1. Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases
BIOS 6660
Hung-Chun (James) Yu
Shaikh Lab, Department of Pediatrics, University of Colorado
Denver Genetic Laboratories, Children’s Hospital of Colorado
11/17/2015

2. Human Genetic Diseases
Mutation
Penetrance
Mutation
Frequency
Kaiser J. Science (2012) 338:

3. Human Genetic Diseases
Complex Disorder
Polygenic, many genes.
Low penetrance/effect size.
Multifactorial, environmental, dietary.
Examples: heart diseases, diabetes, obesity, autism, etc.
Mendelian Disorder
Monogenic (mostly).
Full or high penetrance/effect size.
Examples: sickle cell anemia (HBB) and cystic fibrosis (CFTR).

4. Complex Diseases Multiple causes, and polygenic.
Multiple genetics factors with low penetrance individually.
Coronary artery disease
Coriell Institute for Medical Research.

5. Mendelian Diseases
Veltman J.A. et al. Nat. Rev. Genet. (2012) 13:

6. Mendelian Diseases Dominant Inheritance
U.S. National Library of Medicine.

7. Mendelian Diseases Recessive Inheritance
U.S. National Library of Medicine.

8. Exome Sequencing Focusing on exons or coding regions of genes Exons
Bamshad, MJ., et al. Nat. Rev. Genet. (2011) 12:
Exons
Complementary
Baits

9. Exome Sequencing 3,000,000,000bp (3Gb) human genome
~45% repetitive sequence
~25% genic region
~2% exonic, coding region
20,000 – 30,000 human genes
3,000 – 5,000 disease genes
~4,000 human genetic diseases (OMIM)
114 medically actionable (treatable) genes
Michael O. Dorschner., et al. Am J Hum Genet : 631–640.

10. Exome Sequencing ~40Mb (coding) or 60Mb (coding + UTRs) Gene
Read Coverage
Individual Reads

11. Mendelian Diseases Identified by Exome Sequencing
Timeline
Gilissen C. et al., Genome Biol. (2011) 12:228.

12. Mendelian Diseases Identified by Exome Sequencing
Kym M. Boycott et al. Nature Reviews Genetics (2013) 14:681–691

13. Types of Variation
What kind of variation/mutation can be detected by Exome Sequencing?
SNV (single nucleotide variation)
Small InDel, (insertion/deletion <25bp)
Large InDel, CNV (copy number variation)
Possible, but not reliable.
Aneuploidy (loss/gain of entire chromosome)
Possible.
Translocation
Difficult and not reliable.
Complex rearrangement
Very difficult.

14. Exome Variants SNV (single nucleotide variation)
Synonymous: (1) Silent.
Nonsynonymous: (1) Missense. (2) Nonsense. (3) Stop-loss. (4) Start-gain. (5) Start-loss. (6) Splice-site.

15. Exome Variants Small InDel (insertion/deletion <25bp) Frameshift
In-frame
NHGRI Digital Media Database (DMD),

16. Variant and Population Frequency
Novel/Private variant
Never been reported before.
Rare variant
Minor allele freq. (MAF) < 1%.
Databases
dbSNP (NCBI):
1000 Genomes:
ESP (NHLBI):
ExAC:

17. Exome Variants
How to analyze enormous amount of variants in any given exome?
Gilissen C. et al. Eur. J. Hum. Genet. (2012) 20:
Private/Novel ~
Rare, MAF<1%
~ ,000
Protein altering
~4, ,000
Coding/splice-site
~10, ,000
All
~20, ,000

18. Exome Analysis Strategies
Gilissen C. et al., Eur. J. Hum. Genet. (2012) 20:
Male
Female
Affected
Heterozygous
carrier
Sex-linked
heterozygous
carrier
Mating
Consanguineous
mating
Sequenced individual

19. Trio-based Exome sequencing
Family trio
Both unaffected parents and an affected patient.
Why using trio?
Every inheritance model can be tested
Economical, efficient, single case required.
Access to samples.

20. Trio-based Exome sequencing
Autosomal dominant
de novo
Autosomal recessive
Homozygous * * * * *
Autosomal recessive
Compound heterozygous
X-linked recessive
Hemizygous in male
Gene
Male
Female
Affected
Heterozygous
carrier
Sex-linked
heterozygous * * * * X Y X X * * X Y

21. Trio-based Exome sequencing
Candidate Genes/Variants
Rare (~500-2,000) or novel (~ ) protein altering variants
Plus, variants that fit inheritance model
Rare Variant
Novel Variant
Dominant
de novo
0 ~ 2
Recessive
Compound Heterozygous
0 ~ 20
0 ~ 3
Homozygous
X-linked
0 ~ 10
0 ~ 5

22. Case 1 Clinical information
Case 1 was the result of a non-consanguineous union and he presented to care at four months of age with a seizure disorder, hypotonia and developmental delay. The patient underwent a left parietal craniotomy and partial resection of the frontal cortex without complete resolution of the seizure disorder.
Initial laboratory studies included an elevated homocysteine and methylmalonic acid and a normal vitamin B12 level. Complementation analysis of the patient’s cell line placed the patient into the cblC class. Severe developmental delay, infantile spasms, gyral cortical malformation, microcephaly, chorea, undescended testes, megacolon.
Sequencing and deletion/duplication analysis (microarray) the MMACHC gene was negative in both skin fibroblasts and peripheral blood.

