Translational reading frame determines the pathogenicity of C-terminal frameshift deletions in MeCP2: an alternative therapeutic approach

Reviewer #1 (Public review):

Summary:

The authors scrutinized differences in C-terminal region variant profiles between Rett syndrome patients and healthy individuals and pinpointed that subtle genetic alternation can cause benign or pathogenic output, which harbors important implications in Rett syndrome diagnosis and proposes a therapeutic strategy. This work will be beneficial to clinicians and basic scientists who work on Rett syndrome, and carries the potential to be applied to other Mendelian rare diseases.

Strengths:

Well-designed genetic and molecular experiments translate genetic differences into functional and clinical changes. This is a unique study resolving subtle changes in sequences that give rise to dramatic phenotypic consequences.

Weaknesses:

There are many base-editing and protein-expression changes throughout the manuscript, and they cause confusion. It would be helpful to readers if authors could provide a simple summary diagram at the end of the paper.

Reviewer #2 (Public review):

Summary:

This study by Guy and Bird and colleagues is a natural follow-up to their 2018 Human Molecular Genetics paper, further clarifying the molecular basis of C-terminal deletions (CTDs) in MECP2 and how they contribute to Rett syndrome. The authors combine human genetic data with well-designed experiments in embryonic stem cells, differentiated neurons, and knock-in mice to explain why some CTD mutations are disease-causing while others are harmless. They show that pathogenic mutations create a specific amino acid motif at the C-terminus, where +2 frameshifts produce a PPX ending that greatly reduces MeCP2 protein levels (likely due to translational stalling) whereas +1 frameshifts generating SPRTX endings are well tolerated.

Strengths:

This is a comprehensive and rigorous study that convincingly pinpoints the molecular mechanism behind CTD pathogenicity, with strong agreement between the cell-based and animal data. The authors also provide a proof of principle that modifying the PPX termination codon can restore MeCP2-CTD protein levels and rescue symptoms in mice. In addition, they demonstrate that adenine base editing can correct this defect in cultured cells and increase MeCP2-CTD protein levels. Overall, this is a well-executed study that provides important mechanistic and translational insight into a clinically important class of MECP2 mutations.

Weaknesses:

The adenine base editing to change the termination codon is shown to be feasible in generated cell lines, but has yet to be shown in vivo in animal models.

Reviewer #3 (Public review):

Summary:

Guy et al. explored the variation in the pathogenicity of carboxy-terminal frameshift deletions in the X-linked MECP2 gene. Loss-of-function variants in MECP2 are associated with Rett syndrome, a severe neurodevelopmental disorder. Although 100's of pathogenic MECP2 variants have been found in people with Rett syndrome, 8 recurrent point mutations are found in ~65% of disease cases, and frameshift insertions/deletions (indels) variants resulting in production of carboxy-terminal truncated (CTT) MeCP2 protein account for ~10% of cases. Many of these occur in a "deletion prone region" (DPR) between c.1110-1210, with common recurrent deletions c.1157-1197del (CTD1) and c.1164_1207del (CTD2). While two major protein functional domains have been defined in MeCP2, the methyl-binding domain (MBD) and the NCoR interacting domain (NID), the functional role of the carboxy-terminal domain (CTD, beyond the NID, predicted to have a disordered protein structure) has not been identified, and previous work by this group and others demonstrated that a Mecp2 "minigene" lacking the CTD retains MeCP2 function suggesting that the CTD is dispensable. This raises an important question: If the CTD is dispensable, what is the pathogenic basis of the various CTT frameshift variants? Prior work from this group demonstrated that genetically engineered mice expressing the CTD1 variant had decreased expression of Mecp2 RNA and MeCP2 protein and decreased survival, but those expressing the CTD2 variant had normal Mecp2 RNA and protein and survival. However, they noted that differences between the mouse and human coding sequences resulted in different terminal sequences between the two common CTD, with CTD1 ending in -PPX in both mouse and human, but CTD2 ending in -PPC in human but -SPX in mouse, and in the previous paper they demonstrated in humanized mouse ES cells (edited to have the same -PPX termination) containing the CTD2 deletion resulted in decreased Mecp2 RNA and protein levels. This previous work provides the underlying hypotheses that they sought to explore, which is that the pathological basis of disease causing CTD relates to the formation of truncated proteins that end with a specific amino acid sequence (-PPX), which leads to decreased mRNA and protein levels, whereas tolerated, non-pathogenic CTD do not lead to production of truncated proteins ending in this sequence and retain normal mRNA/protein expression.

