Compound heterozygous variants in RAB34 in a rare skeletal ciliopathy syndrome

Skeletal ciliopathies are a heterogenous group of congenital disorders characterized by multiple internal abnormalities, and distinct radiographic presentation. Pathogenic variants in at least 30 cilia genes are known to cause skeletal ciliopathies. Here we report a fetus with an atypical skeletal ciliopathy phenotype and compound heterozygous variants in the RAB34 gene. The affected fetus had multiple malformations, including posterior neck edema, micrognathia, low‐set and small ears, auricular hypoplasia, cleft lip and palate, short extremities, and a combination of rarely occurring pre‐ and postaxial polydactyly. Genome sequencing identified compound heterozygous variants in the RAB34 gene: maternal c.254T>C, p.(Ile85Thr), and paternal c.691C>T, p.(Arg231*) variants. Only the paternal variant was present in the unaffected sibling. Evidence in the literature indicated that Rab34−/− mice displayed a ciliopathy phenotype with cleft palate and polydactyly. These features were consistent with malformations detected in our patient supporting the pathogenicity of the identified RAB34 variants. Overall, this case report further expands genetic landscape of human ciliopathy syndromes and suggests RAB34 as a candidate gene for skeletal ciliopathies.

narrow thorax, short ribs, short tubular bones, metaphyseal irregularities, hypoplastic pelvis with trident acetabulum, brachydactyly with coneshaped epiphyses, and polydactyly.In addition to skeletal findings, diverse non-skeletal manifestations are often present and include renal cystic dysplasia, retinopathy, and malformations of the central nervous system, heart, and genitalia. 2There is broad clinical variability in skeletal ciliopathies, and phenotypes range from the most severe lethal abnormalities to milder conditions compatible with survival.Pathogenic variants in at least 30 genes are known to cause skeletal ciliopathies, and most of them are inherited in an autosomal recessive manner. 1,3ere are two major types of cilia: motile cilia, located on respiratory cells, sperm cells, and embryonic node cells; and immotile, also known as primary cilia.The primary cilia are microtubule-based sensory organelles located on the cell surface and are involved in regulating cell migration, signaling, tissue development, and organogenesis.The majority of the genes associated with skeletal ciliopathies encode proteins of the intraflagellar transport machinery or dynein motor components. 3b GTPases have recently emerged as potential contributors among the proteins involved in cilia formation.Rab34 is a small GTPase that is essential for promoting ciliary membrane biogenesis in the intracellular ciliogenesis pathway. 4In vivo studies in the Rab34 knockout mouse (Rab34 À/À ) model revealed an important role of Rab34 in ciliary vesicle formation and Hedgehog signaling. 5Rab34 À/À mice display early lethality and phenotypic features consistent with ciliopathy. 5,6Four individuals with homozygous missense variants in RAB34 and features of oralfacial-digital syndrome (OFDS) have been described by Bruel et al. 7 In this study, we report a patient with compound heterozygous variants in RAB34 and a complex malformation syndrome, including cleft lip and short-rib polydactyly.We present detailed clinical and phenotypic features of the fetus and discuss our findings in the context of malformations identified in previous RAB34 studies.6)) for genomic studies of rare disorders and skeletal dysplasias.Informed written consent for participation in the study and for the publication of the clinical data and clinical images was obtained from the parents.

