Lecture 1 - Central Dogma of Molecular Biology

Replication (DNA Synthesis)

Site: Nucleus of Eukaryotic Cells, Nucleoid Region of Prokaryotes

Timing: occurs during Interphase (S Phase)

Principles of DNA Synthesis (Replication)

1.​ Semiconservative: after replication, each DNA helix contains one original (parental) strand and one
newly formed strand.
2.​ Simultaneous: Both parental DNA strands serve as templates simultaneously.
3.​ Bi-directional: from the origin of replication, the process occurs in both directions of the uncoupling
DNA helix.
4.​ Direction: occurs from the 5’ → 3’ direction.
5.​ Semi-discontinuous: one strand of DNA (Leading) is replicated in one continuous strand from 5’ → 3’,
while the other (Lagging) is replicated in fragments which are later connected.

Steps of DNA Synthesis

1.​ Separation of the two strands at multiple origins (ori) of replication, which are rich in A–T sequences,
to form replication bubbles.
a.​ Multiple sites are formed to decrease the time the needed to complete replication.
2.​ Unwinding and separation of the helix bidirectionally at each replication fork.
a.​ Topoisomerase unwinds and relaxes the supercoiled DNA
b.​ DNA Helicase cleaves the interstrand Hydrogen bonds
c.​ Single-strand binding proteins (SSBs) bind to each base to keep them from rejoining.
3.​ RNA primer is synthesized by the primase activity of DNA Polymerase ɑ (alpha).
a.​ Primer is a short RNA sequence that provides a starting point for DNA.
4.​ RNA primer is elongated by DNA polymerases until another RNA stretch is found.
a.​ DNA polymerases read from the 3’ → 5’ direction and synthesis the new strand from 5’ → 3’
b.​ Leading Strand: uses DNA polymerase ε (epsilon), replicating continuously.
c.​ Lagging Strand: uses DNA polymerase (delta), replicating discontinuously.
i.​ Multiple Primers are used to form Okazaki fragments.
5.​ Proofreading: Exonuclease activity of DNA polymerase ε and proofread from 3’ → 5’.
a.​ If an error is found, these polymerases hydrolytically replace the incorrect nucleotide.
6.​ RNA primer is excised by RNAse H and FEN 1 (Flap Endonuclease 1)
7.​ DNA polymerase replaces the RNA primer by DNA nucleotides from 3’ → 5’
8.​ DNA Ligase connects the fragments using ATP.

DNA Structure DNA vs. RNA

1.​ Sugar, Phosphate, N. Base 1.​ Uracil only in RNA; Thymine only in DNA
2.​ Double Helix 2.​ DNA → Deoxyribose, RNA → Ribose
3.​ Phosphodiester Bonds (3’ and 5’) 3.​ DNA is found in chromosomes
4.​ Hydrogen Bonds between Base Pairs 4.​ RNA is found in the cytoplasm and nucleolus
5.​ Anti-Parallel 5.​ RNA is single–stranded, DNA is double-helix
 Transcription (RNA Synthesis)

Why is RNA used instead of DNA to synthesize proteins?

-​ DNA is too complex
-​ DNAse, which hydrolyses DNA, is present in the cytosol.

Transcription: the process of copying DNA → RNA, using only one template (anti–sense) strand of DNA.
-​ It does not need a primer to start, and can involve multiple RNA polymerases.

Begins with the binding of RNA polymerase II to the promoter region that comes before the gene:
-​ TATAAA sequence (TATA box): most common, 25–35 base pairs upstream of the gene.
-​ CCAATC and GGGCGG sequences: determine transcription frequency, located 35–200 base pairs
upstream of the gene, with specific binding proteins.
1.​ TFII–D recognizes and binds to the TATA box
2.​ TFII–F brings RNA Polymerase II to the promoter region, to binds to TFII–D
3.​ TFII-H binds to RNA Polymerase II, using its helicase activity to cleave hydrogen bonds.
a.​ Transcription starts ∼25 base pairs downstream of the TATA box.

Transcription Complex: forms a transcription bubble with RNA Polymerase II, the DNA template, and the
mRNA strand. This bubble is ∼20 base pairs long, and the DNA reforms once it moves along.

Chain elongation: RNA Polymerase moves freely on the DNA template, cleaving two phosphates of
ribonucleotide triphosphates (NTPs), removing pyrophosphates (PPi), and using this energy to covalently bind
nucleotides together in the 5’ → 3’ direction.

Elongation continues until a termination region is reached, recognized by RNA polymerases.

-​ Termination Region: AAUAAA sequence located in the 3’ region of the mRNA, followed by U residues.
-​ Endonuclease: cleaves the RNA just downstream of the termination sequence.
Areas of a Gene undergoing Transcription:
1.​ Promoter
2.​ Transcribed Sequence:
a.​ Upstream Leader Sequence [not translated]
b.​ Protein Coding Sequence [translated region, with exons and introns]
c.​ Downstream Trailing Sequence [not translated]
3.​ Termination Region of U residues

Post-Transcriptional Modification of mRNA

In Prokaryotes, Translation is coupled with Transcription, and so protein synthesis occurs while mRNA is still
being created, and no modification of RNA is needed due to the lack of transport.
-​ Lifespan of Prokaryotic mRNA: about 2 mins.

In Eukaryotes, mRNA is immature after transcription, due to the presence of non–coding introns between
coding regions (exons). It needs modifications for transport and translation.
1.​ Capping: immediately after synthesis, the 5’ end of the pre-mRNA is modified by addition of a
7-methyl-guanosine (7mGppp) cap, for ribosome recognition, stability, and protection from enzymes.
2.​ Poly–A Tail: addition of 100–250 Adenine nucleotides to the 3’ end of the mRNA, to aid in transport
and determine its lifespan.
3.​ Splicing: removing introns and connecting ‘splicing’ exons together using small nuclear RNA
molecules (spliceosomes) to form a continuous protein coding message.
-​ Lifespan of Eukaryotic mRNA: about 10 hours.
 Translation (Protein Synthesis)

Translation: interpreting mRNA code into proteins, using triplet codons to code for a specific amino acid read
by ribosomes, tRNAs, and rRNAs.
-​ Ribosomes: machines of protein synthesis, which hold mRNA, amino–acyl tRNA and form peptide
bonds between amino acids, all to translate mRNA → polypeptide chain
-​ Large Subunit: contains P site (for peptide chain), A site (for amine acid), and E site (Exit)
-​ Small Subunit: contains the mRNA binding site
-​ tRNA: a clover shaped RNA molecule which carries amino acids → ribosome. It is responsible for the
specificity of the codon system through its anticodon loop.
-​ tRNA is activated by a specific amino–acyl–tRNA synthetase, which charges tRNA with the
corresponding amino acid at the 3’ end of the tRNA molecule.

Initiation: Initiator Methionyl-tRNA, small ribosomal unit, and mRNA form an initiation complex, followed by the
formation of a functioning ribosome when the large ribosomal unit binds to the initiator complex.
-​ Polysome: a cluster of ribosomes simultaneously acting on a single mRNA strand.

Elongation: a cyclic process, which includes…

-​ Binding of Aminoacyl-tRNA to the A site of the ribosome.
-​ Peptide bond formation: between the amine group of AA in the A site and the carboxylic group of the
amino acid in the P site. This results in the transfer of the growing peptide to the tRNA in the A site.
-​ Translocation: the tRNA from the A site moves to the P site, freeing the A site to accept a new
aminoacyl-tRNA to continue the elongation cycle.

Termination: A termination codon of mRNA (UAA, UAG, UGA) appears in the A site with releasing factor (RF)
recognizing it, releasing all components of translation (mRNA, tRNA, ribosomal units, polypeptide chain).
 Lecture 2 - Chromosomal Basis of Inheritance

Definitions:
-​ Gene: basic unit of heredity; codes for a specific trait.
-​ Locus: the specific location of a gene on a chromosome.
-​ Genome: all DNA of a cell.
-​ Somatic cell: all body cells except reproductive cells.
-​ Gamete: reproductive cells (i.e. sperm & eggs).
-​ Chromosome: elongate cellular structure in the nucleus composed of DNA and protein.
-​ Diploid (2n): cellular condition of two homologous chromosomes.
-​ Haploid (n): cellular condition where each chromosome type is represented by only one chromosome.
-​ Homologous Chromosome: chromosome of the same size, shape, carrying the same type of genes.
-​ Chromatid: one of two duplicated chromosomes connected at the centromere.
-​ Centromere: region of chromosome where microtubules attach during mitosis and meiosis.

Chromosomes: each species has a characteristic set [karyotype, number, morphology] of the chromosomes
that compose its genome. The 46 chromosomes of human somatic cells constitute 23 pairs (diploid)
-​ Autosomes: 44 autosomes in the human genome comprising 22 pairs. Each is numbered from 1–22 in
decreasing order of length, with each homologous pair of both maternal/paternal origin.
-​ Sex Chromosomes (Gonosomes): involved in sex determination, with different morphologies.
-​ X and Y chromosomes are not homologous, but both contain the pseudoautosomal region.

Chromosomal Structure: each chromosome has a constriction at the centromere, a short (p – petite) arm and
a long (q – grande) arm. During Cell Division, two identical chromatids will be connected at the centromere.
-​ Classification of chromosomes based on centromere location
1.​ Metacentric (Ch. 1, 2, 16, 19, 20) : centromere is perfectly centered; i.e. p arm = q arm
2.​ Submetacentric (Ch. 2, 4, 5, 6–12, 17, 18, X): centromere is off center; p arm < q arm
3.​ Acrocentric (Ch. 13–15, 21, 22, Y): centromere is at the end; p arm <<< q arm
-​ Denver Classification, based on length and centromere location
A.​ Group A (1–3): Large, Metacentric/Submetacentric
B.​ Group B (4, 5): Large, Submetacentric
C.​ Group C (6–12, X): Medium, Submetacentric
D.​ Group D (13–15): Medium, Acrocentric
E.​ Group E (16–18): Short, Metacentric/Submetacentric
F.​ Group F (19, 20): Short, Metacentric
G.​ Group G (21, 22, Y): Short, Acrocentric

Chromosomal Nomenclature:
1.​ Chromosome is identified with a Number from 1-22, or X/Y.
2.​ Each Chromosome is divided into 2 arms (p, q).
3.​ Each arm is divided into regions starting from the centromere → outwards.
4.​ Each Region is divided into bands, identified with a number.

How to write a cytogenetic karyotype [e.g. 46, XY, del(15q13) OR 47, XX + 21]
1.​ Total Number of Chromosomes 5.​ P or Q arm
2.​ Sex Chromosomes 6.​ Region
3.​ Abnormality 7.​ Band
4.​ Chromosome Number 8.​ Subband
Chromosomal Identification: chromosomes can be identified by their size, shape, and position of the
centromere, and the specific G-banding patterns produced by Giemsa Stain.
-​ Karyogram: photographic arrangement of a complete set of chromosomes of a cell or organism.
-​ G Banding: banding procedure that produces patterns unique to each chromosome, that can help to
match homologous pairs in a karyotype. G Banding is the most common procedure, where trypsin is
added to digest chromosomal proteins and then Giemsa stain.
-​ Cells used in a Chromosomal Study
-​ Pre-Birth: Amniocentesis or Chorionic Villus Sampling
-​ Post–Birth: Lymphocytes, Skin Cells, Biopsies, Tumor Cells.

Fluorescent In–Situ Hybridization (FISH): a special technique allowing special visualization that traditional
cytogenetics cannot see. It is used for Microdeletion, Supernumerary Markers, and Aneuploidy Syndromes.
-​ Mechanism: a DNA probe is labeled with a fluorochrome, which after the probe hybridizes with a target
on a chromosome, allows the region of hybridization to be visualized with a fluorescence microscope.
-​ Centromeric Probes: bind to the centromere, used for diagnosis of Aneuploidy Syndromes.
-​ Chromosome-Specific Unique Sequence Probes: bind to specific loci, used to identify
microdeletion and microduplication syndromes.
-​ Whole Chromosome Paint Probes: bind to a whole chromosome, useful for subtle
translocations and small supernumerary markers or rings.

Mitosis
-​ Interphase (resting phase): non–mitotic portion of the cell cycle, composed of…
-​ G0: Dormant Phase, where non–divisible cells arrest (e.g. neurons)
-​ G1 Phase: Normal Cell Function and Growth
-​ S Phase: for DNA Replication
-​ G2 Phase
-​ Prophase: chromosomes condense; centrioles move to ends;
spindle fibers form.
-​ Prometaphase: nuclear membrane dissolves; chromosomes
attach the centromere to spindle fibers via the kinetochore
protein complex.
-​ Metaphase: chromosomes are most seen along the middle
and are attached to the centriole.
-​ Anaphase: centromere of each chromosome divides
longitudinally; daughter chromatids separate to opposite poles
of the cell (46 chromatids at each pole).
-​ Telophase: chromatids arrive at the poles and 2 nuclear
membranes form.
-​ Cytokinesis: cell cytoplasm separates.

