Understanding of the pathogenesis of axonal guidance diseases in recent years has improved greatly, and many molecular genetic conditions related to these pathologies have been found. Among them, there is strong interest in Horizontal Gaze Palsy with Progressive Scoliosis (HGPPS; OMIM 607313), firstly described by Dretakis and Kondoyannis in 1974 in consanguineous Greek pedigrees  , and subsequently reported both in consanguineous pedigrees and unrelated parents of many different ethnicities  .
2. Clinical and Genetic Aspects
HGPPS is a rare, congenital autosomal recessive disorder   in which affected individuals are characterized by the absence of conjugate horizontal eye movementswith preserved vertical gaze, variable convergence, and progressive scoliosis. This last disability, starting in infancy or childhood and adolescence, is the most common reason for medical advice in patients with HGPPS and is the most serious condition in terms of function and appearance by which affected patients undergo surgical intervention early in life     . No other associated neurological or behavioral abnormalities have been highlighted. To date, 39 different mutations, including missense, nonsense, frameshift, and splice site mutations, in the human ROBO3 gene, have been described and related to HGPPS    . This gene is a member of the Roundabout (ROBO) gene family that controls neurite outgrowth, growth cone guidance, and axon fasciculation. ROBO3 gene, encompassing 28 exons and located on chromosomal region 11q23-25, encodes an axon-guidance protein of 1386 amino acid, analogous to mouse Rig1/Rob3, involved in the promoting of midline crossing of neurons in the medulla during brain development   . This proteinis predicted to contain an extracellular segment with five immunoglobulin-like domains (Ig1-5) and three fibronectin III-like domains (FnIII1-3), a transmembrane segment (Tm), and an intracellular segment with three cytoplasmic signalling motifs (CC0-3) (Figure 1)  . Human ROBO3 shares homology with the superfamily of immunoglobulin transmembrane receptors important in axon guidance and neuronal migration, including decussation of developing nerve fiber tracts in the brainstem   . Slit proteins 1-3, a family of secreted chemorepellants, are ligands for ROBO proteins and Slit/ROBO interactions regulate myogenesis, leukocyte migration, kidney morphogenesis, angiogenesis, and vasculogenesis in addition to neurogenesis (Figure 2). ROBO3 gene mutations are causative of the lack, or at least reduction, of crossing of the descending corticospinal and ascending lemniscal sensory tracts in the medulla    -  . Standard MRI T1- and T2-weighted imaging findings consist of pons and cerebellar peduncles hypoplasia, absence of the facial colliculi, butterfly configuration of the medulla and a deep midline pontine cleft    . Although scoliosis in HGPPS patients is usually severe, it remains unclear whether the physiopathology of scoliosis is musculoskeletal or neurogenic. Playing ROBO3 a critical role for hindbrain axons to appropriately cross the midline  , a neurogenic mechanism has been postulated by Jen et al. in 2004  . Nevertheless to date, since no genotype-phenotype correlation has not been elucidated in HGPPS, possibly because of intra-familial variability of the cardinal features    , and whereas HGPPS with scoliosis has been described without detectable mutations in ROBO3 gene   , it is not possible to state that scoliosis is linked to ROBO3 mutations  .
Figure 1. ROBO3 receptor structure. ROBO3 receptor contains an extracellular segment with five immunoglobulin-like domains (Ig1-5) and three fibronectin III-like domains (Fn III 1-3), a transmembrane segment (Tm), and an intracellular segment with three cytoplasmic signalling motifs (CC0-3).
Figure 2. Interacting Proteins for ROBO3 gene. Slit proteins 1-3 and Gpr125 are ligands for ROBO proteins. In particular, Slit proteins 1-3 are a family of secreted chemorepellants and Gpr125 belongs to the family of Adhesion G protein-coupled receptors (GPCRs).
