Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases

BIOS 6660 Hung-Chun (James) YuShaikh Lab04/28/2014

Human Genetic Diseases

Penetrance vs Frequency

Kaiser J. Science (2012) 338:1016-1017.

Human Genetic DiseasesComplex 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.

Complex Diseases

Multiple causes, and polygenic.Multiple genetics factors with low

penetrance individually.

Coriell Institute for Medical Research.https://cpmc1.coriell.org/genetic-education/diagnosis-versus-increased-risk

Coronary artery disease

Mendelian Diseases

Veltman J.A. et al. Nat. Rev. Genet. (2012) 13:565-575.

Mendelian Diseases

Dominant Inheritance

U.S. National Library of Medicine. http://ghr.nlm.nih.gov/

Mendelian Diseases

Recessive Inheritance

U.S. National Library of Medicine. http://ghr.nlm.nih.gov/

Exome Sequencing

Bamshad, MJ., et al. Nat. Rev. Genet. (2011) 12:745-755.

Exome Sequencing~40Mb (coding) or 60Mb (coding

+ UTRs)

Mendelian Diseases Identified by Exome SequencingTimeline

Gilissen C. et al., Genome Biol. (2011) 12:228.

Mendelian Diseases Identified by Exome Sequencing

Rabbani, B., et al. (2012) J. Hum. Genet. 57:621-632.

By mid-2012, ~100 genes identified. By mid-2013, >150 genes identified.

Types of VariationWhat 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.

Exome VariantsSNV (single nucleotide variation)• Synonymous: (1) Silent.• Nonsynonymous: (1) Missense. (2)

Nonsense. (3) Stop-loss. (4) Start-gain. (5) Start-loss. (6) Splice-site.

http://upload.wikimedia.org/wikipedia/commons/6/69/Point_mutations-en.png

http://www.web-books.com/MoBio/Free/Ch5A4.htm

Exome VariantsSmall InDel (insertion/deletion

<25bp)• Frameshift• In-frame

NHGRI Digital Media Database (DMD), http://www.genome.gov/dmd/

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): http://www.ncbi.nlm.nih.gov/SNP/

• 1000 Genomes: http://www.1000genomes.org/

• ESP (NHLBI): http://evs.gs.washington.edu/EVS/

Exome VariantsHow to analyze enormous

amount of variants in any given exome?

Gilissen C. et al. Eur. J. Hum. Genet. (2012) 20:490-497.

~20,000 - 200,000

~10,000 - 30,000

~4,000 - 15,000

~100 - 300

Coding + splice-site

Protein altering

Private/Novel

All

Exome Variants

Bamshad, MJ., et al. Nat. Rev. Genet. (2011) 12:745-755.

Exome Analysis Strategies

Gilissen C. et al., Eur. J. Hum. Genet. (2012) 20:490-497.

Male

Female

Affected

Heterozygous carrier

Mating

Consanguineous mating

Sex-linkedheterozygous carrier

Exome Analysis StrategiesLinkage• 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

Exome Analysis StrategiesCandidate 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

Exome Analysis StrategiesDe novo

• Sporadic mutation• Germline mutation during meiosis• Dominant inheritance

*

Exome Analysis StrategiesDouble-hit

• Unaffected parents are heterozygous carries

• Parental sequence info is very helpful• Recessive inheritance.Compound Heterozygous Homozygous

#*

*

* *

**#

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.

Trio-based Exome sequencing Autosomal dominant

De novo

Autosomal recessive Compound

heterozygous HomozygousMale

Female

Affected

Heterozygous carrier

Sex-linkedheterozygous carrier

X-linked dominant De novo

X-linked recessive Hemizygous in

male

X

*Y XX

XY

*

Trio-based Exome sequencingCandidate Genes/Variants• Protein altering variants• Rare or novel variants• Variants that fit each inheritance

model Rare Variant

Novel Variant

Dominant De novo 0 ~ 1 0 ~ 1

Recessive

Compound Heterozygous 0 ~ 20 0 ~ 3

Homozygous 0 ~ 20 0 ~ 3

X-linked 0 ~ 10 0 ~ 5

Case 1Clinical 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.

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.

Case 2Clinical 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.

Case 2

Case 3Clinical 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.

Case 3

FeatureCombined methylmalonic aciduria and homocystinuria.Severe developmental delay, infantile spasms, gyral cortical malformation, microcephaly, chorea, undescended testes, megacolon

Case 3Patient's older

sister as a summer student in Shaikh Lab

Monster Maxhttp://www.maxwatson.org/

Data for Case Study3 trios• 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.

Exome NGS Workflow

BWA(Burrows-Wheeler

Aligner)

SAMtools

FASTQ2x90bp

SAMFilter unpaired,

unmapped reads

BAMFilter PCR duplicates

artifact

BCFFilter based on Phred

score, mapping quality, read depth,

etc.

VCF

?

VCF FormatVCF (Variant Call Format)

http://www.1000genomes.org/wiki/Analysis/Variant%20Call%20Format/vcf-variant-call-format-version-41

## Meta-information lines

FILTER, INFO, FORMAT

# Header line

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

VCF FormatFORMAT

GT: Genoetype.

0/0: Homozygous normal

0/1: Heterozygous variant

1/1: Homozygous variant

PL: the Phred-scaled genotype likelihoods (>0).

0/0 0/1 1/1

174 ,0 ,178

GQ : Genotype quality (1-99)

Question ?