| Genetic analysis
Genomic DNA was extracted from chorionic villus (CVS) of the fetus, peripheral blood samples from the parents, and saliva from the unaffected sibling.Routine clinical genetic analysis performed on fetal CVS sample showed normal male karyotype and normal CNV findings (VeriSeqTM PGS Kit (Illumina)). 8Clinical exome sequencing done on family trio using TruSight One Expanded Sequencing Panel (6704 genes, Illumina) showed no pathogenic variants.Genome sequencing on family trio was performed at Clinical Genomics, Science for Life Laboratory (Stockholm, Sweden).Fetal and parental DNA samples were sequenced on Illumina NovaSeq 6000 (Illumina) with median coverage of 30Â.Bioinformatic methods and variant calling pipeline have previously been described. 9Initial analysis of genome sequencing data included clinical skeletal dysplasia panel curated according to PanelApp, 10 Nosology of Genetic Skeletal disorders, 1 and in-house panel for skeletal ciliopathies. 3Extended research analysis included all genomic variants ranked according to the inheritance pattern, variant effect on the protein, location in the genomic sequence (prioritized for coding exons ±20 bp of intronic sequence), pathogenicity score, and a minor allele frequency (MAF) of <0.01 according to public and local databases.Segregation analysis was done using Sanger sequencing.

| Clinical findings
The affected fetus was the second child of a healthy nonconsanguineous Japanese couple.The pregnancy was initiated by in vitro fertilization procedure, and two fertilized eggs were transferred.At GA 9+4, the mother was diagnosed with vanishing twins.
The ultrasound of the remaining fetus at GA 13+2 showed multiple abnormalities, including posterior neck edema (nuchal translucency 5.6 mm), micrognathia, low-set ears, auricular hypoplasia, bilateral cleft lip and palate, short extremities and polydactyly, and atrioventricular septal defect (Supplementary Figure 1).Multiple abnormalities were seen in the follow-up scans and the family opted for termination of pregnancy (TOP) at GA 17+5.The autopsy results were consistent with the ultrasound findings and are summarized in Figure 1.

| Genetic findings
The initial analysis of genome sequencing data of the affected fetus and his parents did not reveal any candidate variants in all known skeletal dysplasia genes.Extended research analysis revealed compound heterozygosity for two variants in the RAB34 gene (NM_031934.6,hg19) in the fetus: maternal g.17:27042878A>G, c.254T>C, p.(Ile85Thr), and paternal g.17All evaluated candidate variants are summarized in Supplementary Table 1.
In this study, we report an affected fetus with biallelic variants in the RAB34 gene and a skeletal ciliopathy phenotype accompanied by syndromic features including cleft lip, palate, and polydactyly.These malformations are consistent with those described in Rab34 knockout mice embryos. 5,6Furthermore, the missense variant p.(Ile85Thr) in the affected fetus is located in highly conserved amino acid sequence, which is a characteristic region of small Rab GTPases, and is conserved among several Rab proteins. 11The reference isoleucine is a hydrophobic amino acid with a C-branched structure that is more restricted in the protein conformation.Isoleucine residues are important for ligand binding and protein stability, and the substitution to a hydrophylic threonine would likely have an impact on protein function. 12Based on the strong functional evidence in the literature and absence of biallelic loss-of-function variants in gnomAD database (https://gnomad.broadinstitute.org/)compound heterozygous variants in RAB34 were deemed to be likely pathogenic in the affected fetus.
RAB34 belongs to the family of small Rab GTPases that have an important function in multiple cellular processes, such as cellular differentiation, transport, autophagy, and regulation of signaling pathways.Rab34 is a Golgi-associated GTPase that has been shown to play a role in ciliogenesis in vitro and in vivo.A transgenic Rab34 À/À mouse model generated by Xu et al. 5 showed reduced cilium density in several tissues, including limb buds and the neural tube.Rab34 +/À mice were phenotypically normal, however homozygous Rab34 mutants died at birth.Embryos exhibited classical ciliopathy features, such as polydactyly, cleft lip and palate, and in some cases, exencephaly.Whilst no morphological defects were seen in the heart, lungs, (OMIM #175700), and preaxial polydactyly (OMIM #174700). 1,14e combination of pre-and postaxial polydactyly seen in the affected fetus in our case is a rare manifestation, and might be indicative of alterations in Hh signaling.OMIM search for clinical synopses with both pre-and postaxial polydactyly returns 21 known genetic entities, 19 of which are ciliopathies. 15Altogether, this suggests that RAB34 is a candidate gene for human syndromic ciliopathy.
While this study was under review, Bruel et al. 7  A combination of hyper-rare human case studies supported by transgenic animal models in the literature provides essential information for the functional roles of key human developmental genes.In summary, this case report contributes to expanded knowledge on human ciliopathy syndromes and provides a basis for further elucidation of RAB34 role in human embryogenesis.

