ONLINE SUPPLEMENTARY MATERIAL

Retinal characteristics of the congenital disorder of glycosylation PMM2-CDG

Dorothy A Thompson 1

Ruth J Lyons 1

Isabelle Russell-Eggitt1

Alki Liasis1

Herbert Jägle2

Stephanie Grünewald3

(1) Clinical and Academic Department of Ophthalmology, Great Ormond Street Hospital for Children, UCL ICH London, UK.

(2) University Eye Clinic, Regensburg, Germany.

(3) Metabolic Department, Great Ormond Street Hospital for Children, UCL ICH, London, UK.

Address for correspondence

Dorothy Thompson

Clinical and Academic Department of Ophthalmology,

Great Ormond Street Hospital for Children

Great Ormond Street

WC1N 3JH

UK

Retinal pathways

Accumulating evidence shows that the choice of activated pathway depends upon the background lighting level, i.e. the networks are photodynamic (Volgyi et al., 2004;Bloomfield & Volgyi, 2009). For example, scotopically, when background lighting is dim, information from rod photoreceptors are pooled together at the on-bipolar cell level to improve sensitivity, whilst photopically, in daylight, one photoreceptor may contact one on- and one off- bipolar cell, to improve visual acuity(Eggers & Lukasiewicz, 2011). Functionally some pathways are slow, taking detours via the amacrine cells in the inner retina and depending upon chemical synapses; others take the signal quickly and directly from rod into cone networks to ganglion cells using gap junctions(Stockman et al., 1995;Bloomfield & Dacheux, 2001;Bloomfield & Volgyi, 2009),arrowed in Main Paper Figure 1.

N-glycoproteins in the outer plexiform layer of the retina

Synapses crucial for signal transmission between photoreceptors and on-bipolar cells,containN-glycoproteins,such as MGluR6 and nyctalopin(Audo et al., 2012). N-glycoproteins known to affect the generation of the b-wave are highlighted in Supplemental Table 1.

In response to light the photoreceptor cell and on-bipolar cell membranes change polarisation. Supplemental Figure 1illustrates the distribution in the outer plexiform layer (OPL) and the roleof known N-glycoproteins(*) in this process.

Amino acid changes caused by patient genotype

The amino acid changes caused by the individual genotypes are described in Supplemental Table 2.

Fast and slow rod pathways may be separated by scotopic 15Hz flicker stimulation

Method

Scotopic 15 Hz flicker ERGs, (14.93Hz), to 10 flash strengths presented in ascending order from -3.0 to 0.5 log scot trollandswere recorded from dark adapted eyes to preferentially stimulate slow and fast rod pathways respectively (Stockman et al., 1995). ERGs to each 15Hz stimulus were acquired within a 402ms time window containing 6 cycle periods and response magnitude and phase were determined by Fourier analysis. Response waveform significance was estimated using the method proposed byMeigen and Bach (Meigen & Bach, 1999).

Result

One mildly affected patient 8) tolerated scotopic 15 Hz flicker stimulation which separates input of slow and fast rod pathways. The raw flicker data shows the expected phase reversal arrowed, which has been associated with cancellation of out of phase slow and fast signals, arrowed in Supplemental Figure 3. Fourier analysis of the scotopic 15Hz flicker data showed significant data in both low and high flash strength ranges, and the expected phase shift, though there was a lack of phase change to low flash strength.

LEGENDS Supplementary material

Supplementary Figure 1

Distribution of N-glycoslylated proteins and changes in the OPL in response to light

The distribution of the main N-glycosylated proteins arrayed on the photoreceptor and on-bipolar cell terminals, in the OPL, is shown. In the dark Ca2+ enters the rod and causes glutamate release. Released glutamate binds to metabotropic glutamate receptor mGluR6, which activates an intracellular cascade involving Gαo and Gb5, leading to the closure of the TRPM1 cation channel which hyperpolarises the on-bipolar cell. In light the photoreceptor hyperpolarises, glutamate release slows and the consequent disruption of the mGluR6 binding opens the TRMP1 to depolarise the on-bipolar cell and give rise to the b-wave.

Supplementary Figure 2

Scotopic 15Hz flicker ERGs from patient 8) show the expected cancellation of response as rod signals transition from slow to fast rod pathways at higher flash strengths. The response phase and magnitude are graphically shown to highlight the reversal 0.03 td.s/m2, and are plotted with control data and those of c1 and c2. The fast pathway phase dynamics of the patients are normal. For patients with lower magnitudes the slope of the phase change with increasing flash intensities flattens for the slow pathway and the transition to the fast pathway tend to be shifted to higher intensities.

Supplemental Material REFERENCE LIST

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