Indication of Chromosomal Analysis:

1.​ Presence of a Chromosomal Disorder (e.g. Down Syndrome).
2.​ Presence of Four Features → Physical or Mental Retardation, Malformations, or Dysmorphogenesis.
3.​ Features of two or more syndromes in one patient.
4.​ Malformations are known to have a high association with a chromosomal disorder.
5.​ Child with non–specific dysmorphism without a specific diagnosis.
6.​ Pregnancy in a woman of advanced age
7.​ Familial history of known/suspected chromosomal abnormality
8.​ Fertility Problems, Still Birth, and Neonatal Death (Miscarriage)
 Lecture 3 - Chromosomal Abnormalities

Numerical Anomalies

Polyploidy: addition of a complete haploid set (23) of chromosomes, with two conditions in humans:
-​ Triploidy (3n → 69 chromosomes): most frequently occurs from fertilization of an ovum by two sperms,
or the fertilization of a diploid gamete and a normal gamete (due to anaphase abnormality).
-​ Tetraploidy (4n → 92 chromosomes): occurs from the failure of the zygote to divide after it replicates
its chromosomes; i.e. the failure of progression to the first mitotic division, so replication happens twice.

Aneuploidy: most common and significant human chromosome disorder, occuring in 3–4% of all pregnancies.
-​ Trisomy (46 → 47): the addition of a chromosome, rarely compatible with life, except for…
1.​ Down’s Syndrome (Trisomy 21): Growth/Mental retardation, Craniofacial abnormality, Cardiac
defects, High Risk of Infection & Thyroid Dysfunction.
2.​ Patau Syndrome (Trisomy 13): Mental retardation, Only One Cerebral Hemisphere,
Polydactyly, Heart Defects, Deafness, Eye Defects
3.​ Edward’s Syndrome (Trisomy 18): deformed ears, small mouth/jaw/neck, prominent sternum
and occipital part of the skull.
4.​ Triple X Syndrome (XXX): short stature, webbed neck, primary amenorrhea, no ovaries.
5.​ Klinefelter Syndrome (XXY): infertile male, small testis, gynecomastia, ↓ male characteristics
6.​ Jacob’s Super Male Syndrome (XYY)
-​ Monosomy: always fatal, except for Turner Syndrome (only X)

Non–disjunction: failure of the chromosomes to disjoin properly during one of the Meiotic Divisions
-​ During Meiosis I: gametes containing both paternal and maternal members of the pair.
-​ During Meiosis II: gametes contain both copies of either the paternal or maternal chromosome.
-​ After Zygote Formation: non–disjunction in a mitotic division can result in Mosaicism.

Mosaicism (Mixoploidy): caused by postzygotic non–disjunction or an anaphase lag, where two or more cell
lines are present in one person, resulting in either numerical or structural mosaicism.

Structural Anomalies: result from chromosome breakage, followed by healing in an abnormal combination.

Balanced: no change in the amount of essential genetic material, but lead to abnormal gamete formation.
1.​ Insertion: segment removed from one chromosome is inserted into a different chromosome.
2.​ Inversion: when two breaks occur, and
the middle piece is flipped and
reattached.
-​ Paracentric Inversions do not
include the centromere
-​ Pericentric Inversions involve
the centromere
3.​ Translocation
-​ Reciprocal: breakage of two non–homologous chromosomes and exchange of the segments
-​ Robertsonian: involves two acrocentric chromosomes that fuse and lose their short arms.
-​ Balanced Robertsonian: fusion of two acrocentric chromosomes → 45 ch.
-​ Unbalanced Robertsonian: when a translocated chromosome is inherited → 47 ch.
-​ Risks of Robertsonian Translocation: some translocations increase risk of Down Syndrome
Unbalanced: there is a gain/loss of essential chromosome segment
-​ Deletion: loss of a chromosome segment (partial monosomy).
-​ E.g. Cri du Chat Syndrome [46, XY, del(5p15], resulting in a cat-like cry and hypertelorism
-​ E.g. Wolf–Hirschhorn Syndrome [46, XY, del(4p16)]
-​ E.g. Turner Syndrome [46, X, del(Xq22.1)]
-​ Duplication: less harmful, addition of a segment (partial trisomy) leading to phenotypic abnormalities
-​ Ring: when a chromosome undergoes two breaks and
reunites in a ring structure, expected to be mitotically
stable if the centromere is preserved within the ring
(e.g. Turner Syndrome)
-​ Isochromosomes: chromosome in which one arm is
missing, and the other is duplicated (mirrored)
-​ Isochrome Syndromes are most common for the
X chromosome in Turner Syndrome.

Chromosomal Abnormalities in Turner Syndrome Chromosomal Abnormalities in Down Syndrome

-​ Numerical: 45, X -​ Non–Disjunction: 47, XX/XY, +21
-​ Structural: -​ Translocation
-​ Ring [r(X)] -​ Mosaicism
-​ Isochrome [i(Xq)]
-​ Deletion [del(Xq12)] Recurrence Risk after 1st Pregnancy → +1%

Symbols used in describing a Karyotype

Term Explanation Example

p Short Arm

q Long Arm

cen Centromere

del Deletion 46, XX, del(1)(q21)

dup Duplication 46, XY, dup(13)(q14)

i Isochromosome 46, X, i(X)(q10)

r Ring Chromosome 46, XX, r(21)

t Translocation 46, XY, t(2;4)(q21;q21)

rob Robertsonian Translocation 46, XY, rob(14;21)(q10;q10), +21

inv Inversion 46, XY, inv(9)(p12q12)

der Derivatives 46, XY, der(14;21)(q10;q10), +21

 Lecture 4, 5 - Mendelian Inheritance

Alleles: location of the gene in the chromosome, as an alternative form of the same genetic locus.
-​ Both Maternal and Paternal alleles code for any inherited trait.
Homozygous: an organism with two identical alleles.
Heterozygous: presence of different alleles at one or more loci on homologous chromosomes.
Hemizygous: only one allele from only one parent (e.g. X and Y chromosomes)

Pedigree

-​ Male: Square
-​ Female: Circle
-​ Gender Unknown: Diamond
-​ Pregnancy: Diamond with a P in the middle
-​ Abortion: Triangle
-​ Unaffected: Unshaded shape
-​ Affected: Shaded shape
-​ Carrier: ½ shaded
-​ Sex–Linked Carrier: Circle + Dot
-​ Proband (Patient, Index Case): arrow pointing
to the patient, not necessarily affected
-​ Deceased: Diagonal Line on the individual
-​ Consanguineous marriage: double lines
-​ Divorce: two diagonal lines
-​ Twins: Line originates from the same point
-​ Monozygotic twins: line connects them

Patterns of Mendelian Inheritance: trait/disorder determined by a gene on an autosome or sex chromosome

Autosomal Dominant: only one copy needs to be inherited for the patient to be affected
-​ Vertical Transmission: all generations are affected, usually transmitted without skipping (no carriers)
-​ Male → Male transmission as a key feature.
-​ Likelihood of Inheritance: 50% Diseased, 50% Healthy for each pregnancy.
-​ Autosomal Dominant Inheritance can show…
-​ Pleiotropy: a gene may give rise to unrelated effects → different systemic manifestations.
-​ Tuberous Sclerosis (benign tumors) + learning difficulties, epilepsy, facial rash.
-​ Marfan Syndrome, with thumb sign, ectopia lentils, aortic dilation, and scoliosis.
-​ Variable Expressivity: variation in the degree of severity and symptoms
-​ Polycystic Kidney Disease: can develop renal failure early, or no real impairment.
-​ Neurofibromatosis: can have only cafe-au–lait spots, or much more severe expression.
-​ Reduced Penetrance: when a person has a diseased gene with no diseased phenotype.
-​ Retinoblastoma: cancer in the eyes in children
-​ Treacher Collins Syndrome: ear, eyes, facial bone deformities
-​ New Mutations: sometimes AD traits appear in individuals without any family history of the
disorders, due to exposure to a mutagen or a new random mutation.
-​ Achondroplasia: short limbed dwarfism, in which the parents have normal stature.
Autosomal Recessive: both copies of diseased alleles
must be inherited for the disease to be present.
-​ Likelihood of Inheritance: 50% Carrier, 25%
Diseased, 25% Healthy for each pregnancy.
-​ Both Parents must be carriers or diseased,
usually with skipped generations and related
parents.
-​ E.g. Sickle Cell Anemia, Microcephaly,
Albinism, Cystic Fibrosis

Lyonization: process of inactivation of one X chromosome in a female, occurring randomly in each cell where
each X is active in about ½ of all embryonic cells. If X inactivation is not random, the active X chromosome in
most cells may bear a mutant allele → female will exhibit some of the signs of a disease.

X–Linked Recessive: transmitted through carrier females to affect

their sons (males almost exclusively)
-​ Females must be homozygous, Males are Hemizygous
(only one X chromosome)
-​ Affected Females → produce a carrier or normal daughter
OR affected or normal son
-​ Affected Males → always produce a carrier daughter
-​ E.g. Duchenne Muscular Dystrophy, color blindness,
G6PD, Hemophilia

X–Linked Dominant (uncommon): manifest in heterozygous

females, as well as male hemizygous males.
-​ Affected Males → transfer the trait to all daughters, and to none of his sons.
-​ Heterozygous Mother can transfer to both sons and daughters (50% risk per pregnancy)
-​ E.g. XLDD (abnormal pigmentation, conical teeth), Vitamin D Resistant Rickets

Y Linked: genes that differentiate the embryo into male, encoding testis–specific spermatogenesis factors.
-​ Affected Males will always and only have affected sons (e.g. hairy pinna)

Sex Influenced Traits: some autosomal traits are expressed more frequently in one sex than others.
-​ For Example, Gout affects more males than females
-​ Sex Limited Traits: occurs only in one sex due to anatomical differences (uterine/testicular defects)
 Lecture 6 - Non-Mendelian Inheritance

Non–Mendelian (Non–Classical) Inheritance: genetics that don't follow Mendel’s Laws, utilizing several
different mechanisms including…

Genomic Imprinting: epigenetic phenomenon where different clinical

features result from the expression of a gene either from the paternal or
maternal chromosome (only one parent of origin) via DNA methylation.
-​ 80–100 human genes are known to be imprinted, occurring in
all or some specific somatic cells.
-​ DNA regions involved are known as Differentially Methylated
Regions (DMRs), including Imprinting Control Regions (ICRs)
across imprinted domains (Methylated [Silenced] → Imprinted)
-​ E.g. Chromosome 15 Disorders such as
-​ Prader–Willi Syndrome: regions (SNRPN gene) on Ch15 are only active from father’s allele
-​ Angelman Syndromes: regions (UBE3A gene) on Ch15 are only active from mother’s allele

Uniparental Disomy: when an individual inherits both

homologs of a chromosome pair from one parent.
-​ Uniparental Isodisomy: from non–disjunction in
Meiosis II, where both are identical.
-​ Uniparental Heterodisomy: from Meiosis I
non–disjunction → homologues are different.
-​ If the single active copy of the gene is
lost → no gene product → disease.
-​ E.g. Prader–Willi Syndrome and
Angelman Syndrome.

Mitochondrial Inheritance: few but some significant diseases are caused by mitochondrial DNA (mtDNA)
mutations, which has a higher rate of spontaneous mutation than nuclear DNA.
-​ mtDNA is inherited only through the oocyte, and affects both males and females from the maternal
lineage, but with variable expression due to the difference of how many mitochondria are affected.
-​ Homoplasmy: when all mtDNA in different mitochondrial is identical
-​ Heteroplasmy: when there are two populations (one mutated) of mtDNA.
-​ Organs most susceptible to mitochondrial mutation are the CNS, Skeletal Muscle, and Heart
-​ E.g. MELAS and MERRF

Anticipation: if the onset of disease occurs at an earlier age in offspring than in the parents, or if disease
increases in severity over generations.
-​ Repeat Expansion: mutation that increases the number of times that a DNA sequence is repeated,
resulting in improper protein functioning, and potentially anticipation
-​ E.g. Myotonic Dystrophy (50–1000 CTG repeats), Fragile X–Syndrome (CGG repeats), and
Huntington Disease (due to CAG repeats)

Mosaicism: an individual or tissue can consist of more than one cell line due mitotic error.
-​ Somatic Mosaicism: less severe single–gene disorder, confined to a particular body segment.
-​ E.g. Mosaic Neurofibromatosis, Cancer
-​ Gonadal Mosaicism: when the parents are normal, and genetic tests are normal, but more than one
child is affected with a genetic condition.
 Lecture 7 - Pharmacogenetics

Pharmacogenetics: describes the influence of individual genes on the efficacy and side effects of drugs, via
metabolic pathways which can affect individual responses to drugs, both in therapeutic and adverse effects.
-​ Pharmacogenomics: describes the interaction between drugs and the genome (multiple genes).
-​ Essentially, different people react differently to different drugs.

Principles of Drug Metabolism:

1.​ Intake into Body 3.​ Distribution to intended site 5.​ Metabolism of the Drug
2.​ Absorption into Circulation 4.​ Drug–Cell Interaction 6.​ Excretion

Many Drugs undergo biochemical modification to increase their solubility → easily excreted:
-​ Conjugation: Union of Carbohydrate (CHO) + Glucuronic Acid (e.g. Morphine, Codeine)
-​ Acetylation: introduction of an acetyl group (e.g. Sulfonamide, Isoniazid)

Excretion of Different Drugs (usually in the liver) varies with different drugs
-​ Some are Oxidized → CO2 → Exhaled via the Lungs
-​ Some are Excreted → Kidneys → Urine
-​ Some are Excreted → Bile → Feces

Genetic Variation: inherited variation at the DNA level → functional variation

in gene products that play essential roles in determining the variability in both
therapeutic and adverse reactions.
-​ Sequence Differences can be in Coding Regions (exons), Regulatory
Regions, Introns, 5’ or 3’ Flanking Regions, etc.
-​ If DNA sequence variation occurs in the coding portion (exons) or
regulatory regions, it is likely to result in variation of the product
through alteration of the function, activity, or level of expression.
-​ Polymorphism: the presence of two or more gene forms in a
population (>1% saturation)
-​ Rare Variants: alleles present in less than 1% of population.
-​ Personalized Medicine: using genetic info. to select the most appropriate therapies at a correct dose.

