PROF. OLADAPO ASHIRUM.B.B.S, MS, PHD. HCLD/CC(ABB-USA), FASN, FNSEM, OFR

DEVELOPMENTS AND LANDMARKS OF GENETIC TESTING IN WEST

AFRICA

48th Annual General Meeting and Scientific Conference

SOGON ASABA 2014.

ASSISTED REPRODUCTIVE TECHNOLOGY (ART)

Conception requiring the complex handling or manipulation of male and female gametes in-vitro to facilitate pregnancy

LANDMARKS IN ART

1969 Sperm Capacitation Chang & Yamagimachi

1976 IVF in London Steptoe & Edwards 1978 IVF baby London Steptoe & Edwards

(First IVF Baby – Louise Brown) 1977 LHRH Schally & Guileman 1978 FSH +ve feedback Ashiru & Blake 1980 IVF in Melbourne, Australia Carl Wood 1982 IVF in Virginia Jones 1984 IVF in Lagos, Nigeria Ashiru & Giwa-

Osagie– 1986/1989

1992 ICSI Jacques Cohen

LANDMARKS IN PGD

Jacobson CB & Barter RH 1960 - 1st prenatal diagnosis

Saiki et al 1985 – PCR Alan Handyside and Robert Winston 1989 –

Embryo Biopsy Verlinsky Y et al 1999 – Polar body testing

LANDMARKS IN PGD

1990: Sexing Human Preimplantation Embryo, & Polar Body Analysis for Mendelian Disease; - First PGD Baby Born

1993: FISH Analysis for Sexing and for PGD of Aneuploidies

1996: PGD for Chromosomal Translocations 1999: PGD for Late Onset Common Disorders

with Genetic Predisposition

2000: Pre-implantation HLA Typing

2002: 1st Thousand PGD Babies Born

2002 onwards: Many technological

developments CGH, aCGH, NGS, SNP,qPCR

LANDMARKS IN PGD

TYPES OF PREIMPLANTATION GENETIC TESTING

Preimplantation Genetic Diagnosis (PGD)– Targeted genetic testing of known

genetic abnormality in the couple:Single gene disorderStructural chromosomal

abnormality

Preimplantation Genetic Screening for Aneuploidy (PGS)– Screening for numerical abnormality of select

chromosomes: 13, 18, 21, X, Y, etc Preimplantation Gender Determination or

Gender Selection

TYPES OF PREIMPLANTATION GENETIC TESTING

INDICATIONS FOR PGD

Autosomal dominant disorders– One of the couple is a carrier of the

genetic defect (e.g., Huntington Disease or dwarfism)

Autosomal recessive disorders:– Both couple are carriers of the genetic

defect (e.g., Sickle Cell Disease)

INDICATIONS FOR PGD

X-linked disorder– One of the couple is a carrier of a x-linked genetic

defect (e.g., Hemophilia) Human leukocyte antigen (HLA) matching Structural chromosomal abnormality

– One of the couple is a carrier of a chromosome abnormality (translocation, inversion, deletion, insertion)

INDICATIONS FOR PGS (ANEUPLOIDY SCREENING)

Advanced maternal age Recurrent pregnancy loss Repeated IVF failure Severe male factor infertility

INDICATIONS FOR GENDER SELECTION

One of the couple is a carrier of x-link disorder

Family balancing

• May be largely diagnostic• No paternal/meiosis II/post-zygotic information• May require later follow-up testing (d3/d5)

BIOPSY TECHNIQUES

• Accounts for meiosis II errors• No paternal/post-zygotic information• Most expensive option diagnostically

• Biopsy most detrimental• Chromosomal mosaicism common• Associated with Low implantation

• Highly recommended• 3-10 (?) cells (high accuracy/reliability)• Significance of mosaicism?• Least expensive per patient (diagnosis)• May require vitrification

BIOPSY OBJECTIVE

Methods of Genetic Analysis: PGD single gene disorder

– Whole genome amplification– Polymerase chain reaction (PCR)– Direct detection of specific mutation– Genotyping of linked polymorphic markers to

ensure: Bi-parental inheritance Infer the inheritance of normal copy of the gene (Allele)

Methods of Genetic Analysis: Structural chromosomal abnormality

Fluorescent in situ hybridization (FISH)

PCR-based testing– Whole genome amplification– Genotyping of linked polymorphic markers

METHODS

FISH Gender Determination

Orange: X chromosomeGreen: Y chromosome

FISH Aneuploidy Screening

Normal Male Normal Female

Trisomy 21 Male Red: chromosome 13Cyan: chromosome 18Green: chromosome 21Purple: chromosome XYellow: chromosome Y

ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (aCGH)

• A molecular cytogenetic technique

• Detects 'copy number changes' of chromosomes on a genome

• Embryo DNA is compared with a known normal DNA specimen utilising thousands of specific genetic markers

• More accurate results (fewer false normal or false abnormal results)