In this manuscript, they evaluate missense variants, in-frame deletions, and frame shift deletions within the DPR from the aggregated Genome Aggregated Database (gnomAD) and find that the "apparently" normal individuals within gnomAD had numerous tolerated missense variants and in-frame deletions within this region, as well as frameshift deletions (in hemizygous males) in the defined region. All of the gnomAD deletions within this region resulted in terminal amino acid sequences -SPRTX (due to +1 frameshift), whereas nearly all deletion variants in this region from people with Rett syndrome (from the Clinvar copy of the former RettBase database) had a terminal -PPX sequence, due to a +2 frameshift. They hypothesized that terminal proline codons causing ribosomal stalling and "nonsense mediated decay like" degradation of mRNA (with subsequent decreased protein expression) was the basis of the specific pathogenicity of the +2 frameshift variants, and that utilizing adenine base editors (ABE) to convert the termination codon to a tryptophan could correct this issue. They demonstrate this by engineering the change into mouse embryonic stem cell lines and mouse lines containing the CTD1 deletion and show that this change normalized Mecp2 mRNA and protein levels and mouse phenotypes. Finally, they performed an initial proof-of-concept in an inducible HEK cell line and showed the ability of targeted ABE to edit the correct adenine and cause production of the expected larger truncated Mecp2 protein from CTD1 constructs.

The findings of this manuscript provide a level of support for their hypothesis about the pathogenicity versus non-pathogenicity of some MECP2 CTT intragenic deletions and provide preliminary evidence for a novel therapeutic approach for Rett syndrome; however, limitations in their analysis do not fully support the broader conclusions presented.

Strengths:

(1) Utilization of publicly available databases containing aggregated genetic sequencing data from adult cohorts (gnomAD) and people with Rett syndrome (Clinvar copy of RettBase) to compare differences in the composition of the resulting terminal amino acid sequences resulting from deletions presumed to be pathogenic (n+2) versus presumed to be tolerated (n+1).

(2) Evaluation of a unique human pedigree containing an n+1 deletion in this region that was reported as pathogenic, with demonstration of inheritance of this from the unaffected father and presence within other unaffected family members.

(3) Development of a novel engineered mouse model of a previously assumed n+1 pathogenic variant to demonstrate lack of detrimental effect, supporting that this is likely a benign variant and not causative of Rett syndrome.

(4) Creation and evaluation of novel cell lines and mouse models to test the hypothesis that the pathogenicity of the n+2 deletion variants could be altered by a single base change in the frameshifted stop codon.

(5) Initial proof-of-concept experiments demonstrating the potential of ABE to correct the pathogenicity of these n+2 deletion variants.

Weaknesses:

(1) While the use of the large aggregated gnomAD genetic data benefits from the overall size of the data, the presence of genetic variants within this collection does not inherently mean that they are "neutral" or benign. While gnomAD does not include children, it does include aggregated data from a variety of projects targeting neuropsychiatric (and other conditions), so there is information in gnomAD from people with various medical/neuropsychiatric conditions. The authors do make some acknowledgement of this and argue that the presence of intragenic deletion variants in their region of interest in hemizygous males indicates that it is highly likely that these are tolerated, non-pathogenic variants. Broadly, it is likely true that gnomAD MECP2 variants found in hemizygous males are unlikely to cause Rett syndrome in heterozygous females, it does not necessarily mean that these variants have no potential to cause other, milder, neuropsychiatric disorders. As a clear example, within gnomAD, there is a hemizygous male with the rs28934908 C>T variant that results in p.A140V (p.A152V in e1 transcript numbering convention). This pathogenic variant has been found in a number of pedigrees with an X-linked intellectual disability pattern, in which males have a clear neurodevelopmental disorder and heterozygous females have mild intellectual disability (see PMIDs 12325019, 24328834 as representative examples of a large number of publications describing this). Thus, while their claim that hemizygous deletion variants in gnomAD are unlikely to cause Rett syndrome, that cannot make the definitive statement that they are not pathogenic and completely benign, especially when only found in a very small number of individuals in gnomAD.

