Utility of Microarrays in Molecular Genetics

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1 Utility of Microarrays in Molecular Genetics Madhuri Hegde, Ph.D., FACMG Associate Professor Senior Director Department of Human Genetics Emory Genetics Laboratory Emory University School of Medicine Atlanta, GA

2 Mutation spectrum Hunter syndrome (IDS mutations) 80% small mutations 20% deletions/duplications Duchenne Muscular Dystrophy (DMD) 30% small mutations 65-70% deletions/duplications Small mutations (nucleotide substitutions, small indels) Larger mutations (exon and multi-exon dels and dups) Cystic Fibrosis (CFTR) >90% small mutations <10% deletions/duplications Krabbe disease (GALC) 35-50% deletions/duplications 50-65% small mutations

3 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 Outline 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

4 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

5 Rare Disorders 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.

6 Traditional Methods to Detect Deletions/Duplications Southern blot Laborious and time consuming Multiplex PCR Limited primarily to X-linked genes in males MLPA Subject to interference from polymorphisms Depends on single probe Real-Time PCR Requires optimization for each locus analyzed

7 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.

8 Comprehensive mutation analysis in a clinical diagnostic setting- Use of targeted comparative genomic hybridization (CGH) array for the detection of single- and multiexon gene deletions and duplications Tayeh et al, 2009, Genetics in Medicine

9 Multi gene targeted array Comprehensive testing.. One gene/ one assay concept

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

11 Targeted Array CGH Chr21 HLCS CBS

12 250+ Genes (58 Disorders) Represented on the Targeted CGH Microarray Metabolic disorder genes (28 disorders, 35 genes) Argininosuccinate Lyase Deficiency (ASL) Biotinidase Deficiency (BTD) Carnitine/acylcarnitine translocase Deficiency (SLC25A20) Carnitine Palmitoyltransferase II Deficiency (CPT2) Carnitine Uptake Defect (SLC22A5) Lysosomal storage genes (20 disorders, 20 genes) Alpha-Mannosidosis (MAN2B1) Beta-Mannosidosis (MANBA) CitrullinemiaType 1 (CTLN1) Fabry Disease Alpha-Galactosidase (GLA) Glycogen Storage Disease Type II (Pompe disease) (GAA) Citrullinemia (ASS1) Krabbe disease (GALC ) Epimerase UDP-galactose-4-epimerase (GALE) Galactokinase (GALK1) Galactosemia (GALT) Glutaric Aciduria Type I; GA-1 (GCDH) Glutaric Aciduria Type II (Multiple Acyl-CoA Dehydrogenase Deficiency (ETFA, ETFB and ETFDH) Homocystinuria (CBS) 3-Hydroxy-3-Methylglutaryl CoA Lyase (HMGCL) Isovaleric Acidemia (IVD) Long-Chain hydroxy Acyl-CoA Dehydrogenase Deficiency; LCHAD (HADHA) Maple Syrup Urine Disease - BCKD Complex (DBT, BCKDHA and BCKDHB) MSUD: E3-deficient maple syrup urine disease (DLD) Medium-Chain Acyl-CoA Dehydrogenase (ACADM) 3-Methylcrotonyl-CoA Carboxyl Deficiency; 3-MCC (MCCC1 and MCCC2) Methylmalonic Aciduria (MMAA and MMAB) Methylmalonlyl CoA Mutase (MUT) Ketothiolase Deficiency (ACAT1) Ornithine Transcaramylase deficiency (OTC) Phenylalanine Hydroxylase (PAH) Propionic Acidemia (PCCA and PCCB) Trifunctional Protein Deficiency (HADHA and HADHB) Tyrosinemia type I (FAH) Very Long-Chain Acyl-CoA Dehydrogenase (ACADVL) Metachromatic leukodystrophy (ARSA) Mucopolysaccharadosis type I (IDUA) Mucolipodosis type II (GNPTAB) Mucopolysaccharadosis type IIIA (SGSH) Mucopolysaccharadosis type IIIB (NAGLU) Mucopolysaccharidosis type IIIC (TMEM76) Mucopolysaccharadosis type IIID (GNS) Mucopolysaccharadosis type IVB (GLB1) Mucopolysaccharadosis type VI (ARSB) Mucopolysaccharadosis type VII (GUSB) Nieman Pick A/B (SMPD1) Nieman Pick C (NPC2) Sandhoff Disease (HEXB) Tay Sachs disease (HEXA) Other disorders (10 genes) Autosomal Dominant Optic Atrophy (OPA1 and OPA3) Chromosome 9q subtelomeric deletion syndrome (EHMT1) Congenital Heart Defects (NKX2.5) Connexin 26 (GJB2) Connexin 30 (GJB6) Cystic Fibrosis (CFTR) Fragile X syndrome (FMR1) Rett syndrome (MeCP2) Mowat-Wilson Syndrome (ZEB2)

