The Genetics of Obesity in Saudi Arabian Population

316 Pages·2014·11.39 MB·English

The Genetics of Obesity in Saudi
Arabian Population

Haya Al-Saud

Thesis Submitted for the degree of Doctor of Philosophy

Department of Genomics of Common Disease
School of Public Health
Faculty of Medicine
Imperial College London
December 2013
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Copyright Declaration
Copyright Declaration
‘The copyright of this thesis rests with the author and is made available under a Creative
Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy,
distribute or transmit the thesis on the condition that they attribute it, that they do not use it
for commercial purposes and that they do not alter, transform or build upon it. For any reuse
or redistribution, researchers must make clear to others the licence terms of this work’
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Abstract
Abstract

Obesity is a highly heritable and genetically heterogeneous disorder. Reports have shown
that the number of obese individuals will project to worldwide high levels in the next
couple of years.

In this study I tried to identify missing heritability in obesity by looking at extreme
phenotypes that are likely to be enriched with rare variants. This will improve the power of
their discovery by identifying the loci that may reveal more rare variants. I employed the
strategy of using “extreme” sub-groups of obese patients to identify known and novel loci
for obesity in the population of Saudi Arabia. Two different approaches were used. The
first was to investigate the contribution of copy number variation (CNV) to obesity in Saudi
adults/children that have mental retardation, dysmorphic features and obesity or morbid
obesity: for adults, this was defined as body mass index (BMI) ≥ 30 and ≥ 40 kg.m−2
respectively; for children, BMI respectively above the 90th and 97th percentiles. The second
was to look for known and new Mendelian forms of obesity in multiplex consanguineous
families in children that have extreme obesity (z-BMI above 97th centile).

Genome-wide genotyping was performed using the Affymetrix SNP 6.0 Chip platform
(AFFYMETRIX, Santa Clara, CA) to discover novel copy number variants using
PennCNV. I also used a whole exome sequencing in combination with the detection of runs
of homozygosity (ROH) and linkage analysis to screen for known monogenic and possibly
novel obesity genes in multiplex Saudi consanguineous families.

Two rare obesity causing CNVs were identified from this study. The first was the known
chromosome 16p11.2 heterozygous deletion that has been previously shown to be
associated with obesity. The second is a novel rare homozygous CNV deletion that
encompasses TRIB3 gene that has been previously associated with diabetes. A chromosome
16p11.2 duplication was also found within the known chromosome 16p11.2 deletion. This
duplication helped in narrowing down the obesity-causing region within the chromosome
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Abstract
16p11.2 deletion region. Additionally, rare CNV duplication with a size above 500kb were
found to be slightly enriched (P=0.017) in cases versus controls.

In this study we have also shown the presence of a novel homozygous missense MC4R
mutation in combination with a heterozygous missense LPIN3 mutation within the same
family. The carriers of MC4R mutation have developed an extreme obesity phenotype on
the other hand the carriers of LPIN3 mutation also carried a heterozygous MC4R mutation
and were found to be lean suggesting a reverse effect of the LPIN3 gene on the
development of obesity. In another family a heterozygous deletion was found in a possibly
novel obesity-causing gene CHRNA7. This gene mainly mediates fast signalling
transmission at synapses and is found particularly in the lateral hypothalamus that regulates
food appetite.