(2) The authors focus exclusively on deletions within the "DPR", they define as between c.1110-1210 and say that these deletions account for 10% of Rett syndrome cases. However, the published studies that are the basis for this 10% estimate include all genetic variants (frameshift deletions, insertions, complex insertion/deletions, nonsense variants) resulting in truncations beyond the NID. For example, Bebbington 2010 (PMID: 19914908), which includes frameshift indels as early as c.905 and beyond c.1210. Further specific examples from RettBase are described below, but the important point is that their evaluation of only frameshift variants within c.1110-1210 is not truly representative of the totality of genetic variants that collectively are considered CTT and account for 10% of Rett cases.

(3) The authors say that they evaluated the putative pathogenic variants contained within RettBase (which is no longer available, but the data were transferred to Clinvar) for all cases with Classic Rett syndrome and de novo deletion variants within their defined DPR domain. Looking at the data from the Clinvar copy of RettBase, there are a number (n=143) of c-terminal truncating variants (either frameshift or nonsense) present beyond the NID, but the authors only discuss 14 deletion frameshift variants in this manuscript. A number of these variants have molecular features that do not fall into the pathogenic classification proposed by the authors and are not addressed in the manuscript and do not support the generalization of the conclusions presented in this manuscript, especially the conclusion that the determination of pathogenicity of all c-terminal truncating variants can be determined according to their proposed n+2 rule, or that all of the 10% of people with Rett syndrome and c-terminal truncating variants could be treated by using a base editor to correct the -PPX termination codon.

(4) The HEK-based system utilized is convenient for doing the initial experiments testing ABE; however, it represents an artificial system expressing cDNA without splicing. Canonical NMD is dependent on splicing, and while non-canonical "NMD-like" processes are less well understood, a concern is whether the artificial system used can adequately predict efficacy in a native setting that includes introns and splicing.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors scrutinized differences in C-terminal region variant profiles between Rett syndrome patients and healthy individuals and pinpointed that subtle genetic alternation can cause benign or pathogenic output, which harbours important implications in Rett syndrome diagnosis and proposes a therapeutic strategy. This work will be beneficial to clinicians and basic scientists who work on Rett syndrome, and carries the potential to be applied to other Mendelian rare diseases.

Strengths:

Well-designed genetic and molecular experiments translate genetic differences into functional and clinical changes. This is a unique study resolving subtle changes in sequences that give rise to dramatic phenotypic consequences.

Weaknesses:

There are many base-editing and protein-expression changes throughout the manuscript, and they cause confusion. It would be helpful to readers if authors could provide a simple summary diagram at the end of the paper.

We thank Reviewer #1 for their encouraging comments. As suggested, we will include a summary figure of the genetic changes we have made, and the resulting expression and phenotypic consequences.

Reviewer #2 (Public review):

Summary:

This study by Guy and Bird and colleagues is a natural follow-up to their 2018 Human Molecular Genetics paper, further clarifying the molecular basis of C-terminal deletions (CTDs) in MECP2 and how they contribute to Rett syndrome. The authors combine human genetic data with well-designed experiments in embryonic stem cells, differentiated neurons, and knock-in mice to explain why some CTD mutations are disease-causing while others are harmless. They show that pathogenic mutations create a specific amino acid motif at the C-terminus, where +2 frameshifts produce a PPX ending that greatly reduces MeCP2 protein levels (likely due to translational stalling) whereas +1 frameshifts generating SPRTX endings are well tolerated.