23. Case 1

24. Case 1
9News Colorado: Student joins first-grade class via web (May 15, 2011)

25. Case 1
Monster Max
Patient's older sister as a summer student in Shaikh Lab

26. Case 2 Clinical information
The patient was a 7-month-old boy when first evaluated. He was diagnosed with BPES by a pediatric ophthalmologist. In addition to blepharophimosis, ptosis, and epicanthus inversus normally associated with BPES, he had cryptorchidism, right hydrocele, wide-spaced nipples, and slight 2–3 syndactyly of toes.
Clinical testing demonstrated a normal karyotype (46,XY), and normal FISH studies for 22q11.2 deletion, Cri-du-Chat (5p deletion) syndrome. Thyroid function was normal. Further, normal 7-dehydrocholesterol level was used to rule out Smith–Lemli–Opitz syndrome. Sanger sequencing and highresolution CNV analysis with Affymetrix SNP 500K arrays did not identify a FOXL2 mutation.

27. Case 2
A-D: 2-month old. note blepharophimosis, ptosis, epicanthus inversus (A), posteriorly angulated ears with thickened superior helix and prominent antihelix (B), and slight 2–3 syndactyly of toes in addition to overlapping toes (C, D)
E-F: 3.5-year old. Following oculoplastic surgery to correct ptosis; note right-sided preauricular ear pit (F, indicated by arrow).
G-I: 12-year old. Note the recurrence of ptosis (L>R), arched eyebrows, abnormal ears, thin upper lip vermilion, small pointed chin, downsloping shoulders, and wide- spaced and low-set nipples.

28. Case 3 Clinical information
The proband is a nine year old girl who presented with microcephaly, unilateral retinal coloboma, bilateral optic nerve hypoplasia, nystagmus, seizures, gastroesophageal reflux, and developmental delay including not yet saying specific words (at 29 months old).
On exam, she has microcephaly with a normal height, a down-turned upper lip, and fingertip pads. A karyotype and CGH analysis have been normal. Kabuki (KMT2D and KDM6A) and Angelman (UBE3A and MECP2) syndromes were suspected in this patient.

29. Case 3

30. Exome NGS Workflow Genomic DNA Exome capture Library construction
Sequencing QC
Sequence read processing
Mapping
SAM
BAM
Variant calling
Annotation (General)
Annotation
(In-house)
Filtering
Inheritance test, candidate genes, ect.
Exome Sequencing
Mapping and variant detection
Variant prioritization
Exome Enrichment
Illumina Sequencer
Galaxy/FASTX Toolkit
Galaxy/BWA
Galaxy/Samtool
Galaxy

31. Exome analysis Workflow (this class)
FASTQ sequence
2x90bp (paired-end)
Variant determination
BCF
Filter based on Phred score, mapping quality, read depth, etc.
Mapping to genome
SAM
Conversion
Conversion
VCF
BAM
QC: Filter duplicates, artifacts, and unpaired or unmapped reads,
100 genes
“Mini” Exome ?
BWA
(Burrows-Wheeler Aligner)
SAMtools

32. Data for Case Study 3 trios VCF files “Mini” Exome
A total of 3 families/cases.
Each family/case includes both unaffected parents and an affected patient.
VCF files
Generated from 2x90bp paired-end Exome sequence reads, and at ~50X coverage
Reads mapped to human GRCh37/hg19 and then familial variants calls made in VCF format
“Mini” Exome
100 genes with/without known disease association.
Validated causative genes and randomly selected disease genes or non-disease genes.

33. VCF Format Variant Call Format FILTER, INFO, FORMAT # Header line
## Meta-information lines
FILTER, INFO, FORMAT
# Header line

34. VCF Format FORMAT GT: Genoetype. 0/0: Homozygous normal
0/1: Heterozygous variant
1/1: Homozygous variant
PL: the Phred-scaled genotype likelihoods (>0).
0/ / /1
, ,178
GQ : Genotype quality (1-99)

35. Annotation Tools Annotate variants with useful information
Mutation effect
Population frequency
Clinical association
Genomic sequence and protein domain
Pathogenicity prediction
Gene expression, protein interaction.
…..and many many more.
SeattleSeq:
VEP (Variant effect Predictor):
ANNOVAR:

36. Question ?