Come gather 'round peopleWherever you roamAnd admit that the watersAround you have grownAnd accept it that soonYou'll be drenched to the bone.If your time to youIs worth savin'Then you better start swimmin'Or you'll sink like a stoneFor the times they are a-changin'.

THE NEXT GENERATION:Microarray and Beyond

Karyotype

Resolution: >7-10 Million Base Pairs

(7-10 Mb)

Resolution: < 0.5 Million Base Pairs

(< 500 kb)

Chromosomal Microarray(CMA)

Microdeletion Syndromes

DiGeorge 22q11 Deletion 3.5Mb

Miller Dieker 17p13.3 deletionPrader Willi 15q11-13 deletion 4MBSmith Magenis

17p11.2 deletion 5Mb

Wolf Hirshhorn

4p16.3 deletion 1.9Mb

Williams-Beuren

7q11.23Deletion 1.5Mb

Non-Syndromic Micro Del /Dups

15-20% yield by CMA in children with unexplained developmental delay/ID, and congenital anomalies compared to ~3% with karyotype

16p11.2 Autism 0.55Mb

1q21.1 ID, microcephaly, cardiac, cataracts

0.8Mb

16p13.11 Autism, ID, and schizophrenia

0.8Mb

ID: Intellectual Disability

Velo Cardio Facial Syndrome

Postnatal Studies

Structural Anomalies

Array Adds Significant Clinically Relevant Information in Cases With Normal Karyotype

Structural Anomaly Fiorentino 6.1 %Rosenfeld /Shaffer 6.6 %Schwartz 5.7 %NICHD 6.0 %

7q11.23 microduplication syndrome

Amniocentesis:Karyotype: 46,XYArray: 1.39 Mb gain in 7q11.23

Van der Aa, et al. Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome. European Journal of Medical Genetics 2009

Left Foot Right Foot

Differential Diagnosis• Aase Syndrome• Diamond-blackfan Syndrome• DOOR Syndrome• Duane-radial Syndrome (DR Syndrome)• Fanconi Anemia (Pancytopenia-dysmelia Syndrome)• Fetal Hydantoin Syndrome (Dilantin Embryopathy)• Goodman Syndrome• Holt-Oram Syndrome• Hypomelanosis Of Ito• IVIC Syndrome• Juberg-hayward Syndrome• Lacrimo-auriculo-dento-digital Syndrome (LADD Syndrome) (Levy-hollister Syndrome)• Mesomelic Dysplasia (Werner Type)• Nager Syndrome• Normal Variant : Isolated Anomaly• Poland Syndrome (Pectoral Muscle Aplasia-syndactyly)• Thalidomide Embryopathy• Townes-brocks Syndrome• Trichorhinophalangeal Dysplasia Type (Langer Gidieon Syndrome)• Trisomy 13• Trisomy 22• VATER Association

Mutation

Sequencing

Triphalangeal thumb with Polysyndactyly

• Mutation in SHH gene

• Mutations in the Sonic hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (ZRS, located within the gene LMBR1), commonly called the ZRS), cause limb malformations

Sequencing Analysis

You’re pregnant andYou must know the sex Deep sequencing Ma,

It’s all the rage !

BH 2012 SC

By Indications for Testing

Indication Total Clinically Relevant

95% CI

AMAN=1966

34 (1.7%) 1.2 – 2.4

Positive ScreenN=729

12(1.6%) 0.9 – 2.9

Clinically Relevant Information Seen by CMA andReported to Patients in Cases with

Normal Karyotype

Recurrent CNVs That Have The Potential To Cause Neurocognitive Impairment

Occurred in approximately 1 in 125 (0.8%) cases sampled for AMA or positive screening

Deletions N Nl US1q21.17q11.23

11

10

15q11.2 2 215q13.2q13.3 1 116p11.2 3 216p12.1 1 016p13.11p12.3 3 116p13.11 5 317q12 6 122q11.2 11 3

Duplications N Nl US

1q21.115q11.2q13.115q13.2q13.3

411

211

16p13.11p12.3 2 116p13.11 4 317q1222q11.21

32

22

Conclusion

Based on the increased detection of clinically relevant  abnormalities in both structurally normal and abnormal pregnancies, chromosomal microarray analysis (CMA) should be transitioned to become the first tier test for invasive prenatal cytogenetic diagnosis.

