Oral–facial–digital syndrome type 1 [OFD1 (MIM 311200)] is a developmental condition belonging to the heterogeneous group of oral–facial–digital syndromes (OFDSs).1,2 It occurs in 1 out of 50 000 live births worldwide, with 75% of the cases being apparently sporadic.3 This condition is characterized by malformations of the face, oral cavity and digits. Facial anomalies include frontal bossing, hypertelorism, prominent root of the nose with a large funnel, pseudo-cleft in the upper lip and variable degrees of alopecia. Malformation of the oral cavity include short, thick frenulae, a cleft or high-arched palate, supernumerary or malpositioned teeth and a multilobulated tongue. Digital anomalies include brachydactyly, syndactyly, clinodactyly, camptodactyly, polydactyly and hypoplastic thumbs. There is usually a high degree of phenotypic variability in this syndrome.1,2 It has been shown that the central nervous system is affected in 50% of the cases.4,5 Most of the clinical features overlap with other OFDSs; however, the X-linked dominant pattern of inheritance and the polycystic kidney disease are typical of OFD1 and differentiate it from the other types of OFDS.1,2,6 Renal impairment can be present at birth or develop later on, often leading to renal failure that requires dialysis and/or renal transplantation in late childhood or adulthood.7,8 This syndrome is caused by a heterozygous mutation in the OFD1 gene, mapped to cryptogenic band Xp22.2–22.3 on the X chromosome.9,10 This gene encodes a 1011-amino acid protein that localizes to the centrosome and basal body of cilia required for primary cilia formation and left–right symmetry.11–13 The OFD1 protein has five predicted coiled-coil domains, which are important for protein interaction, and a lissencephaly homology (LisH) domain possibly involved in microtubule regulation and cell migration.14,15 This protein showed widespread expression in different tissues, including those of the pancreas, kidney, skeletal muscle, liver, lung, placenta, brain and heart.14 This protein has a crucial role in human development and causes prenatal lethality in males.1 To date, the spectrum of mutations of the OFD1 gene extends to the phenotypic spectrum to include macrocephaly, intellectual disability, and ciliary dyskinaesia in patients with type 2 Simpson–Golabi–Behmel syndrome and Joubert syndrome.16–18
Patient and methods
We ascertained an Emirati female patient with features of OFD1. Blood samples were collected in ethylenediaminetetraacetic acid tubes after obtaining informed written consent from the parents. Genomic DNA was extracted using the Flexigene DNA extraction kit (Qiagen Gmbh, Hilden, Germany) following the manufacturer’s instructions. Primers were designed using Primer3 software version 0.4.0 (Howard Hughes Medical Institute, Chevy Chase, MD, USA) (http://frodo.wi.mit.edu/) covering the coding and splice site regions of the OFD1 gene. All coding exons were amplified by polymerase chain reaction and subsequently sequenced using the BigDye Terminator kit v3.1 (Applied Biosystems, Carlsbad, CA, USA) on a 3130xl Genetic Analyzer system (Applied Biosystems). DNA chromatograms were inspected and analysed based on the complementary DNA sequence in accordance with the GenBank entries NM_003611.2 using the Sequencing Analysis® version 5.3 software (Applied Biosystems) and ClustalW2 (European Molecular Biology Laboratory–European Bioinformatics Institute, Hinxton, UK) (www.ebi.ac.uk/Tools/msa/clustalw2/). To evaluate the influence of the c.2757+1delG mutation on splicing signals, in silico prediction was carried out using the scan program (The University of Western Ontario, London, ON, Canada) (https://splice.uwo.ca/)19,20 along with the Human Splicing Finder software version 2.4.1 (Ensembl, Hinxton, UK) (www.umd.be/HSF/).21 The prediction was performed based on reference sequence ENST00000340096.
The parents were not related but were from the same tribe of United Arab Emirates (UAE) origin. In the family history the mother had a sister with an intellectual disability. The parents had two children, the first of whom was a healthy female and the second of whom is the case presented here. The pregnancy of this second child was complicated by bleeding in the first 3 months of gestation. The delivery was normal and birth weight was 2.395 kg. She was assessed in the Genetic Clinic (Tawam Hospital, Al Ain) at 8 months of age. Examination revealed a weight of 7.620 kg (< 50th centile), a length 67.20 cm (< 10th centile) and a head circumference of 42.60 cm (10th centile). There were several dysmorphic features, including widely spaced eyes, a tented and thin upper lip, a bifid and lobulated tongue, a midline cleft palate, short, thick frenulae and a serrated gingival margin. Both hands were small but there were no digital anomalies. The child was hypotonic with some degree of head lag. Detailed ophthalmological examination was normal. Ultrasound of the kidneys was normal. Magnetic resonance imaging (MRI) of the brain showed agenesis of the corpus callosum and hypoplastic vermis.
