SMN expression on RNA level in different SMA phenotypes during MN

As the disease cause, SMN expression was investigated first. Samples of every cell line in each respective cell population were investigated by qRT-PCR. Data were normalised as well as graphically plotted relative to total RNA expression.

In fibroblasts, SMN expression in control lines r12 and COII.2 was almost equal (Fig. 45 A).

SMA I patient line ML17 exhibited the least amount of SMN mRNA. Astonishingly, discordant family members showed an inconsistent SMN expression rate: Whereas asymptomatic ML11 and ML13 as well as SMA III ML12 displayed a moderate SMN expression (~40-60% relative to r12), the other lines asymptomatic ML101 and SMA III ML14 and ML102 nearly reached the expression level of healthy controls (75-100% relative to r12). Grouping this data according to the phenotypic background gave a more consistent view in which SMA I patients expressed only ~28% SMN mRNA and SMA III and asymptomatic discordant family members ~68-74%

SMN mRNA relative to controls (Fig. 45 B). Thus, only SMA I demonstrated a very significant difference in SMN mRNA expression.

SMN expression in iPSCs conveyed a more homogeneous impression concerning single cell line expression (Fig. 45 C). Control lines r12 and COII.2 demonstrated the overall highest SMN expression. Moreover, SMA I line HGK1 showed the lowest amount of SMN expression which differed significantly from the control r12 amount. Discordant family 1 members HGK13 and HGK16 exhibited almost half the control amount (~50% relative to r12) reflecting the genetic background with three SMN2 copies. Since siblings of discordant family 2 possessed four SMN2 copies, expectedly the SMN expression rate was higher (60-80% relative to r12) than in other SMA phenotypes, however for most of the iPSC lines still distinctly lower than in control lines. Summarising these results in respect of the phenotype represented the same picture as in fibroblasts (Fig. 45 D). Controls expressed the highest amount of SMN whereas SMA III and asymptomatic siblings almost equally expressed ~65% relative to controls still rendering a significant decrease. SMA I depicted the lowest amount (27% relative to control) indicating very significant difference.

In smNPCs, both control lines r12 and COII.2 displayed the overall strongest SMN expression (Fig. 45 E). Again, SMA I line HGK1 showed a significantly lower amount when compared to control r12. The discordant family members basically revealed a stronger SMN expression rate (70-90% relative to r12) except for HGK21.8 with 46%. When the four phenotype classes were compared, they exhibited a picture congruent with the previous phenotype summaries (Fig. 45 F): A high SMN expression in controls, a significantly lower expression in SMA I (26%

relative to controls) and a moderate significant SMN expression diminution in SMA III and asymptomatic groups (~65% relative to controls).

Analyses of MN culture samples revealed the highest SMN expression in control line r12 (Fig. 45 G). The least SMN expression was found in SMA I HGK1 and exceptionally in SMA III HGK13 being significantly different relative to r12. On the other hand, the remaining SMA III and asymptomatic individuals expressed a medium up to higher SMN amount (~43%-85%

relative to r12) which was not significant. However, the graph represented only data from a

single MN differentiation run. Once samples of the two other runs were processed, the pooled data might give a more conclusive picture of SMN expression in MN cultures.

Nevertheless, grouped data in the phenotypic overview (Fig. 45 H) showed a firm SMN expression in healthy control r12 proving significantly higher than SMA I affected which delivered just approx. 25% expression rate. In contrast, in SMA III and asymptomatic samples SMN expression rate reached a medium level of ~56% and ~68%, respectively (relative to r12) marking only expression in SMA III as significantly diminished.

Taken together, SMN mRNA expression differed between healthy controls, SMA I, SMA III and asymptomatic discordant siblings according to their SMN2 copy number. Cell line-specific variances were balanced to a conclusive phenotypic summary in which SMA I principally expressed roughly a quarter of the control amount (~26%) resulting in a significant difference.

There was evidently a diminished SMN expression in SMA III or asymptomatic groups detectable in comparison to healthy controls so that variance in SMN expression was often significant, On average, in the four phenotypic classes SMA III and asymptomatic discordant expressed ~65% and ~68%, respectively.

Fig. 45: Quantitative analyses of SMN mRNA expression levels in different cell populations via qRT-PCR from healthy controls, SMA I and SMA III affected and asymptomatic discordant siblings. (A) In fibroblasts, control lines r12 and COII.2 expressed most SMN mRNA whereas SMA I ML17 the significantly least amount. Discordant family members showed a variant expression partially lower than controls, partially almost equal. (B) Fibroblast SMN expression in four phenotype classes exhibited no significant differences between SMA III and asymptomatic siblings in comparison to control lines. However, SMA I expression ranged very significantly lower.

(C) In iPSCs, control line r12 displayed a significantly higher SMN expression towards SMA I line HGK1. Discordant family members individually revealed a significantly reduced SMN expression on medium level. (D) In phenotypic classes, the highest SMN expression was found in healthy controls whereas SMA I patient demonstrated a significantly decreased SMN amount. SMN reduction in discordant family members was significant, yet reaching a moderate expression level (~65%). (E) In smNPCs, SMN expression level was significantly lowered in SMA I line HGK1 relative to control line r12 whereas in individual discordant family members SMN amount was not significantly diminished nonetheless appeared still lower than control levels. (F) Phenotypic summary disclosed a similar picture as in previous cell populations leaving SMA I as group with the significantly lowest SMN amount.

Still, SMN expression was significantly scaled down in SMA III and asymptomatic siblings. (G) In MN cultures (day 27), the significantly lowest SMN amount was exceptionally detected in SMA III HGK13 although SMA I HGK1 showed diminished SMN expression as well. The SMN expression rates of other discordant family members

ranged at an insignificant medium level mostly. (H) Consistently, phenotypic overview depicted in SMA I and SMA III a significant reduction relative to control r12. Contrarily, the SMN expression of asymptomatic siblings comprised a not significant medium range. Expression was normalised to total RNA levels and significance calculated relative to r12 and healthy controls, respectively (Kruskal-Wallis test, p <0.05 = *, p <0.01 = **). Error bars represented ± SEM of technical duplicates (n=2). MN culture displayed data of one MN differentiation run (n=1). Note that control COII.2 was excluded in MN culture analyses.

7.5.2 PLS3 expression on RNA level in different SMA phenotypes during MN