Isoniazid (INH) Inactivation: drug used in TB treatment, it is rapidly absorbed spiking in the blood, and is
slowly reduced as the drug is inactivated by N–acetyl Transferases and excreted.
-​ Rapid Inactivators: plasma drug levels fall rapidly after an oral dose → can’t be utilized.
-​ Results in Higher Failure Rate, Needing Larger Doses (Hydralazine → Hypertension) and
(Dapsone → Leprosy), and Liver Damage
-​ Slow Inactivators: plasma drug levels remain high for some time after an oral dose → accumulation.
-​ NAT2 is the gene variant responsible for the Slow Inactivation Variant. It is a homozygous allele
coding for the liver enzyme N–acetyl Transferase. (NAT1 → normal).
-​ Results in Systemic Lupus Syndrome, Polyneuritis (Vitamin B6 Deficiency), Hematological
Adverse Reactions, Increased Bladder Cancer Risk, Increased Adverse Drug Effects.

SuccinylCholine Sensitivity: used in anesthesia for short term apnea and muscle relaxation for intubation.
-​ It is destroyed by pseudocholinesterase (CHE), coded for by the pCHE gene.
-​ When apnea persists (1/2000 patients), CHE works slower than normal and the anesthetist has to
maintain respiration until drug effects have worn off.
Glucose–6–P Dehydrogenase Deficiency: G6P DH is used in RBC glycolysis (Hexose Monophosphate
Shunt) to maintain Glutathione (RBC stability) in the present of oxidizing agents.
-​ Point mutations occur in G6PD gene → ↓ enzyme production or catalytic activity → hemolytic anemia.
-​ It is an X–linked Recessive Trait, common in the mediterranean due increased malarial
resistance, resulting in very dark/black urine, jaundice, and ↓ RBC count/hemoglobin → fatal.
-​ Multiple drugs can induce hemolysis → Antimalarial Drugs, Sulfa Drugs, Fava beans (Favism).

Coumarin (Warfarin) Metabolism: class of anticoagulants used to prevent blood clots such as in DVT.
-​ Mutations occur in the CYP2C9 Gene (→ CYP2C9*2 and CYP2C9*3 Variants)→ decreased
metabolism → increased risk for bleeding. a lower dose is used to maintain the target ratio.

Thiopurine Methyltransferase: toxic substances used for immunosuppression to patients with autoimmune
disorders, leukemia, and organ recipients (e.g. 6–mercaptopurine, 6–thioguanine, azathioprine), with liver
damage and leukopenia as side effects. Azathioprine is reported to cause toxicity in 10–15% of patients
-​ ⅔ of patients who have toxicity → TPMT gene Mutations, responsible for Thiopurine Methylation.

Debrisoquine Metabolism: used for hypertension, certain variants (common in British/European people)
cause reduced hydroxylation and overaction of the drug → hypotension, caused by CYP2D6 (AR) variant.
-​ CYP2D6 (AR) variation plays a role in metabolizing over 20% of prescribed drugs, such as B-blockers.

Dihydropyrimidine Dehydrogenase (DPYD): a rate limiting enzyme, which catabolizes 5–fluorouracil

(chemo– therapeutic drug), measured in peripheral blood mononuclear cells/gene testing (pre–chemotherapy).
-​ DPYD deficiency → severe 5FU–associated toxicity, so measurement ensures no mutation.

Malignant Hyperthermia: a rare anesthesia

complication, resulting in Hyperthermia (up
to 42.5ºC → brain damage) and Muscle
rigidity during anesthesia. It occurs when
Halothane (anesthetic) + Succinylcholine
(muscle relaxant) are both given during
anesthesia, treated with vigorous cooling.
-​ Susceptibility: autosomal dominant
(1/10,000), most commonly in
Ryanodine Receptor (RYR1) gene.
-​ Prediction: in vitro muscle
biopsy to test Halothane +
Caffeine response.

Maturity–Onset Diabetes of the Young: monogenic form of diabetes characterized by young onset (before
25) due to beta–cell dysfunction. Many patients are misdiagnosed with Type 1 Diabetes and given insulin.
-​ HNF1A Gene: Autosomal Dominant Inheritance, causing MODY → 4x better (increased) response to
sulfonylureas, so patients can transfer from insulin injections → sulfonylurea tablets.

Neonatal Diabetes: most frequently due to mutations in KCNJ11 or ABCC8 genes, which encode Kir6.2 and
SUR1 subunits on ATP–sensitive Potassium channels in pancreatic β–cells → prevent K–ATP channel closure,
by reducing the response to ATP → no insulin secretion triggered → diabetes and lifelong insulin required.
-​ Most patients can be treated well with sulfonylurea tablets instead of insulin, which bind sulfonylurea
receptor subunits of the K–ATP channel to cause closure → triggered insulin secretion.
-​ High–Dose sulfonylurea therapy → improved glycemic control → reduced complications later.
 Lecture 8 - Genetic Variants

Mutation/Variant: alteration or change in the genetic material (genome), of two types…

-​ Classic ‘fixed’ Mutation: permanent mutation
-​ Dynamic (unstable) Mutation: unstable; can change size in successive generations
-​ Mutations drive evolution, but can also be pathogenic, occurring…
-​ Spontaneously through errors in DNA replication and repair (most common)
-​ Through exposure to mutagenic agents

Polymorphism: variants with no obvious effect on the phenotype, occurring in more than 1% of the population.
-​ Polymorphisms → common variants that do not result in disease (not harmful/pathogenic mutations).
-​ Single Nucleotide Polymorphisms (common) → one nucleotide changes & doesn’t cause a problem.
-​ Short Tandem Repeat Polymorphisms (STRPs) → used in paternity testing

Classifications of Mutations

Location: differentiates mutations based on where they occur

-​ Germline Mutation: occurs in egg/sperm gametes → inherited mutations, present in all body cells
-​ They cause early–onset congenital diseases, such as Sickle Cell Anemia, Thalassemia, and CF.
-​ Somatic Mutation: occurs in body cells → acquired mutations, causing disease depending on the cell.
-​ They cause adult–onset diseases, such as cancer.
 Molecular Alteration: differentiates mutations based on what their form.

Coding DNA: affect polypeptides.

1.​ Substitutions (Point Mutations): replacement of
a single nucleotide by another (most common).
-​ Transition: Purine → Purine (A/G) OR
Pyrimidine → Pyrimidine (C/T) (common).
-​ Transversion: Purine ↔ Pyrimidine
a.​ Synonymous (Silent): mutation does not
alter the amino acid, especially in the third
position of the codon due to the
degeneracy of the genetic code (Different
Codon → Same AA).
b.​ Non–Synonymous: mutation leads to
alteration of the polypeptide (Diff. Codon → Different AA). It is less frequent than synonymous
mutations → abnormal function, w/ disease or lethality.
i.​ Missense: different AA → altered protein, but can be…
-​ Conservative: no change in
function, due to chemically
similar AA substitution.
-​ Non–Conservative: change in
protein function, due to
chemically different AA → gross
reduction/complete loss of
biological activity
ii.​ Nonsense: substitution that leads to stop codon generation (UAA, UAG, or UGA),
resulting in premature termination → truncated protein. Nonsense Mediated Decay can
help prevent harmful truncated proteins.
2.​ Frameshift Mutations: most important, most result in a stop codon → truncated protein.
a.​ Deletions: loss of 1+ nucleotide, resulting in disruption of the reading frame (not a multiple of 3).
Larger Deletions → Partial/Whole Gene Deletions → Loss of Function or Expression
b.​ Insertions: addition of 1+ nucleotide, resulting in disruption of the reading frame (if not a
multiple of 3). Larger Insertions → Partial/Whole Gene Duplications

Non–Coding DNA: affect the level of gene expression

-​ Promoter/Enhancer Mutations: occurs in promoter sequences (DNA only), decreasing affinity of RNA
polymerase to a promoter site → reduced production of mRNA → reduced production of a protein.
-​ Splice–Site Mutations: occurs at intron–exon boundaries (splice–site), altering splice signals (mRNA
only) → aberrant splicing → loss of a coding sequence → production of abnormal proteins.
-​ They can occur in the Donor Site (GT) or
Acceptor Site (AG): result in exon skipping
and intron retention, activation of cryptic
(pseudo–)splice sites → partial/complete
deletion of an exon.

Trinucleotide Repeat Mutations: dynamic mutations,

becoming more unstable as it expands in size. Above a
certain repeat number → unstably transmitted, increasing
in repeat number (e.g. Huntington’s Disease).
Functional Effects of Mutations on Proteins
-​ Loss of Function Mutations → reduced activity or complete loss of gene products, such as in familial
hypercholesterolemia (e.g. tumor suppressive genes).
-​ Haplo–Insufficiency mutations → ½ normal gene product levels
-​ Gain of Function Mutations → increased gene expression or new function of a gene product, such as
in Huntington’s Disease and Achondroplasia (e.g. oncogenes)
-​ Dominant Negative Mutations → mutant gene in the heterozygous state interferes with the function of
the normal gene → loss of protein activity/function (e.g. Osteogenesis Imperfecta, Marfan Syndrome).

Mutations – Inheritance
-​ Homozygous Mutations: two identical mutations on both chromosomes,
at the same location/gene → causes autosomal recessive disorders,
passed on by asymptomatic carriers (e.g. sickle cell disease).
-​ Compound Heterozygous (CH) Mutation: two different inherited
mutations on each chromosomes, at different locations but within the
same gene → defective protein (→ AR Disorders)
-​ De Novo Mutations: new mutations in a gamete, associated with
advanced paternal age, that are passed on causing Noonan Syndrome,
Achondroplasia, Intellectual Disability, Autism, etc.

Genotype–Phenotype Correlation: the same gene can undergo different mutations at different sites to
produce different phenotypes (diseases), such as the receptor tyrosine kinase gene RET → can result in…
-​ Hirschsprung Disease (Loss of Intestinal Innervation): due to loss of function nonsense mutations.
-​ Familial Medullary Thyroid Carcinoma, Multiple Endocrine Neoplasia 2: due to gain of function
missense mutations.

Causes of Mutations
1.​ Induced: by exposure to mutagens (environmental agents), such as chemicals (formaldehyde,
benzene, food additives), and natural or artificial ionizing radiation.
-​ Ionizing Radiation (X–Rays, Gamma Rays, Cosmic Rays): cause ss/ds breaks, chromosome
breaks and rearrangements → reach all cells of the body (including gametes).
-​ Non–Ionizing Radiation (UV Radiation): causes pyrimidine dimers (covalent bonds between
adjacent pyrimidines), which air unable to properly pair, causing substitution → skin cancer.
2.​ Spontaneous: naturally occuring, through errors in DNA replication or chromosomal division.

Nomenclature: all variants should be described at the most basic (DNA) level.
-​ c. → stands for coding DNA sequence mutation
-​ m. → stands for Mitochondrial sequence mutation
-​ p. → stands for Protein sequence mutation
-​ Nucleotide: Number:Base>Base → shows the
position and affected base (e.g. 1601G>A)
-​ Amino Acid: AA:Number:AA → shows the codon
number, AA switch, and type (e.g. Arg534Gln)
 Lecture 9 - DNA Damage and Repair

DNA is constantly being altered by various processes, including cell division. DNA replication is usually an
accurate process, but errors can occur (1 x 10–7). Maintenance of genetic information requires constant repair.
-​ All living organisms have mechanisms to repair DNA damaged. The ATM protein (“Guardian of the
Genome”) is involved in sensing DNA damage, and mutations can cause ataxia telangiectasia.

DNA Damage: an abnormal change in the chemical structure of DNA (10,000–1M nucleotides/cell/day).
-​ DNA Mutations: a change in the sequence of base pairs.
-​ Sources of DNA Damage
-​ Endogenous Sources:
1.​ Alkylation: addition of ethyl/methyl to Guanine
2.​ DNA Base Mismatching during replication or mitotic stress
3.​ Hydrolysis and Oxidation (by ROS): e.g. the OH– radical is the most reactive, converting
G to T and damaging DNA, proteins, and lipids.
4.​ Depurination
5.​ Demethylation
6.​ Spontaneous Deamination: leads to C → U Conversion
-​ Exogenous Sources (Mutagens): Radiation, Chemicals, ROS (cause stalled replication fork →
single/double strand breaks), Heavy Metals, Air Pollutants, Inflammatory Responses, and
Smoking (causes heavy methylation)

DNA Repair: the process where a cell identifies and corrects damage in DNA, via signalling pathways that
allow cell–cycle arrest (at G0) to give more time for repair (DNA proofreading), correcting 99.9% of errors.
-​ If damage is irreparable, cells can initiate apoptosis, or mutations may arise from the damage.
-​ Consequences of Failed DNA Repair: Genomic Instability, Apoptosis, or Senescence (aging),
predisposing the person to immunodeficiency, neurological disorders, and cancer.
-​ Short Term: physiological dysfunction, genomic instability, apoptosis, abnormal growth,
defective signaling pathways, impaired protein/gene expression, and decreased cell division.
-​ Long Term: Aging, Cancer, Hereditary Disorders (if in germ cells), and genetic divergence.

