Compound Heterozygous ROBO3 Mutation in Two Siblings Presenting with Horizontal Gaze Palsy without Scoliosis: Case-Based Review

Abstract

Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare, autosomal recessively inherited disorder characterized by a congenital absence of conjugated horizontal eye movements with progressive scoliosis developing in childhood and adolescence. HGPPS is caused by mutations of the ROBO3 gene that disrupts the midline crossing of the descending corticospinal and ascending lemniscal sensory tracts in the medulla. We present two siblings, 5-year-old and 2-year-old boys with HGPPS, from non-consanguineous parents. The older brother was brought for the evaluation of moderate psychomotor retardation. He had bilateral horizontal gaze palsy with preserved vertical gaze and convergence. Scoliosis was absent. Cranial MRI showed brainstem abnormalities, and diffusion tensor imaging showed absent decussation of cortico-spinal tracts in the medulla. Clinical diagnosis of HGPPS was confirmed by sequencing of ROBO3 gene, IVS4–1G > A (c.767–1G > A) and c.328_329delinsCCC (p.Asp110Profs*57) compound heterozygous variations were found, and segregated in parents. The younger boy was first reported at 16 months of age and had the same clinical and neuroradiological findings, unlike mild psychomotor retardation. ROBO3 gene analysis showed the same variants in his brother. Our cases show the importance of evaluating eye movements in children with neurodevelopmental abnormalities and looking for brainstem abnormalities in children with bilateral horizontal gaze palsy.

Introduction

Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare autosomal recessive disorder characterized by the congenital absence of conjugate horizontal eye movements, preservation of vertical gaze and convergence, and progressive scoliosis developing in childhood and adolescence. ¹ Among the diseases with horizontal gaze palsy, HGPPS can be distinguished from other etiologies with its specific neuroradiological findings.

In this article, we aimed to present two siblings with congenital horizontal gaze palsy without scoliosis. Both siblings have distinctive radiological features of HGPPS, and early diagnosis is possible with typical MRI and diffusion tensor imaging (DTI) findings.

Background

Scoliosis with progressive horizontal ophthalmoplegia was first reported by Crisfield in four siblings in 1974. ¹ Jen et al described a pathogenetic association between HGPPS and ROBO3 gene mutations in 2004 and mapped ROBO3 to 11q24.2. ² This gene is a member of the Roundabout ( ROBO ) gene family that controls neurite outgrowth, growth cone guidance, and axon fasciculation. ³ ROBO3 expression varies according to brain development period, abundantly expressed in fetal human basis pontis in 15th to 19th gestational weeks before midline crossing of commissural axons, and downregulated after crossing. ^{2 4} Animal studies showed that ROBO3 expression is more prominent on developing commissural axons in the spinal cord, hindbrain, cerebellum, and midbrain but not expressed in the anterior commissure or corpus callosum. ^{4 5} These findings suggest that ROBO3 plays a role in midline crossing; thus, it has been shown that descending corticospinal tracts and ascending sensory tracts could not perform midline crossing in HGPPS. It was proposed that the ROBO3 receptor would inhibit repulsive signaling of ROBO1/2 receptors in commissural axons before they cross the midline, thereby facilitating axonal extension toward the floor plate. ⁴ ROBO3 has also been suggested to potentiate DCC -mediated attraction. ⁶

Clinical Manifestations

The oculomotor signs of HGPPS comprise the congenital absence of horizontal gaze under the conditions of conjugate gaze at smooth pursuit, saccades, and vestibulo-ocular or optokinetic responses. Commissural defects of the midbrain are probably responsible for horizontal gaze palsy. ⁵ In the majority of cases, adduction is preserved under the condition of convergence. Vertical eye movements are mainly unaffected. ^{7 8} Visual fields, pupil function, accommodation, and anterior–posterior segments of the eye are unremarkable. Visual acuity is reported not to be grossly impaired. ⁹ Nystagmus presents in many patients and is mostly horizontal and pendular with low amplitude. ^{7 10 11}

Scoliosis is one of the major components of HGPPS. Scoliosis progresses rapidly, and surgical treatment may be required within the first decade.

