PROF. OLADAPO ASHIRU M.B.B.S, MS, PHD. HCLD/CC(ABB-USA), FASN, FNSEM, OFR DEVELOPMENTS AND LANDMARKS OF GENETIC TESTING IN WEST AFRICA 48 th 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 LHRHSchally & Guileman 1978 FSH +ve feedbackAshiru & Blake 1980 IVF in Melbourne, Australia Carl Wood 1982 IVF in Virginia Jones 1984 IVF in Lagos, NigeriaAshiru & Giwa-Osagie – 1986/ ICSI Jacques Cohen

LANDMARKS IN PGD Jacobson CB & Barter RH st 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: 1 st 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 disorder Structural 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 chromosome Green: Y chromosome

FISH Aneuploidy Screening Normal Male Normal Female Trisomy 21 Male Red: chromosome 13 Cyan: chromosome 18 Green: chromosome 21 Purple: chromosome X Yellow: 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 % 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)

SUMMARY FOR PGS (N= 32) 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

FAMILY BALANCING – SEX SELECTION (N=26) No difference in the proportion of male and female embryos

SUMMARY OF PREGNANCY RATE

SUMMARY OF CLINICAL PREGANCY RATE

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 !