DNA Repair Mechanisms

Direct Reversal: single step process for simple damages, without using a template.
1.​ Demethylation: repairs methylation, using the O6–methylguanine–DNA methyltransferase enzyme.
2.​ Photoreactivation (in bacteria): repairs pyrimidine dimer bonds, via a photoreactivating enzyme that
bind to the dimer and absorbed visible light to photorepair the UV–induced lesion.

Single Strand (ssDNA) Repair (Remove + Repair): multistep, using undamaged strand as a template.
1.​ Nucleotide Excision Repair (NER): Mutated DNA strand is cut around the nucleotide by
endonucleases, then removed by exonucleases. Missing Patch is replaced with the correct nucleotides
by DNA Polymerase, and the new section is fused with the strand via DNA Ligase.
-​ Removes bulky lesions like thymine dimers and lesions from UV and Chemo agents.
2.​ Base Excision Repair (BER): a single base is removed by DNA glycosylase from the AP
(apurinic/apyrimidinic) site, and replaced with the correct base using DNA Polymerase and Ligase.
3.​ Mismatch Repair (MMR): MMR proteins are recruited to a mismatched DNA site, and form a complex
with an MMR enzyme, which removes the incorrect sequence. DNA polymerase + DNA Ligase
resynthesis the correct DNA sequence and fuse it to the strand.
-​ Corrects base mismatches during replication & insertion–deletion loops (IDLs) from slippage.
Double Strand (dsDNA) Repair Mechanisms
-​ Homologous Recombination (HR):
between two sister chromatids, the ends of
double breaks are lost due to degradation,
then end processing and homologous
recombination result in accurate damage
repair using information from the sister
chromatid.
-​ Non–Homologous End Joining (NHEJ):
post separation of sister chromatids, the ends
of the break are lost due to degradation and
then regioned, resulting in DNA deletion due
to lack of a template.
-​ The NHEJ Mechanism is more error prone and dangerous due to deletion of nucleotides.

Trans-Lesion DNA synthesis: involves specialized

DNA polymerases (TLS Polymerase) which are able
to skip DNA lesions and continue synthesis, as if
replication was halted to repair the incorrect
nucleotides (‘stalled’), it may lead to double breaks.
It essentially skips repair to avoid synthesis halting
and further issues.
-​ If incorrect nucleotides were incorporated,
they would become mutations and contribute
to tumor formation and disease, but could
also contribute to evolution and genetic
divergence.

Disorders of DNA Repair

Types of DNA Repair Mechanism Gene Disorders

Colorectal Cancer via

Base Excision
Removal of abnormal bases MUTYH MUTYH–associated
Repair (BER)
polyposis

Nucleotide Excision Removal of thymine dimers and

ERCC Xeroderma Pigmentosum
Repair (NER) large chemical adducts

Nijmegen Breakage
NBS
Syndrome
Removal of dsDNA breaks by
Post-Replication
homologous recombination or BLM Bloom Syndrome
Repair
Non–Homologous end–joining
Breast Cancer and Fanconi
BRCA 1/2
Anemia

Mismatch Repair Corrects mismatched based Colorectal Cancer (Lynch

MSH & MLH
(MMR) caused by replication errors Syndrome - HNPCC)
Ataxia Telangiectasia: autosomal recessive ATM gene mutation, with 7;14
translocation or 7, 14 pericentric inversion in t-lymphocytes, resulting in
defects in ATM and TP53 genes (responsible for sensing DNA damage and
regulating dsDNA repair proteins).
-​ Clinical Features: Conjunctival Telangiectasia, Ophthalmoplegia
(eye muscle weakness), Immunodeficiency, Ataxia/Chorea (poor
control & abnormal movement), Radiation Sensitivity, Leukemia, and
Lymphoma.

Xeroderma Pigmentosum: autosomal recessive ERCC gene mutation, due to a defect in NER resulting in
worsening of dimers as a result of UV radiation.
-​ Clinical Features:
-​ Extreme Light Sensitivity (freckling, hyperpigmentation,
cancers [BCC, SCC, Melanoma])
-​ Neurological Symptoms: deafness, ataxia, spasticity (rigid
muscles), and mental disability
-​ Corneal clouding and other ocular tumors.

Fanconi Anemia: autosomal recessive BRCA mutation in dsDNA homologous recombination repair defect.
These lesions are recognized and repaired by the Fanconi anemia (FA) proteins.
-​ Diagnosis is achieved via chromosomal breakage assay and tests.
-​ Clinical Features: café au lait macules, short stature, radial ray anomalies, pancytopenia (due to it
mainly affecting bone marrow), aplastic anemia, renal malformations, and cancers such as
Myelodysplasia, SCC, and Uterine Cancers
 Lecture 10 - Molecular Techniques I

Genetic Testing: a branch of medical investigation involving analysis of body tissues to investigate DNA, RNA,
and proteins for mutations associated with a genetic condition.
-​ Information gained allows diagnosing, starting treatment and/or prevention strategies for a disease, and
allows for more awareness for the patient regarding their & their families medical status
-​ Genetic Testing requires a cell with a nucleus (no RBCs)

Important Indications for Genetic Testing

1.​ Positive Family History: inherited genetic diseases, SIDS, adult–onset diseases (CVD, dementia,
cancer, diabetes), or couples who want to ensure genetic match before having kids.
2.​ Developmental Delay and Congenital Malformations
3.​ Reproductive Issues: recurrent abortions, consanguinity, high–risk births, etc.
4.​ Infectious Diseases: e.g. Chlamydia, HIV/AIDS, Hepatitis B/C, or parasites such as Toxoplasma
gondii or Giardia lamblia

Methods of Genetic Testing

Biochemical Tests: examine proteins rather than genes, are are useful for…
-​ Inborn Errors of Metabolism (IEM) diagnoses
-​ Assessment of Defective proteins associated w/ other genetic disorders
-​ Screening for open neural tube defects and determining aneuploidy risk.

Cytogenetics: examining chromosomes for abnormalities via karyotyping or FISH, using leukocytes
-​ Plays a major role in testing for Leukemia in Bone Marrow Cells.

Karyotype: a snapshot of all the chromosomes of a cell, it provides a genome-wide image to confirm
numerical and structural chromosomal anomalies.
-​ Steps of Karyotyping
1.​ Sample Collection: from blood, fetal cells, or bone marrow cells
2.​ Cell separation and culture of dividing cells to stimulate mitosis (takes 2–3 days).
3.​ Cell Cycle arrest by incubation with Colcemid to arrest the cell in Metaphase
4.​ Chromosomal isolation via addition of a hypotonic solution to induce cell bursting
5.​ Staining with Giemsa stain and chromosomal analysis with a microscope.

Fluorescence in situ hybridization (FISH): determines gene translocation between chromosomes, deletions,
insertions, or amplifications, with the use of fluorescent DNA probes to target specific chromosomal locations
with complementary sequences. Different FISH probes include…
-​ Centromeric probes: bind to DNA sequences in and
around the centromere of a specific chromosome, and are
used to diagnoses common aneuploidy syndrome (trisomies
13, 18, 21).
-​ Chromosome-Specific Unique-Sequence Probes: bind to
a single specific locus, and are used to diagnose tiny
submicroscopic deletions and duplications (e.g. HER2
overexpression → breast cancer).
-​ Whole-Chromosome Paint Probes: a cocktail of probes
obtained from different parts of a particular chromosome,
used for subtle translocations.
Polymerase Chain Reaction (PCR): amplifies a single DNA sequence (specific genes) millions of times via
exponential amplification (8 → 16 → 32 → 64 → 128 → 256…)
-​ Requires: double stranded DNA, dNTP Nucleotides, nucleic acid primers, heat stable TAQ polymerase,
and specific nucleic primers (forwards and backwards).

Primer: highly specific to the gene, they are short

nucleic acids strands (~20 bases) that serve as a
starting point for DNA synthesis.

PCR Steps
1.​ Template DNA is broken (denatures) via
heating at 94ºC for 1–2 minutes.
2.​ Heat-Stable Primers bind (anneal) at 45–72ºC
(depending on the primer) for 30–60 seconds.
3.​ New Strand Elongation at 72ºC depending on
the polymerase (1 min → 1000 nucleotides)
a.​ 25–30 cycles are usually enough for
most applications (31 cycles → over a
million molecules)

Types of PCR I:
-​ Reverse Transcriptase PCR: a segment of the coding gene is amplified from mRNA (mRNA → DNA).
Therefore, only coding regions of DNA are amplified.
-​ Quantitative Real Times (RT) PCR: the amplified product is linked to a fluorescent reporter molecule
which is measured at each cycle, so we can see the reaction undergoing allowing for optimizing the
efficiency of amplification at each cycle.

Types of PCR II:

-​ Multiplex PCR: amplification of multiple targets in a single PCR experiment (requiring 2+ sets of
specific primers that work in the same temperature range ) to save time.
-​ Nested (RT–)PCR: increase the specificity of DNA amplification by reducing (cutting out) the
non–specific amplificated portion of DNA. First, regular PCR is performed including some nucleotides
round the desired gene, then another PCR occurs on the inner desired gene only.
-​ This avoids pseudogenes when amplifying a polymorphic gene, which may give false results.
-​ E.g. Ch. 11 Codon 6 of the Hemoglobin β chain, where only mutations will be amplified.

Electrophoresis: negatively charged DNA fragments move towards a positively charged electrode on
electrified gel, but they move at different speeds, which allows separation based on fragment length (bands).
-​ Comparing the size and position of the DNA bands can determine the size/quantity of amplified DNA
fragments produced by PCR.

Applications of PCR
-​ Clinical: DNA fingerprinting (paternity tests, crime scene analysis, archeological finds), diseases.
-​ Research: gene closing, expression, genetic mapping, real-time PCR, DNA sequencing, ancient DNA.
 Lecture 11 - Molecular Techniques II

Molecular Genetics: study of the molecular structure of RNA, its cellular activities, and its influence in
determining the overall makeup of the organism.
-​ Pleiotropy: a gene that controls many phenotypic characteristics
-​ Allelic Diseases: different diseases due to different mutagens in the same gene.
-​ Genetic Heterogeneity: a disease with different genetic causes
-​ Allelic Heterogeneity: diseases as a result of different mutations of the same gene
-​ E.g. any mutation of the CFTR gene → Cystic Fibrosis
-​ Locus Heterogeneity: disease as a result of mutations in different genes
-​ E.g. mutations of NIPBL, SMC1A, OR SMC3 Genes → Cornelia de Lange Syndrome
-​ Polygeny: multiple genes control the expression of a certain clinical characteristic (e.g. height).
-​ Penetrance: likelihood of a genetic variant to trigger a specific symptom (if the genotype shows or not).
-​ Expressivity: type/extent of expression of the clinical features

Dosage Analysis: Microarray, MLPA

DNA–Array (Chromosomal Microarray, CMA): a quantitative,

molecular cytogenetic technique that helps to detect a small
chromosome changes (<5 Mb), such as microdeletion or
microduplication syndromes.
-​ CMA resolution is around 1 Kb (highest among current
methods)
-​ Indications: diagnose the cases of Developmental Delay
(DD) Intellectual Disability (ID), Congenital anomalies, and
Dysmorphic Features, particularly with a normal karyotype.
-​ With normal analysis, only 7–10% of DD/ID cases
can be diagnosed (CMA → 15-20%).

Limitations of a Microarray
-​ No evidence of a balanced (no loss) rearrangement, e.g. balanced translocation and inversion
-​ Does not provide information about the position of a duplication (appears more with deletion)
-​ Cannot detect tiny duplications and deletions of DNA segments within a single gene
-​ Doesn’t provide information about point mutations
-​ Limited evidence of mosaicism (cannot detect mosaicism that is <20–25%)

Types of CMAs
-​ Single Nucleotide Polymorphism (SNP) Array: shows heterozygous (one copy) or homozygous (both
copies) deletion and insertions of regions of genes.
-​ Array Comparative Genomic Hybridization (CGH): compares patient DNA with a control to detect
variations in amount of DNA (large scale deletions or duplications).

DiGeorge/Velocardiofacial Syndrome: a 3–Mb microdeletion on Ch. 22 (22q11.2), which is usually sporadic

and affect approximately 1 in 4000 births. Manifestations are usually due to the loss of the TBX1 and COMT
genes in the 22q11.2 region, with COMT contributing to an increased risk of behavioral and mental issues.
-​ Inheritance: Autosomal Dominant, but is 90% de novo (usually sporadic)
-​ Manifestations: heart malformations, thymic and parathyroid hypoplasia, cleft palate, typical facies.
MLPA (Multiplex Ligation–Dependent Probe Amplification): variation of the Multiplex PCR that permits
amplification of multiple targets with one primer, allowing detection of large deletions or duplications (>50 bp).
-​ Uses: deletions/duplications of exons or whole genes, point mutations, and RNA expression.