While motor development is delayed in many patients, cognitive functions are usually normal. ^{7 8 12} Seizure is not an expected finding. Muscle tonus, strength, deep tendon reflexes, hearing, speech, sensory examination, and coordination are normal. ⁸

Torticollis, which has been reported in several infants with HGPPS, can also be a reason for discovery. ⁷

Diagnosis

If a patient presents with the absence of conjugate horizontal eye movements bilaterally and preservation of vertical gaze and convergence, it should raise the suspicion of HGPPS. Congenital bilateral horizontal gaze palsy is expected to be related to structural anomalies rather than acquired etiologies. Neuroimaging findings of HGPPS are specific and consist of hypoplasia of pons and cerebellar peduncles, absence of the facial colliculi, butterfly configuration of the medulla, and a deep midline pontine cleft. ^{13 14 15} DTI and DTI tractography have to be performed to evaluate the corticospinal pathways and to confirm the presence of uncrossed corticospinal tracts. If parents can notice horizontal gaze palsy, scoliosis couldn't be the first reason to present. Although the presence of scoliosis is supportive for diagnosis, it may not be seen in the early period of the disease. The diagnosis should be verified by the analysis of the ROBO3 gene.

Management

Treatment interventions are mainly supportive. Treatment options for scoliosis are physiotherapy, three-dimensional correction using thermoplastic braces, and corrective spine surgery. The goal of ophthalmologic care is to restrain amblyopia to achieve better visual acuity despite the known oculomotor disorders.

Prognosis

It has been highlighted that scoliosis begins as early as the second year of life, continuing to progress during childhood and even after skeletal maturity. Since scoliosis is the most striking outward sign, the ophthalmologic symptoms are often relegated to a position of secondary importance. This leads to delayed care for the nystagmus and any potential amblyopia and permanent poor vision. ¹⁶

Although neurological developmental stages may be normal in HGPPS, mild-moderate psychomotor retardation has been reported in some cases.

Case 1

A 2-year-old boy presented with complaints of inability to walk independently and speak. He was born at 42 weeks gestation to a 19-year-old primigravida mother by caesarean section for the failure of labor progress. Prenatal, natal, and postnatal periods were uneventful. His birth weight was 2,180 g, compatible with intrauterine growth retardation. Parents were non-consanguineous. The mother had mild scoliosis that has not required medical attention. Family history was otherwise negative for ocular and musculoskeletal diseases. The patient had developmental delay, smiling socially at 4 months, holding his head at 8 months, gazing at objects at 12 months, sitting unsupportedly at 13 months, and standing with support at 19 months of age. He could only crawl and stand briefly but could not walk without support at 24 months. He could not utter meaningful words and displayed no social gestures. He had eye contact but did not respond when he was called by name and did not receive commands. He did not have swallowing or chewing difficulties. His head circumference was 49 cm (−0.2 standard deviation score [SDS]), weight 13 kg (0.19 SDS), and height 87.5 cm (−0.18 SDS). There was no dysmorphism. Pupils were reactive, and there was full vertical motility but a complete lack of abduction of either eye and diminished adduction of both eyes. Examination of cranial nerves was normal. Deep tendon reflexes were brisk, and plantar responses were flexor bilaterally. Scoliosis was absent. There was no organomegaly. Other system examinations were normal.

Brain MRI revealed brainstem hypoplasia with decreased pons and medulla volumes. MRI additionally demonstrated a deep midline cleft dorsally along the pontine tegmentum and ventral aspect of the medulla ( Fig. 1C ). DTI tractography confirmed the presence of uncrossed corticospinal tracts (ipsilateral ascending and descending connectivity in the brainstem) ( Fig. 2A, 2B ).

Fig. 1.

Upper series: ( A ) axial T1-weighted and ( B ) sagittal T2-weighted images demonstrate clefts at pons ( arrows ) and ( C ) axial T1-weighted image shows butterfly appearance at medulla oblongata ( arrowheads ) of the older brother at 3 years of age. Lower series: ( D ) axial T2-weighted image demonstrates the cleft at pons ( arrow ), ( E ) sagittal T2-weighted image demonstrates thin corpus callosum ( star ) and flattened brainstem ( arrow ), and ( F ) axial T2-weighted image shows butterfly appearance at medulla oblongata ( arrow ) of the younger brother at 16 months of age.

Fig. 2.

Tractography showed the presence of uncrossed corticospinal tracts ( A ) from anterior to posterior and ( B ) from posterior to anterior views of the older brother at 5 years of age, and ( C ) inferior to superior view of the younger brother at 2 years of age.

HGPPS was diagnosed with clinical and neuroradiological findings. The diagnosis was confirmed as a result of the detection of (NM_022370.4) c.767–1G > A (IVS4–1G > A) and c.328_329delinsCCC (p.Asp110Profs*57) compound heterozygous variations in the ROBO3 gene ( Figs. 3 , 4 5 , 6 ). ROBO3 gene sequencing analysis was performed by using Illumina Miseq Next Generation sequencer, and pathogenicity was evaluated using Alamut Visual, ClinVar, and HGMD Professional (Human Genome Mutation Database) databases. Segregation analysis of family was confirmed by Sanger sequencing. The mother was carrying heterozygous c.328_329delinsCCC variant and the father heterozygous IVS4–1G > A variant.

