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L11
INTRODUCTION TO THE HUMAN CHROMOSOME
1. Define the following terms relating to chromosome morphology: sister
chromatids, centromere, p arm, q arm, telomere and kinetochore.
2. Define homologs. Describe genes and alleles in relationship to homologs.
3. Define autosomes and sex chromosomes, gametes and somatic cells. Describe
the chromosomal basis of gender determination in humans.
4. Define and distinguish the characteristics by which chromosomes are classified.
Be certain to use the following terms: metacentric, submetacentric, acrocentric
and telocentric.
5. Define the significance of mitosis and differentiate the phases and the
significant events in each phase.
6. Define the significance of meiosis and differentiate the phases and significant
events in each phase including the sub-phases of metaphase I.
7. Define crossing over including its effect on the alleles located on homologous
chromosomes.
Eukaryotic Chromosome
Chromosome – distinguish between species and enable transmission
of genetic information, facilitating reproduction and maintenance of
a species (linear with ≈ 1010 bp in length)
 Genes interspersed along the chromosome with origins of replication about
every 100,000 bases
 Made of a single molecule of DNA complexed with proteins (histones)
 Compacted into the cellular nucleus
Occur in sets of two identical sister chromatids
Centromere – contains recognition sites for kinetochore proteins
Telomeres – specialized repetitive sequences that “cap” each end
(maintained by telomerase)
Types of DNA Sequence
Heating denatures DNA, causing the double helical structure to
come apart
Slow cooling allows for the stands to reassociate at a rate dependent on
the unique and repetitive sequences contained within the DNA
 60-70% of the human genome – composed of single (low copy number) DNA
sequences
 30-40% of the human genome – moderately to highly repetitive DNA sequences
(that are not transcribed) (e.g., satellite and interspersed sequence DNA)
Nuclear Genes
Nuclear DNA – ≈3×109 bp / 25,000-30,000 genes
Genes – largely unique DNA sequence that codes for a
polypeptide with a cellular function or in combination with
other polypeptides to form a functional unit (e.g., enzyme,
hormones, receptors)
Distributed variations between the chromosomal regions:
Heterochromatic regions – contains genetically inert non-coding
sequences
Centromeric regions – contain non-coding sequences
Sub-telomeric regions – highest gene density
Nuclear Genes
Multigene family – code for proteins of similar functions,
arising by gene duplication and subsequent divergence
Classic gene family – high degree of DNA sequence similarity
Gene superfamily –limited sequence homology but are functionally
related, sharing similar structural domains
Exons – coding sequences (remain after splicing)
Introns – non-coding intervening sequences (spliced out before
translation)
Pseudogenes
Pseudogenes – DNA sequences that very closely resemble an
expressed gene (but is not generally expressed)
Glucocerebrosidase (GBA) gene – codes for a lysosomal enzyme that
degrades glycosylceramide → glucose and ceramide
 GBA pseudogene (ΨGBA) – located 16 kb downstream of function GBA gene
 Arise through a duplication event inserted into the genome but is inactive due to a mutation in the
coding or regulatory elements of the gene
 Insertion of a cDNA sequence (through reverse transcriptase activity) without promoter sequences
for expression
Extragenic DNA
Extragenic DNA (“junk DNA”) – non-coding sequence with
evolutionary conservation, playing a role in gene regulation
Tandem DNA repeats:
 Satellite DNA – repetitive DNA sequences clustered around the centromeres and
kept separate from the main DNA sequences
 Mini-satellite – terminal telomeric sequences that maintain the integrity of the
chromosomal ends, possessing hypervariable-short tandem repeats (for DNA
fingerprinting)
 Microsatellites – nucleotide repeats located throughout the genome that are
associated with disease (arise by slip strand mispairing or unequal crossover)
Extragenic DNA
Interspersed repetitive DNA sequences:
 Short interspersed nuclear elements (SINEs) – short repeated DNA sequences of
<500 bp that rely on transposons for expression
 ALU repeats – 300 bp repeats with sequence similarity to a signal recognition particle from protein
synthesis that is cut by Alu I restriction enzyme
 Long interspersed nuclear elements (LINEs) – long repeated DNA sequences of
≈6,000 bp
 LINE-1 (L1) – repeated 6,000 bp sequence that codes for a reverse transcriptase
 Both are implicated in inherited disease
miRNA – repress gene expression through RNA/DNA hybrids, exhibiting
many widespread functions (e.g., apoptosis, tumorigenesis, cell
regulation, organogenesis)
Mitochondrial DNA (mtDNA)
Mitochondrial DNA – 16.6 kb / 37 genes
Two types of rRNA and 22 types of tRNAs with 13 subunits of enzymes
(e.g., CYT-b and CYT-oxidase) involved in energy production, using
oxidative-phosphorylation
Maternally inherited
Cytogenics
Cytogenics – the study of chromosomes and cell
division
During cell division, DNA is maximally contracted
and arranged into sister chromatids
 Chromatids join at the central core (centromere
region), diving the chromosome into p and q
movement
 Kinetochore – proteins that assemble at the
centromere, allowing for spindle fiber (microtubule)
attachment and subsequent separation during
replication
 Proteins: CenH3 (a specialized histone), microtubules
(tubulins), motor proteins (dynein and kinesin)
Morphology of Chromosomes
Telomeres – repetitive TTAGGG DNA
sequences
 Normal cells: steadily decrease in length, allowing
for 50-60 cell divisions
 Tumor cells: increased telomerase activity to
increase cell survival
Classified by the position of the
centromere:
 Metacentric – located near the center of the p
and q arms
 Submetacentric – located in an intermediate
position
 Acrocentric – located at the terminal end (and
may have satellites)
Karyotyping
Chromosome-specific band patterns:
Euchromatin – light staining active genes
Heterochromatin – dark staining non-coding DNA
Diploid (2N): 22 autosomal pairs and 1 sex pair
One member of each pair is derived from each pair
– haploid (N)
Homologs – members of a pair of chromosomes
Nomenclature
At any given gene location:
Chromosome number: 1-22
Chromosome arm: p or q
Chromosome band: 1-X
Abnormality examples: +/- for gain or loss
Normal: 46, XY and 46, XX
Male with trisomy 21 (Down’s Syndrome): 47,
XY+21
Female with Cri du Chat Syndrome: 46, XX,
del 5p
Translocation: 46, XY, t(2;4)(p23;q25)
Karyotype
Symbol
Explanation
cen
centromere
del
deletion
46, XX, del(1q21)
dup
duplication
46, XY, dup (13q14)
fra
fragile site
i
isochrome
46, X,i(Xq)
inv
inversion
46, XX, inv(9p12q12)
ish
in situ hybridization
r
ring chromosome
46, XY, r(21)
t
translocation
46, XY, t(2;4)(q21,q21)
ter
terminal end
pter or qter
/
mosaicism
46,XY/47,XXY
