Trio Exome Sequencing: Improving the Chance of a Genetic Diagnosis in Rare Disorders

Anna De Grassi- - Dept. Biosciences, Biotechnologies and Biopharmaceutics,

University of Bari --

Mitocon Conference - June 2015 - Bologna

Exome and Mutations

Personal exome cost: 1,500$; Times required: 6 weeksExon

Capture and Sequencing

Genome

Exome repositories host the exome of tens thousand individuals

Technically Mutations are DNA sequence differences between each person and a “reference”

Reference exon

Exon of Person 1

Exon of Person 2

A C

T

G

mutation

The Exome is the set of ALL the DNA portions (exons) encoding PROTEINS:

EXOME: 70M bases for ~30,000 proteins

Number and Class of Mutations

There are around 120,000 mutations between EACH person and the reference(~40,000 mutations within Protein Coding Exome and the others in the flanking sequence)

Which mutation(s) make the phenotypic difference between people? Why?Mutations are only data and not information!

Reference exon

Exon of Person 1

mutation

Hom.mutation

Het.mutation

Het.mutation

Het.mutation

Exon of Person 1

Exome sequencing also reveals the class of each mutation

Detecting Rare Disease Causing MutationsTo reduce the number of candidate mutations in a patient, we must filter them:

Discard mutations that are common in the human populations: 8,000 retained

Exome + Flanking sequence Total mutations: 120,000 40,000 80,000

Het. mutations: 7,500

Hom. mutations: 500 Genes with putative compound het.

mutations: 2,300

“Brute” filtering:- Remove exon flanking sequences- Remove non protein coding exons- Remove synonymous mutations

- Remove “benign” protein changes

- Literature check- Mutation confirmation

- Verify that the protein does not work properly

Genetic diagnosis in

< 9-26% patients

Improving Detection: Is It Possible?In principle, 85% disease causing mutations should be in protein coding exons

Rabbani, Journal of Human Genetics (2014) 59, 5–15

Disease causing mutations maybe are in the exome (or in the flank. reg.), but they were discarded by “brute” filtering

Can we discard mutations more properly?

Probably YES, if we also sequence the exome of both parents (TRIO):

1) Parents are genetically similar to child 2) Parents are healthy

Hom. mutations

Kid Mom Dad

x x x

Het. mutations (de novo) xx

Het. mutations (compound) x

x

x x

and so on…

and so on…

and so on…

ok discard

Mom Dad Mom Dad

Project and Literature TimingMitocon funding

(5 trios)Delivery of DNA from 3 trios (Besta->USA)

August 2014 December 2014

Raw data delivery (USA->Uniba)

February 2015

Pipeline Development & Candidate Mutations

Today

800 patients

The idea is becoming a diagnostic standard

Our Three TriosChildren with suspected mitochondrial disease without genetic diagnosis

Kin

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p

Sample name

Trio Member SexAge at onset

Onset Clinical Features Neuroradiology Muscle FibrosExome

Seq                     

A1 A Mother F          

A2 A Father M          

A3 A Child F 5 mo Psycho-motor delay

Epilepsy, hypotonia, ophthalmoplegia, microcrania, psychomotor delay, failure to thrive, disorder of swallowing and breathing, hypercalciuria, lactic acidosis

involvement of the basal ganglia, thalamus, subtalami

CI=2% CR Neg Monaco

B1 B Mother F          

B2 B Father M          

B3 B Child F BirthRespiratory failure, epilepsy

Respiratory failure, epilepsy, anemia, dysphagia, hypotonia. Deceased

thin corpus callosum

CIII=28% CIV=33

%CR Neg BGI

C1 C Mother F          

C2 C Father M          

C3 C Child M First months Epilepsy

Hypotonia, epilepsy, deafness, myoclonus, hypospadias

not done CI=27% CR Neg Monaco

No disease causing mutations detected in the mitochondrial genome and in the exome

Bioinfo Pipeline for Trio AnalysisBioinfo pipeline written ad hoc and applied to each trio independently

Raw data

- Quality checking- Mapping to reference

Calling of mutations:hom. or het. in >= 1 trio member

Annotation of mutations:location, consequence,

frequency in human populations

Selection of rare mutations:(< 0.5% or unknown)

Detection of kid/parents combinations compatible with

a monogenic disease

Hom. De novo het. Compound het.

