Assessment of the clinical impact of sequence variants in the FMR1 gene Pubblico
Collins, Stephen Charles (2010)
Published
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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