Example
Stages of the Cell Cycle
Interphase (G1+S+G2): lasts ≈16-24 hours
 Gap 1 (G1) – duplication of cellular contents
 Synthesis phase (S) – duplication of the 46
chromosomes
 Gap 2 (G2) – checks for errors and makes repairs
Non-dividing cells arrest in G0
Mitosis – replication of somatic cells,
producing 2 identical haploid daughter cells
Cytokinesis – division of the cytoplasm
Mitosis
Prometaphase (prophase):
 Formation of 2 centrioles with microtubules, moving to the opposite poles of
the cells
 Condensation of the chromosomes into sister chromatids
 Disintegration of the nuclear membrane
 Attachment of the microtubules to the chromosomal centromeres
Metaphase – chromosomes align at the metaphase plate using the
spindle apparatus from the centrioles
Anaphase – division of the centromeres and separation of the sister
chromatids to opposite poles of the cells
Telophase – development of new nuclear envelopes around
independent chromosomes
Meiosis
Meiosis – cellular division during gamete formation (in
spermatocytes and oocytes), occurring in 2 stages
 Meiosis I – reductional division, deriving a haploid number of chromosomes
 In male X and Y segments, tips of the homologous short arms pair at the pseudoautosomal
regions
 Meiosis II – a mitotic cell division with a haploid number of chromosomes
Synaptonemal complex – protein structure that forms between
homologous chromosomes (2 pairs of sister chromatids), mediating
chromosome pairing, synapsis, and recombination
Recombination – crossing-over between non-homologous
chromatids, creating recombinant chiasmata
Meiosis – Stages:
Leptotene – condensation of chromosomes
Zygotene – alignment of homologous chromosomes along a
synaptonemal complex
Pachytene – tight coiling of each pair of chromosomes (which can
lead to bivalent crossing over between non-homologous
chromosomes or chiasmata)
Diplotene – separation of chromosomes still attached at the
chiasmata
 May have 1-3 chiasmata depending on the size, generating diversity
Diakinesis – continued separation of homologous chromosomes and
condensations
Meiosis – Outcomes:
Division of chromosomes so that each child receives a maternal
and paternal set, preventing identical divisions (1/223 probability)
Crossing over generates diversity through gene shuffling,
alternating genomic regions from each parent
L12-13
CHROMOSOMAL ABNORMALITIES I AND II
1. Contrast/compare aneuploidy and polyploidy.
2. Using the proper nomenclature, identify the most common aneuploidy
conditions
3. Identify mitotic and meiotic nondisjunction and the effects of each.
4. Delineate mosaicism and explain how it effects phenotypic expression of
a chromosomal disorder
5. Distinguish between the following chromosomal aberrations: reciprocal
and non-reciprocal translocations, Robertsonian translocation, insertion,
deletion, paracentric and pericentric inversions, ring chromosome and
isochromosome. Summarize the potential complications, if any, that
may occur at mitosis and/or meiosis for each aberration.
6. Define chimerism and differentiate between the two most common
causes/forms.
Types of Chromosomal Disorders
Chromosomal disorders – most do not result in live birth, leading to
spontaneous abortions
Single gene defects – Mendelian genetic disorders generating
nuclear and mitochondrial gene defects (which typically result in live
birth)
Multifactorial disorders – most common disorders that lead to
fewer congenital malformations, resulting in live births that typically
manifest later during adulthood
Somatic cell genetic disorders – arise after birth in somatic cells,
commonly causing cancers or tumors that are not heritable
Nomenclature
Locus – designates the position or location of a gene sequence
on a chromosome
Alleles – different versions of the gene at a specific locus
 Homozygosity – having the same allele of a given gene locus on both chromosomes
 Heterozygosity – having different alleles at the gene locus of a chromosome
Polymorphism – significant variability in non-coding regions
results from a mutation or random alteration in DNA, presenting
2+ sequence variants in a population with a frequency >1%
Karyotyping – Process:
Isolation of WBCs from a peripheral blood sample, culturing onto a
medium containing PHA to stimulate cell division and growth
 Addition of colchicine, preventing mitotic spindle formation to arrest cell
division during metaphase (for maximal visibility)
Cells are places in a hypotonic solution to stimulate cell lysis on a
prepared slide
 Digestion with trypsin and subsequent staining with Giemsa, creating unique
banding patterns of light and dark bands
 Euchromatin – light staining of active genes
 Heterochromatin – dark staining of inactive genes
Analysis of “metaphase spread” for karyotyping
Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization (CGH) – reveals the loss or
gain of chromosomal regions in test samples relative to normal
controls
Test and reference sample DNA is labeled and hybridized with green and
red fluorochromes, respectively, on either metaphase chromosomes or
an array of BAC clones
Green chromosomal areas have gained areas while red
chromosomal areas have lost areas
Abnormalities
Abnormalities arise as a consequence of errors in chromosome replication
and division (identified through karyotyping and molecular methods)
 Numerical:
 Aneuploidy – deviations from normal 46, XY (e.g., monosomy, trisomy, tetrasomy)
 Polyploidy – gain of another complete set of chromosomes (e.g., triploidy, tetraploidy)
 Structural – result from the breaking and rejoining of segments into a different
configuration
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Translocations: reciprocal, Robertsonian
Deletions
Insertions
Inversions: paracentric, pericentric
Rings
Isochromosomes
 Mixoploidy: mosaicism, chimerism
Non-disjunction
Arise from an error in chromosomal separation during meiosis I
or meiosis II
Meiosis I error – gamete contains both homologs of 1 chromosomal pair
Meiosis II error – gamete contains 2 copies of 1 homolog
 Mosaicism – an embryo with 2 cell types in very early zygotes during mitosis
Cause: unclear
Hypothesis:
Problem with spindle formation in aging females
Recombination failure in some of the female’s fetal primary oocytes
Non-disjunction
MONOSOMY
TRISOMY
Absence of a single
chromosome due to an error
during anaphase
Presence of an extra
chromosome
Survival: Turner Syndrome 45, X
Survival (60%): Down Syndrome
47, XY, +21
 Patau Syndrome 47, XY, +13
 Edwards Syndrome 47, XY, +18
Aneuploidy – Trisomy:
Patau Syndrome – presence of an extra CHR-13 (trisomy 13),
causing a deficit in growth and development
Edwards Syndrome – presence of an extra CHR-18 (trisomy 18),
causing distinctive malformations of the craniofacial area,
hands, and feet
Craniofacial area: low-set and malformed ears, micrognathia, small
mouth with an unusually narrow palate, upturned nose, narrow eyelid
folds due to palpebral fissures, widely spaced eyes due to ocular
