SNP arrays in routine

cytogenetic diagnosis

Illumina User Meeting

Milan september 13, 2010

Boris Keren

Genetic Department

Pitié-Salpêtrière Hospital

Paris

Mental retardation etiologies

Rauch et al. (2006)

Clinical guidance in MR/MCA

- non specific phenotype

- phenotype suggesting a known chromosomal pathology

- phenotype suggesting a known non chromosomal pathology

- non specific phenotype

- phenotype suggesting a known chromosomal pathology

Constitutional cytogenetics

- phenotype suggesting a known non chromosomal pathology

Clinical guidance in MR/MCA

Non specific phenotype

karyotype

- whole genome analysis

- low resolution (~10 Mb)

FISH

- Targeted analysis

- Higher resolution (2Mb-100kb)

Phenotype suggesting a known

chromosomal pathology

Increasing cytogenetic sensitivity

Increasing resolution

Keeping a whole genome analysis

CNV detection on arrays

Cytogenetics on array

Array CGH

SNP Arrays

- 2 informations on each marker :

- Copy number

- SNP genotype

Analysis : BeadStudio

AA

AB

BB

Clustering

Clustering

Clustering

Clustering

Clustering

X analysis

Copy number loss

A

B

Homozygous deletion

Mosaic copy number loss

Copy number gain

AAA

AAB

BBB

ABB

Mosaic copy number gain

Tetrasomy

BBBB

ABBB

AABB

AAAB

AAAA

cnvPartition

Verification: FISH

RP11-17J3

Cytogenetic diagnosis in mental

retardation

karyotype +

FISH

known chromosomal

pathology ? non specific phenotype

karyotype

SNP Array

2007-2008 : HumanCNV370-Duo and HumanCNV370-Quad

2009-2010 : HumanCytoSNP-12

CNV validation

CNV

Coding sequence

DGV

transmission

yes

no

stop

stop

Pathogenic CNV

no

no

yes

yes stop

603 probands

2514 CNV

142 CNV

53 CNV

61 unverified CNV

Interpretation problems

CNV

Coding sequence

DGV

transmission

yes

no

stop

stop

Pathogenic CNV

no

no

yes

yes stop

MiR ?

regulation sequence ?

Autosomal recessive

diseases ?

Complex diseases ?

De novo polymorphism

Interpretation problems

Presymptomatic diagnosis of high cancer risk

Interpretation problems

Carier status of dystrophinopathy

Interpretation problems

Diagnosis in a mother of hereditary neuropathy with liability to pressure palsie

Interpretation problems

Heterozygous deletion of EVC

Interpretation problems

Homozygous deletion of UTG2B17

Interpretation problems

Nov 2009

Information to the patient ?

Interpretation problems

Heterozygous intragenic

duplication of PARK2

Interpretation problems

Deletion 1q21.1

Deletion 15q13.3

Duplication 16p11.2

« copy neutral LOH »

consanguinity

consanguinity

- Low interest in diagnosis. Known autosomal recessive gene in the LOH ?

- Genetic counseling: if the consanguinity was unknown

- Research : homosigozity mapping

Patient

- 17 yo

- mental retardation

- cerebellar ataxia

- hypertrophic cardiomyopathy

- growth retardation

- seizures

Chromosome 16 isodisomy

Patient

- 17 yo

- no neonatal signs

- mental retardation

- growth retardation

- seizures

- obesity

karyotype : de novo inv(16)

Chromosome 15 isodisomy

Problems :

- Father lost to follow up

- Problem : gel in favor of a deletion

- FISH

Mother

Mother

All the 1676 heterozygous SNPs on the patient’s chromosome 15 had the same

phenotype on the mother’s chromosome 15 -> Maternal uniparental disomy

Mother

Patient

- 9 yo

- mental retardation

- microcephaly

- seizures

- vermis hypoplasy

Segmental mosaic upd(11)

Beckwith-Wiedemann ???

- Methyl sensitive qPCR : molecular diagnosis of BWS

- problem : she doesn’t have a BWS phenotype at all

- another anomaly detected: TUBA 1A mutation

Uniparental disomies

- Interest in diagnosis : real but uncommon 7 UPDs for ~800 patients with MR and or MCA.

- Interpretation may be uneasy

Beckwith-Wiedemann

- known upd : leukocytes

Beckwith-Wiedemann

- known upd : tongue biopsy

Beckwith-Wiedemann

- unconclusive qPCR

- in our lab: 2 other patients with unconclusive MS-qPCR and UPD confirmed with SNP array

- suspected 11p15 trisomy on MS-qPCR, FISH unconclusive

Beckwith-Wiedemann

SNP arrays allow to:

- detect trisomies in a more sensitive way than FISH

- detect UPDs in a more sensitive way than MS-qPCR

- estimate the size and the mosaic rate of the anomaly

Can be used in complement of MS-qPCR or MS-MLPA

Beckwith-Wiedemann

• Call rate = 0.998

• p10 GC = 0.69

• Control dashboard ok

Quality control

Softwares

Using the Illumina cluster file

Softwares

Clustering SNP with the patients of the same technique

?

– CNV analysis on microarray : now used in routine diagnosis for patients with mental retardation

– Difficulties in interpreting CNVs : – Pathogenic significance

– Unexpected anomalies

Summary

– Benefits : – No control DNA

– LOH : consanguinity, UPDs diagnostic

– High troughput

– Flaws : – quality controls

– Must process many samples at the same time to cluster SNP

– Not as used as Agilent array CGH.

Illumina chips vs Array CGH in

constitutional cytogenetics

Cytogénétique

Patricia Leite

Eric Fonteneau

Génétique Médicale

Delphine Héron

Aurélia Jacquette

Sandra Whalen

Plateforme P3S

Wassila Carpentier

Florent Soubrier

Centre Mutualisé

Génétique Constitutionnelle

GHU-Paris Est

Jean-Pierre Siffroi

CRICM INSERM U975

Christel Depienne

Caroline Nava

Alexis Brice

Cytogénétique

Sandra Chantot-Bastaraud

Marie-France Portnoï

Génétique Médicale

Lydie Burglen

Sandrine Marlin

Alexandra Afenjar

Pitié-Salpêtrière

Armand-Trousseau Hospitals

Explorations fonctionnelles

Irène Netchine

Sylvie Rossignol

Salah Azzi