Duchenne Muscular Dystrophy (DMD): most common and severe form of muscular dystrophy, caused by
mutations in the DMD (Dystrophin) Gene at Xp21.2, affecting 1 in 3,500 males.
-​ DMD Gene (>2 Mb): encodes a protein, with is part of the dystrophin–glycoprotein complex (DGC) that
bridges the inner cytoskeleton and the ECM. Mutations may cause DMD, BMD, or Cardiomyopathy.
-​ Inheritance: X–Linked Recessive
-​ Clinical Features of DMD:
1.​ Progressive Muscular Weakness between the ages of 3–5.
2.​ Abnormal Gait with Positive Gowers sign (difficulty getting up from the floor).
3.​ Calf pseudohypertrophy (replacement of muscle fibers by fat and CT).
4.​ Wheelchair dependence by 11/12 years old.
5.​ Deterioration leading to lumbar lordosis, joint contracture, and cardiorespiratory failure.

Becker Muscular Dystrophy (BMD): milder condition due to mutations in the same gene (1 in 20,000 males).
-​ Clinical Features: very similar but a much less aggressive disease course; mean onset of 11 years.

Sequencing Methods: Sanger Sequencing, NGS, WES, WGS

Sanger Sequencing: gold standard in mutation detection when you know the target you are looking for, based
on selective ‘chain termination’ reactions.
-​ Uses: Specific Mutations in a Specific Gene, analysing small genes (not more than 27 exons, such as
the CFTR gene), screening familial mutations, confirming NGS detection, confirming de novo mutations
by testing the mutation on the parent.

Necessary Components:
-​ PCR Product
-​ Primer specific to the target
-​ DNA polymerase
-​ Free nucleotides
-​ Dideoxynucleotides (ddNTPs): used to stop
the DNA at random places so you can figure
out the DNA sequence.
-​ Puffer Solution
Cystic Fibrosis: from a mutation in the CFTR Gene at 7q31.2, affecting 1 in 2,000–3,000 people of western
european origin, 1 in 15,000 african americans, and 1 in 31,000 asian americans.
-​ Inheritance: Autosomal Recessive (one of the most common).
-​ Clinical Features: chronic lung disease caused by recurrent infections with secondary cardiac failure,
pancreatic failure (malabsorption), nasal polyps, rectal prolapse, cirrhosis, and diabetes.

Whole Exome Sequencing (WES): focuses on identifying disease–causing DNA variants within the 1.5–2%
of the human genome (exomes only), recommended in cases of suspected hereditary diseases which cannot
be clearly assigned to one known genetic syndrome based on clinical features (no target).
-​ Advantages: identifies very rare genetic causes of disease, novel genes, blended phenotypes (2+
disorders in a single patient → mixed clinical features), and phenotypic expansion of syndromes.

Whole Genome Sequencing (WGS): sequences the entire

genome, able to detect CNV smaller than three exons,
balanced chromosomal rearrangements, can analyse
mitochondrial DNA, can reveal triplet repeat expansion, but is
difficult due to data storage and interpretation.
-​ Indications; more useful in critically ill neonates when
time is short, otherwise similar to WES.

Benefits of WES/WGS: short–term clinical diagnosis, and

active management changes (modifications to meds, procedures, or treatment), reproductive information for
couples, potential impact on other family, cost–effective.
 Lecture 12 - Dysmorphology

Classification of Genetic Disorders

1.​ Single Gene Disorders → Autosomal Dominant/Recessive, X–Linked Diseases.
2.​ Chromosomal Abnormalities.
3.​ Multifactorial Disorders due to gene/environmental interaction.

Clinical Approach to a Genetic Disorder

1.​ History: Personal, Developmental, Prenatal, Natal, Postnatal, and
Obstetric Histories, Positive Family history of any similar condition, and
a systemic review of all relevant patient history.
2.​ Pedigree Analysis: most commonly used, showing relationships among
family members, degrees of relationships, and which family members
are affected/carriers of a disease.
3.​ Examination:
a.​ Growth Parameters: Height, Weight, Head Circumference (BMI)
b.​ Systems Examination: checking bodily systems
4.​ Dysmorphology Assessment
5.​ Investigations: defined by the anomalies observed, and include
chromosomal, metabolic, and molecular studies, or more general tests
such as scans, urine analysis, or biopsies.

Dysmorphology Assessment

Dysmorphology: study of human congenital malformations (birth defects), especially those involving anatomy.

Head
-​ Microcephaly: Head Circumference below 3 SD of the Mean, caused by early skull fusion.
-​ Macrocephaly: Head Circumference above 3 SD of the Mean
-​ Brachycephaly: an infant skull deformity characterized by a lower-than-normal ratio of the skull's length
to its width (flat occipital lobe), such as in Down Syndrome.

Eye
-​ Normal: eyelids are positioned so the lateral canthus (corner) is ~1 mm higher than the medial canthus.
-​ Upwards Slanting: slanting into the middle (e.g. Down Syndrome)
-​ Downwards Slanting: slanting away from the middle
-​ e.g. Treacher Collins Syndrome
-​ Hypertelorism: abnormal increased distance between the eyes
-​ Hypotelorism: abnormal decreased distance between the eyes

Ear
-​ Low Set Ears: minor anomaly involving an ear set below its normal
location, when the helix (upper end) of the ear is set below the plane
of both inner canthi (inner corners of the ear).
-​ Microtia: underdeveloped outer ear
-​ Anotia: missing one/both ears
-​ Protruding Ears: more than 2 cm from the head
Mouth & Mandible
-​ Macrostomia: Large Mouth -​ Micrognathia: Small Mandible
-​ Microstomia: Small Mouth -​ Retrognathia: Receding Mandible
-​ Macroglossia: Large Tongue -​ Prognathism: Protruding Mandible

Hands & Fingers

-​ Arachnodactyly (‘Spider Fingers’):
abnormally long and slender fingers
& toes (e.g. Marfan Syndrome)
-​ Brachydactyly: short fingers and toes
(e.g. Down Syndrome)
-​ Polydactyly: extra fingers/toes that are
present at birth (preaxial → thumb;
postaxial → pinky)
-​ Syndactyly: fused digits
 Lecture 13 - Birth Defects

Birth Defects: condition existing at/before birth, regardless of the cause (inherited or acquired
[environmental]).

Causes of Birth Defects

-​ Genetic: Chromosomal, Single Gene Disorders, or Multifactorial (genetic + environmental factors).
-​ Environmental Factors

Congenital Abnormalities: condition existing at birth, and often before, or that develops during the first moth
of life (neonatal disease) → can be genetic or acquired.
-​ Major Congenital Defects: serious structural abnormalities with an adverse outcome on functional or
social acceptability of the individual
-​ Minor Congenital Defects: simple abnormality, with only a small risk of serious consequences, and
are of neither medical or cosmetic importance.

Structural Abnormalities Incidence (% of those with Birth Defects)

Spontaneous Miscarriage
80–85% ; 25%
(First Trimester; Second Trimester)

Major Abnormality apparent at birth 2–3%

Major Abnormality apparent later 2%

Minor Abnormality 10%

Death in Perinatal Period 25%

Death in the first year of life 25%

Death at 1–9 Year of Age 20%

Mortality: high incidence of major and minor abnormalities observed in early spontaneous miscarriages.
-​ First Year of Life: 25% of all deaths are due to major structural abnormalities
-​ From 1–10 Years: 20%
-​ From 10–15 Years: 7.5%

Examples of Major Congenital (Structural) Abnormalities

-​ Cardiovascular: Ventricular or Atrial Septal Defect (V/ASD), PDA, Tetralogy of Fallot
-​ CNS: Hydrocephaly (due to ↑ CSF), Microcephaly, Anencephaly (no skull vault), Neural Tube Defects
-​ GIT: Cleft Lip/Palate, Esophageal Atresia (closed off esophagus), Imperforate Anus
-​ Urogenital: Bilateral Renal Agenesis, Infantile Polycystic Kidney
-​ Limb: Transverse Amputation (no limb)

Examples of Minor Congenital (Structural) Abnormalities

-​ Preauricular Pit/Tag -​ Lip pits
-​ Epicanthic Fold -​ Single Palmar Crease; Fifth Finger Clinodactyly
-​ Lacrimal Duct Stenosis (can’t cry) -​ Syndactyly between the Second and Third toes
-​ Brushfield Spots of the Iris -​ Supernumerary Nipple
 Classifications and Mechanisms of Birth Defects

Single Abnormalities
-​ Malformation: arrested or misdirected development or a tissue early in gestation, with a primary
(endogenous) and intrinsic cause (e.g. Congenital Heart Defects, Cleft Lip, Neural Defects).
-​ Most involve only one organ, with multifactorial inheritance (both genetic and environmental),
and so its inheritance and recurrence risk cannot be determined.
-​ Disruption: external factors (e.g. ischemia, infection,
trauma) that disrupt normal development (secondary and
extrinsic → not genetic), with the breakdown of tissue
-​ Deformation: abnormal mechanical force which distorts
an normal developed structure.
-​ Causes: Oligohydraminos (not enough Amniotic
Fluid) or abnormal uterine structure can lead to
hip dislocation or positional talipes (club foot).
-​ Dysplasia: abnormal cell organization in a single tissue,
caused by single gene defects
-​ E.g. Skeletal Dysplasia, Ectodermal Dysplasia
(affect teeth, hair, nails, glands)

Multiple Abnormalities
-​ Sequence: occur as a consequence of an event cascade initiated by a single primary factor.
-​ E.g. Potter Sequence, which results from oligohydramnios (not enough Amniotic Fluid)
-​ Reduced Urinary Output → Oligohydramnios → Fetal Compression → Squashed Face,
Hip Dislocation, Club Foot, Pulmonary Hypoplasia → Death
-​ Syndrome: recognizable pattern of abnormalities with a single known underlying cause
-​ E.g. Chromosomal Abnormalities (Trisomy), Single Gene Disorder (Van der Woude Syndrome).
-​ Down Syndrome: Mongolian Features, Single Palmar Crease, Foot Abnormalities, etc.
-​ Van der Woude Syndrome: Cleft Lip/Palate/Uvula, Lower Lip Pit, Missing Teeth
-​ Association: malformations that tend to occur together by chance (no known cause), such as VATER
Syndrome (Vertebral, Anal, Tracheo–Esophageal, and Renal/Radial Ray Abnormalities)
 Lecture 14 - Prenatal Diagnosis

Prenatal Diagnosis: testing the mother of fetus before birth to determine the risk of abnormalities and supply
patients with information so that they can make informed choices during pregnancy.
-​ Goals of Prenatal Diagnosis
1.​ Providing reassurance to at–risk families when results are normal
2.​ Providing risk information to couples for whom pregnancy termination is an option
3.​ Allowing couples to prepare for the birth of an affected child
4.​ Helping the health-care professional to plan delivery, management, and care of the infant when
a disease is diagnosed. Congenital anomalies account for 20–25% of prenatal deaths.

Indications for Prenatal Diagnosis

-​ Significant Risk of Disease: positive family history of
chromosomal abnormalities, single gene disorder, or
neural tube defect (aka previously affected family).
-​ A serious condition to justify termination of pregnancy.
-​ Advanced Maternal Age: increased risk for
non–disjunction aneuploidy (Trisomy 21).

Screening: test used to identify individuals with a higher chance

of a health concern (higher false positive risk).
Diagnostic: a test used to confirm a diagnosis (usually more
expensive or invasive).

Methods of Prenatal Diagnosis

-​ Non–Invasive: Ultrasound, Maternal Blood (Serum), Maternal Blood (with fetal cells), Fetal
Echocardiography, Pre–implantation Genetic Diagnosis.
-​ Invasive: Chorionic Villus Sampling, Amniocentesis, Percutaneous Umbilical Blood Sampling,
Embryoscopy & Fetoscopy.

Maternal Blood (Serum):

-​ ɑ–Fetoprotein (AFP): arises from the yolk sac and fetal liver in the 6th week of gestation, with the
highest concentration in the second trimester, then falling progressively.
-​ Maternal Serum ɑ–Fetoprotein (MSAFP): utilized to determine fetal AFP levels due to small
amounts leaking to amniotic fluid, crossing the placenta to mother’s blood.
-​ Causes of ↑ MSAFP: incorrect gestational age, threatened abortion, multiple pregnancy, Fetal
Abnormalities (neural tube, abdominal wall, skin, renal defects, Turner Syndrome).
-​ β–HCG: used to test for pregnancy, as it is detectable ~1 week after conception. It is used with other
biomarkers to detect chromosomal abnormalities (↑ β–HCG + ↓ AFP → Down Syndrome)
-​ Very High (↑↑) β–HCG → Trophoblastic Diseases (e.g. choriocarcinoma), while the absence of a
fetus on ultrasound + ↑ β–HCG → Hydatidiform Mole.
-​ Estirol: Estradiol (E2) is the dominant estrogen in non–pregnant women, with E3 undetectable.
-​ E3 levels increase 1000x in pregnancy due to Dehydroepiandrosterone (DHEA) production by
fetal adrenal gland, converted to E3 by placental aromatase enzyme.
-​ ↓ E3 → Down Syndrome, Adrenal Hypoplasia, or Anencephaly.
-​ Inhibin–A: made by placenta/corpus luteum, ↑ Inhibin–A → Down Syndrome, Preterm Delivery.
-​ PAPP–A: ↓ PAPP–A → Trisomies 13, 18, 21, small for gestational age (SGA), or stillbirth.
-​ ↑ PAPP–A levels are seen in fetuses large for gestational age (LGA).
Serum Biomarkers: biomarkers used to assess fetal and maternal health, diagnosing maternal (Gestational
Diabetes, Pre–eclampsia, Trophoblastic Disease) & fetal conditions (Down Syndrome, Neural Tube Defects).
-​ Fetal Assessment: ensures satisfactory growth in utero, with factors causing fetal growth restriction
including poor maternal nutrition, placental
insufficiency, and fetal abnormalities.