hypertelorism
Hands and feet: overlapped, flexed fingers, webbing of the 2nd and 3rd
toes, clubfeet
Aneuploidy – Trisomy:
Downs Syndrome – presence of an extra CHR-21 (trisomy 21),
causing intellectual and developmental disabilities
Development: flattened nose and face, upward slanting eyes, single
palmer crease, increased toe creases, widely separated 1st and 2nd toes
Risk: increases with maternal age (36 years)
 Non-disjunction in maternal meiosis I
 Robertsonian translocations – a break between 2 acocentric chromosomes near the
centromeres with subsequent fusion of the longer arms (through centric fusion)
Aneuploidy – Partials:
Cat Eye Syndrome (Schmid-Fraccaro Syndrome) – results from
an inverted duplication of the long arm inv dup 22q11, causing
coloboma of the iris, anal atresia with fistula, downslanting
palpebral fissures, heart and renal malformations
Normal or near-normal mental development
Wolf-Hirschhorn Syndrome (WHS) – results from a partial
deletion of the short arm 4p-, causing severe development
delays and characteristic facial appearance
85-90% of cases occur due to de novo deletion
Aneuploidy – Partials:
Cri du Chat Syndrome – results from a deletion of the short arm
del5p, causing a “cat-like” cry from an underdeveloped larynx
Others: severe cognitive and motor delays, behavioral problems,
unusual facial features, small head with wide eyes
90% of cases occur due to de novo deletion
DiGeorge Syndrome – results from a deletion of the long arm
22q11.2, causing developmental defects
Developmental defects: palatal defects, conotruncal heart defects,
abnormal ear exams, hypocalcemia, microcephaly, mental retardation
Polyploidy
Created non-viable fetuses that are either spontaneously
aborted or die shortly after birth
Triploidy – 69
Tetraploidy – 92
Causes:
Retention of a polar body in oocyte division
Formation of a diploid sperm
Fertilization of an oocyte by 2 sperm (dispermy)
Translocations
Translocations – transfer of genetic material from 1 chromosome to
another due to the breaking and exchanging of fragments
 Can be an equal balanced or reciprocal exchange, preserving all chromosomal
material (e.g., CHR-11 ↔ CHR-22)
 Identified by FISH
Robertsonian translocation – reciprocal translocation in which the
breakpoints are located at or near the centromere of 2 acocentric
chromosomes, creating a large metacentric chromosome and a
much smaller fragment (e.g., CHR-13, -14, -15, -21, -22)
 Can produce balanced or unbalanced bivalent, trivalent, or quadrivalent
translocations, leading to normal translocations, partial trisomy, or partial
monosomy
Translocations – Quadrivalent:
The pattern of chromosome segregation (adjacent or alternate)
determines whether the gametes have a balanced or unbalanced
complement of genetic material
2:2 segregation – segregation of the quadrivalent during the later
stages of meiosis I
 If alternate chromosomes segregate to each gamete, the gamete will carry a
normal or balanced haploid complement
 With fertilization, the embryo will either have normal chromosomes or carry a
balanced arrangement
3:1 segregation – segregation of 3 chromosomes to 1 gamete with
only 1 chromosome to the other gamete, leading to an unbalanced
trisomy or monosomy
Translocations
Downs syndrome – can result from a balanced Robertsonian
translocation to cause a partial 21 trisomy of 13q21q or 14q21q
Parents with one child with a partial trisomy 21 from a balanced
location may be at risk for a 2nd child with Down Syndrome
 Translocation tracing – identifies translocation carriers in a family
All gametes will be disomic for 21 or nullsomic for 21
66% of cases occur due to de novo deletion
Deletions
Deletions - result in a loss of genetic material, generating a
monsomic region ( >2% deletion of a haploid genome is lethal)
Large chromosomal deletions: Cru du Chat Syndrome (4p del)
and Wolf-Hirschorn Syndrome (5p del)
Seen under a light microscope
Submicroscopic deletions: Angelman Syndrome (15p del) and
Prader-Willi Syndrome (15p del)
Seen by high resolution prometaphase cytogenic and FISH
Insertions
Insertions – when one segment of a chromosome is inserted
into another chromosome
Balanced – if moved from another chromosome and inserted
Unbalanced – if lost from another chromosome and inserted
 50% chance of inheriting the insertion or deletion randomly
Inversions
Inversions – two-break rearrangement within a single
chromosome where the segment is reversed in position
Pericentric inversion – includes the centromere
 Crossover during meiosis I: inversion loop forms, resulting in 1 duplication of the
non-inverted segment and deletion of the other end (while the other has the
opposite arrangement with the inversion chromosome)
Paracentric inversion – does not involve the centromere
 If crossover loop occurs, the products are an acentric fragment or dicentric (which
fails to undergo mitosis and leads to zygote failure)
Others
Isochromosomes – loss of 1 chromosomal arm with the
duplication of the other (2p’s or 2q’s)
Ring chromosome – created when a strand break occurs at both
ends of the same chromosome, generating “sticky ends” that
form a ring (while the material from the ends is deleted)
Mixoploidy
Mosaicism – individual with 2+ somatic chromosome numbers
derived from the same zygote
Chimerism – presence in an individual of 2+ distinct cell lines of
different genetic origin
Dispermic chimeras – result of double fertilization of 2 ova which fuse
to 1 embryo
 Hermaphrodite – fusion of different sex embryos (XX/XY)
Blood chimeras – exchange of blood between twins in utero
Uniparental Disomy
Uniparental disomy –
presence of 2 homologous
chromosomes inherited from
only 1 parent
Isodisomy – parent passes on 2
copies of the same chromosome
(through non-disfunction in
meiosis II)
Heterodisomy – parent passes on
1 copy of each homolog (through
non-disfunction in meiosis I)
Abnormalities – Sex Chromosome:
Gender specific:
Female abnormalities – variation in the number of X chromosomes
 Turner Syndrome (45, X) and Triple X Syndrome (47, XXX or 48, XXXX)
Male abnormalities – variations in the X and Y number
 Klinefelter Syndrome (47, XXY or 48, XXXY or XY/XXY mosaic) or XYY Syndrome (47,
XYY) of “super-males”
Instability Syndromes
Rare single gene (autosomal recessive) syndromes where there
is a characteristic cytogenic abnormality, generating a broad
molecular defect (and increased risk of cancer)
Bloom Syndrome – defect in DNA ligase, increasing somatic
recombination and sister chromatid exchange
ICF Syndrome – deficiency of 1 of the DNA methyltransferases that
maintains pattern of genome methylation, leading to centrometric
instability and immunodeficiency
E.g., Fragile X Syndrome
L14
PAT TERNS OF INHERITANCE: MENDELIAN
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Define the following basic genetics terms: dominant allele, recessive allele, phenotype, genotype, mutation, premutation and wild type.
