utility of microarrays in molecular genetics · biotinidase deficiency (btd) beta-mannosidosis...

Utility of Microarrays in Molecular Genetics

Madhuri Hegde, Ph.D., FACMGAssociate Professor

Senior DirectorDepartment of Human Genetics

Emory Genetics LaboratoryEmory University School of Medicine

Atlanta, GA

Mutation spectrum

Duchenne Muscular Dystrophy (DMD)

30% small mutations

65-70% deletions/duplications

Hunter syndrome (IDS mutations)

80% small mutations

20% deletions/duplications

Cystic Fibrosis (CFTR)

>90% small mutations

<10% deletions/duplications

Small mutations (nucleotide substitutions, small indels)

Larger mutations (exon and multi-exon dels and dups)

Krabbe disease (GALC)

35-50% deletions/duplications

50-65% small mutations

• Specialize in molecular testing for rare and ultra rare disorders

• Use of CGH arrays for comprehensive mutation detection

• CGH array>>> focus on DMD (addressing challenges in detection)

• Reduce cost

• Eliminate labor intensive steps by implementing multiplexing capabilities and cost effective platforms

• Use of blood spots and saliva- Early detection and need to characterize mutations accurately for inclusion in clinical trials for therapy and inclusion in registries

Outline

Comprehensive mutation analysis

• Full gene sequence analysis to detect mutations in disease genes is common place in molecular diagnostic laboratories

• Current technology allows rapid development and implementation of sequencing assays

• Since mutation identification is of paramount importance for diagnosis confirmation, genetic counseling, risk assessment and carrier screening, efforts must be made to perform comprehensive mutation analysis

• As we enter the era of rare disorder testing the true mutation spectrum for many rare disorders is not known.• Comparative Genomic Hybridization (CGH), which has undergone rapid development in the last few years, offers is a powerful alternative to the current methods used for detecting large deletions and duplications. • Carrier testing for family members at risk for carrying the mutations and prenatal testing could be offered following identification of a deletion or duplication.

Rare Disorders

Traditional Methods to Detect Deletions/Duplications

Southern blotLaborious and time consuming

Multiplex PCRLimited primarily to X-linked genes in males

MLPA Subject to interference from polymorphismsDepends on single probe

Real-Time PCRRequires optimization for each locus analyzed

Disadvantages of current technologies to detect deletion/duplication

• Methods dependant on hybridization of a single probe

• Testing for autosomal disorders and X‐linked disorders in females is difficult– Duplications are not easily detected

• These methods are time consuming, laborious and do not accurately detect all types of mutations. Suitable for testing in males.

Comprehensive mutation analysis in a clinical diagnostic setting- Use of targeted comparative genomic hybridization (CGH) array for the detection of single- and multi-

exon gene deletions and duplications

Tayeh et al, 2009, Genetics in Medicine

Multi gene targeted array

Comprehensive testing…………..

One gene/ one assay concept

Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 Chr11 Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr21 Chr22 ChrX

Targeted Array CGH

Targeted Array CGHChr21

HLCS

CBS

Metabolic disorder genes (28 disorders, 35 genes) Lysosomal storage genes (20 disorders, 20 genes)Argininosuccinate Lyase Deficiency (ASL) Alpha-Mannosidosis (MAN2B1)

Biotinidase Deficiency (BTD) Beta-Mannosidosis (MANBA)

Carnitine/acylcarnitine translocase Deficiency (SLC25A20) CitrullinemiaType 1 (CTLN1)

Carnitine Palmitoyltransferase II Deficiency (CPT2) Fabry Disease Alpha-Galactosidase (GLA)

Carnitine Uptake Defect (SLC22A5) Glycogen Storage Disease Type II (Pompe disease) (GAA)

Citrullinemia (ASS1) Krabbe disease (GALC )

Epimerase UDP-galactose-4-epimerase (GALE) Metachromatic leukodystrophy (ARSA)

Galactokinase (GALK1) Mucopolysaccharadosis type I (IDUA)

Galactosemia (GALT) Mucolipodosis type II (GNPTAB)

Glutaric Aciduria Type I; GA-1 (GCDH) Mucopolysaccharadosis type IIIA (SGSH)

Glutaric Aciduria Type II (Multiple Acyl-CoA Dehydrogenase Deficiency (ETFA, ETFB and ETFDH) Mucopolysaccharadosis type IIIB (NAGLU)