Conditions Detected with Maternal Serum Screening

-​ Neural Tube Defects: occur in about 1.5/1000
pregnancies, with ↑ AFP in amniotic fluid & serum.
-​ Anencephaly: failure of closure at the
cranial end of the neural tube
-​ Spina Bifida: failure of closure at the
caudal end of the neural tube
-​ MSAFP screening identifies ~90%
of Anencephaly and ~80% of Spina Bifida, with amniotic fluid AFP and elevated
acetylcholine esterase confirming (not ultrasound).
-​ Down’s Syndrome: occurs in about 1/800 pregnancies, diagnosed using the classic triple screen
(MSAFP, β–HCG, and uE3) sometimes combined with Inhibin–A (Quadruple Screen). Levels of these
analytes change during pregnancy, so correct gestational age is necessary for correct interpretation.
-​ In the Late First Trimester → PAPP–A, β–HCG, and Ultrasound for Nuchal Translucency are
used for assessment of Down Syndrome (Nuchal Translucency is ↑ in ~70% of Down’s S.).
-​ Abnormal results MUST be followed with karyotyping and invasive tests to confirm diagnosis.
-​ Invasive Testing carries a 0.5–1.0% risk of pregnancy loss.

Non–Invasive Prenatal Testing/Screening (NIPT/S): uses cell free DNA from placental trophoblastic cells,
detected after 7+ weeks of gestation. NIPT may show incorrect (discordant) results due to…
-​ Analytical Issues: failure of extraction or amplification or a very low fetal fraction.
-​ Placental Mosaicism: can produce a false negative.
-​ Twins: second gestational sac/vanishing twin → a
dead twin’s DNA might be used instead.
-​ CNVs or Maternal Mosaicism: e.g. SCA (Sex
Chromosome Aneuploidy)

Special Problems in Prenatal Diagnosis

1.​ Failure to obtain a sample or culture failure
2.​ An ambiguous or unexpected chromosome result
3.​ Ultrasonographic ‘soft marker’
 Lecture 15 - Oncogenetics

Differentiation between Genetic and Environmental Factors in Cancer

-​ Family Studies: clustering of cancer cases within families supports a genetic contribution to cancer.
-​ Risk varies according to the age of onset (earlier the age at diagnosis → greater the risk).
-​ Twin Studies: the risk of two twins both developing breast cancer are low, suggesting that
environmental factors are more important than genetic factors.
-​ Disease Association: the association of blood groups with disease suggests a possible genetic
contribution to disease etiology, such as an association with blood group A and gastric cancers.

Cancer Genetics: All cancers are genetic, but not all of them are inherited. Most cancer mutations occur due
to exposure to controllable environmental factors, and are usually not inherited.
-​ However, some are born with increased risk of cancer due to the inheritance of an altered gene
important for cell growth and DNA repair (e.g. BRCA1/2 → Breast, Ovarian, and Prostate Cancer).
-​ Cancer may be seen to skip generations due to reduced penetrance or the two hit hypothesis.
-​ Non–Inherited (Somatic) Cancers may also affect multiple members of a family due to shared
environmental factors (e.g. smokers, unhealthy eating habits, housing issues, etc.)

Cancer Predisposition Genes

-​ Oncogenes: mutated forms of normal genes (protooncogenes) that have key roles in cell growth and
differentiate pathways → uncontrolled growth by excessive cellular proliferation. (Dominant Effect)
-​ Tumor Suppressor Genes: control cell proliferation and constitute the largest group of hereditary
cancer genes, including DNA repair genes which detect/repair genetic damage. (Recessive Effect).
-​ Two Hit Hypothesis: loss of both alleles of a tumor suppressor gene through two independent
mutation events (e.g. inheritance + additional somatic mutation) → cancer.

Inherited Cancer Syndromes

-​ Hereditary Non–Polyposis Colorectal Cancer (Lynch Syndrome): 1:1000 incidence, causing ~5%
of all colorectal cancers, with a characteristic family–like incidence of colorectal cancer at a young age
(45–60 years old), mostly in the right section of the colon. (Males → 70% Risk; Females → 50%)
-​ HNPCC → autosomal dominant, affecting DNA mismatch repair genes MLH1 or MSH2 (90%).
-​ In addition, women with HNPCC have an additional risk (60%) for endometrial cancer.
-​ Extracolonic Manifestations: Stomach (13%), Ovarian (12%), and Small Intestinal cancers.
-​ Familial Adenomatous Polyposis (FAP): an inherited disorder resulting in the development of
thousands of benign growths (polyps) of the large bowel as early as childhood, with a high risk of
carcinomatous change of the polyps → 90% of affected individuals develop bowel cancer.
-​ FAP → Autosomal Dominant in APC, A. Recessive in MUTYH.
-​ Extracolonic Manifestations: Congenital Hypertrophy of Retinal Pigment Epithelium (CHRPE),
Osteoma of the Mandible and Skull, Epidermoid Cysts, Desmoid, Turcot’s Syndrome
-​ Breast Cancer: most common cancer in women (1:9 incidence in women), with 15–20% of affected
women having a positive family history, with greater risk from closer relatives, earlier age at diagnosis,
paternal history of breast cancer, or additional occurrence of ovarian cancer.
-​ BRCA1 Mutations: on chromosome 17q, with ↑ risk of breast, ovarian, and prostate cancers.
-​ BRCA2 Mutations: on chromosome 13q, with ↑ risk of breast and prostate cancers in men.
-​ Prostate Cancer: a common cancer affecting men, with 15% of affected individuals having a similarly
affected relative, resulting in a 2–5x higher risk for prostate cancer of male relatives at a younger age.
-​ Genes: Hereditary Prostate Cancer 1 (HPC1), HPC2, BRCA1, BRCA2.
Preventing Genetic Cancers: Vaccination, Healthy Diet/Weight, Exercise, Avoidance of Tobacco, Predictive
Tests in cases of Positive Family History.

Genetic Counseling in Familial Cancer: recognition of individuals with an inherited susceptibility to cancer
usually relies on taking a careful family history for suggestive features, such as…
-​ Several close (1st or 2nd Degree) relatives with a common or related cancer
-​ Bilateral tumors in paired organs (e.g. both breasts, ovaries, testicles)
-​ Tumors in two different organ systems in the same individual
-​ Two family Members with the same rare cancer

Requirements of a Screening Test for Persons at Risk for a Familial Cancer-Predisposing Syndrome or
at Increased Risk for the Common Cancers:
-​ The test should detect a malignant or premalignant condition at a stage before it is producing
symptoms, with high sensitivity and specificity.
-​ The treatment of persons detected by screening should improve the prognosis.
-​ The benefit of early detection should outweigh potential harm from the screening test.
-​ The test should preferably be non-invasive, as most at-risk individuals require long-term surveillance.
-​ Adequate provision for prescreening counseling and follow-up should be available.
 Lecture 16 - Hemoglobinopathies

Hemoglobin (Hb): found in RBCs, it is used to transport O2 to tissues (1 Hb Molecule → 4 O2 Molecules)

-​ Structure: Heme, a porphyrin ring with a central Fe2+ atom, and Globin, a quaternary protein.

Types of Hemoglobin Structure % in Adults

Gower I 𝛇2ϵ2 (Zeta 2, Epsilon 2)

Embryonic Gower II ɑ2ϵ2 (Alpha 2, Epsilon 2)

Portland I 𝛇2𝛄2 (Zeta 2, Gamma 2)

Fetal HbF ɑ2𝛄2 (Alpha 2, Gamma 2) <1%

HbA ɑ2β2 (Alpha 2, Beta 2) 97–98%

Adult
HbA2 ɑ2δ2 (Alpha 2, Delta 2) 2–3%

Adult Hemoglobin (HbA): responsible for O2 Binding in the Lung and

transport to tissues for use in aerobic metabolism. The alpha (141 amino
acids) and beta (146 amino acids) globin chains are very similar.

Hemoglobin Synthesis: via a complex series of steps, it continues in

the cell throughout its early development in bone marrow.
-​ Site: Heme → mitochondria and cytosol of immature RBCs,
Globin → Ribosomes
-​ Ch. 11 (β–globin cluster): contains genes for synthesis
of epsilon, gamma, delta, and beta globin.
-​ Ch. 16 (ɑ–globin cluster): contains genes for formation
of zeta and alpha globin

Disorders of Hemoglobin
-​ Structure Globin Variants: defects from a single AA substitution in ɑ or β chains (e.g. sickle cell).
-​ Synthesis of Globin Chain Disorders: quantitative defects (e.g. the thalassemias).

Sickle Cell Disease (HbSS): An autosomal recessive blood disorder with rigid abnormal sickle shaped RBCs
due to a point mutation in the β–globin gene (chromosome 11).
-​ As a result of this mutation, Glutamine (charged) → Valine (non–polar), resulting in a sticky coating on
RBCs that makes them stick to one another → sickle shaped. (Lifespan → 20 days).
-​ Normal RBCs: Disc-shaped, distortable, with a lifespan of 120 days.
-​ Sickle Cell Symptoms: Anemia, Jaundice, Delayed Growth, Hand–Foot Syndrome, Sickle Cell Crisis
-​ Sickle Cell Crisis → episodes of pain with varying frequency and intensity, followed by
remission, commonly occurring in the lower back, leg, abdomen, and chest.
-​ Diagnosis: CBC (shows anemia), Blood Film (shows sickle cells), Hb Electrophoresis (HbS variant).
-​ Treatment: prophylactic penicillin and hydroxyurea to increase HbF.

Sickle Cell Trait (HbAS): found in heterozygous carriers, these people are protected against malaria and do
not develop the sickle cell disease (found in 40% of africans and 9% of african americans).
-​ Increased Risk for sudden death during exercise, vaso–occlusion from severe hypoxia.
Thalassemias: the most common group of inherited disorders, they are quantitative defects with reduced rate
of one or more globin chains, classified based on the globin chain(s) that are synthesised in reduced amounts.

Pathophysiology of Thalassemia: imbalance of globin chains → more free globin chains → precipitate →
hemolysis of RBCS → Hemolytic anemia, with hyperplasia of bone marrow.

Genetic Basis of Alpha Thalassemia: each cell has four copies of the ɑ–globin, with each responsible for ¼
production of ɑ–globin. So, there are four possible mutation states…
-​ Loss of One Gene → Silent Carrier
-​ Loss of Two Genes → Thalassemia Minor (trait)
-​ Loss of Three Genes → Hemoglobin H (accumulation of β–globin → precipitation → HbH)
-​ Loss of Four Genes → Hemoglobin Barts (no alpha chains produced, only gamma chains)

Hemoglobin Barts (ɑ0 Thalassemia [𝛄4] → Most Severe): all four ɑ–globin alleles are deleted or
dysfunctional, and Hb is formed of four gamma globin chains. This is also called Hydrops Fetalis.
-​ Characteristics: Fetal Onset of Edema, Ascites, Pleural and Pericardial Effusions, and Severe
Anemia, leading to congestive heart failure → tissue hypoxia and death.
-​ Prenatal Diagnosis: by ultrasound at 22–28 weeks.
-​ Death in the neonatal period is almost inevitable.

Hemoglobin H (HbH disease [β4 > 25%]): a result of deletion or dysfunction of 3 of the four ɑ–globin alleles.
-​ Unlike Hemoglobin Barts, patients may survive into adulthood.
-​ Characteristics: infants with microcytic hypochromic hemolytic anemia and hepatosplenomegaly, with
thalassemia–like bone changes in ⅓ of affected individuals.

β–Thalassemia: due to underproduction of the β–globin chain, usually due to splice site 5’GT or 3’AG of the
introns (most common in the mediterranean), with two possible mutations:
-​ Reduced β–globin Chains (β+) → Thalassemia Minor (Trait): no signs or symptoms, usually with
microcytic hypochromic anemia which can be confused with iron deficiency anemia.
-​ Absent β–globin Chains (β0) → Thalassemia Major (Cooley’s Anemia): present after 6 months as
increased ɑ–globin chains → unstable precipitates → damage and hemolysis, with severe transfusion
dependent anemia and expansion of bone marrow (uneven face and skull).
-​ δ β–Thalassemia: Underproduction of both δ and β–globin chains, caused by extensive deletions
involving the δ and β–globin genes.

Diagnosis of Thalassemia Major

-​ CBC and Blood Film: show low CBC and Hb Levels, Microcytosis, elevated reticulocytes, bilirubin and
urobilinogen levels, with pale nucleated RBCs, and marked anisocytosis.
-​ X-Ray: shows thinning and widening of the marrow space due to overactive bone marrow, with areas of
osteoporosis in some long bones and deformed phalanges.
-​ Hb Electrophoreses: rise in HbF, light increase in HbA2.

Treatment of Thalassemia: none recommended

-​ Regular Transfusions, Chelating Agent (desferrioxamine),
Splenectomy, and Bone Marrow Transplants may be performed.