In summary, this thesis presents multiple novel CNVs and genes that are found as
promising obesity causing variants. It is hoped that the work from this thesis may
contribute towards elucidation of the role of CNVs and novel genes in the development of
obesity.
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Table of Contents
Table of Contents
Copyright Declaration ................................................................................................................................... 2
Abstract .............................................................................................................................................. 3
Table of Contents ............................................................................................................................. 5
List of Tables ..................................................................................................................................... 9
List of Figures ................................................................................................................................. 12
Acknowledgements ...................................................................................................................... 21
Declaration of originality ........................................................................................................... 22
Publications .................................................................................................................................... 23
Abbreviations ................................................................................................................................. 24
1 Introduction ........................................................................................................................... 27
1.1 Frequency of obesity and how it became a pandemic .................................................. 27
1.2 Morbidity and mortality of obesity ..................................................................................... 27
1.3 Physiological basis of obesity ............................................................................................... 28
1.4 Environmental Factors ............................................................................................................ 31
1.5 Heritability of obesity .............................................................................................................. 32
1.6 Genetic basis of obesity ........................................................................................................... 32
1.6.1 Monogenic obesity .............................................................................................................................. 32
1.6.2 Syndromic forms of obesity ............................................................................................................ 35
1.6.3 Polygenic obesity ................................................................................................................................. 38
1.6.4 Genome-wide association studies ................................................................................................ 40
1.7 The role of copy number variation in the development of obesity ......................... 44
1.7.1 Copy number variation in the human genome ....................................................................... 45
1.7.2 Mechanism of CNV formation ........................................................................................................ 50
1.7.3 Methods of CNV prediction and validation ............................................................................... 51
1.7.4 The role of copy number variation in the development of obesity ................................ 52
1.8 Strategy to identify monogenetic determinants of obesity ........................................ 53
1.8.1 Gene discovery ..................................................................................................................................... 54
1.9 Overall Aim .................................................................................................................................. 63
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Table of Contents
1.10 Specific objectives ..................................................................................................................... 63
2 Materials and Methods ....................................................................................................... 65
2.1 Study subjects ............................................................................................................................. 65
2.1.1 Mental retardation and extreme obesity in Saudi adult and child cases and controls
65
2.1.2 Saudi consanguineous families with extreme obesity ......................................................... 66
2.1.3 Saudi WES normal controls............................................................................................................. 68
2.1.4 Replication samples ........................................................................................................................... 68
2.2 DNA extraction ........................................................................................................................... 69
2.3 Genotyping arrays ..................................................................................................................... 69
2.4 Prediction of rare obesity causing CNVs ........................................................................... 70
2.4.1 Data normalisation and quality control ..................................................................................... 72
2.4.2 Generation of Log R Ratio and B-allele frequency from Affymetrix data..................... 73
2.4.3 CNV prediction algorithms used to detect CNVs from different Affymetrix platforms
74
2.4.4 Filtration and merging of adjacent CNVs .................................................................................. 76
2.4.5 Removal of spurious CNV calls and identification of overlapping/neighbouring
genes 76
2.4.6 Identification of obesity candidate CNVs .................................................................................. 77
2.4.7 CNV validation using different techniques ............................................................................... 78
2.4.8 Assessment of Global Burden of Large CNVs ........................................................................... 84
2.5 Prediction of Monogenic obesity genes: ............................................................................ 84
2.5.1 Linkage Analysis .................................................................................................................................. 86
2.5.2 Whole Exome Sequencing ............................................................................................................... 92
2.5.3 Generation of variant calls from whole exome sequencing data using genome quest
95
2.5.4 PCR-based validation of candidate genes identified from whole exome sequencing
98
2.5.5 Sequencing ........................................................................................................................................... 103
3 Monogenic Forms of Obesity in Consanguineous Saudi Families with Extreme
Obesity ........................................................................................................................................... 106
3.1 Introduction ............................................................................................................................. 106
3.2 Whole genome SNP array quality control ...................................................................... 107
