Pathogenesis of novel FMR1 mutations in fragile X syndrome Open Access

Myrick, Leila Khoogar (2014)

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Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability (ID) and also the leading monogenic cause of autism spectrum disorder. In most cases, FXS is caused by a trinucleotide repeat expansion within the FMR1 gene that causes transcriptional silencing and loss of the encoded protein, FMRP. FMR1 missense mutations that disrupt FMRP function are also expected to cause FXS, however, in over two decades since FMR1 was discovered, only a single pathological missense mutation has been reported (I304N). Recently we identified two novel variants, G266E and R138Q, in males with ID who tested negative for repeat expansion. To determine if these variants are pathological, we infected Fmr1 KO cells with either G266E-FMRP or R138Q-FMRP lentivirus. We found that G266E behaves like a functional null and is unable to rescue any of the functions associated with FMRP's canonical role as a postsynaptic translation regulator. Specifically G266E failed to rescue AMPAR trafficking, associate with polyribosomes, or bind mRNA. We also modeled the G266E mutation onto crystallographic data of FMRP's KH1-KH2 domain and found this mutation is likely to cause significant structural disruption. Conversely, R138Q-FMRP rescued all the phenotypes associated with translation regulation. However, when neuronally expressed in transgenic dfmr1-deficient Drosophila, R138Q did not rescue synaptic overgrowth at the neuromuscular junction, suggesting this mutation specifically impairs FMRP's presynaptic function. Electrophysiological and biochemical studies confirmed the presynaptic defect and revealed that R138Q-FMRP is unable to modulate action potential duration through loss of interaction with presynaptic BKCa channels. In order to determine if R138Q causes any structural changes, we solved the crystal structure for the amino terminal domain where R138Q resides. We did not find any significant conformational changes between wild-type or R138Q crystal structures, however we unexpectedly discovered a novel RNA binding domain instead. In conclusion, G266E is a pathological null mutation while R138Q is a partial loss-of-function mutation that confers isolated loss of FMRP presynaptic function. These results separate the pre- and postsynaptic functions of FMRP and demonstrate investigational and clinical utility of screening for conventional FMR1 mutations in individuals with ID.

Table of Contents

Chapter 1. Introduction and Background 1

1.1 Fragile X syndrome 2

1.1.1 Clinical description 2

1.1.2 Genetics of FXS 4

1.2 Fragile X Mental Retardation Protein (FMRP) 6

1.2.1 Structure and domains 6

1.2.2 KH domains 7

1.2.3 RGG domain 9

1.2.4 Amino-terminal domain 11

1.2.5 FMRP Function 12

1.3 Conventional FMR1 Mutations 15

1.3.1 Gross and small FMR1 deletions 15

1.3.2 FMR1 mutation screening 17

1.3.3 FMR1 point mutations 19

1.3.4 Proposed research 22

Chapter 2. Fragile X syndrome due to a G266E missense mutation 28

2.1 Abstract 29

2.2 Introduction 30

2.3 Methods 32

2.3.1 Cloning and lentivirus production 32

2.3.2 Cell culture 32

2.3.3 AMPA receptor trafficking 32

2.3.4 Polyribosome profiling and western blotting 33

2.3.5 RNA co-immunoprecipitation and quantitative RT-PCR 34

2.3.6 List of primer sequences 35

2.4 Results 36

2.4.1 Identification of a patient with a novel FMR1 missense mutation (G266E) 36

2.4.2 Functional analysis of mutant G266E-FMRP 37

2.4.3 Structural analysis of mutant G266E-FMRP 39

2.5 Discussion 39

Chapter 3. An independent role for presynaptic FMRP revealed by R138Q FMR1 missense mutation associated with intellectual disability and seizures 50

3.1 Abstract 51

3.2 Introduction 52

3.3 Methods 55

3.3.1 Patient description 55

3.3.2 Constructs 57

3.3.3 Animals 58

3.3.4 Cell culture 59

3.3.5 AMPA receptor trafficking 59

3.3.6 Polyribosome profiling and western blotting 59

3.3.7 RNA co-immunoprecipitation and quantitative RT-PCR 60

3.3.8 Drosophila neuromuscular junction immunostaining and analysis 62

3.3.9 Electrophysiology slice preparation 63

3.3.10 Action potential recordings and analysis 63

3.3.11 Protein expression and purification 64

3.3.12 Binding Assay 65

3.3.13 List of primer sequences 66

3.4 Results 66

3.4.1 Identification of ID patient with R138Q missense mutation 66

3.4.2 R138Q mutation does not affect postsynaptic functions of FMRP 67

3.4.3 R140Q mutation impairs presynaptic function in Drosophila 69

3.4.4 R138Q mutation impairs presynaptic function in mouse central neuron 70

3.4.5 R138Q mutation disrupts FMRP interaction with BKCa channels 71

3.5 Discussion 73

3.5.1 Summary of results 73

3.5.2 Insights into FXS pathophysiology from FMR1 missense mutations 74

3.5.3 BKCa channel dysfunction in ID and seizures 74

3.5.4 Amino-terminal domain and FMRP function 76

Chapter 4. Crystal structure of wild-type and R138Q mutant FMRP amino terminal domain 88

4.1 Abstract 89

4.2 Introduction 90

4.3 Methods 92

4.3.1 Protein expression and purification 92

4.3.2 Protease digestion and mass spectrometry 93

4.3.3 Crystallography 94

4.4 Results and Discussion 95

4.4.1 FMRP amino terminal region forms an integral domain containing two tandem Agenet modules and a novel KH module 95

4.4.2 FMRP tandem Agenet domain is structurally similar to other tandem Tudor domains that bind methylated lysine 97

4.4.3 FMRP has a KH0 module that may participate in binding of nucleic acids 98

4.4.4 Functionally relevant mutation of the FMRP amino terminal domain 99

4.5 Summary 100

Chapter 5. Discussion 113

5.1 Summary of Findings 114

5.2 Future Directions 116

5.2.1 Separation of pre- and postsynaptic FMRP function 116

5.2.2 Links between FMRP presynaptic function and seizure susceptibility 117

5.2.3 Amino terminal domain interactions with chromatin and RNA 120

5.3 Significance 122

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