Strengths:

This is a comprehensive and rigorous study that convincingly pinpoints the molecular mechanism behind CTD pathogenicity, with strong agreement between the cell-based and animal data. The authors also provide a proof of principle that modifying the PPX termination codon can restore MeCP2-CTD protein levels and rescue symptoms in mice. In addition, they demonstrate that adenine base editing can correct this defect in cultured cells and increase MeCP2-CTD protein levels. Overall, this is a well-executed study that provides important mechanistic and translational insight into a clinically important class of MECP2 mutations.

Weaknesses:

The adenine base editing to change the termination codon is shown to be feasible in generated cell lines, but has yet to be shown in vivo in animal models.

We thank Reviewer #2 for their positive comments. We agree that an in vivo study demonstrating effective DNA base editing in our CTD-1 mouse model is the obvious next step, and this work is in progress. However, given the ever-increasing use of pre- and neonatal screening for genetic diseases, we felt it important to disseminate our findings as soon as possible. The family pedigree in Figure 3C is a clear demonstration of this need.

Reviewer #3 (Public review):

Summary:

Guy et al. explored the variation in the pathogenicity of carboxy-terminal frameshift deletions in the X-linked MECP2 gene. Loss-of-function variants in MECP2 are associated with Rett syndrome, a severe neurodevelopmental disorder. Although 100's of pathogenic MECP2 variants have been found in people with Rett syndrome, 8 recurrent point mutations are found in ~65% of disease cases, and frameshift insertions/deletions (indels) variants resulting in production of carboxy-terminal truncated (CTT) MeCP2 protein account for ~10% of cases. Many of these occur in a "deletion prone region" (DPR) between c.1110-1210, with common recurrent deletions c.1157-1197del (CTD1) and c.1164_1207del (CTD2). While two major protein functional domains have been defined in MeCP2, the methyl-binding domain (MBD) and the NCoR interacting domain (NID), the functional role of the carboxy-terminal domain (CTD, beyond the NID, predicted to have a disordered protein structure) has not been identified, and previous work by this group and others demonstrated that a Mecp2 "minigene" lacking the CTD retains MeCP2 function suggesting that the CTD is dispensable. This raises an important question: If the CTD is dispensable, what is the pathogenic basis of the various CTT frameshift variants? Prior work from this group demonstrated that genetically engineered mice expressing the CTD1 variant had decreased expression of Mecp2 RNA and MeCP2 protein and decreased survival, but those expressing the CTD2 variant had normal Mecp2 RNA and protein and survival. However, they noted that differences between the mouse and human coding sequences resulted in different terminal sequences between the two common CTD, with CTD1 ending in -PPX in both mouse and human, but CTD2 ending in -PPC in human but -SPX in mouse, and in the previous paper they demonstrated in humanized mouse ES cells (edited to have the same -PPX termination) containing the CTD2 deletion resulted in decreased Mecp2 RNA and protein levels. This previous work provides the underlying hypotheses that they sought to explore, which is that the pathological basis of disease causing CTD relates to the formation of truncated proteins that end with a specific amino acid sequence (-PPX), which leads to decreased mRNA and protein levels, whereas tolerated, non-pathogenic CTD do not lead to production of truncated proteins ending in this sequence and retain normal mRNA/protein expression.