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Table of Contents
3.3 Discovery of Genomic Regions of Interest ..................................................................... 108
3.3.1 Informativeness of SNPs ................................................................................................................ 108
3.3.2 Linkage analysis ................................................................................................................................. 110
3.3.3 Runs of Homozygosity ..................................................................................................................... 128
3.4 Whole Exome Sequencing Variant Discovery ............................................................... 137
3.4.1 WES output and target coverage ................................................................................................ 137
3.4.2 Variant Calling from GenomeQuest ........................................................................................... 139
3.4.3 Filtering and prioritisation of candidate obesity-causing variants .............................. 141
3.4.4 Candidate Variants ........................................................................................................................... 143
3.5 Validation of variants found in candidate obesity genes ......................................... 150
3.5.1 Known monogenic obesity genes ............................................................................................... 151
3.5.2 Potential obesity-causing genes .................................................................................................. 153
3.6 Discussion ................................................................................................................................. 159
3.6.1 Linkage and ROH regions ............................................................................................................... 159
3.6.2 WES variant detection ..................................................................................................................... 161
3.6.3 Linkage analysis and sequencing of the probands .............................................................. 162
3.6.4 Obesity-causing variants ................................................................................................................ 164
3.7 Summary .................................................................................................................................... 170
3.8 Limitations ................................................................................................................................ 170
3.9 Future work .............................................................................................................................. 171
3.9.1 Re-run of whole exome sequencing .......................................................................................... 171
3.9.2 Identification of underlying mechanisms ............................................................................... 171
4 The Contribution of Rare Copy Number Variants to Obesity Susceptibility . 174
4.1 Introduction ............................................................................................................................. 174
4.2 Data normalisation and quality control ......................................................................... 177
4.3 Frequency of copy number variation in cases versus controls.............................. 178
4.4 CNV prediction ......................................................................................................................... 182
4.4.1 Stage I: Exclusion of samples with obesity-related or other known syndromes .... 182
4.4.2 Stage II: Exclusion of all genomic variants found in normal non-diseased samples
185
4.5 Assessment of CNV candidacy ............................................................................................ 186
4.5.1 CNVs shared between two samples including genes.......................................................... 186
4.5.2 CNVs in one sample including genes ......................................................................................... 187
4.5.3 CNVs shared between two samples not including genes .................................................. 187
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4.5.4 CNVs in one sample not including genes ................................................................................. 187
4.6 Creation of University of California, Santa Cruz browser tracks ........................... 191
4.7 Identification of rare CNVs which may disrupt obesity candidate genes ........... 193
4.7.1 CNVs shared between two samples including genes.......................................................... 193
4.8 Investigation and validation of rare CNVs that may disrupt obesity candidate
genes 206
4.8.1 Rare homozygous deleted CNVs ................................................................................................. 206
4.8.2 Rare heterozygous 16p11.2 CNV deletion previously associated with obesity ...... 213
4.9 Genome-wide analysis of large CNVs from the Saudi mental retardation and
extreme obesity cohort ....................................................................................................................... 217
4.9.1 Merging of large CNVs predicted by PennCNV prediction algorithm .......................... 217
4.9.2 Large copy number variants identified in Saudi extreme obesity and mental
retardation cohort ............................................................................................................................................ 221
4.9.3 Assessment of global burden of large CNVs in obesity cases and controls in the
Saudi extreme obesity mental retardation cohort .............................................................................. 224
4.10 Discussion ................................................................................................................................. 225
4.11 Summary .................................................................................................................................... 230
4.12 Limitations ................................................................................................................................ 231
4.13 Future work .............................................................................................................................. 231
4.13.1 Samples ............................................................................................................................................ 231
4.13.2 Use of other CNV detection algorithms ............................................................................... 231
4.13.3 Replication in other Cohorts ................................................................................................... 232
4.13.4 Validation of 16p11.2 obesity-associated GSVs .............................................................. 232
5 Conclusion ............................................................................................................................ 234
References: ................................................................................................................................... 239
Appendix A ................................................................................................................................... 257
Appendix B ................................................................................................................................... 291
Appendix C .................................................................................................................................... 301