13 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

14 DMD gene: MLPA vs CGH Deletion Ex8-13 Hegde et al, Hum Mutation, 2008

15 MLPA

16 MLPA (DMD gene)

17 OGT HD 180K/60K EGL Array

18 DMD Ex 7 del

19 DMD Ex 2 dup

20 DMD Ex 12 dup

21 DMD Ex del

22 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

23 tissue found!

24 Exon 63 dup

25 EmArray Targeted CGH Array ~ close to the intronic breakpoint ( bp) 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

26 OTC exon 2 del- ~700bp

27 Autosomal Recessive disorders NBS genes

28 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

29 Detection of a 11,652 bp deletion in PAH Normal Deletion Deletion encompasses exons 5 and 6 of PAH

30 Sequence of the PAH gene junction fragment Exon 5 sequence Intron 6 sequence

31 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.

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

33 Maple syrup urine disease (MSUD) Duplication 20 year old male with a diagnosis of MSUD Referred for sequencing the BCKD complex genes BCKDHA, BCKDHB and 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 in BCKDHB. A second mutation was not detected Sequence data for the p.v251a looked strange

34 Detection of a ~129kb duplication in BCKDHB Normal Duplication* Duplication encompasses Exons 7 to 9 of BCKDHB

35 Krabbe disease deletion and duplication on opposite 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.

36 Detection of a Common 30kb Deletion in GALC Allele specific PCR is used to detect this common deletion normal 30kb del Kleijer et al. J. Inher. Metab. Dis :

37 Detection of a Common 30kb Deletion in GALC Intron 10 3 end exon 1 Normal Deletion

38 Family with Daughter Affected with Krabbe disease One copy of 30kb del identified in the daughter A 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 normal 30kb del

39 Detection of a ~11,000 bp duplication mutation within GALC gene Intron 10 3 UTR Exon 1 Mother Ex11 3 Daughter Ex15 3 Father Ex11-14 Normal Deletion Duplication* *1 st case of a duplication within GALC Alexander et al, Am J Med Genetics, Submitted

40 Affected Daughter Carries a Deletion and a Duplication of the GALC Gene 11kb dup/neg Intron11 Intron 14/Neg 30kb del/neg Intron 10 3 end Allele 1: 30kb deletion (from intron 10 3 end) Allele 2: 11kb duplication (from intron 10 intron 14)

41 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..

42 XLID

43 Validation of DNA extracted from blood and saliva

44 Using blood spots as a starting material

45 Data from dried blood spots- DMD Newborn/ Infant screening

46 Data from dried blood spots- DMD Newborn/ Infant screening

47 Data from dried blood spots- DMD Newborn/ Infant screening

48 Saliva validation Chr 16: UMOD Saliva Blood

49 Data from dried blood spots - Newborn/ Infant screening - 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.

50 Cytogenetic array>>>>><<<<<<molecular arrays Interpretation

51 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 Points to Remember Data and breakpoints highly dependant on array design- number of probes vs spacing Genes with associated pseudogenes cannot be analyzed on CGH

52 Richards et al, Genetics in Medicine, 2008

53 Acknowledgements Lora Bean Brad Coffee Ephrem Chin Chad Alexander Melissa Lee David Ledbetter Stephen Warren