Given that neither case is derived from multiplex families
Given that neither case is derived from multiplex AVL-301 with affected individuals, principles of Mendelian segregation cannot be applied to implicate the T201S mutation in causing familial CJD. In addition, like many rare gene variants, it is not possible to apply practice guidelines such as the American College of Medical Genetics and Genomics for novel gene variants (Richards et al., 2015) in the case of T201S, due to insufficient data to combine criteria for stratification (Table 1). With T201S in particular, there is lack of segregation, functional, de novo, and computational and predictive data to satisfy the stipulated American College of Medical Genetics and Genomics criteria. Moreover, it should be pointed that highly penetrant PRNP mutations cause disease by unknown mechanisms that result in a conformational structural change, rather than by simple loss- or gain-of-function mechanisms in which functional and computational data can be more tractable to study in cellular models. Interrogation of PrP structure and its perturbations by missense mutations have largely focused on stabilization/destabilization of the native PrP structure. However, these studies using recombinant PrP show that stabilization/destabilization of the native PrPC structure is not consistently observed across all known pathogenic mutations (Liemann and Glockshuber, 1999). Furthermore, observations made from these models may not be applicable to real life, as recombinant PrP is unanchored to the cell membrane and unglycosylated, and certainly could fail to capture all of the folding problems encountered in vivo. Alternatively, the disease-associated mutations may primarily affect the stability of more relevant on-pathway folding intermediates (Hart et al., 2009). As such, the pathogenicity of T201S cannot be completely ruled out based solely on PrP structural considerations, despite the seemingly minor perturbation of the native PrPC structure by the T201S substitution. Nevertheless, we showed that it is possible to produce both qualitative and quantitative estimates of pathogenicity and penetrance for T201S respectively, by harnessing data from multiple lines of evidence specific for prion disease. Different prion strains can propagate in the same host to produce different disease phenotypes and appear to be encoded by distinct abnormal PrP conformations and assembly states (Collinge, 2016, Collinge and Clarke, 2007, Prusiner, 1998). Different human PrPSc isoforms associated with phenotypically distinct forms of human prion disease (molecular strain types) have considerable diagnostic utility and are classified by both the fragment size and ratio of the 3 principal PrP bands seen after protease digestion (Hill et al., 2006, Hill et al., 2003). Variations in the primary sequence of human PrP profoundly affect the ability of the expressed protein to propagate particular prion strains through conformational selection (Collinge, 1999, Collinge, 2001, Collinge, 2016, Collinge and Clarke, 2007, Wadsworth and Collinge, 2011, Wadsworth et al., 2004, Wadsworth et al., 2010). The codon 129 polymorphism (either methionine or valine) determines the ability of wild-type human PrP to propagate particular prion strains in patients with sporadic or acquired forms of prion disease while highly penetrant missense mutations that cause IPD (Mead, 2006), for example, P102L, E200K, and D178N, impose additional conformational preferences for PrP assemblies, resulting in PrPSc molecular strain types that are distinct from those propagated in patients with sporadic or acquired etiologies (Asante et al., 2015, Asante et al., 2009, Hill et al., 2006, Wadsworth et al., 2010, Wadsworth et al., 2006). Immunoblot and glycoform analyses showed that PrPSc from T201S brain tissue resembled that of type 2 sCJD 129MM rather than that seen in highly penetrant PRNP point mutations, for example, E200K. This dissimilarity is further reinforced by the absence of white matter filamentous PrP deposits on neuropathology, although it can be argued that this observation has limited negative predictive value for variants toward the carboxy-terminal of PRNP (Reiniger et al., 2013). PrP structural analyses also suggest that the resulting amino acid substitution is not expected to impart a significant change in PrP conformation.