Distinguish why it is more difficult to determine inheritance patterns in humans than in experimental animals such as fruit flies. Describe how such patterns are ascertained in
humans.
Calculate genetic risk by using a Punnett square.
Define pleiotrophy.
Compare/contrast variable expressivity and penetrance and their clinical presentations.
Explain why most lethal autosomal dominant traits are inherited from a mosaic parent or result from de novo mutations.
What kinds of lethal dominant traits can be directly passed through several generations?
Define and give an example of codominance.
Define consanguinity and explain why it increases the probability of having a child with a genetic disorder.
Define locus heterogeneity and distinguish its effect on genetic inheritance.
Explain why males are more commonly affected by a recessive trait carried on the X chromosome.
Define the tenants of the Lyon hypothesis and recognize the results of these tenants in females. Define skewed inactivation.
Distinguish why it is difficult to determine an X-linked dominant trait from a pedigree.
Recognize the pedigree for a Y-linked trait. Name the two traits inherited via this mode.
Distinguish the clinical presentation of traits inherited via partial sex linkage.
Distinguish between the pedigrees for the following modes of inheritance: autosomal dominant, autosomal recessive, X-linked recessive, X-linked dominant and Y-linked
recessive.
Define multiple alleles and give an example. Calculate genetic risk using a Punnett square.
Define consanguinity and explain why it increases the probability of having a child with a genetic disorder.
Define locus heterogeneity and distinguish its effect on genetic inheritance.
Explain why males are more commonly affected by a recessive trait carried on the X chromosome.
Define the tenants of the Lyon hypothesis and recognize the results of these tenants in females. Define skewed inactivation.
Distinguish why it is difficult to determine an X-linked dominant trait from a pedigree.
Recognize the pedigree for a Y-linked trait. Name the two traits inherited via this mode.
Distinguish the clinical presentation of traits inherited via partial sex linkage.
Distinguish between the pedigrees for the following modes of inheritance: autosomal dominant, autosomal recessive, X-linked recessive, X-linked dominant and Y-linked
recessive.
Define multiple alleles and give an example. Calculate genetic risk using a Punnett square.
Distinguish between the following non-Mendelian patterns of inheritance: anticipation, mosaicism (somatic and gonadic), uniparental disomy
Distinguish between polygenic and multifactorial inheritance.
Compare/contrast the presentations of monogenic and polygenic traits present within a population. Given a specific trait, choose its mode of inheritance.
Determine if a trait is polygenic or multifactorial.
Nomenclature
Mendelian inheritance – single gene traits caused by mutations in nuclear genes
Locus – a segment of DNA occupying a defined position (frequently called “gene”)
 Alleles - alternative variants of a locus/gene
 Wild-type – common allele
 Mutant or variant allele – differ from wild-type due to a mutation
 Haplotype – a given set of alleles at a locus or cluster of loci on a chromosome
 Polymorphism – at least 2 reasonably common loci variants
Genotype – set of alleles that make up an individual’s genome
Phenotype – observable expression of the genotype as a morphological characteristic or biochemical
trait
Single Gene Disorder – determined by the alleles at a given locus
 Homozygous – all alleles the same at a given locus
 Heterozygous – alleles at a given locus are different
 Compound heterozygote – mutant alleles of the same loci present on each chromosome
Pleiotrophy – genes that have 1+ discernible effect
Nomenclature
 Hemizygous – abnormal allele on male X chromosome
Proband – the member of a family who appears to have the disease (index case)
 Kindred – extended family diagramed in a pedigree
 Pedigree – graphical representation of a family tree with constant symbols
 1st degree relative: parents, siblings, and offspring of the proband
 2nd degree relatives: grandparents, aunts, and uncles, etc.
