Chromosome Microarray Analysis (CMA)

Transcription

1 Chromosome Microarray Analysis (CMA) Ina E. Amarillo, PhD FACMG Cytogenomics Lab (Associate Medical Director) Pathology (Assistant Professor)

2 OUTLINE Clinical Indications for Cytogenomics Testing Cytogenomics Tests Karyotype FISH CMA CMA as a Clinical Tool Platforms Analysis Softwares CMA Protocol Analysis Interpretation and Reporting CMA as a Research Tool

3 Cytogenomics Lab Requesting MD (clinical diagnosis, differentials, order appropriate tests) Tests (genetics, enzyme, protein, radiography, etc Genetics cytogenetics, molecular genetics Results to requesting MD MD to Patient (management, therapy)

4 Cytogenomics Testing Cancer HemeOncology, Hemepathology, Oncology Hematopoietic (blood and bone marrow) Solid Tumors (lymph node, solid tissue) fresh or fixed

5 Cytogenomics Testing Constitutional Peripheral Blood, Skin punch, buccal swab (NICU, Peds, Neuro, Clin Gen etc) Prenatal (OB-Gyn) Amniotic Fluid Chorionic Villi Sample Products of Conception (OB-Gyn) tissues from spontaneous abortions villi fetal parts (skin, limbs, etc)

6 Sample Processing QA/QC (proper tube, conditions, no contaminations, correct ordered test) Setup Direct for FISH or CMA Setup for culture (in situ or suspension) Cell harvest Metaphase spread/slide preparation Analysis and Interpretation Sign out (lab director) Release to requesting MD

7 Cytogenomics Tests: Karyotype Analysis Constitutional, Cancer specimen setup harvest slide prep G-banding (Giemsa stain and trypsin) 46,XY,+13 Karyotype; 2n=47 46,XY Karyotype 2n=46 45,X Karyotype; 2n=45

8 Cytogenomics Tests: FISH Analysis Constitutional, Cancer Fluorescence In Situ Hybdridization Constitutional, Cancer unique (BACs, cosmids) repeats duplication (3 copies) deletion (1 copy) 2 centromeres deletion (1 copy) a pair of chromosome 1 Principles of Cytogenetics, 3 rd ed

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10 Resolution of Karyotype, FISH and CMA in detecting genomic changes 2013 ACMG Review Course

11 2013 ACMG Review Course

12 2013 ACMG Review Course

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15 Platforms Nature Biotech 29(6), 2011

16 Analysis Softwares Statistics, Bioinformatics, Engineers Nature Biotech 29(6), 2011

17 CMA in our Lab

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20 cytogeneticist software developers Copy Number gain, loss, neutral Size (Mb) Gene Content Reportable Variant?

21 Protocol and QA/QC steps QA/QC

22 Protocol and QA/QC steps

23 Karyoview Detail View Segment Table View

24 Analysis Karyoview

25 Analysis Detail View

26 CMA Reporting Criteria Size Deletion: 200 Kb Duplication: 500 Kb AOH: 10 Mb Gene Content and relevance Established Clinical Significance below cutoff but significant Variants of Uncertain Clinical Significance Abnormal or Normal

27 Copy Number Loss 22q11.21

28 Processed Data Analysis and Interpretation

29 Processed Data Analysis and Interpretation Copy Number (CN) State CN = 1 Normal: CN = 2 Allele Peaks (A allele frequency) +0.5, -0.5 Normal: +1.0, 0, -1.0 A_ AA B_ AB BB Weighted Log2 Ratio

30 Copy Number Loss ~2.8 Mb on 22q11.21

31 Copy Number Loss ~2.8 Mb on 22q11.21 Analysis and Interpretation Built-in and Custom Tracks

32 22q11.2 deletion syndromes Congenital heart disease (74% of individuals), particularly conotruncal malformations (tetralogy of Fallot, interrupted aortic arch, ventricular septal defect, and truncus arteriosus) Palatal abnormalities (69%), particularly velopharyngeal incompetence, submucosal cleft palate, bifid uvula, and cleft palate Characteristic facial features Learning difficulties (70%-90%) An immune deficiency (regardless of the clinical presentation) (77%), Autoimmune disorders Hypocalcemia (50%) Significant feeding and swallowing problems; constipation w/ or w/o GI anomalies (intestinal malrotation, imperforate anus, and Hirschsprung disease) Renal anomalies (31%) Hearing loss (both conductive and sensorineural) Laryngotracheoesophageal anomalies Growth hormone deficiency Seizures (idiopathic or associated with hypocalcemia) CNS anomalies including tethered cord Skeletal abnormalities (scoliosis with or without vertebral anomalies, clubbed feet, polydactyly, and craniosynostosis) Ophthalmologic abnormalities (strabismus, posterior embryotoxon, tortuous retinal vessels, scleracornea, and anophthalmia) Enamel hypoplasia Gene Reviews