No filter on protein coding exons and splice sites

Regulatory exome and flanking regions: filter for mitochondrial location OR effect on splicing

x x x

10Gb x 3 Few Gene Candidates

Candidate Genes for Trio A

Mutations Single Exome

Trio Exome

Candidate Genes

De novo heterozygous 429 0 -

Homozygous 12 0 -

Compound heterozygous (n.genes)

63 3 CRAT(m+m), DOLK(m+m), PCSK4(m+s)

Mutations Single Exome

Trio Exome

Mito genes

Splicing Effect

Candidate Genes

De novo heterozygous 7003 8 1 1 TIMM22 (i)

Homozygous 429 71 0 1 PAX3 (u)

Compound heterozygous(n.genes)

2252 257 0 2 RPA1(u+u)

Flanking regions (utrs, introns, intergenic)

Based on literature and DB: known disease, protein function, tissue expression, KO mouse, HW-equilibrium

Protein coding exons and splice sites

The First Candidate Gene for Trio AAcyl-CoA+Car

Acyl-Car + CoA

Acyl-Car+CoA

Car

Car

Acyl-CoA + Car

Acetyl-CoA + Car

β oxidation

Pyruvate

CoA

Krebs cycle

Glycolysis

Citrate

Acetyl-Car+ CoA

CoA

Acetyl-CoA

Acetyl-Car

CoA

<<<<<PyruvateLactic Acid

- Fatty Acid- Cholesterol- Ketone Bodies

Citrate

Oxalacetate +

?

Putative Effect of CRAT deficiency:

- Excess of Acetyl-CoA- Lactic acidosis?- Excess of Acetyl-CoA or Acetyl-Car in blood?

?

*

*

CRAT physiological role:

- Remove excess of Acetyl-CoA from mitochondria

(fasting->feeding)

- Avoid PDH inhibition- Avoid CoA synt.

inhibition- Free CoA

CRAT

CRAT Mutations Do Not Look Like “Benign”“Brute” filtering: CRAT mutations are predicted to

be “benign” by two algorithms out of three.

Crystallized structure of CRAT (~600 aa)Mutated aa are <= 4 Å from carnitine

Wild

type

Mut

atio

ns

Tyr 89

Val 548

Cys 89

Met 548

Kin

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y C

iro

L.

Pie

rri

Candidate Genes for Trio B

Mutations Single Exome

Trio Exome

Candidate Genes

De novo 758 2 TSFM (s), ADAMTS17(m)

Homozygous 14 0 -

Compound heterozygous(n.genes)

69 4 DHX38(m+m), CD36(m+m), CLSTN3(m+s), EHBP1L1 (m+s)

Protein coding exons and splice sites

Mutations Single Exome

Trio Exome

Mito genes

Splicing Effect

Candidate Genes

De novo 7594 3 0 0 -

Homozygous 426 80 0 0 -

Compound heterozygous(n.genes)

2440 284 0 4 PTPRD (u+u), CCND1(u+u), HKDC1 (m+u), PCSK6(u+i),

Flanking regions (utrs, introns, intergenic)

The First Candidate Gene for Trio BTSFM (Translation elongation factor) – de novo synonymous mutation

- Main cellular location: mitochondria- Main function: help translating proteins encoded by the mitochondrial

genome- Cell type: almost ubiquitary- Known disease: combined oxidative phosphorylation deficiency-3

An intronic mutation in the other allele?