Consanguineous – a couple who have 1+ common ancestors
Penetrance – probability that a gene will have any phenotypic expression at all (%
of people with the genotype who are affected)
 Reduced penetrance – <100% expression of the disease genotype
Expressivity – the severity of expression of the phenotype among individuals with
the disease causing genotype
 Variable expressivity – severity of disease differs among people with the genotype
Mendelian Inheritance
Unifactorial inheritance – disorders or traits attributed to a
single gene disorder
Autosomal inheritance – a gene that is located on an autosome
Sex-linked inheritance – a gene on an X or Y chromosome
 X pairs with the pseudoautosomal region on Y (vice-versa)
Patterns of inheritance:
Dominant expression – only 1 copy of a mutant gene pair is needed
(HH/Hh)
Recessive expression – 2 copies of the same mutant allele is present (hh)
Autosomal Inheritance
Male and females are equally affected
Dominant inheritance:
 Expressed in both homozygous dominant (HH) and heterozygous (Hh) individuals
 Co-dominance expression of 2 alleles at the same locus (e.g., ABO blood groups)
 Possible incomplete dominance of heterozygotes, leading to less severe symptoms
Recessive inheritance:
 Expressed only in homozygous recessive individuals (hh)
 Not expressed in carriers but can contribute to loss of function as a result of the
mutation in future generations (Hh)
Pseudoautosomal inheritance – exchange of X and Y genetic material,
mimicking autosomal inheritance
 Dyschondrosteosis – dominant mutation in SHOX gene skeletal dysplasia with
forearm deformity and short stature
Onset of Disease
Congenital disorder – recognized at birth
 Fetal disorder – shown by multiple miscarriages in a family (proving reduced fertility)
 Late-onset disorder – can reproduce (but the presence of the allele may be masked by
the death of a parent)
Genetic heterogeneity – diseases that show similar phenotypic variations
 Allelic heterogeneity – related phenotypes as a result of different mutations at the
same locus
 CFTR has 1,400 mutations that all produce a clinical spectrum of the disease (Classic Cystic
Fibrosis), causing progressive lung disease, pancreatic insufficiency, and congenital absence of the
male vas deferens
 Loci heterogeneity – overlapping phenotypes as a result of mutations at different loci
 Retinitis Pigmentosa – causes visual impairment due to degeneration of the photoreceptors
(through autosomal dominance, autosomal résistance, and X-linkage)
Onset of Disease
Phenotypic (or clinical) heterogeneity – distinct phenotypes in
different families with mutations in the same allele, impacting
genetic counseling
Hirschprung Disease – dominant loss of function mutation in RET
(receptor Tyr kinase), causing a failure to develop colonic ganglia defect
in colon motility and constipation
Other RET mutations: multiple endocrine neoplasia type 2A/2B –
inherited cancer of the thyroid and adrenal glands due to unregulated
hyperfunction
LMNA gene – encodes lamin A/C (a nuclear envelope protein)
 Associated with: Emery-Driefuss Muscular Dystrophy, Charcot-Marie-Tooth
Peripheral Neuropathy or Lipodystrophy, Hutchinson-Gifford Progeria
Pedigree
Analysis of gene transmission (diagramed in a Punnett square):
Carrier × Carrier: Rr × Rr – generates RR, 2 Rr, or rr offspring (1:2:1)
Carrier × Affected: Rr × rr – generates 2 Rr and 2 rr offspring (1:1)
Affected × Affected: rr × rr – generates rr only
R
 Influenced by the sex of individual with sex-influenced traits
 HFE gene mutation: Hemochromatosis – affects females by lowering the
dietary intake of iron, lowering alcohol usage, and increased iron loss
during menstruation
Carrier frequency – important in calculating
disease risk → genetic counseling
R
r
r
RR Rr
Rr rr
Consanguinity
Consanguinity – chance of a mutant allele at the same locus
increases if the parents are related (due to possessing a single
common ancestor)
 Measured by the coefficient of inbreeding (F) to identify the identity of
descent
 Xeroderma Pigmentosum – defective DNA repair (commonly in 1st cousin
marriages)
Inbreeding – mating between individuals of an geographically
restricted area (or for cultural reasons), increasing the risk of
obtaining a mutant allele
 Tay-Sachs Disease – autosomal recessive neurological degenerative disorder
resulting in death within 6 months in Ashkenazic Jews (frenquency: 1/30)
Autosomal Dominant Disorders
Huntingtons Disease – completely autosomal dominant disease
Incomplete dominant diseases:
Achondroplasia – skeletal disorder, causing short stature dwarfism
Familial Hypercholesteremia – premature coronary disease
Sex-limited dominant diseases – autosomally transmitted within
one gender
LCGR mutation: Familial Testotoxicosis – male-limited precocious
puberty at 4 years of age
X-linked Inheritance
X-linked inactivation of normal females: random inactivation of
an X in somatic cells, equalizing the amount of product
expressed in each sex
One X active in one set of somatic cells and the other X active in a
second set of somatic cells (e.g., Barr bodies), acting as mosaics for 2
cell populations to manifest possible heterozygotes
Manifestation of heterozygotes or skewed X-linked inactivation:
Hunter’s Syndrome – X-linked lysosomal storage disorder in which cells
can make iduronate sulfatase (active normal X), secreting the enzyme
into the cellular space, while mutant X cells can export the active
enzyme
X-linked Dominance
Regularly expressed in heterozygotes, lacking male-to-male
transmission and affecting all daughters of affected males
Due to X-linked inactivation, almost all females express incomplete Xlinked dominance
Hypophosphatemic Rickets – impaired ability of the kidney tubules to
reabsorb PO4
Rett Syndrome – mutation in a DNA-binding protein (MECP2), causing
the rapid onset of neurological problems (which leads to spastic
children with purposeless flapping of arms and legs and autism)
Mosaicism
“Pure” somatic mosaic – mutant cells are not present in the
gametes, manifesting as a segmental or patchy abnormality (e.g.,
Segmental Neurofibromatous 1 (NF1))
 Depends on when the mutation occurred and what cell lineage it occurred in
“Pure” germline mosaic – mutant cells are restricted to the gametes
(e.g., Hemophilia A/B and Osteogenesis Imperfecta)
 Must rule out autosomal dominant, X-linked disease, and carrier status
Mixed mosaic – could be present in both somatic and gametic cell
lineages (depending on when the embryonic mutation occurred)
Characteristics: Autosomal Recessive
Characteristics: Autosomal Dominant
Phenotype seen in siblings of the proband
Phenotype appears in every generation with each
affected individual having an affected parent
(exceptions low penetrance and new mutation)
Males and females equally affected
Any child of an affected parent has a 50% of disease
Both parents of an affected child are
asymptomatic as carriers of mutation
Phenotypically normal family members do not
