGLU (14). All pathogenic variants described here in the CYP26B1 gene are numbered according to the reference sequence NM_019885.4 (reference genome GRCh38, hg 18)

Protein modelling revealed that both WT and missense variant structures show a small amount of variance in canonical cavity formation. However, a large change in the interior is visible with the p.Val361_Asp382del variant (Fig. 3B, C). p.(Pro118Leu) and p.(Arg234Gln) variants were present right before αB’ and at the start of αG on the structure, respectively. The p.(Pro118Leu) variant resulted in a non-conservative substitution right at an opening used by RA to enter and bind to the enzyme active site. Since leucine is bulky in comparison, we hypothesize that the mutant enzyme is less efficient at binding RA. Conversely, the p.(Arg234Gln) variant is present at one of the putative membrane contact points for this protein and swaps a positively charged membrane contact for an uncharged polar contact. The p.Val361_Asp382del variant involves the end component of αK that contains the ExxR motif and disrupts the first and second beta sheets creating a noncanonical association between β 1–3 and residues near β 2–2 (Fig. 3C).

BioID analysis confirmed previously reported associations with ANKLE2, ATF6, CDKAL1, CYP51A1, ESYT1, FKBP8, GPRC5C, OSBPL8, PDZD8, PKMYT1, and VRK2 proteins—most of which are overrepresented in ER gene ontology (GO) terms (Huttlin et al. 2021). A GO analysis of the 241 CYP26B1 BioID associations also reveals an overrepresentation of biological processes involving ER organization and signalling and homeostasis (Fig. 4A). While not part of driver GO terms, 13 GO terms were related to catabolic processes.

A CYP26B1 proteomic analysis reveals a loss of proximal associations with the p.Pro118Leu variant. Gene ontology analysis of the CYP26B1 proximal associations identified by BioID on the right. B, C Scatter plots comparing proximal associations with the wild-type (WT) and p.Pro118Leu (left) and p.Arg234Gln (right) CYP26B1 proteins. Labelled proteins had a Bayesian false discovery rate (BFDR) ≤ 1%, an average spectral count ≥ 10 in either the WT or mutant CYP26B1 BioID, and ≥ twofold change when comparing the two proteins. Grey lines denote the twofold change thresholds. D Gene ontology analysis of proteins whose proximal associations were altered with the p.Arg234Gln variant

The proteome of WT CYP26B1 and the p.(Pro118Leu) variant was nearly identical, with only a few differences (mostly gained protein associations, Fig. 4B). However, only two GO terms relating to membrane proteins were deemed statistically significant. The p.(Arg234Gln) proteome was also very similar to that of the WT protein, but the variant weakened a greater number of CYP26B1 protein association (Fig. 4C). GO terms linked to the affected CYP26B1 associating proteins included inositol triphosphate calcium release, translation, and regulation of cation channels (Fig. 4D).

When examining the cellular localization of CYP26B1 plasmids alongside an ER membrane marker (calnexin), all constructs exhibited localization to the ER membrane (Fig. 5). The CYP26B1 protein signals appeared granulated with some foci formation in all constructs, with an increased loci formation in the mutants compared to the WT (online supplemental Fig. S6A, B). The expression of unfolded protein response (UPR) genes (BiP, spliced Xbp1, Atf4, Atf6, Ern1, Perk) were tested through quantitative PCR and results showed normal expression relative to the WT (online supplemental Fig. S6C). UPR expression and foci formation (ERN1, BiP, spliced XBP1) were also verified in stably transfected HEK293T/17 (online supplemental Fig. S7A, B) and presented similar results found in NIH/3T3 cells. It was assumed this foci formation represents only an artifact of the technique, possibly due to overexpression and/or aggregation caused by the affinity between CYP26B1 and the 3XFLAG tag.

CYP26B1/FLAG proteins are properly localized. Representative images of immunofluorescence performed on NIH/3T3 cell line transiently transfected with wild-type (WT), p.Val361_Asp382del, p.Pro118Leu, and p.Arg234Gln CYP26B1/FLAG constructs (red). Calnexin (cyan) was used as an ER membrane marker and Hoechst was used for nucleus staining (here shown in yellow). Scale bar = 15 µm. N = 3 independent experiments