Fig. 3.

ROBO3 gene sequencing analysis of the patient for NM_022370.3 c.328_329delinsCCC p.Asp110Profs*57 variant.

Fig. 4.

ROBO3 gene sequencing analysis of the patient for NM_022370.3 c.767–1G > A variant.

Fig. 5.

Electropherogram for NM_022370.3 c.328_329delinsCCC p.Asp110Profs*57 variant.

Fig. 6.

Electropherogram for NM_022370.3 c.767–1G > A variant.

The patient is now 5 years old, and he started to walk at 3 years. He still cannot speak, but he learned how to use body language. Until now, he has not had any seizures. He did not exhibit scoliosis at his current age; however, regular orthopaedic follow-up continues for it.

Case 2

The second case was the sibling of the first case. He was 16 months old when he was reported to our clinic. He could not either walk or speak. He was born at term with a birth weight of 3,160 g. His neurological development was delayed, holding his head at 7 months and sitting at 14 months. He could stand but not walk at 16 months. He could not say any words, but his social response was better than that of his older brother. He was responding when he was called by his name and was receiving commands. His head circumference was 43 cm (0.67 SDS), weight 11 kg (−0.07 SDS), and height 80 cm (−0.34 SDS).

Visual acuity, pupillary reactions, and examination of the anterior and posterior segments of the eyes were normal. Vertical eye movements were intact, but horizontal eye movements were absent with testing saccades and smooth pursuit. The patient could adduct each eye with convergence. There was no nystagmus. Examination of cranial nerves was normal. There was no upper motor neuron, extrapyramidal system, and cerebellar finding. No skeletal abnormalities and scoliosis were observed.

Brain MRI and DTI revealed similar brainstem abnormalities as detected in his brother and thin corpus callosum ( Figs. 1D–F , 2C ). The ROBO3 gene sequence analysis showed the same compound heterozygous variations in his brother. Exome sequencing showed no other pathogenic variants in genes associated with developmental split-brain syndrome (DSBS) and intellectual disability. The patient's diagnosis was delayed because the family could not bring him to our clinic earlier due to the coronavirus disease 2019 (COVID-19) pandemic. Now, he is 2 years old, and he started to walk with assistance with the support of physiotherapy. He can use body language better than his older brother, and he can point with his hand what he wants. He still cannot speak meaningful words, but he can walk independently. He has not had any seizures as well. Scoliosis has not developed in the follow-up until now.

Discussion

Smooth horizontal gaze is controlled by the abducens nucleus. The abducens nuclei are located in the lower part of the pontine tegmentum at the level of the fourth ventricular floor. They are surrounded by the axons of the motor nuclei of the facial nerve, curving around the abducens nuclei to form the internal genu of the facial nerve. Together, the abducens nuclei and facial nerve roots form the facial colliculi: paired prominences of the fourth ventricular floor that are clearly depicted on axial MR images in healthy individuals. The absence of such prominence suggests selective agenesis of the abducens nuclei, thereby explaining congenital horizontal gaze palsy. Normal facial nerve function in patients with HGPPS suggests that the roots of the facial nerve are normally developed. Remarkably, the conformation of the pons in our patients closely resembles that of the early gestational metencephalon, from which the pons is developed. Between gestational weeks 5 and 8, the developing fourth ventricle shows a ventral furrow that deeply indents the posterior aspect of the metencephalon. ¹⁷ This furrow will progressively disappear as dorsomedial nuclei and tracts develop. Abnormal development of the abducens nuclei and MLF could result in the persistence of an abnormally deep ventral fourth ventricular furrow, thus producing the split pons sign that is pathognomonic to HGPPS.