Hereditary Persistence of HbF (ɑ2𝛄2): benign condition in which significant

HbF production continues into adulthood, usually asymptomatic and is due to
deletions of the δ and β–globin genes or point mutations.
 Lecture 17, 18 - Inborn Errors of Metabolism

Metabolism: chemical reactions constituting catabolism and anabolism, facilitated by enzymes.

-​ Inborn Errors of Metabolism: inherited enzyme defects, coined by Sir Archibald Garrod, resulting in
accumulation (toxicity) of the metabolite (reactant) and deficiency of the product.

Major Types of IEMs

-​ Disorders of Intoxication
-​ Disorders of Energy Metabolism
-​ Disorders of Complex Molecules

Disorders of Intoxication

Aminoacidopathies
-​ Phenylketonuria (PKU): Autosomal Recessive disorder that is screened for at birth, it is due to
absence of phenylalanine hydroxylase enzyme or the BH4 Co–factor, resulting in toxic build up of
phenylalanine and low levels of tyrosine, melanin, and dopamine.
-​ Treatment: Low Phenylalanine, Tyrosine Rich Diet, BH4 Supplementation, Pegvaliase.
-​ Homocystinuria: due to cystathionine β–synthetase deficiency and Vitamin B12 Deficiency from
Cobalamin Disorder or Dietary Deficiencies → Accumulation of Homocysteine & Methionine.
-​ Symptoms: tall, thin, scoliosis, poor vision, seizures, and lense sublulation.
-​ Maple Syrup Urine Disease (MSUD): show increase Leucine, Isoleucine, and Valine due to the
absence of branched-chain keto acid dehydrogenase (BCKD), so no forming Acetyl–CoA.
-​ Treatment: Low Branched Chain AA Diet, Thiamine (Vitamine B1), Liver Transplant

Urea Cycle Defects: Ornithine Transcarbamylase Deficiency (OTCD): X–Linked Disorder presenting in the
first week of life with hyperammonemia (no nitrogen removal), resulting in CNS toxicity.
-​ Symptoms: Lethargy, Poor Feeding, Coma, and Death.
-​ Investigation: Hyperammonemia (>400 umol), Elevated Plasma Glutamine, Orotic Acid, Low Citrulline.
-​ Treatment: Low Protein Diet, Arginine Supplementation, Liver Transplant, Nitrogen Scavengers
(Sodium Phenylacetate/Phenylbutyrate, Sodium Benzoate)

Galactosemia: defect in the galactose metabolism pathway → accumulation of toxic Galactose–1–phosphate

presenting within the first weeks of life.
-​ Presentation: Hepatomegaly, Edema, Ascites (Liver Failure), Hypoglycemia, G – Infections, Poor
Feeding, Vomiting, Jaundice, Lethargy, Cataracts.
-​ Diagnosis: Urine Reducing Substance, Decreased Plasma GALT, Increased Galactose–1–Phosphate.
-​ Treatment: Galactose free diet

Disorders of Energy Deficiency

Glycogen Storage Disease Type 1a (Von Gierke Disease): AR due to absent Glucose–6–Phosphatase.
-​ Symptoms: Hypoglycemia with Lactic Acidosis, Hepatomegaly
-​ Treatment: frequent feedings, complex carbs, overnight glucose dip, liver transplant

Oxidative Phosphorylation Disorders: occur within the mitochondria, following any inheritance (+ maternal).
-​ Manifestations: Muscle Weakness, Cardiomyopathy, Liver Failure, Seizure, Encephalopathy, Stroke.
-​ E.g. Mitochondrial Encephalopathy Lactic Acidosis Stroke Like Episodes (MELAS)
 Disorders of Complex Molecules

Lysosomal Storage Disorders: Lysosomes usually contain over 40 hydrolyzing enzymes (acidic pH)
-​ Hurler’s Disease: presents with intellectual disability, short stature, skeletal deformity, corneal opacity,
hepatosplenomegaly, and coarse facial features.
-​ Diagnosis: increased urine mucopolysaccharides, reduced enzyme activity
-​ Treatment: Enzyme Replacement Therapy, Stem Cell Transplants

Peroxisomal Disorders
-​ Zellweger Spectrum Disorder (ZSD): a peroxisome biogenesis disorder with impaired assembly of the
peroxisome → multiple enzyme deficiencies.
-​ Features: Dysmorphic, Hypotonia, Hepatomegaly, Seizure, Bone, Brain, and Renal Changes.
-​ Diagnosis: Increased Plasma very long fatty acid chains, reduced RBC plasmalogens,
increased phytanic and pristanic acid.
-​ No treatment exists.

Levels of Investigations and Examples

-​ DNA: Molecular Tests, such as OTC gene
sequencing
-​ RNA: mainly done for research purposes
-​ Protein: enzyme assays (e.g.
α-L-iduronidase enzyme assay → MPS1)
-​ Substrate/Product: Plasma amino acids
(e.g. Phenylalanine and Tyrosine → PKU)
-​ Organ Damage: Organ Function Tests
(e.g. Liver Function → GSD Type 1a)

IEM Causes of Abnormal Results

-​ Primary: defect in the cycles/origin issue, such as Pyruvate, TCA Cycle, or Oxidative Metabolic Defects
-​ Secondary: defect in the secondary reactions that shift amounts of molecule, such as
Gluconeogenesis Defects, GSD1a, Organic Acidemias, Fatty Acid Oxidation Disorders

Non–IEM Causes of Abnormal Results

-​ Sample Hemolysis (most common cause): incorrect sampling and handling → skewed result
-​ Other: Circulatory collapse, Hypoxic Insult or Hypoventilation, Diarrhea, Sepsis, Hepatic Failure

First–Line Investigations Second–Line Investigations

Non–Specific Specific

Screening Diagnostic

Fast Turn–Around Time Slow Turn–Around Time

Cheap and Readily Available Expensive, Requires special coordination

Easy to interpret Difficult to interpret

Ordered by a GP Ordered by a Specialist

 Lecture 19 - Genetic Counseling

Genetic Counseling: a communication process that deals with the risk of developing or transmitting a genetic
disorder, helping people to understand and adapt to the implications of genetic contributions to disease.
-​ Consultand: an individual who seeks genetic counseling.
-​ Counselor: a healthcare professional, trained in human genetics, whose role is to provide the
consultand with information that helps them understand the medical diagnosis, mode of inheritance,
options available to them, and all of their implications in diagnosis, transmission, and risk.

Genetic Counseling by Clinical Geneticists vs. Genetic Counselors

-​ Clinical Geneticists: focus on the diagnosis and clinical management of the patient
-​ Genetic Counselors: Deliver psychosocial support, explain patterns of inheritance, support family
communication, and help the patient to manage the burden and implications of disease.

Indications for Genetic Counseling

1.​ Pre–Conceptional/Prenatal: advanced maternal age or maternal illness, exposure to teratogens,
current pregnancy with abnormal genetic screening results, identified fetal anomalies, history of
stillbirths or miscarriage, history of a previous birth defect, positive carrier test, infertility, consanguinity.
2.​ Pediatrics: multiple birth defects, global developmental delays, dysmorphic features, unusual growth
patterns, suspected genetic syndrome or metabolic disorder, positive family history.
3.​ Adults: recognized genetic disorder, positive family history, known inherited cancer syndrome or early
onset cardiovascular disease, family history of sudden death, suspected connective tissue disorder.

Steps in Genetic Counseling

1.​ Establishing Diagnosis: involves carrying out a detailed family history, complete clinical examination,
and appropriate investigations.
2.​ Risk Assessment and Estimation of a Recurrence Risk: straightforward if the pedigree is very clear,
but factors such as variable onset age and reduced penetrance can make it more complicated.
-​ Risk Quantification: different ways of quantification (e.g. ratio or percentage), but it is important
to emphasis that the risk applies to each pregnancy independently.
-​ Risk Qualification: communication of the burden (severity) of a diagnosis, such as treatment
options, pain and suffering, and availability of prenatal diagnosis.
3.​ Communication of Relevant Information
4.​ Discussion of Options, including the availability and risks of prenatal diagnostics and reproductive
options, such as pre–implantation genetic diagnosis.
5.​ Communication and Support: essential, and it is important that the counselor be a good listener,
receptive to the patients feelings, and committed to ensuring the full understanding of the consultand.
-​ Important Factors: Setting, Timing, Honesty, Cultural Sensitivity, Resources, and most
importantly being non–directive and non–judgmental of the consultand’s informed choices.

Special Issues in Genetic Counseling

-​ Consanguinity: a relationship between blood relatives (third cousin or closer), which increases the
incidence of congenital malformations, autosomal recessive disorders, and multifactorial disorders.
-​ Incest: a relationship between first–degree relatives, forbidden in almost all cultures, associated with a
very high risk of abnormalities in the offspring.
-​ Non–Paternity: occurs when an individual's presumed father is not in fact their biological father
determined by accident in some genetic tests, where it is recommended to inform individuals
undergoing testing that it might reveal non–paternity before they undergo the test.
 Lecture 20 - Genetic Therapies

Gene Therapy: a technique that uses genetic material in the treatment or prevention of both inherited (e.g.
hemophilia and sickle cell disease) and acquired (e.g. leukemia) disorders.
-​ Goals of Gene Therapies: to prevent the pathogenic events that damage cells, to correct reversible
features, and to repair or change a gene based on pathogenesis (aka protein modification).

Gene Therapy Techniques

1.​ Gene Replacement: introducing a new gene, and the
replacement of a mutant gene with a normal, functional one
(e.g. for loss of genetic diseases like DMD).
2.​ Gene Augmentation: modifying an existing gene’s activity
by introducing short sequences of oligonucleotides.
3.​ Gene Silencing (Knockdown): inactivation of a mutated
toxic gene (e.g. Huntington’s Disease)
4.​ Gene Editing: editing a gene directly, using gene editing
systems (e.g. CRISPR-Cas9, which was discovered as a
bacteria immune defence) to target and precisely cut a gene
to inactivate or replace it.
-​ CRISPR–Cas9 contains Guide RNA sequences
(19–23bp) and an endonuclease enzyme, relying on
non–homologous end joining (NHEJ → error prone) and
homology directed repair (HDR → safe and accurate).

Strategies for Gene Transfer:

-​ Ex–Vivo Gene Therapy: using cultured cells in a lab and reinsertion.
-​ In–Vivo Gene Therapy: direct delivery of genes into the cells of a
specific tissue in the body.

Requirements for Gene Therapy:

-​ Disease: knowledge of pathophysiology, molecular defect/gene, and lack of a good alternative therapy.
-​ Delivery: an appropriate (accessible, long half life and replicative potential) target cell and vector.
-​ Regulation: must be safe and efficacious
-​ Therapy: availability of functional copy of the gene and regulatory components.

Target Cells: ideal cells are stem/progenitor cells, or affected cells.

-​ In PKU → 5% of hepatocyte mass (~1010 cells) is required, or less if gene expression is increased.
-​ In Muscular Dystrophies → therapy needs to be inserted into a significant fraction of myocytes.

Vectors: they are ideally safe, readily made, easily introduced into the target tissue, and would allow for the
expression of the gene of interest for life.
-​ Risks: immunotoxicity, insertional mutagenesis causing
malignancy, inactivation of an essential gene.
-​ Types: Viral Vectors and Non–Viral Vectors

Non–Viral Vectors: Cationic Polymers and Lipid Nanoparticles,

which are simpler and safer with low toxicity and
immunogenicity, allowing for large–scale production, but isn’t
efficient in gene delivery.
Viral Vectors
-​ Adeno–Associated Virus (AAV): ssDNA non–enveloped vector, with serotype-dependent pre–existing
immunity, low inflammatory responses, and high liver transduction.
-​ HD–Adenovirus: dsDNA non–enveloped vector, with serotype-dependent pre–existing immunity, acute
inflammatory responses, and high liver transduction.
-​ Lentivirus: ssRNA enveloped integrative vector, with low pre–existing immunity
-​ Herpes Virus: dsDNA enveloped episomal vector, with a large capacity (30-50kb); neurotropic virus.

Examples of Gene Therapies

Spinraza in Spinal Muscular Atrophy: SMA is caused by mutations in the SMN1 gene, which leads to a
deficiency of SMN protein (Survival Motor Neuron Protein) and motor neuron degeneration. The backup SMN2
gene produces a non-functional version of SMN due to
alternative splicing (it skips exon 7).
-​ Spinraza: an antisense oligonucleotide that binds to
the SMN2 pre-mRNA and modifies its splicing so that
exon 7 is included, resulting in the production of a
functional SMN protein from the SMN2 gene.

Luxturna in Retinitis Pigmentosa: RP is a group of genetic

disorders that lead to the progressive degeneration of photoreceptors,
starting with rods (night vision) and later cones (day/central vision),
causing night blindness, tunnel vision, and eventual complete
blindness, with one of the damaged genes being RPE65.
-​ Luxturna is an AAV2-based gene therapy that delivers a
normal copy of the RPE65 gene into retinal pigment epithelial
(RPE) cells (in vivo) using an adeno-associated virus (AAV),
which restores the production of functional RPE65 enzyme
and reactivates the visual cycle, improving vision.