Homocystinuria (CBS) Mucopolysaccharidosis type IIIC (TMEM76)

3-Hydroxy-3-Methylglutaryl CoA Lyase (HMGCL) Mucopolysaccharadosis type IIID (GNS)

Isovaleric Acidemia (IVD) Mucopolysaccharadosis type IVB (GLB1)

Long-Chain hydroxy Acyl-CoA Dehydrogenase Deficiency; LCHAD (HADHA) Mucopolysaccharadosis type VI (ARSB)

Maple Syrup Urine Disease - BCKD Complex (DBT, BCKDHA and BCKDHB) Mucopolysaccharadosis type VII (GUSB)

MSUD: E3-deficient maple syrup urine disease (DLD) Nieman Pick A/B (SMPD1)

Medium-Chain Acyl-CoA Dehydrogenase (ACADM) Nieman Pick C (NPC2)

3-Methylcrotonyl-CoA Carboxyl Deficiency; 3-MCC (MCCC1 and MCCC2) Sandhoff Disease (HEXB)

Methylmalonic Aciduria (MMAA and MMAB) Tay Sachs disease (HEXA)

Methylmalonlyl CoA Mutase (MUT) Other disorders (10 genes)Ketothiolase Deficiency (ACAT1) Autosomal Dominant Optic Atrophy (OPA1 and OPA3)

Ornithine Transcaramylase deficiency (OTC) Chromosome 9q subtelomeric deletion syndrome (EHMT1)

Phenylalanine Hydroxylase (PAH) Congenital Heart Defects (NKX2.5)

Propionic Acidemia (PCCA and PCCB) Connexin 26 – (GJB2)

Trifunctional Protein Deficiency (HADHA and HADHB) Connexin 30 – (GJB6)

Tyrosinemia type I (FAH) Cystic Fibrosis (CFTR)

Very Long-Chain Acyl-CoA Dehydrogenase (ACADVL) Fragile X syndrome (FMR1)

Rett syndrome (MeCP2)

Mowat-Wilson Syndrome (ZEB2)

250+ Genes (58 Disorders) Represented on the Targeted CGH Microarray

Targeted Array CGH• Gene centric design

• 180K/60K probes tiled on the array

• Average spacing in coding region = ~4 probes/100 bp

• Average spacing in intronic region = 25 bp

• Length of probes ranges from 60 bp

• CGH performed using same sex controls

• Array analyzed using CYTOSURE

• Data masking feature

DMD gene: MLPA vs CGH

Deletion Ex8 - 13

Hegde et al, Hum Mutation, 2008

MLPA

MLPA (DMD gene)

OGT HD 180K/60K EGL Array

DMD Ex 7 del

DMD Ex 2 dup

DMD Ex 12 dup

DMD Ex 12-53 del

DMD (Archived sample)

•A young woman (K.M.) in early pregnancy

• She knew of a history in her mother’s family of DMD.

• Three maternal uncles (2 were identical twins) had been diagnosed with DMD in the 1960s, and died in the 1970s-80s

Don Love, Auckland, NZ

• tissue found!

Exon 63 dup

EmArray Targeted CGH Array

• ~ close to the intronic breakpoint (100‐200bp)• Non contiguous rearrangements • Advantages over MLPA‐ Partial exon disruption (more than one probe per exon), endpoints of large deletions, accurately detect deletions and duplication in males and females.

• Single exon del/dup• Rapid TAT/ Cost• Understand structural elements

OTC exon 2 del- ~700bp

Autosomal Recessive disorders

NBS genes

Phenylketonuria (PKU): Deletion/ point mutation

• 4 month old male

– Elevation of phenylalanine on NBS

– Diagnosis of PKU/hyperphenylalaninemia

– One copy of a c.728G>A (p.R243Q)

– A second mutation was not identified by sequencing

Deletion encompasses exons 5 and 6 of PAH

Normal

Deletion

Detection of a 11,652 bp deletion in PAH

Sequence of the PAH gene junction fragment

Exon 5 sequence

Intron 6 sequence

Maple syrup urine disease (MSUD)‐Non amplification of exon

• 2 year old male with a diagnosis of MSUD–Referred for sequencing the BCKD complex genes BCKDHA, BCKDHB and DBT

–Enzymatic analysis performed by our biochemical genetics laboratory found very low activity in cultured fibroblasts

–No mutations were identified by sequencing the BCKDHA, BCKDHB and DBT genes 

*However, exon 2 of the DBT gene was unable to be amplified.