Findings of Unknown Significance

Variable Expressivity

Variants of Uncertain Clinical Significance

1. Other Cases - known del/dup or Mendelian disorders

OMIM, DECIPHER (Sanger)- known benign CNV

DGV (Toronto), dbVar (NCBI)- comparison with other cases

PubMed, DECIPHER2. Large Databases

ISCAConsortium3. Genomic/Gene Content

- correlates with size/locationUCSC, Ensembl (Sanger)

Variants Of Uncertain Clinical Significance

Counseling Issues

VOUS Pathogenic Likely Benign

2007 Study Classification

94(2.5%)

35(0.9%) -

2012 Classification

57(1.5%)

64(1.7%)

8

As CMA Transitions Into Practice Counseling By Professionals With Knowledge And Expertise In CMA Will Be Required

Mosaic Inversion Balanced Recip Translocation

Marker Other Autosmeal Trisomy

Total

Han 2008 .15 % .15% .50 % .10% .02% .85 %

Chang2012 .3 % .20 % .40% .08% - 1.0 %

Frequency of Findings of Uncertain Significance in Amniocentesis Karyotype

CVS: Confined Placental Mosaicism 1-2%

Berg: Genetics in Medicine 2011

Berg, Genetics In Medicine, June 2011

?

CVS: del16p13.12p13.11

CVS: del16p13.12p13.11 CVS: 2.0 Mb del16p13.12p13.11

Described with Autism Spectrum Disorder (ASD)/Developmental Delay, and seizures

Incomplete penetrance/ Variable Expressivity

40-44

45-49

50-54

55-59

60-64

65-69

70-74

75-79

80-84

85-89

90-94

95-99

100-104

105-109

110-114

115-119

120-124

125-129

130-134

135-139

140-144

0

2

4

6

8

10

12

14

16

18

20

ControlCarrier

Num

ber o

f Cas

esFull Scale IQ difference of 28 or 2 SD

Mean 80SD 15

Mean 108SD 12

Counseling Issues

Long term prospective study of individuals identified with pathogenic CNVs and variants of

uncertain clinical significance

Incomplete Penetrance/ Variable Expressivity

Non Invasive Prenatal Diagnosis of Common Trisomies (13,18,21)

( 1:500 Pregnancies)

Vs

Invasive Diagnosis with Array Analysis(>1:100)

All Patients Should be Counseled about the Relative Advantages and Disadvantages of Each Approach

PRETEST COUNSELING

• Additional information about the health/development of the child

• Findings of uncertain significance• Unanticipated information about the health of a parent• Pre-symptomatic recognition of adult on-set condition• Determination of non-paternity

Should be discussed with the patient prior to testing and an understanding of the patients interest in this information should be explored and documented

Issues To Discuss

Who will/can do this?

Noninvasive Prenatal Diagnosis of a Fetal MicrodeletionSyndrome

David Peters, Ph.D.Tianjiao Chu, Ph.D.Svetlana A. Yatsenko, M.D.Nancy Hendrix, M.D.W. Allen Hogge,M.D. UrvashiSurti, Ph.D. Kimberly Bunce, Ph.D.Mary Dunkel, M.S.Patricia ShawB.S.AleksandarRajkovic, M.D.Magee–Womens Research Institute

GENOMICSNoninvasive Whole-Genome Sequencing of a Human FetusJacob O. Kitzman,1 * Matthew W. Snyder,1 Mario Ventura,1,2 Alexandra P. Lewis,1 Ruolan Qiu,1LaVone E. Simmons,3 Hilary S. Gammill,3,4 Craig E. Rubens,5,6 Donna A. Santillan,7Jeffrey C. Murray,8 Holly K. Tabor,5,9 Michael J. Bamshad,1,5 Evan E. Eichler,1,10 Jay Shendure1 *

Concerns of Increasingly Complex Non-Invasive Fetal Testing

• Uncertain Reassurance• More Uncertain Findings• Scope Creep• Counseling • Ethics of What to Test For