Sanger DNA sequencing revealed a novel heterozygous single-nucleotide deletion, c.2757+1delG, affecting the splice donor site of exon 20, as illustrated in Figure 1. Prediction analyses using bioinformatics tools showed that this deletion affects the authentic splice site leading to the creation of a cryptic splice site at position c.2756 (Figure 2). The erroneous splicing will lead to frameshift and subsequently creation of a premature termination codon at amino acid position 922 of the protein (p.Lys920ArgfsX2).
In this study, we reported a case of OFD1 in an Emirati family. DNA chromatograms showed that both parents harbour the wild-type sequence whereas the child is heterozygous for the c.2757+1delG mutation (Figure 1). This finding suggests that the deletion is a de novo mutation in this child, but it cannot exclude the possibility of mosaicism in the mother, which might occur in OFD1, especially with the history of intellectual disability in this family.22 Based on a splice prediction program, this mutation will abolish the authentic donor splice site of exon 20 resulting in a frameshift and premature termination codon (p.Lys920ArgfsX2). The truncated transcripts will lack the fifth coiled-coil domain and most probably will be subject to nonsense-mediated RNA decay (NMD), as the mutation is located more than 55 nucleotides before the last exon–exon boundary. Relative quantitative expression of OFD1 messenger RNA (mRNA) levels in two different families with Joubert syndrome (JBTS10) showed that only 30–58% of OFD1 expression remained, which suggests that truncated mRNAs are targeted by the NMD pathway.17 Most of the identified mutations in the OFD1 gene led to loss of function due to premature truncation of the protein.10,12,23–26 Moreover, Tsurusaki and his colleagues27 speculated that mutations downstream of exon 17 leading to a longer, truncated transcript might result in a milder form of OFD1.
Genotype–phenotype correlation studies showed a significant association between high-arched/cleft palate and missense and splice site mutations in the studied cases.2,10,24–26 In addition, cystic kidneys are reported to be prevalent in patients with mutations in exons 9 and 12, particularly splice site mutations. Intellectual disability was more predominantly associated with mutations in exons 3, 8, 9, 13 and 16, while tooth abnormalities are more frequently associated with mutations in the coiled-coil domains.2,24–26 The female child in this study showed dysmorphic features and brain abnormalities with no digital anomalies or renal impairments at this age. MRI of her brain showed agenesis of the corpus callosum and hypoplastic vermis, which have been shown to be the most common anomalies of the brain in patients with OFD1.26
Oral–facial–digital syndrome type 1 is known as ciliopathy because the protein is expressed at the base of cilia and is essential for their normal formation.6,11–13 The cilia are involved in cell movement and different chemical pathways required for normal development and function of different part of the body.12,13 Diseases caused by defects in primary cilia displayed a broad spectrum of clinical features, including polydactyly, craniofacial abnormalities, structural brain malformations, situs inversus, obesity, diabetes and polycystic kidney disease, as shown in cases of Bardet–Biedl syndrome and Joubert syndrome.28 Primary cilia play a crucial role in the regulation of various signalling pathways such as sonic hedgehog, Wnt [wingless-type MMTV (mouse mammary tumour virus)] and platelet-derived growth factor, which are involved in vital functions in development and homeostasis.29 Therefore, disruptions of these pathways will affect the normal process of cell proliferation, fate determination and migration and central nervous system patterning.30–34
In conclusion, we report a causative heterozygous splice site mutation affecting exon 20 in the OFD1 gene underlying the clinical features of OFD1 in a female child from the UAE. The child had the typical facial and oral manifestations of the syndrome but lacked the digital anomalies. This finding extends the spectrum of OFD1 mutations and highlights the major role of this gene during human development. The discovery of disease-causing mutations is important for adopting effective prevention and therapeutic approaches that might minimize the burden of genetic conditions in the UAE population.35