L12 Genetic Screening, Testing, Gene Therapy, and Gene Editing

Comprehensive flashcard deck covering pre-pregnancy, prenatal, and newborn genetic screening/testing, genetic testing in adults/children, gene therapy approaches, and CRISPR-Cas9 gene editing based on Module 3 Lecture 12.

What cell type is harvested as the source of genetic material for Pre-Implantation Genetic Diagnosis (PGD)?

Trophectoderm cells obtained from a $5\text{--}6\text{ day}$ blastocyst.

What is the primary limitation of Polar Body Analysis compared to Pre-Implantation Genetic Diagnosis (PGD)?

Polar Body Analysis only evaluates the maternal genome, as polar bodies are formed during oogenesis.

How do placental trophoblast alterations differ between Trisomy 21 and Trisomy 18?

Trisomy 21 causes syncytiotrophoblast hyperplasia (elevating syncytiotrophoblast-derived proteins) and cytotrophoblast hypoplasia, whereas Trisomy 18 causes hypoplasia of both cytotrophoblasts and syncytiotrophoblasts (reducing all associated serum markers).

What are the characteristic serum levels of PAPP-A and $\beta$-hCG in first-trimester screening for Trisomy 21?

PAPP-A levels are decreased, while free/total $\beta$-hCG levels are increased.

What ultrasound measurement shown in the image is evaluated during first-trimester genetic screening, and how is it altered in Trisomy 21 and Trisomy 18?

Nuchal Translucency (NT), which measures the fluid-filled area at the back of the fetal neck; it is increased (larger area) in both Trisomy 21 and Trisomy 18.

Based on the MSAFP distribution graph, what condition causes elevated Maternal Serum Alpha-Fetoprotein (MSAFP) levels, and what causes decreased levels?

Open spina bifida (Neural Tube Defects) causes elevated MSAFP levels, whereas Trisomy 21 and Trisomy 18 cause decreased MSAFP levels.

Which four serum markers constitute the Quad screen performed during the second trimester?

Maternal Serum Alpha-Fetoprotein (MSAFP), $\beta$-human Chorionic Gonadotropin ($\beta$-hCG), unconjugated estradiol (uE3), and Inhibin A.

What is the key clinical advantage of the Integrated Screen over single-trimester screening?

By integrating markers from both the first and second trimesters, the detection rate for Trisomy 21 and Trisomy 18 rises above $90\%$.

What is the biological source of fetal cell-free DNA (cfDNA) detected in maternal serum?

Apoptotic placental cells release fetal genomic DNA fragments into the maternal circulation.

What invasive procedure is illustrated, when is it typically performed, and what is its associated risk of fetal loss?

Amniocentesis; it is performed at $15\text{--}20\text{ weeks}$ of pregnancy and carries a fetal loss risk of $0.1\text{--}0.5\%$.

What invasive diagnostic procedure is shown, and what is its primary advantage over amniocentesis?

Chorionic Villus Sampling (CVS); its primary advantage is earlier diagnostic capability (performed at $10\text{--}11\text{ weeks}$ of pregnancy).

What three criteria must a disease fulfill to be included in a State Newborn Screening Panel?

1) A well-understood natural history, 2) existing treatments that significantly alter patient outcomes after early detection, and 3) a reliable (sensitive and specific) screening test.

How do Diagnostic testing and Predictive genetic testing differ regarding patient presentation?

Diagnostic testing is performed on a symptomatic patient to confirm or rule out a specific condition, whereas Predictive testing is performed on an asymptomatic patient to assess future disease risk.

What distinguishes Pre-dispositional testing from Pre-symptomatic predictive testing?

Pre-dispositional testing assesses an increased risk or susceptibility for disease (e.g., BRCA1/2, LDLR mutations), whereas Pre-symptomatic testing determines whether an asymptomatic person will definitely develop a high-penetrance autosomal dominant disease (e.g., Huntington Disease).

What physical maneuver is illustrated in the diagram, and with which genetic disorder is it associated?

Gowers Sign (using hands to push on legs to stand up), associated with Duchenne Muscular Dystrophy.