DBT exon 2 (7.5Kb)(two hispanic families)

Maple syrup urine disease (MSUD)‐ Duplication

• 20 year old male with a diagnosis of MSUD– Referred for sequencing the BCKD complex genes BCKDHA, BCKDHBand DBT

– Patient had elevation of the branched chain amino acids leucine, isoleucine and valine, as well as alloisoleucine in plasma

– One copy of c.752T>C (p.V251A) change in Exon 7 was detected inBCKDHB. A second mutation was not detected

–Sequence data for the p.V251A looked strange

Detection of a ~129kb duplication in BCKDHB

Normal

Duplication*Duplication encompasses

Exons 7 to 9 of BCKDHB

Krabbe disease‐ deletion and duplication onopposite alleles

• Caused by a deficiency of the enzyme galactocerebrosidase. 

• Mutations in the GALC gene cause a deficiency of the enzyme galactocerebrosidase. 

• Deletion of segment of GALC starting in intron 10 and extending through 3’ end of the gene (30kb del) is the most common mutation representing ~35%‐50% of mutant European alleles.

Allele specific PCR is used to detect this common deletion

normal

30kb delKleijer et al. J. Inher. Metab. Dis. 1997. 20:587-594

Detection of a Common 30kb Deletion in GALC

exon 1

Detection of a Common 30kb Deletion in GALC

Intron 10 – 3’end

NormalDeletion

Family with Daughter Affected with Krabbe disease

normal

30kb del

One copy of 30kb del identified in the daughterA second mutation was not identified by DNA sequencing

One copy of the 30kb del identified in the mother

No mutation was identified in the father

Intron 10 – 3’UTR Exon 1

Detection of a ~11,000 bp duplication mutation within GALC gene

NormalDeletionDuplication*

Father

Daughter

Mother

*1st case of a duplication within GALCAlexander et al, Am J Med Genetics, Submitted

Ex11 - 14

Ex15 – 3’

Ex11 – 3’

Affected Daughter Carries a Deletion and  a Duplication of the GALC Gene

Allele 1: 30kb deletion (from intron 10 – 3’end)

Allele 2: 11kb duplication (from intron 10 – intron 14)

11kb dup/NegIntron11 – Intron 14/Neg 30kb del/Neg

Intron 10 – 3’end

Array Based Testing for Other Disorders

• X‐linked Intellectual disability (XLID)‐ 93 genes

• Cystegen‐13 genes for polycystic kidney disease

• Inherited cancer genes

• Neuromuscular disorders

• Congenital disorders of glycosylation

• Many more…..

XLID

Validation of DNA extracted from blood and saliva

Using blood spots as a starting material……

Data from dried blood spots- DMD Newborn/ Infant screening

Data from dried blood spots- DMD Newborn/ Infant screening

Data from dried blood spots- DMD Newborn/ Infant screening

Saliva validation

Saliva

Blood

Chr 16: UMOD

- The targeted CGH array can detect deletions and duplications in DNA isolated directly from dried blood spots and saliva.

- DNA recovered from 3mm dried blood spot punches is of a sufficient amount and quality for several targeted array CGH assays.

- Whole genome amplified DNA generated with phi29 does not perform as well in the detection of deletions and duplications in the DMD gene, because of a higher background, though deletions were called correctly when compared to DNA isolated directly from blood.

Data from dried blood spots -Newborn/ Infant screening

Cytogenetic array>>>>><<<<<<Molecular arrays

Interpretation

•Duplications can be difficult to interpret- Important to compare to clinical presentation

•Duplications are difficult to confirm- qPCR, breakpoint PCR

•Intronic changes need confirmation (inversions)

•Deletion (in frame / out of frame)

•Limits of detection>>> reflex to whole genome array

•Mosaicism

•Analysis algorithms

•Data and breakpoints highly dependant on array design- number of probes vs spacing

•Genes with associated pseudogenes cannot be analyzed on CGH

Points to Remember

Richards et al, Genetics in Medicine, 2008

Acknowledgements

Lora BeanBrad Coffee

Ephrem ChinChad Alexander

Melissa LeeDavid LedbetterStephen Warren