APPROACHES TO MEASURING THE FREQUENCY OF ACHONDROPLASIA AND HYPOCHONDROPLASIA CAUSING FGFR-3 MUTATIONS IN HUMAN SPERM

APPROACHES TO MEASURING THE FREQUENCY OF
ACHONDROPLASIA AND HYPOCHONDROPLASIA CAUSING FGFR-3
MUTATIONS IN HUMAN SPERM
A Thesis
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Master of Science
The Interdepartmental Program
In Veterinary Medical Sciences
through
The Department of Comparative Biomedical Sciences
by
Andrew T. Daters
B.S., Washington and Lee University, 1997
August 2002

---

ACKNOWLEDGEMENTS
I would like to first thank my parents who gave me the utmost encouragement and
support through all of my academic endeavors. I would also like to thank my patient wife,
Callie, without whom the real funding of this project would not be possible. Finally, I
would like to thank Dr. Vince Wilson who was the only one out there willing to take a
down on his luck student under his wings. And to the one who sat with me from page 1
until the end and gave up many afternoon walks, I would like to thank my dog, Goose.
ii

---

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................ ii
LIST OF TABLES .......................................................................................................... v
LIST OF FIGURES......................................................................................................... vi
ABSTRACT.................................................................................................................... viii
CHAPTER 1. INTRODUCTION ................................................................................ 1
CHAPTER 2. FIBROBLAST GROWTH FACTORS ................................................ 3
CHAPTER 3. FIBROBLAST GROWTH FACTOR RECEPTORS ........................... 5
CHAPTER 4. FIBROBLAST GROWTH FACTOR RECEPTOR 3 .......................... 12
CHAPTER 5. SKELETAL DYSPLASIAS ..............................................................................15
5.1 ACHONDROPLASIA .................................................................................................................15
5.2 HYPOCHONDROPLASIA .........................................................................................................18
CHAPTER 6. MUTATIONS AND DISEASE............................................................ 22
CHAPTER 7. APPROACH ......................................................................................... 33
7.1 APPROACH AT THE HYPOCHONDROPLASIA SITE...........................................................34
7.2 APPROACH AT THE ACHONDROPLASIA SITE...................................................................35
CHAPTER 8. MATERIALS AND METHODS .......................................................... 40
8.1 NEEDLE-IN-A-HAYSTACK ASSAY .......................................................................................40
8.2 HYPOCHONDROPLASIA SITE................................................................................................41
8.2.1. MUTANT PCR AND RESTRICTION ENDONUCLEASE (PCR/RE)
SELECTION OF THE HYPOCHONDROPLASIA SITE ......................................................41
8.2.2 LCR AND PAGE OF THE HYPOCHONDROPLASIA SITE ................................................42
8.3 ACHONDROPLASIA SITE........................................................................................................42
8.3.1 MUTANT PCR AND RESTRICTION ENDONUCLEASE (PCR/RE)
SELECTION OF THE ACHONDROPLASIA SITE ...............................................................42
8.3.2 LCR AND PAGE OF THE ACHONDROPLASIA SITE ........................................................43
CHAPTER 9. RESULTS.............................................................................................. 45
9.1 RESULTS OF THE PCR/RE/LCR OF THE HYPOCHONDROPLASIA SITE.........................45
9.2 INCORPORATION OF A MspI RESTRICTION SITE..............................................................46
9.3 DESIGN OF THE SECOND VERSION OF THE LCR ASSAY AT SITE 1620 .......................53
9.4 LCR RESULTS FOR SITE 1620.................................................................................................53
9.5 DESIGN OF THE THIRD VERSION OF THE LCR ASSAY AT SITE 1620...........................67
9.6 ALLELE SPECIFIC PCR RESULTS FOR SITE 1138...............................................................72
iii

---

CHAPTER 10. DISCUSSION ..................................................................................... 85
CHAPTER 11. CONCLUSIONS................................................................................. 90
REFERENCES................................................................................................................ 92
VITA ............................................................................................................................... 101
iv

