Genetic diagnosis and identification of novel genes in neuromuscular disorders using next ...

331 Pages·2012·31.22 MB·English

Institut de Génétique et de Biologie Moléculaire et cellulaire
CNRS-INSERM-Université de Strasbourg

THÈSE

Présentée en vue de l’obtention du grade de

Docteur de l’université de Strasbourg

Discipline : Sciences du vivant
Aspects moléculaires et cellulaires de la biologie

Par
Nasim VASLI

Genetic diagnosis and identification of novel genes in
neuromuscular disorders using next generation sequencing

Soutenue le 17 Décembre 2012, devant la commission d’examen :

Mr. Le Docteur Jocelyn Laporte Co-directeur de Thèse
Mr. Le Professeur Jean-Louis Mandel Co-directeur de Thèse
Mme. Le Professeur Hélène Dollfus Rapporteur interne
Mr. Le Docteur Christophe Béroud Rapporteur externe
Mr. Le Docteur Richard Redon Rapporteur externe
Acknowledgements
I would like to thank my jury members, Professor Hélène Dollfus, Dr. Christophe Béroud, Dr. Richard
Redon and Professor Jamel Chelly for accepting to read and to evaluate my PhD work.
My biggest thank is for my major supervisor, Dr Jocelyn Laporte. I’ve never forgotten His helps from even
the days before coming to France and starting my PhD until the last days in the lab. I’ve learnt many
things from him and I had very nice days in his lab during past four years. I would like to thank him for
giving me the opportunity to work with him in his team on a very exciting project and helping me for my
future life.
The second person that I would like to thank is my co-supervisor, Professor Jean-Louis Mandel. For me
he is a live genetics text book and I’ve learnt many things from him. I would like to thank him for helping
and supporting me to come to France, to continue my PhD project and my future life.
I would like to thank my friends and colleges in MTM team. We had very nice days together and I’ve
never forgotten our lab meetings, several non-scientific activities and birthday parties in atrium of third
floor of IGBMC.
I would like to acknowledge Bernard Jost, Serge Vicaire and Muriel Philipps in Microarray and
Sequencing Platform of IGBMC. I would like to thank especially Bernard who was really helpful person
and he was always accessible for answering to my questions.
I would like to thank warmly Stephanie Le Gras in sequencing and bioinformatics platform of IGBMC.
During past four years, Stephanie was one of the persons who helped me a lot in bioinformatic aspects
of my project. She was a very kind and helpful person. Although it was not her responsibility to help me
for the projects that were performed in private companies, she put a lot of efforts in helping me and I’ve
never forgotten her helps.
I sincerely thank Dr. Christos Gavriilidis for being helpful for correction of my thesis manuscript.
I would like to thank Dr Valérie Biancalana from Hôpital civil of Strasbourg who was really helpful for
performing experiments and giving us the clinical data of our patients.
I would like to thank Dr. Jean Muller for giving me the access to ALAMUT software and helping us for
VaRank program.
I sincerely thank all people from IGBMC, clinicians, our collaborators and patients who helped me to
work on my PhD work during these four years and to finish it.
And finally I would like to thank my family, especially my lovely husband. Without him and his supports,
this PhD project could not be possible.
Abbreveations
aCGH: array comparative genomic hybridization
CNM: Centronuclear myopathy
CNV: Copy number variation
cPAL: combinatorial probe anchor-ligation
DNB: DNA nano-balls
HTS: High-throughput sequencing
Indel: Insertions-Deletions
MAPKKK: MAP kinase kinase kinase
MLPA: multiplex ligation-dependent probe amplification
MPS: Massively parallel sequencing
NGS: next generation sequencing
NMD: Neuromuscular disorder
NMJ: Neuromuscular junction
PI domain: Phosphoinositide- binding domain
SNP: Single-nucleotide polymorphism
SNV: Single Nucleotide Variation
SVA: Sequence Variant Analyzer
SV: Structural variation
XLMTM: X-linked myotubular myopathy
List of figures
Figure 1: Schematic representation of affected structures in NMDs. 1
Figure 2: Some clinical and histopathological features in patients with XLMTM. 6
Figure 3: Some clinical and histopathological features in patients with AD DNM2 -related CNM. 8
Figure 4: Some clinical and histopathological features in patients with AR BIN1 -related CNM. 9
Figure 5: Some clinical and histopathological features in patients with RYR - related CNM. 12
Figure 6: Solid-phase capture and enrichment. 16
Figure 7: Solution-phase capture and enrichment. 17
Figure 8: Microdroplet PCR. 18
Figure 9: Illumina sequencing technology. 19
Figure 10: Sequencing by ligation method using in SOLiD sequencer from Applied Biosystems. 20
Figure 11: Pyrosequencing using in Roch/454 platform. 22
Figure 12: Semiconductor sequencing using in Ion torrent platform. 23
Figure 13: The complete genomics sequencing technology. 25
Figure 14: Good quality and bad quality DNAs. 31
Figure 15: Number of targeted nucleotides in 3 different captures kits. 32
Figure 16: Number of unique overlapping reads or coverage, using 3 different kits. 33
Figure 17: Numbers of non- covered, totally covered and targeted regions, using 3 different kits. 34
Figure 18: Number of detected SNVs and Indels, using 3 capture kits. 35
Figure 19: Quality score for each nucleotide in reads with 40 base pairs length. 36
Figure 20: Micro deletion detection. 38
Figure 21: coverage for targeted nucleotides and exons. 52
Figure 22: The NGS reads, direct sequencing result and implicated domains and amino acid conservation
through evolution for patient C (AHJ97) with c.490_491del, p.Met164ValfsX24 variant in ZAK.
55
Figure 23: Expression level of ZAK in different tissues. 57
Figure 24: RT-PCR results for AHJ97 (first lane for each couple of primers) and two control negatives
without any variations in ZAK (second and third lanes for each couple of primers). 58
Figure 25: Sequence coverage for targeted nucleotides for AHJ97. 63
Figure 26: Pedigree of ABJ family. 68
Figure 27: Sequence coverage for targeted nucleotides in ABJ75 and ABJ79. 69
Figure 28: The variations in PDE4DIP gene detected by exome sequencing. 72
Figure 29: PDE4DIP variations segregation in ABJ family. 73
Figure 30: Sanger sequencing results for MYO5B and TTN genes in 314-1 and his parents. 81
Figure 31: Position of missense, GTT>CTT, V>L change in exon 168 of TTN. 81
Figure 32: The predictions showing the disruption of donor splice site due to missense, GTT>CTT, V>L
change in exon 168 of TTN. 82
Figure 33: Sanger sequencing results for CLIP1 and FLYWCH1genes in 314-1 and his parents. 84
Figure 34: comparison between whole genome and exome sequencing. 100