In this manuscript, they evaluate missense variants, in-frame deletions, and frame shift deletions within the DPR from the aggregated Genome Aggregated Database (gnomAD) and find that the "apparently" normal individuals within gnomAD had numerous tolerated missense variants and in-frame deletions within this region, as well as frameshift deletions (in hemizygous males) in the defined region. All of the gnomAD deletions within this region resulted in terminal amino acid sequences -SPRTX (due to +1 frameshift), whereas nearly all deletion variants in this region from people with Rett syndrome (from the Clinvar copy of the former RettBase database) had a terminal -PPX sequence, due to a +2 frameshift. They hypothesized that terminal proline codons causing ribosomal stalling and "nonsense mediated decay like" degradation of mRNA (with subsequent decreased protein expression) was the basis of the specific pathogenicity of the +2 frameshift variants, and that utilizing adenine base editors (ABE) to convert the termination codon to a tryptophan could correct this issue. They demonstrate this by engineering the change into mouse embryonic stem cell lines and mouse lines containing the CTD1 deletion and show that this change normalized Mecp2 mRNA and protein levels and mouse phenotypes. Finally, they performed an initial proof-of-concept in an inducible HEK cell line and showed the ability of targeted ABE to edit the correct adenine and cause production of the expected larger truncated Mecp2 protein from CTD1 constructs.

The findings of this manuscript provide a level of support for their hypothesis about the pathogenicity versus non-pathogenicity of some MECP2 CTT intragenic deletions and provide preliminary evidence for a novel therapeutic approach for Rett syndrome; however, limitations in their analysis do not fully support the broader conclusions presented.

Strengths:

(1) Utilization of publicly available databases containing aggregated genetic sequencing data from adult cohorts (gnomAD) and people with Rett syndrome (Clinvar copy of RettBase) to compare differences in the composition of the resulting terminal amino acid sequences resulting from deletions presumed to be pathogenic (n+2) versus presumed to be tolerated (n+1).

(2) Evaluation of a unique human pedigree containing an n+1 deletion in this region that was reported as pathogenic, with demonstration of inheritance of this from the unaffected father and presence within other unaffected family members.

(3) Development of a novel engineered mouse model of a previously assumed n+1 pathogenic variant to demonstrate lack of detrimental effect, supporting that this is likely a benign variant and not causative of Rett syndrome.

(4) Creation and evaluation of novel cell lines and mouse models to test the hypothesis that the pathogenicity of the n+2 deletion variants could be altered by a single base change in the frameshifted stop codon.

(5) Initial proof-of-concept experiments demonstrating the potential of ABE to correct the pathogenicity of these n+2 deletion variants.

Weaknesses:

(1) While the use of the large aggregated gnomAD genetic data benefits from the overall size of the data, the presence of genetic variants within this collection does not inherently mean that they are "neutral" or benign. While gnomAD does not include children, it does include aggregated data from a variety of projects targeting neuropsychiatric (and other conditions), so there is information in gnomAD from people with various medical/neuropsychiatric conditions. The authors do make some acknowledgement of this and argue that the presence of intragenic deletion variants in their region of interest in hemizygous males indicates that it is highly likely that these are tolerated, non-pathogenic variants. Broadly, it is likely true that gnomAD MECP2 variants found in hemizygous males are unlikely to cause Rett syndrome in heterozygous females, it does not necessarily mean that these variants have no potential to cause other, milder, neuropsychiatric disorders. As a clear example, within gnomAD, there is a hemizygous male with the rs28934908 C>T variant that results in p.A140V (p.A152V in e1 transcript numbering convention). This pathogenic variant has been found in a number of pedigrees with an X-linked intellectual disability pattern, in which males have a clear neurodevelopmental disorder and heterozygous females have mild intellectual disability (see PMIDs 12325019, 24328834 as representative examples of a large number of publications describing this). Thus, while their claim that hemizygous deletion variants in gnomAD are unlikely to cause Rett syndrome, that cannot make the definitive statement that they are not pathogenic and completely benign, especially when only found in a very small number of individuals in gnomAD.

(2) The authors focus exclusively on deletions within the "DPR", they define as between c.1110-1210 and say that these deletions account for 10% of Rett syndrome cases. However, the published studies that are the basis for this 10% estimate include all genetic variants (frameshift deletions, insertions, complex insertion/deletions, nonsense variants) resulting in truncations beyond the NID. For example, Bebbington 2010 (PMID: 19914908), which includes frameshift indels as early as c.905 and beyond c.1210. Further specific examples from RettBase are described below, but the important point is that their evaluation of only frameshift variants within c.1110-1210 is not truly representative of the totality of genetic variants that collectively are considered CTT and account for 10% of Rett cases.