transmit the disease phenotype to their children
(exceptions low penetrance or variable expressivity)
Parents may be from consanguinous mating
(especially if the condition is rare in
the general population)
Males and females equally likely to transmit
phenotype to children of either sex
Recurrence risk is ¼ for sibling of the proband
Isolated cases may be due to a new mutation
(especially if fitness is low)
Characteristics: X-linked Recessive
Characteristics: X-linked Dominant
Incidence of traits is much higher in males
than females
Affected males with normal mates:
No affected sons with all daughters affected
Heterozygous females are unaffected (but
Offspring of carrier females (males and females equally)
may express the condition depending on
have a 50% chance of inheriting disease
X-linked inactivation pattern)
Affected males transmit to all daughters
and each carrier daughter has a 50%
chance of transmitting to her sons
No male to son transmission
Carrier females transmit the trait while
affected males in a pedigree are related via
female relatives
A high % of isolated cases are due to new
mutations
Affected females are 2X as common as affected males
and tend to have milder disease and variable expression
of phenotype
Unstable Repeat Expansions
Generation of expanded repeats
beyond a normal range can lead to
a disease phenotype:
 Mechanism: “slipped mispairing” – a
DNA replication error
 Repeat CAG (poly Q): CAGCAGCAG
 Repeat CCG (poly R): CCGCCGCCG
All conditions have neurological
symptomology
Parental bias – anticipated
susceptibility
 Founder effect – loss of genetic
variation that occurs when a new
population is established by a very
small number of individuals
Transmission: autosomal, recessive,
or X-linked
Differences:
 Length and base sequences of the
repeated unit
 Number of repeats in a normal,
presymptomatic or affect individual
 Location of the repeated unit within
gene
 Pathogenesis of disease
 Degree to which instability occurs in
mitosis and meiosis
 Parental bias where expansion occurs
Unstable Repeat Expansions
Huntingtons Disease – autosomal dominant neuropathic
degeneration of the cortex and striatum, leading to death (repeat
CAG (poly Q) >39 expansions)
Appears at earlier ages when transmitted by an affected male
Premutation: 29-35 poly Q expansions
Other poly Q diseases: Spinobulbar Muscular Atrophy (X-linked)
and Spinocerebellar Ataxias (autosomal dominant)
Fragile X Syndrome (Xq.27.1) – improper condensation of
chromatin during mitosis (repeat CCG (poly R) >1000 expansions)
Premutation: 60-200 poly R expansions
Unstable Repeat Expansions
Myotonic Dystrophy – autosomal dominant (through the
mother) disorder, causing myopathy, cataracts, and
hypogonadism (repeat CTG (poly L) >2,000 expansions)
Premutation: 38-54 expansions
Friedreich Ataxia (FRDA) – autosomal recessive expansion in the
mitochondrial intron of frataxin, causing uncoordinated limb
movements, cardiomyopathy, speech difficulties, and scoliosis
(repeat AAG (poly K) >100 expansions)
Genetic Modification
Modifier – a gene that alters the phenotype in a non-allelic gene
Cystic Fibrosis: explanation for pulmonary insufficiency
Mannose binding lection 2 (MBL2) – prevents phagocytosis induced
through complement activation on the surface of carbohydrates
Transforming growth factor-β1 (TGFβ1) – promotes lung fibrosis and
scarring after infection
β-Thalassemia: coinheritance of α-Thalassemia is linked to
milder disease expression, reducing the amount of excess αchain produced to offset the β-chain imbalance in hemoglobin
Gene Pleiotrophy
Pleiotrophy – genes with 2+ discernible effects or a gene that
expresses effects on multiple aspects of physiology and anatomy
Marfan Syndrome – autosomal dominant of Fibrillin 1 gene in
connective tissue, causing abnormalities in the skeleton, eye,
and cardiovascular system
Others:
Cystic Fibrosis – affects the sweat glands, lungs, and pancreas
Osteogenesis Imperfecta – affects the bones, sclera, and teeth
Albinism – affects pigmentation and optic fiber development
L15
PAT TERNS OF INHERITANCE: MITOCHONDRIAL
1. Identify the organization and structure of mitochondrial genome
DNA and give examples of what type of proteins it encodes.
2. Distinguish homoplasmy and heteroplasmy.
3. Understand maternal input (transmission) on disease
4. Identify the interaction between nuclear genome and mitochondrial
genome.
5. Identify role of mitochondrial in disease (ex: mutations in mtDNA
genome LHON, MERRF) and mutations in nuclear DNA (Leigh
Syndrome, secondary OXPHOS disorders)that result in
mitochondrial dysfunction.
6. Identify examples of nuclear gene defects that cause mitochondrial
disease.
Mitochondria
Cross-talk:
 Nuclear genes – provide mitochondrial
proteins (1,500) which can have mutations,
conveying mutant proteins to the
mitochondria (e.g., heme biosynthesis,
protein import and assembly, mtDNA
transcription/replication)
 >60% of diseases result from nuclear gene
mutations of proteins
 Mitochondrial genes – encode 13 proteins
functioning in respiratory complexes
 15% of diseases result from mitochondrial gene
mutations
Mitochondria
Compartmentalization:
 Cytosol: glycolysis
 Innermembrane: Fatty-acyl CoA transport &
oxidative phosphorylation
 Matrix: TCA cycle & β-oxidation
Oocyte production: small number of mother’s
mitochondria are randomly selected to enter
the early egg cells
 Bottleneck effect – random selection can contribute
a greater ratio of mutant mitochondria per egg
(which replicate), increasing the possibility of
heteroplasmy in the child
 If the level of mutant mitochondria exceeds a certain
threshold  mitochondrial dysfunction
Map of Disease
MELAS – Mitochondrial Myopathy
and Encephalomyopathy  lactic
acidosis and stroke-like symptoms
KSS – Kearns-Sayre Syndrome
LHON – Leber’s Hereditary Optic
Neuropathy
NARP – Neuropathy, Ataxia, and
Retinis Pigmentosa
MERRF – Myoclonic Epilepsy with
ragged red fibers
LSP – Leigh Syndrome
mtDNA transcription and replication
Mitochondrial Disease
Mitochondrial Myopathy – subclass of disease with
neuromuscular deficiency
Symptoms: poor growth, muscle weakness and loss of
coordination, visual problems, hearing problems, learning
disabilities, heart disease, liver disease, kidney disease,
gastrointestinal disorders, respiratory disorders, neurological
problems (e.g., seizures and neurodevelopmental disorders),
autonomic dysfunction, dementia, diabetes mellitus
Mitochondrial Disease
LHON – Leber’s Hereditary Optic Neuropathy – causes painless,
bilateral and subacute visual failure
 Diagnosis: visual findings of legal blindness (which significantly impacts quality of
life)
 Mutation: m.3460G>A, m. 11778G>A, or m.14484T>C
 Affect different Complex I genes
 Treatment: provision of visual aids and occupational therapy with support
 At-risk: alcoholics and smokers
LSP – Leigh Syndrome – causes heterogeneous respiratory chain
complex defects
 Mutation: NDUFA12 (nuclear) of Complex I or ATP6 (mitochondrial) of Complex V