Figure 35: Schematic filtration workflow for genome as well as exome datasets. 101
List of tables
Table 1- Different types of neuromuscular disorders in three affected structures and number of known
genes and chromosomal loci with unidentified genes. 2
Table 2- Implicated genes in CNM. 4
Table 3- List of papers showing the gene identification in myopathies by NGS. 28
Table 4- Some studies using different strategies for finding causal genes/mutations. 43

Table 5- List of genes selected for targeted sequencing of 76 genes. 51

Table 6- Statistics regarding to NGS reads for targeted sequencing of 76 genes. 51

Table 7- Statistics regarding to nucleotide coverage for targeted sequencing of 76 genes. 52

Table 8- Statistics regarding to NGS reads for targeted sequencing of 2500 genes. 53

Table 9- Number of different types of exonic variants in patients C, D and F for targeted sequencing of
2500 genes. 54

Table 10- Number of exonic variants after filtration in patients C, D and F for targeted sequencing of
2500 genes. 54

Table 11- Statistics regarding to NGS reads for trio sequencing. 62

Table 12- Statistics regarding to nucleotide sequencing coverage for trio sequencing. 62

Table 13- Statistics regarding to detected SNVs and Indels. 63

Table 14- Two homozygous variants detected in AHJ97. 64

Table 15- Sporadic cases sequenced in different sequencing platforms. 66

Table 16- Statistics regarding to NGS reads, depth of coverage and detected SNVs and Indels in CNM
sporadic cases. 66

Table 17- Common genes with different variations in at least four CNM sporadic samples. 67

Table 18- Statistics regarding to NGS reads for ABJ79 and ABJ75. 68

Table 19- Statistics regarding to nucleotide coverage for ABJ79 and ABJ75. 69
Table 20- Statistics regarding to detected SNVs and Indels. 70

Table 21- Compound heterozygous variations in ABJ75 and ABJ79. 71
Table 22- Variations in PDE4DIP detected and shown in new version of Exome variant Server. 73
Table 23- Statistics regarding to genome and exome coverages for ABJ79 and ABJ68. 74

Table 24- Statistics regarding to numbers of detected variations in ABJ79 and ABJ68. 75

Table 25- Compound heterozygous variations in ABJ75. 76

Table 26- Statistics regarding to genome and exome coverages for AIZ family. 78

Table 27- Statistics regarding to numbers of detected variations in AIZ family. 78

