Assessment of the clinical impact of sequence variants in the FMR1 gene Open Access

Collins, Stephen Charles (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/44558d88m?locale=en
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Abstract


Abstract
Assessment of the clinical impact of sequence variants in the FMR1 gene
By Stephen Charles Collins
Fragile X syndrome, the most common inherited form of developmental delay, is
typically caused by trinucleotide repeat expansion in FMR1. However, little is known
about the clinical significance of sequence variants in FMR1. Only three pathogenic
sequence variants in FMR1 have previously been described: one missense mutation, one
splice site mutation, and one frameshift nonsense mutation. While these variants were
detected in patients with classic fragile X-like features, it is possible that other mutations
may result in a more subtle developmental phenotype. Therefore, to assess the clinical
impact of FMR1 sequence variants, we performed DNA sequencing on large populations
of patients with either a fragile X-like phenotype or general developmental delay.
Through the use of novel high-throughput technologies, namely array-based sequencing
and massively parallel sequencing, we were able to sequence FMR1 in significantly more
patients than was previously feasible. Notably, we detected no pathogenic FMR1
sequence variants in 51 fragile X-like patients. However, in 963 patients with
developmental delay, we detected one missense variant and three promoter variants, all of
which show evidence of a possible functional effect. If a functional effect is verified,
these variants would represent the second FMR1 missense mutation and the first three
FMR1 promoter mutations to cause developmental delay. These data suggest that
sequence variants in FMR1 may indeed be a significant contributor to the heterogeneous
etiology of developmental delay.

Table of Contents


Table of Contents

1. Introduction..................................................................................................................1
1.1 Intellectual Disability: Diagnosis, Prevalence, and Impact................................1
1.2 Etiologies of Intellectual Disability.......................................................................2
1.3 Fragile X Syndrome...............................................................................................4

1.3.1 History of Fragile X Syndrome....................................................................5

1.3.2 Fragile X Syndrome Phenotype...................................................................9

1.3.3 Diagnosing Fragile X Syndrome................................................................13
1.3.4 Managing Fragile X Syndrome..................................................................16
1.4 FMR1.....................................................................................................................18
1.4.1 FMR1Promoter..........................................................................................21
1.4.2 FMR1: CGG Repeat...................................................................................25
1.4.3 Conventional Mutations in FMR1 ............................................................29
1.5 FMRP....................................................................................................................34
1.6 ASFMR1................................................................................................................43
1.7 Proposed Research...............................................................................................45
2. Point mutations in FMR1 are not a major cause of fragile X syndrome..............50
2.1 Introduction..........................................................................................................50
2.2 Subjects and Methods..........................................................................................52
2.2.1 Subjects and Samples..............................................................................52
2.2.2 FMR1LR-PCR........................................................................................54
2.2.3 Sequencing-by-hybridization..................................................................56
2.2.4 Variant Detection and Confirmation.....................................................56

2.2.5 Western Blotting......................................................................................57
2.3 Results...................................................................................................................57
2.3.1 Sequence Accuracy..................................................................................57
2.3.2 Novel FMR1 Sequence Variants.............................................................59
2.3.3 Array-based Deletion Detection..............................................................59
2.4 Discussion.............................................................................................................59
3. Development of Pooled-template Massively Parallel Sequencing for the
Identification of Novel Sequence Variants in a Disease Gene................................64
3.1 Introduction..........................................................................................................64
3.2 Material and Methods.........................................................................................65
3.2.1 Genomic DNA Samples...........................................................................65
3.2.2 LR-PCR Targeting of FMR1..................................................................66
3.2.3 LR-PCR Amplicon Pooling.....................................................................67
3.2.4 Target Library Construction..................................................................68
3.2.5 Genome Analyzer Sequencing and Analysis.........................................68
3.2.6 Variant Detection.....................................................................................69
3.2.7 Variant Confirmation..............................................................................70
3.3 Results...................................................................................................................70
3.3.1 Illumina GA Performance.......................................................................70
3.3.2 GA Sequencing of FMR1 in a Single Individual...................................70
3.3.3 Variant Detection.....................................................................................72
3.4 Discussion.............................................................................................................75

4. Novel missense and promoter variants in FMR1 are associated with
developmental delay...................................................................................................80
4.1 Introduction..........................................................................................................80
4.2 Material and Methods.........................................................................................82
4.2.1 Clinical Population..................................................................................82
4.2.2 Genomic DNA Samples...........................................................................83
4.2.3 Massively-Parallel Sequencing...............................................................83
4.2.4 Control Genotyping.................................................................................83
4.2.5 In Silico Analysis......................................................................................85
4.2.6 Clinical Assessment..................................................................................85
4.2.7 Luciferase Assays.....................................................................................86
4.2.7.1 Plasmid Construction..................................................................86
4.2.7.2 Cell Culture and Transfections...................................................86
4.2.7.3 Luciferase Assays.........................................................................87
4.3 Results...................................................................................................................87
4.3.1 Sequence Variants in FMR1...................................................................87
4.3.2 Characterization of the Novel Missense Variant p.R138Q..................98
4.3.2.1 Identification and Preliminary Functional Evaluation............98
4.3.2.2 Clinical Evaluation....................................................................101
4.3.2.3 Patient Pedigree.........................................................................102
4.3.3 Novel Variants in the FMR1 Promoter................................................105
4.3.3.1 Identification and Preliminary Functional Evaluation..........105
4.3.3.2 Effects on Promoter Activity.....................................................108

4.3.4 Noncoding Variants in FMR1...............................................................108
4.4 Discussion...........................................................................................................110
5. Polymorphic missense and frameshift variants in the putative ASFMR1-encoded
protein refute its proposed role in fragile X syndrome........................................116
5.1 Introduction........................................................................................................116
5.2 Methods and Results..........................................................................................117
5.3 Discussion...........................................................................................................125
6. Concluding Remarks...............................................................................................127
6.1 Summary.............................................................................................................127
6.2 Future Directions...............................................................................................130
6.3 A final word........................................................................................................133
7. References.................................................................................................................135












Figures

1.1. History of Fragile X Syndrome....................................................................................6
1.2. Diagram of FMR1......................................................................................................19
1.3. Conservation of the FMR1 promoter..........................................................................22
1.4. Diagram of FMRP......................................................................................................37
1.5. Diagram of the overlapping locus of ASFMR1 and FMR1........................................44
1.6. Mammalian conservation of the putative ASFMR1 ORF protein..............................46
2.1. Diagram of FMR1.......................................................................................................55
2.2. FMRP absent in patient harboring a 5'UTR deletion in FMR1..................................61
3.1. Base-by-base error rate for pooled-template MPS......................................................73
3.2. Error-prone bases are common sites of false positives...............................................76
4.1. Functional implications of the p.R138Q missense change.........................................99
4.2. Clinical implications and inheritance of the p.R138Q missense change..................103
4.3. Novel variants in the FMR1 promoter......................................................................106
4.4. Luciferase assays of the variant FMR1 promoters. ..................................................109
5.1. Diagram of ASFMR1 and FMR1...............................................................................118
5.2. Mammalian conservation of the putative ASFMR1 ORF protein.............................120
5.3. Missense and truncating variants found in the ASFMR1 protein.............................124





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