F I G U R E 1
Fetus with atypical RAB34-related skeletal ciliopathy.(A) Postmortem photographs of the affected fetus (TOP GA 17+5).Note bilateral cleft lip and palate, auricular hypoplasia, microretrognathia, narrow thorax, rhizomelic shortening of the limbs, bilateral pre-and postaxial polydactyly of hands and feet.(B) Postmortem radiographs.Note handlebar clavicles, mild thoracic narrowing, mildly delayed vertebral ossification, underdeveloped lower part of the ilia, rhizomelic shortening of the long bones and lack of metaphyseal flaring.(C) Magnified photographs of hands.Note 7 digits with preaxial polysyndactyly and postaxial polydactyly of both hands.(D) Magnified photographs of feet.Note 6 digits with preaxial polydactyly of the right foot, and 7 digits with preaxial polydactyly and postaxial polysyndactyly of the left foot.[Colour figure can be viewed at wileyonlinelibrary.com] liver, or neural tube, the lethality of Rab34 À/À mice suggested functional defects in the vital organs.More detailed studies of Rab34 role in skeletal morphogenesis in vivo are complicated by the early lethality of Rab34 À/À mice.Loss of Rab34 was suggested to affect the Hedgehog (Hh) signaling pathway in mice embryos.Primary cilia are essential for Hh signaling, which is a key pathway regulating limb patterning in early development. 13Members of the Hh signaling pathway, especially Sonic hedgehog (Shh) and Indian hedgehog (Ihh), are crucial for appropriate skeletal morphogenesis.Shh signal transduction is mediated by GLI2/3 transcription factors.The alterations in Gli2-FL (active form) to Gli3-R (repressor form) ratio seen in Rab34 mutant mice leads to impaired Hh signaling, which, in turn, leads to polydactyly.In humans, loss-of-function variants in GLI3 are associated with polydactyly and polysyndactyly phenotypes, including Pallister-Hall syndrome (OMIM #146510), Greig-Cephalopolysyndactyly syndrome

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I G U R E 2 RAB34 variants in the affected family.(A) Pedigree of the family.Half-filled symbols indicate carrier status for RAB34 variants.(B) RAB34 variants in the affected fetus (II-2) and his healthy brother (II-1).Nucleotide changes are indicated by arrows, ref-reference nucleotide.(C) Evolutionary amino acid conservation of the identified variants (according to UCSC, https:// genome.ucsc.edu/).Amino acid residues affected in the fetus are indicated by arrows.[Colour figure can be viewed at wileyonlinelibrary.com] described four individuals with oral-facial-digital syndrome (OFDS) and biallelic pathogenic variants in RAB34.The general pattern of limb and facial malformations resembled that of our patient.However, the oral anomalies were more complex in the individuals reported by Bruel et al.OFDS encompasses a heterogenous group of clinical entities, but only OFDS type 2 (OMIM #252100) and type 4 (OMIM #258860) are classified as genetic skeletal disorders. 1Malformations observed in OFDS type 4 are similar to those seen in another skeletal ciliopathy, short-rib-thoracic dysplasia (SRTD) type 4. The pattern of skeletal features (narrow thorax, handlebar clavicles, polydactyly, and underdeveloped iliac bones) in the affected fetus in our study is consistent with an unclassifiable SRTD.Thus, it is possible that pathogenic variants in RAB34 lead to a spectrum of ciliopathy phenotypes, including OFDS and SRTD, highlighting the importance of meticulous clinical phenotyping in heterogeneous diseases.