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Pathogenesis of novel FMR1 mutations in fragile X syndrome

Myrick, Leila Khoogar (2014)
Dissertation (156 pages)
Committee Chair / Thesis Adviser: Warren, Stephen
Committee Members: Bassell, Gary ; Dunham, Christine ; Jin, Peng ; Lah, James J
Research Fields: Biology, Neuroscience; Biology, Genetics
Keywords: Fragile X Syndrome; missense mutations; FMRP; amino terminal domain; FMR1 sequencing; presynaptic function
Program: Laney Graduate School, Biological and Biomedical Sciences (Neuroscience)
Permanent url: http://pid.emory.edu/ark:/25593/gsd2k

Abstract

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 neurons......... 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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