The differential diagnosis of HGPPS embraces several congenital and acquired disorders of eye movement. Congenital diseases included in the differential diagnosis are Duane retraction syndrome, Möbius syndrome, HOXA1, HOXB1, DCC, and MACF1 gene mutations, and others. Clinical and neuroimaging findings can differentiate these entities from HGPPS. Duane retraction syndrome consists of a congenital abduction deficit of the eyeball accompanied by the retraction of the globe on attempted adduction and by upshoots or downshoots of the affected eye on adduction. Pathological and imaging findings reveal aplasia or hypoplasia of the abducens nuclei and nerve. ¹⁸ Möbius syndrome is a congenital disorder characterized by facial diplegia, paralysis of lateral gaze movements, and other cranial nerve palsies. It is usually associated with cranial and musculoskeletal anomalies. MRI reveals brainstem hypoplasia with the absence of the facial colliculi. ¹⁹ Bilateral sensorineural deafness is the main phenotypic feature that distinguishes the HOXA1 -related syndromes from HGPPS, and scoliosis has never been reported with HOXA1 disorders. ^{20 21} Hereditary congenital facial paresis (HCFP) belongs to the congenital cranial dysinnervation disorders. HCFP is characterized by the isolated dysfunction of the seventh cranial nerve and can be associated with hearing loss, strabismus, and orofacial anomalies. The only causative gene for HCFP is HOXB1 (17q21; HCFP3). Möbius syndrome shares facial palsy with HCFP but is additionally characterized by the limited abduction of the eye(s). ²² Biallelic DCC mutations have been identified in DSBS, which shares several features with HGPPS. ^{23 24} Differential diagnosis can only be possible with genetic analysis due to similar phenotypic features. Similar brainstem malformations can be seen in patients with MACF1 mutations, but normal cortical thickness and loss of severe intellectual disability, axial hypotonia, spasticity, and seizures were inconsistent with MACF1 mutations in our cases. ²⁵ No pathogenic variants of DCC , MACF1 , HOXA1 , and HOXB1 genes were found in exome sequencing in the younger brother, which supported that the phenotype was strongly associated with the ROBO3 mutation. Since horizontal gaze palsy is generally not noticed by families and even physicians in HGPPS cases, a differential diagnosis with acquired causes of horizontal gaze palsy such as ischemic, hemorrhagic, demyelinating, and tumoral lesions of pons may be required.

Scoliosis is the most frequent reason for medical advice in patients with HGPPS because of major functional limitation, pulmonary compromise, and pain. It often requires surgical intervention. The spinal deformity is often observed in early childhood or even in the first year of life with an average age at diagnosis being 4.3 year ^{9 16 26} and is rarely reported in adolescence. ²⁷ Neonatal scoliosis has also been reported. ²⁸ Although scoliosis enacts a substantial medical problem for all affected individuals, it remains unclear whether the physiopathology of scoliosis is musculoskeletal or neurogenic. Since ROBO3 is necessary for hindbrain axons to appropriately cross the midline, ²⁹ a neurogenic mechanism has been postulated by Jen et al, ² even though, currently, it is undefined how and why ROBO3 mutations during development cause progressive scoliosis after birth and in light of our knowledge, it is not possible to state that scoliosis is linked to ROBO3 mutations. ³⁰ In our two patients, scoliosis did not develop until now. Since scoliosis may develop in the late period, close follow-up of the cases is required. However, it is known that scoliosis may not be seen in every patient with ROBO3 mutation. There are not enough data that scoliosis can be prevented with a regular physical therapy program. Since no specific variants have been identified in cases with ROBO3 mutations that do not progress with scoliosis, we think that whether or not scoliosis will develop in our case can only be understood during the follow-up period.

In our patients, ROBO3 sequencing analysis revealed compound heterozygosity for (NM_022370.4) c.328_329delinsCCC (p.Asp110Profs*57) and c.767–1G > A (IVS4–1G > A) variants ( Figs. 5 , 6 ) and segregated in parents. The c.328_329delinsCCC (p.Asp110Profs*57) variant is a deletion–insertion variant leading to a premature stop codon at position c.110 that changes the reading frame. This variant has not been previously reported in silico prediction databases and in the literature. The variant was not found in gnomAD exomes and gnomAD genomes. According to the American College of Medical Genetics (ACMG) recommendations, it should be classified as a new pathogenic variant. The c.767–1G > A (IVS4–1G > A) variant is in a canonical splicing region, and this change disrupts RNA splicing. This variant has been reported as likely pathogenic in ClinVar database, but its minor allele frequency has not been found in gnomAD exomes and gnomAD genomes. According to ACMG recommendations, it is classified as pathogenic. To date, 76 different mutations, including missense, nonsense, frameshift, splicing, gross deletion, small deletion, small insertion, and small indel mutations, and 174 variants have been reported according to the mastermind database. ³¹ The most common mutations are missense mutations c.955G > A (p.E319K) and c.2108G > C (p.R703P). ²⁶ 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. ⁷ 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, ³² 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. ³³ While the c.328_329delinsCCC (p.Asp110Profs*57) variant takes place in the second exon, the (c.767–1G > A) (IVS4–1G > A) variant takes place in the fourth intron. The mutations are highly diverse, and indistinguishable phenotypes result from ROBO3 nonsense, frameshift, splice site, or missense mutations, supporting a complete loss of ROBO3 function. ³³

Conclusion

This study reported the two cases of Turkish patients with HGPPS disorder carrying a novel ROBO3 gene mutation. Reports from the literature indicate that there is a significant delay in HGPPS diagnosis. So, it is important to evaluate eye movements in children with neurodevelopmental abnormalities and to look for brainstem abnormalities in children with bilateral horizontal gaze palsy.