33 22q11.2 deletion syndromes 40 OMIM and 66 RefSeq genes Gene Reviews

34 Cytogenomics Tests for 22q deletions FISH Probe Coverage

35 22q Duplication CN=3 AAA AAB ABB BBB

36 22q duplication syndromes 3-Mb or 1.5-Mb proximal tandem duplication. generally mild and highly variable intellectual disability/learning disability delayed psychomotor development growth retardation, and/or hypotonia apparently normal - The high frequency with which the 22q11.2 duplication is found in an apparently normal parent of a proband suggests that many individuals can harbor a duplication of 22q11.2 with no discernible phenotypic effect

37 Deletion/Duplication Hotspots

38 Summary of Deletion/Duplication

39 AOH Absence of Heterozygosity (aka LCSH long contiguous stretch of homozygosity)

40 AOH (absence of heterozygosity) CN=2 (copy neutral) AA AB BB

41 Identity by Descent (IBD) - Consanguinity

42 Identity by Descent (IBD) - Consanguinity Total: ~210 Mb MULTIPLE REGIONS WITH ABSENCE OF HETEROZYGOSITY (AOH) IDENTIFIED ON CHROMOSOMES 1, 2, 4, 5, 8, 11, 15, and 18

43 Consaguineous Matings and Coefficient of Inbreeding Chart Proportion of Genes in Common Coefficient of inbreeding of child (F) in percentage Size of LCSH (Mb) Degree of Type of Mating Relationship MZ twins 1 100% 3000 Parent-Child 1 1/2 25 % 750 Brother-sister (including dizygotic twins) 1 1/2 25% 750 Brother-half sister 2 1/4 12.5% 375 Uncle-niece or auntnephew 2 1/4 12.5% 375 Half uncle-niece 3 1/8 6.25% First cousin 3 1/8 6.25% Double first cousins 2 1/4 12.5% 375 Half first cousins 4 1/ % First cousings once removed 4 1/ % Second cousins 5 1/ % more common, lesser meiotic recombination, higher inbreeding coefficient higher occurrence of autosomal recessive genes and disorders ~210 Mb Thompson and Thompson. Genetics in Medicine. Edition 7 th

44 Uniparental Disomy (UPD) Single Chromosome/Segment Imprinted Genes? Known UPD?

45 Classification of Variants Benign Pathogenic Disease Causing Uncertain Clinical Significance Databases (UCSC, DGV, DECIPHER, ISCA) Published Research or Case Reports Gene Function

46 DATABASES

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48 ~ Mb BENIGN Gain/Loss Variant on 16p11.2

49 ~ Mb BENIGN Gain/Loss Variant on 16p11.2

50 16p region benign and pathogenic PATHOGENIC GAIN/LOSS BENIGN GAIN AND LOSS centromere

51 CNV smaller than the reporting cutoff AUTS2 - autism susceptibility candidate 2 (19 exons)

52 CNV smaller than the reporting cutoff AUTS2 - autism susceptibility candidate 2 (19 exons) Firth et al 2009 AJHG

53 CNV smaller than the reporting cutoff AUTS2 - autism susceptibility candidate 2 (19 exons)

54 Variants smaller than the cutoff (7q11.22; Kb intronic/exonic deletion ) Amarillo et al 2014

55 Variants smaller than the cutoff (7q11.22; Kb intronic/exonic deletion )

56 Variants smaller than the cutoff (7q11.22; Kb intronic/exonic deletion )

57 Variants in gene-desert region: position effect Amarillo et al 2013

58 Variants of Uncertain Clinical Significance areas of genomics research smaller than cutoff but no relevant publications or reports smaller than cutoff but relevant studies in model animal organisms intronic copy number changes chromosome rearrangements and gene disruptions biomarker discovery gene discovery

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60 THANK YOU!