• Error rate of 2% compared to 5 - 10% of FISH

aCGH versus Conventional CGH

• Better resolution (as low as 5 – 10 kilobytes of DNA sequences)

• Cloned DNA fragments with exact chromosomal location replaces reference metaphase spread

• Better detection of aberrations

• Better identification of micro deletions and duplications

• Increases the possibility of mapping changes directly onto the genomic sequence

ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (aCGH) - METHOD

Same principle as conventional CGH Labelling of DNAs (control and test) with 2 different

'flurophores' (green and red) Green flurophore (cyanine 3) for test / patient and red

flurophore (cyanine 5) for control / reference are used as 'probes'

Competitive cohybridisation of probes onto nuclei acid targets (cloned genomic fragments (BACs / plasmids), cDNAs or oligonucleotides) with thousands of genetic markers.

Digital imaging / specialised microscopes to capture and quantify fluorescence intensities of hybridised flurophores.

ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (aCGH) – RESULT

Interpretation of results. Ratio of the fluorescence intensities is proportional to the ratio

of 'copy number of DNA sequences' in the test and reference genomes.

Altered Cy 3 : Cy 5 ratio indicates a loss or gain of the patient DNA at that specific genomic region

If Cy 3 : Cy 5 ratio is equal on one probe (equal intensities of the flurophores), patient's genome is interpreted as having equal quantity of DNA as in the reference sample.

Array CGH remains the ‘gold standard’ for PGS with the greatest clinical experience to date

RCTs show great promise for PGS Numerous RCTs are ongoing Relatively easy to set up Use of a reference laboratory recommended

as a first step

ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (aCGH)

DETECTION OF WHOLE CHROMOSOME ANEUPLOIDIES IN SINGLE CELLS

Trisomy 13 male(47,XY,+13)

Sequencing Results

24Sure Results

PGD/PGS SINCE 2009 (MART DATA)

Chromosomes Aneuploidy (CA) Single gene disorders (Sickle cell – SC) Family balancing (sex selection – FB)

So far over 75 cases have been done

SUMMARY OF REASONS FOR PGD/PGS (N=75)

CA FB SC0%

5%

10%

15%

20%

25%

30%

35%

40%

45%43%

32%23%

SUMMARY FOR PGS (N= 32)

Normal Abnormal No Result

0%

10%

20%

30%

40%

50%

60%

26%

59%

15%

SUMMARY OF EMBRYO BIOPSED FOR CHROMOSOMES ANEUPLIODY (N=300)

59% of embryos biopsied were abnormal

15% No result rate

? Number and quality of cells tested

SICKLE CELL DIAGNOSIS (PGD) N = 17

41% of biopsied embryos were homozygous and heterozygous normal

34% of embryos undiagnosed

Normal Abnormal No Result0%

5%

10%

15%

20%

25%

30%

35%

40%

45% 41%

25%

34%

SUMMARY OF EMBRO BIOPSED FOR SICKLE CELL DIAGNOSIS

(N= 133)

FAMILY BALANCING – SEX SELECTION (N=26)

Male Total Female0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

47%

100%

47%

SUMMARY OF GENDER ANALYSIS IN FAMILY BALANCING

No difference in the proportion of male and female embryos

SUMMARY OF PREGNANCY RATE

CA FB SC0%

10%

20%

30%

40%

50%

60%

70%

32%

59%

9%

SUMMARY OF CLINICAL PREGANCY RATE

CA (%) FB (%) SS (%)0

5

10

15

20

25

30

35

30

35

9

Clinical Rate Series2

SETTING UP A PGD LAB

Two ways

IVF centre and PGD centre in the same institute – preferred

Transport PGD

ORGANIZATION OF THE PGS/PGD CENTRE

Highly successful IVF unit Patients need genetic and specific PGD counselling Biopsy performed by trained embryologist Diagnosis performed by molecular biologist/cytogeneticist Patient information leaflets and consents Excellent communication between IVF centre and

diagnosis lab Join the PGD Consortium

WHAT MAKES A GOOD PGD CENTRE

COMMUNICATION

Excellent IVF Platform

Excellent Diagnostics Laboratory

Integration of Services

Commitment to Follow-up

TRANSPORT PGD

FUTURE OF PGD/PGS

• PGD–New technology to allow diagnosis

of more disorders–Whole genome amplification–SNP microarrays, array-CGH,

quantitative real time PCR (qPCR)–Blastocyst biopsy and vitrification

• PGS–Randomised controlled trials to see if valid

procedure with clinical significance–NOT cleavage stage biopsy, NOT FISH–Try polar body or trophectoderm biopsy–SNP, array-CGH, or sequencing

FUTURE OF PGD/PGS

BEETHOVEN

“I RECOGNIZE NO SUPERIORITY IN MANKIND OTHER

THAN GOODNESS.”

THE TEAM

THANK YOU

FOR LISTENING!