 Others: MTTV, MTTK, MTTW, or MTTL1 of mitochondrial tRNA proteins
 Inheritance: X-linked recessive and autosomal recessive
Mitochondrial Disease
Mitochondrial depletion syndromes:
Alpers Syndrome (4A) and MNGIE (4B):
 DNA polymerase gamma-2 (PLOG2) – c-terminal polymerase domain with an nterminal exonuclease domain for proof-reading function and increased fidelity of
mtDNA replication
 Mutations in exonuclease function: high frequency of randomly distributed
mutations in the mtDNA genome (due to decreased polymerase activity)
 Mutation: A467T and repeat CAG (poly Q) expansion
Progressive External Opthalmoplegia – autosomal dominant and
recessive mutation in PLOG
Mitochondrial Disease
KSS – Kearns-Sayre Syndrome – causes opthalmoplegia, pigment
degeneration of the retina, and cardiomyopathy (with associated
facial and muscle weakness, deafness, small stature, and increased
CSF protein)
 Mutation: mtDNA deletions in the muscle mitochondria (e.g., MTTL1)
MERRF – Myoclonic Epilepsy with ragged red fibers – causes an
elevation in pyruvate and lactate due to deficiencies in the Complex I
(NADH-CoQ reductase) and IV (cytochrome c oxidase) subunits
 Mutation: 8344A>G of MTTK (80-90%), MTTL1, MTTS1/2, MTTF, or MTND5
NARP – Neuropathy, Ataxia, and Retinis Pigmentosa – causes sensory
neuropathy, muscle weakness, ataxia, and vision loss
 Mutation: ATP6 (a subunit of ATP synthase) (70-90%)
Mitochondrial Disease
MELAS – Mitochondrial Encephalomyopathy – causes muscle
weakness, recurrent headaches, loss of appetite, vomiting, and
seizures resulting in brain damage (with associated lactic acidosis)
 Mutation: Complex I (NADH-CoQ reductase) and tRNA MT-TL1 (80%), MT-TH,
or MT-TV
MNGIE – Mitochondrial Neurogastrointestinal Encephalopathy
Syndrome (POLIP Syndrome) – autosomal recessive mutation in
TYMP (thymidine phosphorylase) (100%), causing poor GI mobility,
pseudo-obstruction with peristalsis, and subsequent malabsorption
 Mutation: Complex IV (cytochrome c oxidase)
Acquired Mitochondrial Disease
Polymerase gamma (POLG) – replicates mitochondrial DNA
(similar to reverse transcriptase in HIV)
Antiretroviral drugs will also inhibit POLG in AIDS patients, stopping the
replication of host mitochondrial DNA and leading to a decreased
number of organelles and function → metabolic acidosis
Treatment: IVF of donated female eggs with affected mother’s maternal
genome and father’s sperm creates an embryo with no mitochondrial
disease
L16
PAT TERNS OF INHERITANCE: MULTIFACTORIAL
1.Distinguish between polygenic and
multifactorial inheritance.
2.Describe how polygenic traits present
within a population. Give examples.
3.Describe the tests to determine if a
trait is polygenic or multifactorial.
Complex Disease Inheritance
Non-genetic factors (e.g., the environment)
play a crucial role in disease causation:
 Multifactorial or complex inheritance pattern
 Family members have more shared gene
patterns (2-4%) and environmental exposures
than an individual chosen at random from the
population
 Gene-to-gene interactions: polygenicity of
multigenic effects – additive or synergistic
amplification by genes at multiple loci
 Gene-to-environment interactions: raise or
lower susceptibility, triggering disease
acceleration
Examples: common congenital
malformations or acquired childhood and
adult diseases
Complex Disease Inheritance
Qualitative traits – presence or absence of a genetic disease
Quantitative traits – measurable physiological or biochemical
quantities (e.g., height, blood pressure, serum cholesterol
concentration, and BMI)
Relative risk ratio (λr) – measured of familial aggregation by
comparing the frequency of the disease in the relatives of an
affected proband with its prevalence in the general population
𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑖𝑛 𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑟𝑜𝑏𝑎𝑛𝑑
λ𝑟 =
𝑝𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑔𝑒𝑛𝑒𝑟𝑎𝑙 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛
Value of λr = 1 (low): indicates that a relative is no more likely to develop
the disease than any other individual in the population
Genetic Studies
Gene mapping studies identify genes and associated coincidence of
mutation:
 Concordance – 2 related individuals with the same disease (through inheritance or
shared environmental factors)
 Can occur even when the 2 affected relatives have different predisposing genotypes (with a
“phenocopy” or “genocopy” of the disease)
 Discordant – only 1 member has the disease in a comparison of 2 family members (in
which the unaffected doesn’t possess the predisposing genotype or has not
experienced the “trigger” for the disease)
Case control studies compare patients with disease with selective criteria
that direct the choice of individuals without the disease (controls):
 Ascertainment bias – family with the disease can report better than a control family
 Recall bias – family with affected members that are more motivated to report
Genetic Studies
Selective criteria: occupation, environmental
exposure, geographic location, ethnicity
Unrelated family members act as controls:
 Spouses matching in age, ethnicity, location, and
exposure
Alleles in Common with the Proband
RELATIONSHIP
PROPORTION
Monozygotic twins
1
1st degree relative: dizygotic twin, sibling, or parent
½
2nd degree relative: grandparent
¼
3rd degree relative
⅛
Twin Studies
Digozytic (DZ) twins – 2 eggs fertilized
upon conception, sharing 50%
commonality between alleles
 Greater concordance for disease in MZ
twins compared to same sex DZ twins
presents a strong argument for genetic
influence in disease development
Monozygotic (MZ) twins – split from the
same fertilized egg and, thus, sharing
identical genes (0.3%)
 Allows for comparison of identical
individuals raised in different environments
 Disease concordance of <100% presents a strong
argument for the influence of non-genetic factors
(e.g., exposure to infection, dietary differences,
somatic mutations, aging, X-linked inactivation in
females)
Limitations:
 MZ share the same genotype but not the
exact same gene expression due to somatic
rearrangements or X-linked inactivation
 Environmental exposure will not be the
exact same into adulthood
 Measurement of concordance give an
average estimate: relative predisposing
factors may differ and skew interpretation
Ascertainment bias:
 Volunteer-based ascertainment – one twin
recruits the other after diagnosis of the
disease
 Population-based ascertainment –
recognized first as twins before the health
assessment
Multifactorial Inheritance
Multifactorial inheritance –
combination of several genes or
variants
Variety of environmental
conditions: prenatal exposure in
utero, childhood environment, and
adult environment
Polygenic (quantitative traits) –
exhibit a Gaussian distribution
 Position of the graph peak and shape
are governed by the mean (μx) and the
variance (σx2)
 Mean (μx) – arithmetic average of the values
