PMC3154129_01
Patient 1 at the age of 8 years, after a bilateral medial rectus recession at the age of 2.5 years. A-F show the eye position at different attempted movements. The eye position at upgaze is shown in A. B depicts the eye position at right gaze. C shows the eyes in primary position. The eye position in left gaze, downgaze and under the condition of convergence is demonstrated in D, E, F, respectively.
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Figure 1 shows his eyes at the age of 8 years under different gaze conditions.
Pedigree diagrams of the three participating families. Circles represent females, squares males and rhombi offspring of unknown sex. Filled symbols indicate clinically affected individuals. HGPPS-patients are marked by arrows: patient 1 (II.1) and patient 2 (II.2) in A, patient 3 (II.1) in B and patient 4 (II.1) in C. All patients were born to consanguineous parents.
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The parents from Saudi Arabia were first cousins (Figure 2B). The first son of parents of Turkish origin who were first cousins (Figure 2C) was referred at the age of three months because of his head turn to the right and nystagmus, which had intermittently been recognized since he was ten weeks old.
PMC3154129_02
Patient 2 at the age of six years. Eye position at primary gaze, at attempted left gaze, and under the condition of convergence is shown in A, B, and C, respectively.
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Figure 3 shows her eye movements at the age of 6 years.
cDNA analysis for c.3319A>C mutation of the ROBO3 gene. Reverse transcription (RT)-PCR showed leaky splicing with a weak wild-type band with an expected size of 576 bp and a stronger band of around 450 bp, yielded by the skipping of exon 22 of the ROBO3 gene, for patient 2 (lane 1). For the heterozygous father (lane 2), RT-PCR showed a stronger wild-type band and a weaker band for the exon 22 skipped product. Sequencing of the different products showed unaltered splicing for the larger product (upper electropherogram) and skipping of exon 22 of the ROBO3 gene (electropherogram below) for the smaller product leading to a frameshift and premature stop codon on the protein level.
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The mutation led to the skipping of exon 22 of the ROBO3 gene (Figure 4).
PMC3154129_03
Pedigree diagrams of the three participating families. Circles represent females, squares males and rhombi offspring of unknown sex. Filled symbols indicate clinically affected individuals. HGPPS-patients are marked by arrows: patient 1 (II.1) and patient 2 (II.2) in A, patient 3 (II.1) in B and patient 4 (II.1) in C. All patients were born to consanguineous parents.
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The parents from Saudi Arabia were first cousins (Figure 2B). The first son of parents of Turkish origin who were first cousins (Figure 2C) was referred at the age of three months because of his head turn to the right and nystagmus, which had intermittently been recognized since he was ten weeks old.
Eye motility in patient 3. The eyes are shown at upgaze (A), at right gaze (B), in primary position (C), left gaze (D) and downgaze (E).
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Vertical movements were normal (Figure 5).
Sequencing chromatogram of the mutation c.2769_2779del11, 2779+1_+20del20 of the ROBO3 gene. The homozygous deletion of 31 bp of the ROBO3 gene in patient 3 includes the splice donor site of exon 17 and most probably leads to an altered splicing.
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Direct sequencing revealed a homozygous deletion of 31 bp (c.2769_2779del11, 2779+1_+20del20) spanning the splice donor site of exon 17 of the ROBO3 gene (Figure 6) most probably leading to an altered splicing.
Eye motility in patient 4 at the age of 9 months. Eye position is shown at upgaze (A), primary position (B), right gaze (C), left gaze (D), downgaze (E), and under the condition of convergence (F).
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Figure 7 and Figure 8 show his eye movements at the age of nine months.
Doll's head maneuver in patient 4 at the age of 9 months. Under passive rotation of the head to the right compensatory eye movements to the left can not be observed.
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Figure 7 and Figure 8 show his eye movements at the age of nine months.
Sagittal magnetic resonance imaging scan of patient 4 at the age of 6 months. The frontal lobes are hypoplastic (arrowhead). The corpus callosum is thinned (star) and the brainstem is flattened (arrow).
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MRI at the age of six months shows hypoplasia of the frontal lobes and the corpus callosum (Figure 9).
Sequencing chromatogram of the insertion/deletion mutation c.913delAinsTGC of the ROBO3 gene. The homozygous mutation in patient 4 most likely leads to a frame shift and premature stop codon (p.Ile305CysfsX13).
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Molecular findings: A homozygous ROBO3 insertion/deletion mutation was identified (c.913delAinsTGC; p.Ile305CysfsX13; Figure 10).