Compare integrative viral vectors (e.g., lentivirus) and non-integrative viral vectors (e.g., AAV) regarding transgene durability and genomic risks.

Integrative viral vectors provide stable/permanent transgene expression but carry a risk of insertional mutagenesis; non-integrative viral vectors remain episomal with no genomic disruption, but transgene expression wanes over time.

What safety advantage does Ex Vivo gene therapy offer over In Vivo gene therapy?

Ex Vivo gene therapy permits screening and genetic analysis of modified cells for off-target effects before re-infusing them into the patient.

What is the mechanism and clinical indication for Betibeglogene autotemcel (Zynteglo)?

It is an ex vivo gene replacement therapy using a lentiviral vector to insert a wildtype $\beta$-globin gene into autologous hematopoietic stem cells to treat $\beta$-thalassemia.

Which FDA-approved gene replacement therapy uses an AAV vector in vivo to express micro-dystrophin ($\mu$DMD) in skeletal muscle?

Delandistrogene moxeparvodec (Elevidys) for Duchenne Muscular Dystrophy.

What is the mechanism of action of Inclisiran (Leqvio)?

It is a modified siRNA that targets and degrades PCSK9 mRNA, lowering PCSK9 protein levels to prevent LDLR degradation and increase hepatic LDL-C clearance.

Outline the steps of Chimeric Antigen Receptor (CAR) T Cell Therapy shown in the diagram.

1) T cells are isolated from patient blood, 2) a viral vector delivers a CAR-encoding gene into T cells, 3) T cells express CAR on their surface, 4) CAR-T cells are multiplied and re-infused into the bloodstream, and 5) CAR-T cells target and kill cancer cells.

What is insertional mutagenesis in gene therapy, and what two main pathological outcomes can result from it?

The random integration of a transgene into the host genome by an integrative viral vector, which can cause gene knockout (disrupting essential normal genes) or promoter-mediated oncogene activation (leading to cancer).

What major risk of systemic viral gene therapy was demonstrated by the case of Jesse Gelsinger?

Severe, fatal immune and inflammatory responses triggered by high-dose viral vector administration (adenoviral vector delivering wildtype OTC).

How does the CRISPR-Cas9 system create targeted genomic modifications as shown in the diagram?

Cas9 uses a guide RNA (sgRNA) to bind a specific target DNA sequence and induce a double-strand break (dsDNA break), triggering repair via NHEJ or HR to achieve knockout or gene correction.

What gene-editing strategy and delivery mode were used in the personalized treatment of infant KJ Muldoon?

An Adenine Base Editor (k-abe) system delivered in vivo via liposomes carrying mRNA coding for k-abe and sgRNA to convert A to G, correcting the Q335X mutation in CPS I deficiency.

What is the therapeutic target and editing approach of Exagamglogene autotemcel (CASGEVY) in treating Sickle Cell Disease?

It uses ex vivo CRISPR-Cas9 editing to disrupt the BCL11A enhancer in hematopoietic stem cells, downregulating BCL11A to reactivate fetal hemoglobin (HbF / $\gamma$-globin) expression.

What target gene was edited by He Jiankui in human embryos, and what major safety/biological risks were raised?

The CCR5 gene (to confer HIV resistance); risks included ethical violations, uncertain/mosaic allele editing, and increased overall mortality associated with homozygous CCR5 deletion.

What are the primary advantages and disadvantages of non-viral vectors (e.g., liposomes, nanoparticles) compared to viral vectors in gene therapy?

Non-viral vectors avoid immunogenicity, biosafety concerns, and insertional mutagenesis, but they generally have lower transgene delivery efficiency and higher production costs.

What is the primary purpose and target population of Carrier Genetic Testing?

It tests asymptomatic individuals or couples (often with a family history or specific ethnic background) to assess whether future children are at risk for inheriting a genetic condition such as Cystic Fibrosis.

What sample collection method and laboratory technique are standardly used for Newborn Screening panels $24\text{--}48\,\text{hours}$ after birth?

A heel stick blood sample analyzed via Tandem Mass Spectrometry.