---

LIST OF TABLES
Table 1. FGFR-3 Missense mutations diseases.............................................................. 29
Table 2. Version 1 primer sequences and annealing temperatures
for the PCR/RE selection of the C1620A/G base change ................................ 35
Table 3. Primer sets, annealing temperatures, and PCR product length
of the Version 1 PCR protocol for the Hypochondroplasia site....................... 35
Table 4. LCR Version 1 primers and size in base pairs for the
Hypochondroplasia site.................................................................................... 36
Table 5. Sizes of LCR product, from both sense and antisense strands......................... 36
Table 6. Version 1 primer sequences and annealing temperatures
for the PCR/RE selection of the G1138A/C base change ................................ 38
Table 7. Primer sets, annealing temperatures, and PCR product length
of the achondroplasia site................................................................................. 38
Table 8. LCR assay for the achondroplasia site 1138.................................................... 38
Table 9. Sizes of LCR product, from both sense and antisense strands......................... 39
Table 10. Version 2 primer sequences and annealing temperatures
for the PCR/RE selection of the C1620A/G base change .............................. 45
Table 11. Version 3 primer sequences and annealing temperatures .............................. 51
Table 12. LCR Version 2 primers and size in base pairs ............................................... 56
Table 13. Sizes of LCR product, from both sense and antisense strands....................... 56
Table 14. Amount of mutant DNA combined with 6 µg of wild-type DNA................. 67
Table 15. LCR Version 3 primers and size in base pairs ............................................... 69
Table 16. Sizes of LCR product, from both sense and antisense strands....................... 69
Table 17. Primer sequences and sizes for the allele specific PCR technique ................ 73
Table 18. Primer sequences and sizes for the allele specific PCR technique ................ 83
v

---

LIST OF FIGURES
Figure 1. Schematic representation of the FGFR-3 protein ........................................... 6
Figure 2. Schematic representation of a dimerized FGFR-3 complex........................... 9
Figure 3. Schematic representation of the FGFR-3 protein ........................................... 30
Figure 4. Graphic representation of ACH and HCH mutations..................................... 31
Figure 5. LCR primer design for the detection of mutated sequences........................... 37
Figure 6. Agarose gel of the PCR products and restriction products............................. 47
Figure 7. Agarose gel of the PCR products of primer set S-ZR .................................... 48
Figure 8. Agarose gel of the PCR products of primer set T-YR.................................... 49
Figure 9. Autoradiograph a first version LCR ............................................................... 50
Figure 10. Schematic representation of the incorporation of the restriction
endonuclease recognition sequence .............................................................. 52
Figure 11. Agarose gel of successful nested PCR/RE sequences .................................. 54
Figure 12. Agarose gel of successful PCR with primer set S-MspIR ............................ 55
Figure 13. LCR primer design for the detection of mutated sequences ........................ 57
Figure 14. Autoradiograph of a second version LCR testing the Wt and Mt primers ... 59
Figure 15. Autoradiograph of an LCR test..................................................................... 61
Figure 16. Autoradiograph of an LCR run at 64°C........................................................ 62
Figure 17. Autoradiograph of an LCR testing progressively decreasing
concentrations of oligonucleotide standard T .............................................. 64
Figure 18. Autoradiograph of an LCR run at 62°C........................................................ 66
Figure 19. Autoradiograph of the 2nd version LCR with the selection process ............. 68
Figure 20. Autoradiograph of an LCR run at 65°C........................................................ 70
Figure 21. Autoradiograph of an LCR of the selection process..................................... 74
vi

---

Figure 22. Autoradiograph of LCR selection process using a mutant primer mix ........ 75
Figure 23. Schematic representation of the allele specific PCR at site 1138................. 76
Figure 24. Agarose gel of the test run with the allele specific
wild-type and mutant primers at 60°C .......................................................... 78
Figure 25. Agarose gel of the test run with the allele specific
wild-type and mutant primers at 65°C .......................................................... 79
Figure 26. Agarose gel of the test run with the allele specific
wild-type and mutant primers at 58°C .......................................................... 80
Figure 27. Agarose gel of the test run with the allele specific
wild-type and mutant primers at 59°C .......................................................... 81
Figure 28. Agarose gel of the test run with the allele specific wild-type
and mutant primers at 59°C using ½ the amount of template DNA ............. 82
Figure 29. Agarose gel of the test run with second set of the allele
specific wild-type and mutant primers.......................................................... 84
vii

