Guest Lecture Review

Stem Cells

• Stem cells are the body’s master cells, with the potential to develop into many different cell types.

• 2 general properties:

  • Make copies of themselves

  • Differentiate or develop into more specialized cells

• 2 major types:

  • Embryonic: pluripotent, meaning they can become any cell in the body

  • Adult: limited differentiation, meaning they can only develop into a specific type of cell

  • Induced pluripotent (iPSC): derived by reprogramming somatic cells

Objectives in Stem Cell Study

• Key Foci:

  • Research objectives include developing methods for generating specific cell types from stem cells, understanding stem cell biology, exploring potential for organ regeneration, and investigating the use of stem cells in drug development and disease modeling.

• Classification of Stem Cells:

  • Totipotent Cells: Can give rise to any cell type, including extra-embryonic tissues; e.g., a zygote.

  • Pluripotent Cells: Can become any cell type within the embryo; e.g., ESCs and iPSCs.

  • Multipotent Cells: Limited to differentiating into a set of related cell types; e.g., hematopoietic stem cells from bone marrow.

Cloning

• Understanding Cloning in STEM:

  • Cloning refers to creating a genetically identical copy of an organism or cell.

  • Uses techniques such as somatic cell nuclear transfer (SCNT) and gene cloning.

• Significant Achievements:

  • Major developments include Dolly the sheep, the first mammal cloned from an adult somatic cell (1996).

• Therapeutic Cloning

  • Aims to produce embryonic stem cells that are genetically identical to the patient, reducing rejection risk for transplants.

  • Methods studied include creating blastocysts from somatic cells and harvesting stem cells for therapy.

• Reproductive Cloning

  • The transfer into the uterus with the intent to establish a pregnancy.

  • Offspring is genetically identical to the donor of the transferred nucleus.

• Cloning For Research

  • Cloning animal models of disease

  • Create genetically engineered animals to produce drugs or proteins

  • Produce human antibodies, proteins to fight pathogens

iPSCs as Cloning Alternatives

• Overview of Induced Pluripotent Stem Cells:

  • iPSCs are somatic cells that have been reprogrammed to a pluripotent state.

  • They can generate patient-specific stem cells without creating embryos

• Key Factors in Pluripotent Conversion:

  • 4 transcription factors, Oct4, Sox2, Klf4, and c-Myc, are able to reprogramming cells. Techniques like viral transduction and non-viral methods such as plasmid transfection improve safety and efficiency in generating iPSCs.

• Considerations in Clinical Use:

  • Advantages: Ethical appeal, potential for patient-specific therapies, and a reduced risk of immune rejection.

  • Disadvantages: Mutagenesis risk, potential for tumor formation, and the need for strict control in differentiation to achieve desired cell types.

• Research Strategies for Heart Disease:

  • Research efforts concentrate on cardiac stem cell therapy's role in regeneration, addressing challenges such as delivery methods, cell survival post-transplant, and differentiation into functional heart tissue to restore heart function post-infarction.

• Potential Complications:

  • Tumorigenicity (the formation of tumors)

  • Migration from the site of transplantation

  • Increased immunological incompatibility

  • Death of transplanted cells

Genetic Screening and Testing

Screening Tests

• Purpose: to detect early disease or risk factors for disease in large numbers of apparently healthy individuals, not intended to provide definitive diagnosis

• Population-based screening;

  • Pap smear, mammograms, prostate exam, colonoscopies

• Genetic screening:

  • Newborn, carrier, and prenatal screenings

• Validity of Screening Tests:

  • Sensitivity: ability of a test to correctly identify those who truly have the disease.

    • % of diseased individuals who have positive test results

  • Specificity: the ability of a test to correctly identify those who do not have the disease, thus reducing false positive results.

    • % of non-diseased individuals who have negative test results

  • Positive Predictive Value (PPV): likelihood that someone who tests positive is truly positive.

    • High PPV indicates a small % of false positives among those who test positive

  • Negative Predictive Value (NPV): likelihood that someone who tests negative is truly negative.

    • High NPV indicates a small % of false negatives among those who test negative

Carrier Screening

• Genetic testing of asymptomatic couples for carrier status of common genetic diseases to determine reproductive risks

• Began as ethic bases screening, now is universal

• Challenges:

  • Access to testing: Ensuring equitable access for all populations, particularly underserved communities.

  • Interpretation of results: Variability in understanding and communicating genetic information to patients.

  • Psychological impacts: Addressing the emotional burden of potential risk and decision-making following results.

Preimplantation Genetic Screening (PGS)

• Evaluating embryos during IVF for genetic abnormalities prior to implantation to reduce the risk of inherited conditions.

• Goal: decrease chance of miscarriage or chromosomal abnormalities

Prenatal Screening

• Goal: to identify individuals who are at increased risk to have a child with a genetic condition

• Population:

  • May reduce diagnostic tests in high risk populations

  • May increase diagnostic tests in general populations

• 1st Trimester Screen:

  • 10-14 weeks

  • Utilizes maternal serum markers and ultrasound to assess the risk of chromosomal abnormalities.

• 2nd Trimester Screen:

  • 16-24 weeks

  • Diagnostic for many organ malformations

  • ~15% false positive rate

• Non-Invasive Prenatal Screen:

  • After 8 weeks

  • A blood test that analyzes fetal DNA in the mother's blood

  • Can assess the risk of chromosomal abnormalities

  • Offers a higher detection rate with lower false-positive rates compared to traditional screening methods.