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List of Tables
List of Tables

Table 1-1 Genomic regions encompassing genes involved in the development of obesity and
developmental delay/intellectual disability...................................................................... 37
Table 1-2 Details of 32 BMI and 14 waist-hip ratio (WHR) loci detected from recent GWAS
scans. ................................................................................................................................ 42
Table 1-3 A selection of CNV studies published up to date. ................................................... 49
Table 1-4 Some diseases identified through whole exome. ..................................................... 57
Table 2-1: Summary of Saudi MR and extreme obesity cases and control samples ............... 66
Table 2-2: Frequency of different phenotypic data collected from Saudi MR and
extreme obesity samples ................................................................................................ 66
Table 2-3: Summary of Saudi consanguineous families with extreme obesity - cases and
control samples ................................................................................................................ 68
Table 2-4: Summary of replication samples from King Saud University ............................... 68
Table 2-5: Summary of genotyping arrays used in the study .................................................. 70
Table 2-6: MLPA probes in the P343-C1 AUTISM-1 SALSA MLPA kit from MRC Holland
falling within the chromosome 16p11.2 region ............................................................... 79
Table 2-7: MLPA Hybridisation Reaction Mix................................................................... 80
Table 2-8: MLPA Ligation Reaction Mix ............................................................................... 80
Table 2-9: MLPA Polymerase Reaction Mix .......................................................................... 81
Table 2-10: MLPA Buffer Reaction Mix ................................................................................. 81
Table 2-11: Primer sequences of the 6,905bp TRIB3 homozygous CNV deletion ................. 83
Table 2-12: PCR reaction mix for genotyping the TRIB3 6,905bp homozygous deletion ...... 83
Table 2-13: Capture of WES variants generated from GenomeQuest ..................................... 98
Table 2-14: Candidate genes and families in which they were sequenced .............................. 99
Table 2-15: Primer sequences of CHRNA7 gene ..................................................................... 99
Table 2-16: Primer sequences of FES gene ........................................................................... 100
Table 2-17: Primer sequences of LPIN3 gene ....................................................................... 101
Table 2-18: Primer sequences of MC4R gene........................................................................ 102
Table 2-19: PCR reaction mix for sequencing the MC4R, LIPN3, FES and CHRNA7 genes
........................................................................................................................................ 103
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List of Tables
Table 3-1: Number of missing genotypes removed from each chromosome with more than
10% Mendel error rate ................................................................................................... 108
Table 3-2: Linkage intervals using 6,666 SNPs. Limits of each peak are defined by position
of first adjacent marker with LOD > 0. ......................................................................... 111
Table 3-3: Linkage intervals using 86,000 SNPs. Limits of each peak are defined by position
of first adjacent marker with LOD > 0. ......................................................................... 114
Table 3-4: Linkage intervals using 444,000 SNPs. Limits of each peak are defined by
position of first adjacent marker with LOD >0. ............................................................. 115
Table 3-5: rs12062017 SNP with highest LOD score per family in chromosome 1 ............. 117
Table 3-6: rs17517928 SNP with highest LOD score per family in chromosome 2 ............. 117
Table 3-7: rs496826 SNP with highest LOD score per family in chromosome 3 ................. 117
Table 3-8: rs10051931 SNP with highest LOD score per family in chromosome 5 ............. 118
Table 3-9: rs11994661 SNP with highest LOD score per family in chromosome 8 ............. 118
Table 3-10: rs12351388 SNP with highest LOD score per family in chromosome 9 ........... 118
Table 3-11: rs12365463 SNP with highest LOD score per family in chromosome 11 ......... 119
Table 3-12: rs11074004 SNP with highest LOD score per family in chromosome 15 ......... 119
Table 3-13: rs2367502 SNP with highest LOD score per family in chromosome 17 ........... 119
Table 3-14: rs12150959 SNP with highest LOD score per family in chromosome 19 ......... 120
Table 3-15: rs6062678 SNP with highest LOD score per family in chromosome 20 ........... 120
Table 3-16: Sum of SNPs with highest LOD > 0 score for each chromosome calculated from
the per family linkage analysis ...................................................................................... 120
Table 3-17: Linkage intervals generated from EasyLinkage, Allegro using 444,000 SNPs.
Limits of each peak are defined by position of first adjacent marker with LOD > 0. ... 122
Table 3-18: Runs of Homozygosity generated from PLINK found in cases with extreme
obesity ............................................................................................................................ 129
Table 3-19: ROH generated from AutoSNPa found in cases with extreme obesity .............. 135
Table 3-20: Sequencing metrics ............................................................................................. 137
Table 3-21: GenomeQuest variant calling metrics in each sample ........................................ 139
Table 3-22: Filtering by variant class and the identification of candidate gene variants ...... 143
Table 3-23: Prioritisation of homozygous candidate obesity variants ................................... 143
Table 3-24: Candidate obesity variants generated from WES ............................................... 150
Table 3-25: Gene variants after validation with Sanger sequencing ..................................... 157
Table 4-1: Median size of different types of CNVs detected in this study ............................ 182
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