Libmeldy in Metachromatic Leukodystrophy: mutations in the ARSA gene cause

ARSA enzyme deficiency, reducing sulfatide breakdown and leading to intra–lysosomal
sulfatides accumulation → demyelination and neurodegeneration (lysosomal disease).
-​ Libmeldy: a one-time ex vivo gene therapy designed to treat early-onset MLD in
eligible patients. A patient's blood or bone marrow stem cells are collected →
working ARSA gene is inserted into the stem cells using a lentiviral vector, and
the patient is given libmeldy before re–infusion of the stem cells.

Gene Therapies for the Treatment of Cancer: delivery of genetic elements to the cancer cell or to immune
response ells to correct abnormalities or induce an immune response against the cancer cells.
-​ Corrective strategies may involve replacing defective tumor suppressor genes, suppressing oncogenes,
programming the release of suppressive molecules, and increasing susceptibility to chemotherapy.
-​ Suicide Gene Therapy: inserting suicide genes into cancer cells, activated only with drugs.
 Lecture 21 - Approach to Patients and Families

Multifactorial Inheritance: many genes and their interaction

with environmental factors → phenotype.
-​ They do not follow a simple Mendelian Transmission
Patterns, as they require both a susceptible genetic
background and significant environmental factors to
result in a disease.

Multifactorial Disorders include many traits, as well as most isolated ‘non-syndromic’ malformations, with
recurrence risk influenced by severity, sex, degree of relationship, and the number of affected relatives.
-​ Most Importantly, they include Neural Tube Defects and Cleft Lip/Palate.

Neural Tube Defects: the neural tube closes at the 4th week of
gestation, and NTDs result from the defective close
-​ Upper End NTD → anencephaly or encephalocele.
-​ Lower End NTD→ spina bifida or myelomeningocele.
-​ They can be isolated (most), due to chromosomal
abnormalities, or part of a syndrome.
-​ Periconceptional Folic Acid Supplements → reduces risk
by 70% for a woman with an affected child.
-​ NTDs can be detected prenatally by elevated
amniotic AFP levels.

Cleft Lip/Palate: refers to a gap or split in the upper lip or palate, from a
defect during the development of the face and palate.

Single Gene Disorders: they account for up to 10% of all congenital

abnormalities, running in families (AD, AR, X–Linked), and can be
isolated, affecting single organ systems, or causing multiple congenital
abnormality syndromes affecting multiple systems.
-​ Pedigree Analysis is very useful for determining inheritance.
-​ Essential Information on Single–Gene Disorder includes the
gene, its locus and inheritance, disease mechanism, clinical
features, methods of diagnosis, and its management.

AD Disorders: Neurofibromatosis Type 1, Marfan Syndrome, Achondroplasia, and Noonan Syndrome

Neurofibromatosis Type 1: due to NF1 gene, which codes for

neurofibromin protein, on chromosome 17q11.
-​ Inheritance: Autosomal Dominant, 50% de novo
-​ Disease Mechanism: loss of function variant →
activation of the RAS–MAPK Pathway
-​ Clinical Features: Multiple café au lait sports and
neurofibromas (benign skin tumors), axillary or inguinal
freckling, optic nerve glioma, mild developmental/learning
delay, Lisch Nodules (iris).
Marfan Syndrome: due to the FBN1 gene which codes for fibrillin–1 ECM protein, on chromosome 15q21.
-​ Inheritance: Autosomal Dominant, 25% de novo
-​ Disease Mechanism: Dominant Negative pathogenic variants, resulting in <35% of the normal amount
of fibrillin–1 protein in the extracellular matrix.
-​ Clinical Features: Aortic Root Dilatation, Mitral Valve Prolapse, Pectus Carinatum or Excavatum, Flat
Feet, Joint Laxity, Ectopia lentils, Arachnodactyly
-​ Diagnosis: revised Ghent Criteria, which diagnoses based on features and family history.
-​ Management: Symptom Management, Aortic Complication Prevention with β–adrenergic blockers and
angiotensin–II receptor blockers (ARBs), regular surveillance.

Achondroplasia: dye to the FGF3 gene, which codes for limiting bone growth, on
chromosome 4p16.3.
-​ Inheritance: Autosomal Dominant, 80% de novo
-​ Disease Mechanism: gain of function variant causes overactivation of the
FGFR3 gene → inhibition of chondrocyte proliferation and differentiation.
-​ Clinical Features: Disproportionate short stature, rhizomelic (proximal)
shortening of limbs, trident hands, obstructive sleep apnea, spinal cord
compression
-​ Management: symptomatic, Vosortide can enhance height in children until
growth plates close.

Noonan Syndrome: genes belong to the RAS–MAPK pathway, mainly PTPN11 (50%) and SOS1.
-​ Inheritance: Autosomal Dominant, mostly de novo.
-​ Disease Mechanism: gain of function variant → activation of the RAS–MAPK Pathway
-​ Clinical Features: characteristic facial features
(hypertelorism, down–slanting eyes, low set ears), short
stature, congenital heart disease (male), neck webbing,
pectus deformity, bleeding disorders.
-​ Diagnosis: Gene (PTPN11/SOS1) Sequencing,
Echocardiogram, Renal Ultrasound, bleeding studies.

AR Disorders: Spinal Muscular Atrophy, Cystic Fibrosis

Spinal Muscular Atrophy: due to the SMN1/SMN2 genes on chromosome 5q13. (Inheritance: AR).
-​ Disease Mechanism: due to loss of SMN1, because SMN2 cannot produce enough protein on its own.
-​ Clinical Features: progressive muscle weakness, floppy baby syndrome, frog leg position, impaired
head control, absent deep tendon reflexes, tongue fasciculations.
-​ SMA is divided into: I (early onset, severe) → IV (adult onset).
-​ Diagnosis: motor unit disease on electromyogram, SMN1 sequencing to look for exon 7 deletion.
-​ Management: physical therapy, Spinraza (treats all forms of SMA), Zolgensma (only treats SMA I).

Cystic Fibrosis: due to the CFTR gene (deltaF508 mutation accounts for 70%) on chromosome 7q31.
-​ Inheritance: Autosomal Recessive
-​ Disease Mechanism: loss of function variant, disrupting the function of chloride channels, preventing
the regulation of water → excessively thick secretions and damage.
-​ Clinical Features: chronic pulmonary disease, failure to thrive and malnutrition due to chronic diarrhea
and malabsorption, meconium ileus, male infertility.
-​ Diagnosis: elevated chloride levels in sweat, detection of deltaF508 mutation.
 X–Linked Disorders: Duchenne Muscular Dystrophy, Fragile X Syndrome

Duchenne Muscular Dystrophy: due to the DMD (Dystrophin) gene deletion (65–80%) on chromosome Xp21
-​ Inheritant: X–Linked Recessive
-​ Disease Mechanisms: loss of function; lack of dystrophin → actin is not bound to membrane proteins.
-​ Clinical Features: Muscle Weakness, + Gower Sign, Calf Pseudohypertrophy, Dilated Cardiomyopathy
-​ Diagnosis: elevated creatine kinase, DMD sequencing for gene deletion or point mutations
-​ Management: symptomatic, corticosteroids for muscle strength, dystrophin restoration therapies.

Fragile X Syndrome: due to the FMR–1 (Fragile X Mental Retardation Protein) gene
on chromosome Xq27.3.
-​ Inheritance: X–Linked Triplet Repeat
-​ Anticipation: disease increases in severity and decreases in age of onset with
generations, and FXS occurs due to a pre–mutation which becomes a full
mutation after inheritance (unstable FMR1 alleles).
-​ Clinical Features: intellectual disability, delayed development and autistic
features, long face, prominent forehead/jaw, macroorchidism (enlarged testes),
premature ovarian failure in female carriers.
-​ Fragile X–associated tremor/ataxia syndrome (FXTAS): in male
carriers → progressive intention tremor, ataxia, and parkinsonism.
-​ Diagnosis: increased CGG triplet repeats (55–200 repeats → permutation; >200 repeats → mutation).

Numerical Chromosomal Abnormalities

Patau’s Syndrome (Trisomy 13): Holoprosencephaly, Microcephaly, Microphthalmia,

Polydactyly, Midline Cleft Lip & Palate, Cardiac and Renal Abnormalities, Intellectual
Disability → very poor prognosis.
-​ Inheritance: 75% Maternal Non–Disjunction, 20% Translocation, 5% Mosaicism.

Down Syndrome (Trisomy 21): Hypotonia, Excess Nuchal Skin, Brachycephaly,

Epicanthic Folds, Depressed Nasal bridge, Single Palmar Crease, Clinodactyly
of the 5th Finger, Growth Delay, Leukemia, Short Stature.
-​ Inheritance: 95% de novo (maternal non–disjunction), 4% Robertsonian
Translocation, 1% Mosaicism.
-​ Recurrence Risk: Non–Disjunction → 1% + Maternal Age,
Familial Translocation → 1–3% in Males, 10–15% in Females,
21q21q Translocation → 100%.

Turner Syndrome (XO → SHOX gene): Congenital Lymphedema (hand/feet

swelling), Short Stature, Short Webbed Neck, Aorta Coarctation/Bicuspid Aortic
Valve, Ovarian Failure/Streak Gonads, Horseshoe Kidneys.

Klinefelter Syndrome (47, XXY): karyotype shows an additional X chromosome, with

testosterone from puberty used for treatment, which is beneficial for sex characteristic
developments & osteoporosis prevention.
-​ Clinical Features: tall stature, infertility, azoospermia, testicular atrophy (small
testes), female distribution of fat and body hair, increased risk of male breast
cancer and osteoporosis.
 Structural Chromosomal Abnormalities

Cri du Chat (5p) Syndrome: due to a terminal deletion of the short

arm of chromosome 5 (5p15.2).
-​ Inheritance: mostly sporadic de novo deletions, while 10%
have a parent w/ a balanced translocation.
-​ Clinical Features: characteristic cat–like cry of affected
neonates, growth deficiency, intellectual disability,
microcephaly, hypotonia, dysmorphic features (micrognathia,
epicanthal folds, low–set ears).

DiGeorge/Velocardiofacial (22q11.2 Deletion) Syndrome: due to the TBX1 gene on chromosome 22q11.2.
-​ Inheritance: Autosomal Dominant, with >90% de novo deletions
-​ Disease Mechanism: abnormal development of pharyngeal arches due to haploinsufficiency of TBX1.
-​ Clinical Features: heart malformations, thymic and
parathyroid hypoplasia, typical fallacies,
developmental delay, psychi atric illness
(schizophrenia and autism).
-​ Diagnosis: Hypocalcemia, Hypoparathyroidism,
Immunodeficiency, Growth Hormone Deficiency.
FISH, Microarray, or MLPA show a 3–Mb Deletion.
-​ Management: no vaccines if immunodeficient.

Imprinting Disorders

Prader–Willi Syndrome: genes associated with PWS are normally only expressed from the paternal
chromosome 15 in the PWCR region (genes inherited from the mother are inactivated)
-​ SNRPN gene: nuclear ribonucleoprotein protein, involved in pre–mRNA splicing.
-​ Inheritance: 70% → paternal deletion, 30% → maternal uniparental disomy
-​ Genetic Mechanism: caused by loss of the paternal 15(q11–q13), due to…
-​ Deletion of the paternal inherited chromosome 15(q11–q13) region
-​ Uniparental Disomy of the maternal chromosome 15(q11–q13) region.
-​ Imprinting defect of the paternal chromosome 15(q11–q13) region
-​ Clinical Features: neonatal hypotonia, failure to thrive in infancy, hyperphagia
leading to obesity, short stature, small hands and feet, hypogonadism, narrow
bifrontal diameter, almond-shaped eyes

Angelman Syndrome: genes associated with AS are normally only expressed from maternal chromosome 15.
-​ UBE3A gene: ubiquitin protein ligase production, involved in targeting proteins for degradation.
-​ Inheritance: 70% → maternal deletion, 5% → paternal uniparental disomy, 10% → UBE3A mutation.
-​ Genetic Mechanism: caused by the loss of the maternal chromosome 15(q11–q13), due to…,
-​ Deletion of the maternal 15(q11–q13), including UBE3A.
-​ Uniparental Disomy of the paternal chromosome 15.
-​ Imprinting defect of the maternal 15(q11–q13) region
-​ Clinical Features: severe developmental delay, inappropriate
happy demeanor with frequent laughing, smiling, and excitability,
microcephaly and seizures, and an unsteady/ataxic gait.
Beckwith–Wiedemann Syndrome (BWS) and Silver–Russell Syndrome: two clinically
opposite growth– affecting imprinting disorders, usually depending on the parental origin of
the imprinted chromosome.
-​ Cause: disruptions in gene regulation/expression of imprinted genes, including
growth factors and tumor suppressor genes on chromosome 11p15 → overgrowth in
BWS, growth restriction in SRS.

Beckwith–Wiedemann Syndrome (BWS): tested using methylation analysis, deletion, and

UPD tests.
-​ Clinical Features: Overgrowth Syndrome (macrosomia, macroglossia, abdominal
wall defect, and neonatal hypoglycemia), hemihyperplasia, renal abnormalities, ear
abnormalities, increased risk of embryonal tumors (Hypatoblastoma).
-​ *Associated with IVF technologies*

Silver–Russell Syndrome: also includes potential maternal UPD of ch. 7 (~10%).

-​ Clinical Features: marked growth restriction, normal head size w/ small, triangular
face, body asymmetry, fifth finger clinodactyly.