5. Estado del Arte y Marco Teórico
5.1.9. Caracterización de Organizaciones Solidarias
The enrichment of the rs1990622 risk allele in GRN+ FTLD-TDP cases, as described above, was initially surprising, given that GRN mutations are highly penetrant (Cruts et al., 2006; Gass et al., 2006). Why would a disease risk allele that exerts only a small effect on disease risk (the odds ratio for the risk allele of rs1990622 is 1.64), be overrepresented (or underrepresented) in individuals with a Mendelian disease-causing mutation? One potential explanation is that TMEM106B genotype plays a disease-modifying role in the presence of pathogenic GRN mutations. Such a mechanism is plausible, given the extremely wide range of ages at which GRN mutation carriers develop FTLD symptoms (Benussi et al., 2015). Consistent with this hypothesis, two studies published shortly after the FTLD-TDP GWAS reported that the rs1990622 risk allele is associated with a ~12-13 year earlier age at disease onset in GRN+ FTLD-TDP cases (Cruchaga et al., 2011; Finch et al., 2011). An epistatic interaction between TMEM106B and GRN is further supported by the findings summarized in Chapter 5.1.1, as well as the cellular co-localization of TMEM106B and PGRN within LAMP-1+ organelles (Chen-Plotkin et al., 2012), which mark late endosomes and lysosomes (Lamb et al., 2013). Thus, TMEM106B genotype not only affects risk of developing FTLD-TDP, but also affects the clinical presentation of a Mendelian form of FTLD-
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TDP. Finch et al. (2011) reported that while 19.1% of control individuals are homozygous for the rs1990622 protective allele, only 2.6% of GRN+ FTLD-TDP individuals have this specific genotype (P=0.009) (Finch et al., 2011). While it is unclear whether the protective haplotype simply delays age at onset or reduces the penetrance of the GRN mutations, these results suggest an interaction between TMEM106B and GRN in FTLD-TDP pathogenesis.
As discussed in Chapter 1, the three major genetic causes of FTLD are mutations in the MAPT, GRN, and C9orf72 genes, with C9orf72 being the most common cause of FTLD, ALS, and FTLD/ALS (Tan et al., 2017). Importantly, disease-causing hexanucleotide repeat expansion (HRE) mutations result in TDP-43 pathology (Al-Sarraj et al., 2011), and, similar to GRN mutations, are likely not fully penetrant, given that they are present in some sporadic FTLD cases (Tan et al., 2017) and have been found in neurologically normal individuals of advanced age (Beck et al., 2013; Harms et al., 2013; Majounie et al., 2012). Thus, it is possible that TMEM106B haplotype affects the clinical manifestation of C9orf72-associated FTLD (C9orf72+ FTLD-TDP) in addition to GRN+ FTLD-TDP.
To test this hypothesis, I investigated whether there is an association between rs1990622 genotype and age at onset or age at death in C9orf72+ FTLD-TDP cases (DeJesus-Hernandez et al., 2011; Renton et al., 2011). In both discovery and replication cohorts, I observed a statistically significant association between rs1990622 and both age at death and age at disease onset. Surprisingly, each copy of the rs1990622 risk allele delayed age at death and age at disease onset, which is opposite the direction seen in GRN+ FTLD-TDP (Gallagher et al., 2014). In other words, in the presence of pathogenic C9orf72 HREs, the genotype that increases risk for FTLD-TDP more generally is actually protective against disease manifestation.
These results were specific to C9orf72+ FTLD-TDP, as rs1990622 genotype had no effect on age at death or age at onset in mutation-negative FTLD-TDP. At the same time, I also observed a significant enrichment of the rs1990622 risk allele in C9orf72 HRE carriers, and confirmed the previously published enrichment of the risk allele in GRN+ and mutation-negative FTLD-TDP patients (Gallagher et al., 2014). Importantly, an independent study from another group confirmed
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the enrichment of the rs1990622 risk allele in C9orf72+ FTLD-TDP (van Blitterswijk et al., 2014). This group did not observe an effect of rs1990622 genotype on age at disease onset, but the authors performed their analyses by combining C9orf72+ FTLD and ALS cases, thus potentially precluding the ability to detect a disease-specific effect (van Blitterswijk et al., 2014).
