1. Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases
BIOS 6660
Hung-Chun (James) Yu
Shaikh Lab
04/28/2014

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

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

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
Bamshad, MJ., et al. Nat. Rev. Genet. (2011) 12:

9. Exome Sequencing
~40Mb (coding) or 60Mb (coding + UTRs)

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

11. Mendelian Diseases Identified by Exome Sequencing
By mid-2012, ~100 genes identified.
By mid-2013, >150 genes identified.
Rabbani, B., et al. (2012) J. Hum. Genet. 57:

12. Types of Variation
What kind of variation/mutation can be detected by Exome Sequencing?
SNV (single nucleotide variation)
Small InDel, (insertion/deletion of <25bp)
Large InDel, CNV (copy number variation)
Possible, but not reliable.
Aneuploidy
Same as CNV
Translocation
Possible, but not reliable. Limited.
Complex rearrangement
Not likely.

13. 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.

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

15. Variant and Population Frequency
Novel/Private variant
Never been reported before.
Rare variant
Minor allele freq. (MAF) < 1%.
Polymorphic variant
MAF > 1% (0.01) or 5% (0.05).
Databases
dbSNP (NCBI):
1000 Genomes:
ESP (NHLBI):

16. 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 ~
Protein altering
~4, ,000
Coding + splice-site
~10, ,000
All
~20, ,000

17. Exome Variants
Bamshad, MJ., et al. Nat. Rev. Genet. (2011) 12:

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

19. Exome Analysis Strategies
Linkage
Large family with multiple
affected individuals
Pathogenic variant co-segregate
with disorder.
Homozygosity
Affected patients from
consanguine parents.
Homozygous mutation within a
homozygous stretch in the genome.
Ideal for recessive disorders

20. Exome Analysis Strategies
Candidate genes
Biased approach
Require current biological knowledge
Good for screening or clinical diagnosis of known disorders.
Overlap
Require multiple unrelated individuals with identical disorders.
Monogenic disorders

21. Exome Analysis Strategies
De novo
Sporadic mutation
Germline mutation during meiosis
Dominant inheritance *

22. Exome Analysis Strategies
Double-hit
Unaffected parents are heterozygous carries
Parental sequence info is very helpful
Recessive inheritance.
Compound Heterozygous
Homozygous # * * * * * * #

23. Trio-based Exome sequencing
Family trio
Unaffected parents and an affected patient.
Why we use trio? What can be tested using trio? Advantages?
Economical, efficient, single case required.

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

25. Trio-based Exome sequencing
Candidate Genes/Variants
Protein altering variants
Rare or novel variants
Variants that fit each inheritance model
Rare Variant
Novel Variant
Dominant
De novo
0 ~ 1
Recessive
Compound Heterozygous
0 ~ 20
0 ~ 3
Homozygous
X-linked
0 ~ 10
0 ~ 5

26. Case 1 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 1
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 2 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 2

30. Case 3 Clinical information
Case 3 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. Sequencing and deletion/duplication analysis (microarray) the MMACHC gene was negative in both skin fibroblasts and peripheral blood.

31. Case 3 Feature Combined methylmalonic aciduria and homocystinuria.
Severe developmental delay, infantile spasms, gyral cortical malformation, microcephaly, chorea, undescended testes, megacolon

32. Case 3
Monster Max
Patient's older sister as a summer student in Shaikh Lab

33. Data for Case Study 3 trios VCF files “Mini” Exome
A total of 3 families/cases.
Each family/case includes unaffected parents and an affected patient.
VCF files
Familial variants calls in VCF format, mapped to human GRCh37/hg19.
2x90bp paired-end reads, with ~50X coverage
“Mini” Exome
100 genes with/without known disorder association.
Validated causative genes, plus randomly selected genes.

34. (Burrows-Wheeler Aligner)
Exome NGS Workflow
FASTQ
2x90bp
SAM
Filter unpaired, unmapped reads
BAM
Filter PCR duplicates artifact
BCF
Filter based on Phred score, mapping quality, read depth, etc.
VCF ?
BWA
(Burrows-Wheeler Aligner)
SAMtools

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

36. VCF Format INFO AA : ancestral allele
AC : allele count in genotypes, for each ALT allele, in the same order as listed
AF : allele frequency for each ALT allele in the same order as listed: use this when estimated from primary data, not called genotypes
AN : total number of alleles in called genotypes
BQ : RMS base quality at this position
CIGAR : cigar string describing how to align an alternate allele to the reference allele
DB : dbSNP membership
DP : combined depth across samples, e.g. DP=154
END : end position of the variant described in this record (for use with symbolic alleles)
H2 : membership in hapmap2
H3 : membership in hapmap3
MQ : RMS mapping quality, e.g. MQ=52
MQ0 : Number of MAPQ == 0 reads covering this record
NS : Number of samples with data
SB : strand bias at this position
SOMATIC : indicates that the record is a somatic mutation, for cancer genomics
VALIDATED : validated by follow-up experiment
1000G : membership in 1000 Genomes

37. 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)

38. Question ?