Candidate Genes for Trio C

Mutations Single Exome

Trio Exome

Candidate Genes

De novo 675 3 HSDL2 (s), PHACTR1(m), ZNF28 (f)

Homo/hemyzygous 21 1 HTR2C (m-chrX)

Compound heterozygous(n.genes)

68 2 H2AFY2(s+s), GPR125(s+s)

Protein coding exons and splice sites

Mutations Single Exome

Trio Exome

Mito genes

Splicing Effect

Candidate Genes

De novo 6671 1 0 0 -

Homozygous 461 80 0 0 -

Compound heterozygous(n.genes)

2470 273 3 0 SLC25A10 (f+i), TSFM (i+u), SLC25A2 (m+u)

Flanking regions (utrs, introns, intergenic)

The First Candidate Gene for Trio CSLC25A10 - (frameshift + intron)

- Main cellular location: mitochondria- Main function: transport of dicarboxylic acids across the mitochondrial membrane (Krebs cycle

substrates)- Cell type: almost ubiquitary- Known disease: na

Wt: CCCCTTCGGGCTMut:CCCCTTCAGGCT

intron

New donor splice site

Conclusions and Perspectives

Trio vs Proband Exome:- The number of candidate mutations is lowered without loosing information- “Brute” filtering and control of parent DNA can be avoided- New candidate mutations are pointed out for lab validation

Validation on patient cells:- Trio A: functional assay for CRAT - Trio B: check transcripts for TSFM- Trio C: check transcripts for SLC25A10

Bioinfo Pipeline Optimization

Acknowledgments

- Paola Desideri- Piero Santantonio- Cristina Rebagliati

- Valeria Tiranti- Eleonora

Lamantea- Daniele Ghezzi

- Ciro Leonardo Pierri- Angelo Vozza- Luigi Palmieri- Cesare Indiveri- Vito Porcelli- Giovanni Parisi- Giuseppe Punzi

Other candidate genes

- CLSTN3 (calsitenin 3) - (missense + synonymous)

- Main cellular location: membranes- Cell type: neuronal cells, peripheral nerves, glandular cells,

myocytes- Known disease: na- Main function: help development of excitatory and inhibitory

synapses

Rare missense+

New donor splice siteCheck transcript

- TSFM - (intron + 3’utr)

New acceptor splice site+

New branch point ?Check transcript

- GPR125 - (synonymous + synonymous)

- Main cellular location: membrane- Main function: unknown, receptors of hormones and

neurotransmitters- Cell type: neuronal cells, myocytes- Known disease: na

ESE sites broken+

ESE sites broken/ New ESS sitesCheck transcript

Trio B

Trio C

The Genetic Diagnosis

Top->downBottom->up

Known genetic disease

- Sequencing of few DNA “pieces”

(Mitochondrial genome sequencing)

Genetic Diagnosis

Known disease causing mutation

Putative disease causing mutation

Experiments

Suspect of genetic bases, but unknown genes

Finding a small “piece” of DNA responsible of the disease

Sequencing as many “pieces” of DNA as possible

The Human Reference Genome

Cas Kramer et. al, Leicester University (UK)

- Around 3,000 Mb characters- 130 volumes- Pages printed on both sides- 43,000 characters per page(mitochondrial genome in ½ page)

DNA handbook to “build a human being and let it work”

A SINGLE (Hybrid) Genome sequenced in 10 years (2,000) for 3,000M $

The Reference Genome Sequence

4 different characters (bases), no spaces, no words, no sentences

Exons

(2%)

From Exons to Protein Sequence

Gene

(DNA sequence)

Protein

(amino acid sequence)

Having the sequence of ALL the EXONS means having the sequence of ALL the PROTEINS:

EXOME: 70M bases for ~30,000 proteins

Protein structure and function

Transcript

Class and Number of Mutations

There are around 120,000 mutations between EACH individual and the reference exome(~40,000 mutations within Protein Coding Exome and the others in the Flanking sequence)

Which mutation(s) make the phenotypic difference between individuals? Why?Mutations are only data and not information!

Reference exon

Exon of Person 1

mutation

Exon of Person 1

Hom.alleles

Het.alleles)

Het.alleles

Het.alleles

Exon of Person 1

read

Exome sequencing let us detect the class of each mutation