Table 28- Compound heterozygous variations in 314-1. 80

Table 29- Muscle expression of the proteins and presence of variations in Exome Variant Server, NHLBI
Exome Sequencing (http://evs.gs.washington.edu/EVS/) for detected variations in MYO5B and TTN.
82
Index
Acknowledgements
List of abbreviations
List of figures
List of Tables
1- Introduction 1
1-1- Neuromuscular disorders 1
1-2- Centronuclear myopathies 4
1-2-1- X-linked MTM1-related CNM (myotubular myopathy) 5
1-2-2- Autosomal dominant DNM2-related CNM 7
1-2-3- Autosomal recessive BIN1-related CNM 8
1-2-4- MTMR14-related CNM 10
1-2-5- RYR1-related CNM 11
1-3- Biological questions and aims 12
1-3-1- Aim 1: Gene identification 12
1-3-1-2- Previous methods for gene identification 13
1-3-1-3- Massively parallel sequencing 14
1-3-1-3-2- Template preparation & barcoding 15
1-3-1-3-3- DNA enrichment 15
1-3-1-3-4- High-throughput sequencing 18
1-3-1-3-5- Data analysis 27
1-3-1-4- Massively parallel sequencing for gene identification 28
1-3-2- Aim 2: Improving diagnostic of NMD using NGS 29
Review article: Impacts of massively parallel sequencing for genetic diagnosis of neuromuscular
disorders 30
2- Materials and methods 31
2-1- DNA quality control for NGS 31
2-2- Capture kits 32
2-3- Data analysis workflow 35
2-3-1- Sequence quality control 36
2-3-2- Sequence alignment 37
2-3-3- Variant calling and annotation 37
2-3-4- Variant filtration 39
2-3-4-1- Filtration and ranking based on frequency 39
2-3-4-2- Filtration and ranking based on function 40
2-3-4-3- Filtering the “Black genes” and sequencing errors 40
2-3-4-4- Filtration and ranking based on effect 41
2-3-4-5- Filtration and ranking based on conservation 41
2-3-4-6- Filtration and ranking based on inheritance mode 41
2-3-4-6-1- Recessive mode of inheritance 42
2-3-4-6-2- Dominant mode of inheritance 42
2-3-4-7- Filtration and ranking based on gene function, tissue expression profile and
cellular localization 43
3- Results 44
Results for aim 1: Gene identification in CNM 44
3-1- Patient selection 44
Publication 1: Novel molecular diagnostic approaches for X-linked centronuclear (myotubular)
myopathy reveal intronic mutations 45
A- Introduction 46
B- Aim of study
C- Results
D- Conclusion
E- Original paper
Publication 2: Myotubular myopathy caused by multiple abnormal splicing variants in the MTM1
RNA in a patient with a mild phenotype 47
A- Introduction 48
B- Aim of study
C- Results
D- Conclusion
E- Original paper
Publication 3: Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Great Danes with
Highly Progressive Centronuclear Myopathy 49
A- Introduction 50
B- Aim of study
C- Results
D- Conclusion
E- My contribution
F- Original paper
3-2- Targeted sequencing 51
3-2-1- NGS for 76 selected genes 51
3-2-2- NGS for 2500 selected genes 53
3-2-2-1- Results for patient C (AHJ97) 54
3-2-2-2- Results for patient D (34263) 58
3-2-2-3- Results for patient F (AHH42) 59
3-3- Exome sequencing 59
Publication 4: An integrated diagnosis strategy for congenital myopathies 60
A- Introduction 61
B- Aim of study
C- Results
D- Conclusion
E- Original paper
3-3-1- Trio sequencing for AHJ97 62
3-3-1-1- Recessive scenario due to homozygous change 64
3-3-1-2- Recessive scenario due to compound heterozygous changes 64
3-3-1-3- De novo scenario 65
3-3-2- Exome sequencing for CNM sporadic cases 65
3-3-3- Exome sequencing for ABJ family 68
3-3-3-1- Recessive scenario due to compound heterozygous changes 70
3-3-3-2- Recessive scenario due to homozygous change 73
3-4- Whole genome sequencing 74
3-4-1- Whole genome sequencing for ABJ family 74
3-4-1-1- Recessive scenario due to compound heterozygous changes 76
3-4-1-2- Recessive scenario due to homozygous change 77
3-4-2- Whole genome sequencing for AIZ family (314-1 and parents) 78
3-4-2-1- Recessive scenario due to compound heterozygous changes 80
3-4-2-2- Recessive scenario due to homozygous change 83