3. Spectrum of Mutations in HGPPS
The purpose of our study is to group all HGPPS-related mutations described to date, both pathogenic and uncertain significance. Mutations are listed in Table 1. HGPPS-related mutations occur in all ROBO3 gene exons and exon-intron boundaries, mostly located on the extracellular part of the protein, inherited both in affected members of consanguineous families harboring homozygous ROBO3 mutations and in individuals from non-consanguineous families harboring compound heterozygous mutations  . Most reported ROBO3 mutations are scattered throughout the ROBO3 gene without a specific region or domain that can be considered a hot-spot area for mutations  , although an important accumulation of missense and frameshift mutations in the last exons coding for the C-terminal part of the receptor has been described  . This difference, contrasting with HGPPS-associated variants, suggests a critical role of the extracellular domain in ROBO3 function. The mutations are highly diverse, and indistinguishable phenotypes result from ROBO3 nonsense, frame-shift, splice-site, or missense mutations, supporting a complete loss of ROBO3 function  . Some mutations are predicted to induce a premature stop-codon, associated with expression of a truncated protein or mRNA degradation vianonsense mediated decay (NMD)  . In addition, we report in Table 1 a lot of variants of uncertain pathological significance, described for the first time by Illumina Clinical Services (Laboratory Illumina, 2016). It cannot be excluded that they could cause the pathological phenotype, even though experiments are necessary to determine whether these variations underlie the pathogenesis of HGPPS.
Table 1. Currently known mutations in ROBO3 gene linked to HGPPS.
♦ The nucleotide number where the mutation was located was not mentioned by the authors. ● The codon number was not mentioned by the authors. Mutations in bold were inherited in heterozygous status; mutations in Italics and bold were inherited both in heterozygous status as compound and in heterozygous status. The remaining mutations were inherited in homozygous status. NA = Not available. NR = Not reported by authors.
4. Discussion and Conclusion
ROBO3 is a transmembrane receptor that plays an important role in axon guidance and neuronal migration and is critical for long ascending medial lemniscal and descending corticospinal tracts in the medulla. In fact, in HGPPS contralateral axon projections of inter-nuclear neurons that co-ordinate the activity of oculomotor and abducens neurons fail to form, resulting in defects in horizontal eye movements. Mutations in ROBO3 are associated with noncrossing of selected paths in the central nervous system that are normally subjected to midline crossing during embryonic development  . A clinical misdiagnosis of HGPPS seems unlikely because the association of horizontal gaze palsy and severe scoliosis is considered pathognomonic of HGPPS   . Diffusion tensor imaging (DTI) and DTI tractography have to be performed to evaluate the corticospinal pathways and to confirm the presence of uncrossed corticospinal tracts, being a useful adjunct to the structural magnetic resonance imaging (MRI) in confirming the clinical features suggestive of HGPPS. Since an early diagnosis of HGPPS is possible by analysis of the ROBO3 gene, this is of great importance with respect to genetic counselling of HGPPS families  . A genotypic assessment of ROBO3 mutations presents challenges because relatively little is known about the function of various ROBO3 domains or actions of alternative splice forms of ROBO3 in the human Brainstem    . The action of ROBO3 or its protein product might be inhibited by environmental or epigenetic factors in the developing brainstem; furthermore, a phenotype identical to HGPPS might be caused by abnormalities in ROBO3 splice variant expression. Moreover,although most reported ROBO3 mutations are equally distributed along the ROBO3 sequence, it would be interesting to determine whether specific mutation types are associated with a more disease phenotype and/or whether other disease genes for patients with horizontal gaze palsy with or without scoliosis who do not harbor mutations in ROBO3 are engaged. Consequentially, a Targeted Next Generation Sequencing (NGS) could be useful to elucidate the possible contribute of other genes. In fact, despite the advances in understanding about this condition much remains to be investigated on the consequences of mutations on ROBO3 expression and function. Further detailed functional analyses are necessary to clarify a possible role of the variants of uncertain pathological significance as causative of the disease. In conclusion, we hope that this article will help in molecular screening for the ROBO3 gene and will contribute to enlargement of the ROBO3 gene variation database.
No funding was received for this work.