(3) The authors say that they evaluated the putative pathogenic variants contained within RettBase (which is no longer available, but the data were transferred to Clinvar) for all cases with Classic Rett syndrome and de novo deletion variants within their defined DPR domain. Looking at the data from the Clinvar copy of RettBase, there are a number (n=143) of c-terminal truncating variants (either frameshift or nonsense) present beyond the NID, but the authors only discuss 14 deletion frameshift variants in this manuscript. A number of these variants have molecular features that do not fall into the pathogenic classification proposed by the authors and are not addressed in the manuscript and do not support the generalization of the conclusions presented in this manuscript, especially the conclusion that the determination of pathogenicity of all c-terminal truncating variants can be determined according to their proposed n+2 rule, or that all of the 10% of people with Rett syndrome and c-terminal truncating variants could be treated by using a base editor to correct the -PPX termination codon.

(4) The HEK-based system utilized is convenient for doing the initial experiments testing ABE; however, it represents an artificial system expressing cDNA without splicing. Canonical NMD is dependent on splicing, and while non-canonical "NMD-like" processes are less well understood, a concern is whether the artificial system used can adequately predict efficacy in a native setting that includes introns and splicing.

We thank reviewer #3 for their constructive comments. A number of these relate to our analysis of databases of pathogenic (RettBASE) and non-pathogenic (gnomAD) databases. We disagree with their assertion that we are looking at only a small subset of RTT CTD mutations. We detail 14 different RTT CTDs in the manuscript, but these include the 3 most frequently occurring, which alone account for 121 RTT cases in RettBASE.

We used the original RettBASE database for our analysis, which contained significantly more information than was transferred to Clinvar. We may not have made this sufficiently clear and will remedy this during revision of the manuscript.

We stress that RettBASE contained many non-RTT causing mutations. For this reason, we employed stringent selection criteria to define a high-confidence set of RTT CTD alleles. Importantly, this set was chosen before any investigation of reading frame or C-terminal amino acid sequence. Our stringent set was selected based on three criteria: location within the C-terminal deletion prone region (CT-DPR), a diagnosis of Classical RTT and at least one case where that mutation had been shown to be absent from both parents (i.e. that it was a de novo mutation). This excluded a large number of CTD alleles which fitted the +2 frameshift/PPX ending category as well as some in other categories. There are good reasons to believe that the vast majority of genuinely pathogenic RTT CTD mutations do fall into this class.

Concerning gnomAD CTDs, we chose to restrict our detailed analysis to those which are present in the hemizygous state, to exclude individuals which mask a pathogenic mutation due to skewed X-inactivation. Data from all zygosities are shown in Fig. 3, SF1.

We will revise the manuscript to include tables of all extracted data relevant to this region, from both gnomAD and RettBASE, along with analysis of a less stringent set of RettBASE CTDs for reading frame and C-terminal amino acid sequence. We hope this will make clear the strength of the evidence for our conclusions.

We agree with Reviewer #3 that inclusions of variants in gnomAD does not exclude the possibility that they may cause medical/psychiatric conditions other than RTT. This point is already mentioned in the Discussion, but we plan to emphasise it further. The pedigree included in the paper, as well as others that we have learned of, argue that loss of the C-terminus of MeCP2 has few if any phenotypic consequences, but more cases are needed to robustly assess this conclusion.

We disagree that our HEK cell-based system is not suitable for testing efficacy of reagents for use on endogenous alleles in vivo. The editing process is not dependent on splicing, and we have shown in this manuscript that making this single base change has the same effect on an endogenous knock-in allele (CTD1 X>W) or a cDNA-based transgene (Flp-In T-REx CTD1 + base editing), namely, to increase the amount of truncated MeCP2 produced.