Prenatal diagnosis gene therapy

Why is prenatal diagnosis done?

Inform parents about the fetus; counsel/support reproductive decisions; offer fetal therapy and prevent postnatal complications

Can some conditions be treated in utero?

Yes — in utero or right after birth

His closing line: two things to know

Difference between screening and diagnostics; difference between somatic and germline gene transfer

Screening tests

Ultrasound (NT, anatomic survey), maternal serum marker screening, NIPT (cell-free DNA)

Diagnostic tests

Chorionic villus sampling (CVS) and amniocentesis

Screening gives…

A risk estimate, not a diagnosis; more false positives and negatives

Diagnostic testing gives…

Definitive yes/no ('gold standard')

Screening risk

Low (ultrasound or blood draw)

Diagnostic test risk

Invasive; miscarriage ~1/300–1/500 (CVS or amnio)

Who gets screening?

Population-wide, also used on individual mothers

Who is usually offered diagnostic testing?

High-risk pregnancies: increased aneuploidy risk or single-gene risk (ultrasound finding, family history, positive carrier screen); available to any pregnant woman

1st-trimester serum screening timing

Weeks 11–13+

2nd-trimester (quad) screening timing

Weeks ~15–21+

NIPT timing

From week 10 through delivery

Nuchal translucency timing

Weeks 11–13+

Fetal anatomic survey timing

After 18 wk (best 18–22 wk)

CVS timing

~10–13+ wk

Amniocentesis timing

15+ wk

Why do earlier and later tests differ?

Earlier tests (NT, CVS) come first; later ones need a more developed fetus

Nuchal translucency (NT) measures…

Fluid under the skin behind the fetal neck (nape)

Abnormal NT value

3.0 mm

Increased NT raises risk of…

Aneuploidy and fetal heart defects

NT detection of aneuploid fetuses

~60%

Do most fetuses with increased NT have aneuploidy?

No — weak test, screening only; combine with other tests

Fetal anatomic survey detects…

Structural anomalies: congenital heart defects, facial defects (cleft lip/palate)

Structural anomalies on anatomic survey raise…

Chance of aneuploidy and/or genetic syndromes

Soft markers

E.g., echogenic intracardiac foci — not anomalies but adjust the chance of aneuploidy

Slide 10 and 11 ultrasound examples

Normal four-chamber heart vs VSD; 2D and 3D ultrasound of cleft lip

Maternal serum marker screening

Blood test for fetal/placental proteins and hormones; routine, offered to all pregnant women

Serum screening evaluates chances of…

Down syndrome (T21), trisomy 18, open neural tube defects

How are serum screening results given?

As a 1/### fraction (risk)

His view of serum screening

Modest accuracy, cheap, non-invasive — 'on the way out'

First trimester screen components

NT ultrasound + serum free β-hCG and PAPP-A

Quad screen analytes

AFP, uE3 (unconjugated estriol), hCG, inhibin A

Integrated and sequential screens

Combine 1st and 2nd trimester info: slightly higher detection and/or lower false positives

NIPT evaluates…

Cell-free DNA (cfDNA) in maternal blood

Where does fetal cfDNA come from?

Fetal/placental DNA degraded into fragments in the placenta and released into mother's blood

Fetal vs maternal cfDNA fragment size

Fetal ~300 bp; maternal ~500 bp (separated partly by size)

How is NIPT DNA analyzed?

Next-generation sequencing (billions of bases per run)

NIPT detects

Trisomies 21, 18, 13; monosomy X; large deletions/duplications; common microdeletion syndromes

Can NIPT detect single-base changes?

Experimental (cost and sequencing depth)

NIPT performance for T21

~99% detection, 0.5% false positive rate (1 in 200)

NIPT vs serum screening

Higher detection, lower false positives, higher PPV

Is NIPT recommended for all pregnancies?

Slide conflicts; professional societies now recommend routine NIPT (not all women get it yet)

NIPT cost vs serum screening

Hundreds of dollars vs tens

Why can NIPT fail?

Some pregnancies have little fetal DNA in maternal blood

Is NIPT diagnostic?

No — 'approaching diagnostic' but still a screen

CVS method

Sample of the early placenta; transabdominal (needle through abdomen) or transcervical

Amniocentesis method

Transabdominal sample of amniotic fluid around the fetus

Poll: which test should NOT be done at week 12?