Newborn Screening

• Goal: diagnose genetic disorders early in life to ensure timely intervention and management.

• Process:

  • Blood (from heel-prick) sent to a state-run laboratory

  • Physician notified of abnormal results

  • Physician informs parents and discusses follow-up plan

Diagnostic Testing

• Purpose: To confirm, or determine the presence of disease in an individual suspected of having the disease

• Symptomatic:

  • Used to confirm or rule out suspected genetic disease in a symptomatic individual

  • May provide information for medical management or risk to family members/future offspring

• Asymptomatic/Predictive/Predisposition:

  • Used to determine a healthy person’s predisposition to develop disease

  • Testing usually sought based on family or medical history

Sequencing

• Purpose: reveal order of bases present in the genome of an organism.

• Types:

  • Single Gene Test:

    • one test looks for one condition

  • Multi-Gene Panel:

    • one test looks for many genes/conditions that have the same symptom

  • Exome:

    • Looks for mutations in a large percentage of known disease genes in exonic and flanking intronic regions (1-1.5% of the genome)

    • One test looks for thousands of disorders

    • 25-30% diagnostic yield

  • Genome:

    • Looks for mutations in exon and intronic regions, including non-coding regions

    • 40% diagnostic yield

Newer, Supportive Genetic Tests

• Testing for epigenetic signatures:

  • Variant of uncertain significance (VUS) in a gene involved in transcriptional regulation/chromatin remodeling

• RNA sequencing:

  • Has been done routinely on a clinical basis for hereditary cancer genes for a few years now

  • Can order from certain labs to help determine whether a VUS may be disease-causing

• Deep sequencing for low level mosaicism:

  • Became clinically available within the last year

  • Used for conditions that are always mosaic or may be mosaic

  • Used for parents of a child with apparent de novo variant who want to know more accurate recurrence risk

Clinical Genetics: Diagnosis and Counseling

• 80% of rare diseases are currently known to be genetic.

• Conditions once thought to be environmental, but were proved to be genetic

  • Cerebral Palsy: once thought to be due to birth injury, even though some did not show signs of injury

  • Autoimmune Disorders: previously considered to be entirely immune system-related, research has revealed HLA haplotypes that increase susceptibility and severity.

Evaluation

• Traditional Model:

  • Geneticist + Genetic Counselor

• Newer Models:

  • GC-first or GC-only

    • FHx of genetic disorder

    • Common neurodevelopmental indications

    • Specific phenotype with gene or gene panel available

    • Consenting for exome or genome sequencing

  • Nurse Practitioners or Physician Assistants

    • When physical exam or management is needed

Information to Guide A Diagnosis

• Clinical intake

  • physical exam, PMHx, FHx

• Laboratory/Imaging results

  • X-ray, MRI, sweat test, etc

• Expertise of other specialists

• Genetic testing

Path to a Genetic Diagnosis

• Pathognomonic Findings (least common)

  • A finding specific to a particular diagnosis

• Clinical Diagnostic Criteria (occasional)

  • A written set of symptoms associated with a particular condition

  • Published in the medical literature

• Suggestive Features (sometimes)

• Non-Specific Symptoms (most common)

Considerations for Genetic Testing

• Patient perception of test

• Genetic discrimination questions

• Coping with uncertainty

• Parental guilt

• The process of receiving a diagnosis can be time-consuming and frustrating for families

• Not all individuals seen in Genetics clinic receive a diagnosis

• Impact of genetic diagnosis

Genetics of Vascular Malformations

Vascular Anomalies

• Vascular Malformations

  • Capillary

  • Venous

  • Arteriovenous

  • Lymphatic

    • Generalized Lymphatic Anomaly (GLA)

    • Kaposiform Lymphangiomatosis (KLA)

  • Combined

    • Capillary Venous Lymphatic Malformations (CVLM)

• Vascular Tumors

  • Benign

    • Hemangioma

  • Locally Aggressive

    • Kaposiform hemangioendothelioma (KHE)

  • Malignant

    • Angiosarcoma

Vascular Malformations

• Usually present at birth or in early life

• Can affect different parts of the vascular system:

  • Arteries, Veins, Capillaries, Lymphatics

• Expansive growth

• Often do not involute

• Can become symptomatic at any time in life:

  • Growth spurts, puberty, infection, trauma

• Gene mutations identified in many vascular malformations

• Mutations are in key cell signaling pathways & many are oncogenes (cancer causing) in other tissues

Somatic vs Germline Mutations

• Somatic:

  • Non-germline somatic cells

  • Cannot be inherited

  • Mutation in tumor only

• Germline:

  • Present in egg or sperm

  • Can be inherited

  • Cause cancer family syndrome

  • All cells become affected in offspring

Somatic

• Capillary Malformations:

  • Isolated “port-wine” (strawberry) birthmarks

  • GNAQ activating mutation

• Venous Malformations:

  • Mutations on TIE2 (TEK)

    • over 95% of spontaneous venous malformations

  • Mutations in PIK3CA

    • 30-50% of TEK-negative VMs

• CVLM, CLOVES, & KTS:

  • CLOVES: Combination of vascular, skin, lipomatous overgrowth, and musculoskeletal abnormalities

  • Lipomatous masses cause asymmetric hypertrophy of the trunk and other areas of the body

  • Caused by somatic mutations in PIK3CA gene

    • Mosaic gain-of-function mutations

    • Can be treated with Alpelisib or Rapamycin