As mentioned above, the protective effects of the rs1990622 risk allele in C9orf72+ FTLD- TDP seem counterintuitive, especially given that the risk allele is enriched in C9orf72 HRE carriers. How could a particular genetic variant (representing a particular haplotype) increase risk of developing disease in large cohorts consisting of both Mendelian and non-Mendelian cases (Finch et al., 2011; Van Deerlin et al., 2010; van der Zee et al., 2011), while at the same time protect against the manifestation of disease in individuals with a specific disease-causing mutation? In the case of GRN mutation carriers, the enrichment of the rs1990622 risk allele is consistent with the earlier age at disease onset seen in risk allele carriers – an individual with a GRN mutation is more likely to develop FTLD-TDP at any given age if he/she has the TMEM106B risk haplotype, thus creating ascertainment bias in the clinic. Indeed, the scarcity of protective allele homozygotes among GRN+ FTLD-TDP cases (Finch et al., 2011) suggests that some individuals with the protective haplotype might never present with clinical FTLD symptoms. However, this type of effect cannot explain the C9orf72 result, in which the rs1990622 risk allele is 1) enriched in C9orf72+ FTLD-TDP cases, but 2) appears to delay both age at disease onset and age at death.
Several possible explanations may be compatible with these results. First, we may be observing an artifact of limited sample size. We note, however, that our study, with 30 clinical sites around the world, is very large (and likely the largest possible in this disease), and the differences in local clinical practice would tend to bias towards a negative result. Alternatively, the enrichment of the rs1990622 risk allele in C9orf72 HRE carriers, combined with the protective effects of the risk allele with regards to disease manifestation, may indicate that the TMEM106B FTLD-TDP risk haplotype confers a fitness advantage in individuals with the C9orf72 HRE.
While epistatic interactions have been reported for other Mendelian diseases, specifically cystic fibrosis (Cutting, 2010) and sickle cell disease (Sankaran et al., 2010), the interaction
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between TMEM106B and C9orf72 appears to be a rarer case of “sign epistasis”, in which the phenotypic outcome of the interaction depends on genetic background (Weinreich et al., 2005). Specifically, the TMEM106B risk haplotype appears to be deleterious in the absence of the C9orf72 HRE (thus, its association with increased risk of FLTD-TDP), whereas in the presence of the C9orf72 HRE, it is beneficial. Interestingly, recent cell biological experiments performed in my lab support a sign epistatic relationship for TMEM106B and C9orf72. As will be discussed in more detail in Chapter 5.3, overexpression of TMEM106B in various cell types leads to enlarged LAMP- 1+ late degradative organelles. These organelles fail to acidify properly, resulting in impaired protein degradation and cell death (Brady et al., 2013; Busch et al., 2016; Chen-Plotkin et al., 2012; Stagi et al., 2014; Suzuki and Matsuoka, 2016). However, siRNA-mediated knockdown of C9orf72 rescues these phenotypes (Busch et al., 2016), and it is worth noting that human C9orf72 HRE carriers have reduced C9orf72 protein levels, presumably because the expanded allele cannot be efficiently translated into functional protein (Ciura et al., 2013; DeJesus-Hernandez et al., 2011; Donnelly et al., 2013; Gijselinck et al., 2012; Tran et al., 2015). These results suggest that both TMEM106B and C9orf72 play roles in lysosomal function, and that in the presence of C9orf72 HREs, which reduce C9orf72 protein levels, the TMEM106B risk haplotype (and resulting increased TMEM106B levels, see Chapter 5.2) may help restore lysosomal homeostasis. In summary, evidence from human genetics, as well as cellular biological experiments, support a disease- modifying role of TMEM106B in C9orf72+ FTLD-TDP.
Importantly, the association between rs1990622 genotype and age at onset and/or age at death in both C9orf72+ and GRN+ FTLD-TDP also strongly suggests that the causal variant influencing disease risk and manifestation at the TMEM106B locus is common, since these genetic modifier effects were observed in cohorts of only ~50-100 individuals (Cruchaga et al., 2011; Finch et al., 2011; Gallagher et al., 2014). As mentioned above, the TMEM106B risk SNP alleles are very common in the population; thus, a causal variant in strong LD with the GWAS risk SNPs could potentially explain these genetic modifier effects, whereas a rare causal variant could not. This is an important point, since an alternative interpretation of GWAS is that common variants may
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associate with disease risk as a result of being in low or partial LD with a rare causal variant of strong effect (Gibson, 2012). Thus, it can be difficult to determine a priori whether a GWAS causal variant is in strong LD with the disease-associated SNPs (which is typically assumed, but not proven), or driven by a “synthetic association” due to partial LD with a rare causal variant. However, since the associations of TMEM106B SNPs with FTLD-TDP 1) risk and 2) age at onset and death are likely driven by the same functional variant(s), these genetic modifier effects effectively rule out the possibility of a rare causal variant at this locus.