Amniocentesis

Tests run on a CVS/amnio sample

FISH and karyotype typically; microarray increasingly; NGS as costs fall

Microarray method

Oligonucleotide panels covering all chromosomes; hybridize DNA; fluorescence quantifies extra/missing material — considered diagnostic

Karyotype resolution

~2–3 Mb

FISH resolution

~120–400 kb

Microarray resolution

~500 bp

NGS resolution

1 bp

Slide 21 example of resolution

VCFS/DiGeorge 22q11.2 microdeletion — karyotype, FISH probe (deleted vs normal 22), array, NGS

Pre-implantation genetic testing (PGT)

IVF embryos screened for aneuploidy or single-gene disorders after biopsy

PGT biopsy

Day 3: micro-needle removes 1–2 cells; cells totipotent and equivalent, embryo not harmed

Can IVF embryos be stored?

Yes — frozen indefinitely

PGS stands for and tests for

Pre-implantation genetic SCREENING — aneuploidy

PGS method

Microarray (lower resolution than on CVS/amnio)

PGS detection rate

~98% (he said ~99%) — NOT diagnostic

PGS offered to

IVF couples

Is aneuploidy common in embryos?

Extremely common

PGD stands for and tests for

Pre-implantation genetic 'DIAGNOSIS' — a single-gene disorder the couple is at risk for

PGD method

Family-specific molecular test: DNA from single cells, PCR then sequencing for the mutant allele

Is PGD diagnostic?

No — confirm with CVS or amniocentesis

PGD offered to

Couples at increased risk for a single-gene disorder

Why is PGT not diagnostic?

Aneuploid cells are often lost/'competed away' by wild-type cells; mutations can be excluded in development — biopsy may not match the embryo

Does competing away abnormal cells happen in adults too?

Probably in somatic tissues too (single-cell sequencing)

Robertsonian case: father's karyotype

45,XY,der(14;21)(q10;q10) (39-year-old male)

Robertsonian case: amnio FISH colors

Red = 21, yellow = 14, green = 12

Robertsonian case: amnio FISH result

3 copies of chromosome 21 → translocation Down syndrome

Poll: possible fetal karyotypes from Robertsonian carrier

Normal, balanced carrier, trisomy 21, monosomy 14, trisomy 14, monosomy 21

Why translocation Down in the case?

Fetus inherited fused 14;21 (with 21 material) + normal 21 from father + mother's 21

Robertsonian case 1st IVF cycle

11 oocytes, 7 fertilized, 5 biopsied, 4 normal/balanced; 3 transferred day 4 → no pregnancy

Robertsonian case 2nd IVF cycle

15 oocytes, 10 fertilized, 9 biopsied, 3 normal/balanced all transferred → singleton; CVS at 12 wk showed balanced translocation

Robertsonian case final karyotype

45,XX,der(14;21)(q10;q10) — balanced carrier, phenotypically normal girl at 38 weeks

Why is a 45-chromosome balanced carrier normal?

Dosage of chromosome 21 is still two copies

What rule does the Robertsonian case illustrate?

PGD followed by CVS confirmation

Somatic gene therapy targets

Body tissues: bone marrow, liver, muscle, brain (in development)

Is somatic therapy heritable?

No real chance — considered safer for the population

Germline gene therapy targets

Embryo, eggs, germ cells — heritable

Is germline editing approved in the US?

No — generally forbidden in most countries

Somatic therapy types on slides

Retrovirus, adenovirus, lentivirus, AAV, mRNA, RNAi

Germline therapy type on slides

Nuclear transfer

ADA-SCID

'Bubble boy disease': severe combined immunodeficiency from ADA (adenosine deaminase) mutation; recessive

First person treated by gene therapy

Ashanti DeSilva (ADA-SCID)

ADA-SCID gene therapy method

Normal ADA gene cloned in plasmid → disabled retrovirus → infect patient's T cells → grow in culture → reinfuse

ADA-SCID long-term safety (NEJM 2025)

No harmful effects from retroviral integration so far; similar for lentiviral

How can integration risk be reduced?

Sequence ex vivo treated cells to see where the vector integrated

Retrovirus as vector

RNA virus; reverse transcribed then integrates — risk if it hits an important gene

Lentivirus as vector

Integrates — same concern as retrovirus

CRISPR

Edits the genome (deletions, single-nucleotide edits); high fidelity; possible off-target effects; can bring gene activators to a site

Adenovirus as vector

Usually does not integrate (small risk); considered safer; used in many vaccines

AAV as vector

Generally doesn't integrate; usually for neural cells; a bit safer

mRNA as therapy

Stays in cytoplasm, not reverse transcribed — essentially safe for the genome

RNAi

Double-stranded RNA knocks down gene expression (modified to resist degradation); doesn't integrate

Antisense oligonucleotides

Modified small DNAs that suppress mRNA translation or cause exon skipping; don't integrate