---

ABSTRACT
Achondroplasia and hypochondroplasia are two forms of skeletal dysplasias caused
predominantly by single base mutations in the fibroblast growth factor receptor 3 gene
(FGFR-3). The mutation for achondroplasia is a G1138A/C substitution and the mutation
for hypochondroplasia (occurring about 50% of the time) is a C1620A/G substitution.
Recent genetic studies have shown that spontaneous mutations for achondroplasia and
hypochondroplasia occur exclusively on the paternally derived chromosome, suggesting
that these mutations occur preferentially during spermatogenesis. For unknown reasons,
the mutation rates at these FGFR-3 nucleotides appear to occur at a much higher frequency
than nucleotide specific mutation rates observed in other human genetic diseases.
The purpose of this study was to develop an assay that could detect the frequencies
of achondroplasia and hypochondroplasia causing mutations in human sperm. A Needle-
in-a-Haystack PCR/RE/LCR selection technique has been developed that measures single
base changes, commonly single base substitution mutations, at sensitivities of one mutant
allele in one cell in up to 107 wild-type cells. This technique was modified and designed
for the achondroplasia and hypochondroplasia base sites 1138 and 1620 of the FGFR-3
gene. With the development of this technique, future studies could focus on determining
the frequencies of the mutations in the sperm of fathers of affected children and the
frequencies of the mutations in the sperm of the normal population. These studies will
help elucidate the paternal age effect, have important implications in genetic counseling
and provide a novel method by which to study genetic disease in humans.
viii

---

CHAPTER 1. INTRODUCTION
The fibroblast growth factors are a complex family of mammalian signaling
molecules. Their interaction with their corresponding family of fibroblast growth factor
receptors constitutes a very dramatic cell signaling pathway responsible for many different
molecular processes. Many human disease processes have been linked with faulty growth
factors and/or growth factor receptors. Two such diseases, achondroplasia and
hypochondroplasia, are caused by single base mutations in the fibroblast growth factor
receptor 3 (FGFR-3) gene.
These mutations to the FGFR-3 gene result in the constitutive activation of the
receptor and its subsequent cell signaling pathway. Through a complex series of molecular
events, the insult to FGFR-3 alters the finely tuned process of long bone growth and
development leading to varying forms of short-limb dwarfism.
The incidence of short-limb dwarfism in the population is between 1 in 15,000 and
1 in 40,000 live births. This high degree of incidence makes the FGFR-3 gene one of the
most mutation prone genes in the human genome. In addition to the high rate of mutation,
these mutations in an affected individual are derived from the paternal allele. A paternal
age effect has also been shown for these mutations.
Because the achondroplasia and hypochondroplasia mutations are both derived
from the paternal allele, and because there is an increased incidence with advanced
paternal age, it is believed that germline mutations are responsible for the development of
the disease in offspring. It is not known, however, whether these germline mutations occur
early in the development of sperm stem cells, or later in the processes of spermatogenesis.
1

---

It is also unknown as to whether or not the mutation is found through the entire population
of stem cells, or rather, in an isolated pocket of mutated stem cells.
For these reasons, it would be very beneficial to be able to determine the frequency
of the achondroplasia and hypochondroplasia causing mutations in a population of human
sperm. The ability to compare these frequencies in the sperm of males with affected
offspring, to affected males, and to normal individuals would be very beneficial in
determining the origin and effects of these germline mutations.
This project focused on creating an approach to measuring the frequency of
achondroplasia and hypochondroplasia causing FGFR-3 mutations in human sperm. This
would be accomplished by designing a highly sensitive assay that could detect one mutated
genome in a background population of one million wild-type cells. In future endeavors,
this assay could then be used to measure the frequency of the mutations in the sperm of
fathers with affected offspring, affected individuals, and normal males.
2

---