 Variance (σx2) – measure of the spread of
values on either side of μx
Trait is considered normal or
abnormal depending on how far it
is above or below μx
 Basic statistical theory – when a
quantitative trait is normally distributed
in a population, only 5% of the
population will have measurements
more than 2 standard deviations above
or below μx
 Approximately 68%, 95%, and 99.7% of
observations fall within the mean ± 1, 2, or 3
standard deviations, respectively
Polygenic Inheritance
Binomial expansion: (p + q)(2n): where
p = q = ½ and n = # of loci
 As the number of loci increases, the
distribution increasingly resembles a normal
Gaussian curve
If the trait is determined by 2 equally
frequent alleles at a single locus:
phenotypic distribution of three groups
1:2:1
If the trait is determined by 2 alleles at
2 additive loci: distribution phenotypic
distribution of five groups 1:4:6:4:1
Polygenic Inheritance
Correlation: statistical measure of the degree of the relationship
between 2 parameters
Example: 1st degree relatives share 50% of their genes and should
correlate as 0.5 if polygenic
Physiological parameters with continuous normal distribution:
BP, head circumference, height, intelligence, skin color
Regression of the mean – observed tendency for offspring to
deviate from the mean (due to certain characteristics being
influenced by the environment or by non-additive genes)
Liability/Threshold Model
Liability/threshold model - all of the factors
that influence the development of a
multifactorial disorder (genetic or
environmental) can be lumped together as a
single phenomenon known as liability,
accounting for discontinuous multifactorial
traits
 Population incidence – proposes that a threshold
exists above which the abnormal phenotype is
expressed in a general population
 Familial incidence – proposes that a threshold exists
above which the abnormal phenotype is expressed
among relatives
Curve is shifted to the right for relatives and
the extent of the shift depends on relatedness
Liability/Threshold Model
Deleterious liability – combination of several “bad” genes and
adverse environmental factors
The mean liability of a group can be determined from the incidence
of the disease in the group using the statistics of the normal
distribution, estimating the correlation between relatives
Generates a threshold that, once reached, determines those that
are affected (based on familial risk)
 Examples: cleft lip or palate, club foot, congenital heart defects, hydrocephaly,
neural tube defects, pyloric stenosis
Heritability
Heritability (h2) – defined as a fraction of total phenotypic variance of a quantitative
trait that is caused by genes (not the environment) and measures the extent that
different alleles at a variable number of loci are responsible for variable expression
across a population (by quantifying the role genetic differences play in determining
variability across a quantitative trait in a population)
 No genetic contribution: h2 = 0
 Genetic contribution: h2 = 1
Difficulties:
 Relatives share more than genes: an h2 value alone may not be a good estimate
 Even if h2 is high (closer to 1), it tells you nothing about the mechanism underlying the trait
(i.e., the number of alleles) or how the alleles at the different loci interact
 Cannot be considered in isolated populations
Degree of familial clustering: λ𝑠 =
𝑟𝑖𝑠𝑘 𝑡𝑜 𝑡ℎ𝑒 𝑠𝑖𝑏𝑙𝑖𝑛𝑔𝑠 𝑜𝑓 𝑎𝑓𝑓𝑒𝑐𝑡𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠
𝑟𝑖𝑠𝑘 𝑡𝑜 𝑡ℎ𝑒 𝑔𝑒𝑛𝑒𝑟𝑎𝑙 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛
 Multifactorial diseases are common and pose a significant risk to morbidity and mortality
Identification
Method 1: Linkage Analysis – map single gene disorders by studying
co-segregation of other known genetic markers
 Complications:
 Difficult to look for additive linkages of several genes that minimally contribute to a disease
phenotype in polygenic traits
 Age of onset can impact the genetic status of family members
 Requires excess information (e.g., mode of inheritance, gene frequencies, and penetrance)
that is difficult to acquire
Method 2: Affected-Sib Pair Analysis – look for “identity by descent”
in affected sibling pairs
 Whether the affected siblings inherit a specific allele more or less often than
by mere coincidence (as a random incidence in the population)
Identification
Method 3: Linkage Disequilibrium Mapping – mapping a specific
region of a chromosome expected to contain the disease gene (by
reducing it to a finite area, constructing SNP haplotypes within the
region, defining historial crossover points, and sequencing possible
variants associated with the disease)
 Affected by mating preferences
Method 4: Association Studies – compare frequencies of a specific
variant in affected parents with its frequency in a carefully matched
control group (in a case control study)
 Odds ratio – measures the strength of association (evidence for association if
the frequencies differ significantly)
 Disadvantages (that grant false positives): small sample sizes, weak statistical
support for SNP or loci, low a priori probability of selected SNPs or varients
associated with the disease, and population stratification
Identification
Method 5: Genome Wide Association Studies – compare multiple variants across
the genome in a case control study
 HAPMAP Project – typing of SNPs to different ethnicities in order to show that SNPs are
strongly correlated with SNPs on a nearby genetic region
 Advantages: allows for a comprehensive unbiased scan of the genome that can identify novel
susceptibility factors (by capturing all meiotic events in a population and disease genes with
only small associated risks)
 Identifies multiple interacting disease genes and their respective pathways, providing a comprehensive
understanding of the disease etiology
Associated results: allow for a better understanding of disease susceptibility,
identification of low and high risk genes in a polygenic disease, identification of new
drug targets directly related to disease causation (in personal and proactive
therapy), and susceptibility and expected onset of disease ascertained earlier in life
(for better detection and safer treatment to prevent significant damage or for
administration of therapeutics to prevent the disease entirely)
Multifactorial Disease
Digenic Retinitis Pigmentosa – causes retinal degradation due to
heterozygous mutations in 2 unlinked genes, resulting in photoreceptor
membrane defects
Idiopathic Cerebral Brain Thrombosis – causes clots to form in the venous
system of the brain, leading to catastrophic occlusion and death (5-30%)
 Factors:
 Factor V – Arg 506 Gly substitution increases stability to sustain a longer anticoagulant effect
 Prothrombin – mutation (g.2021G>A) in 3’URT
 Oral contraceptives – contain synthesis estrogen, effecting Factor X and prothrombin
Placental Artery Disease – causes severe preeclampsia, premature
separation of the placenta from the uterine wall, and intrauterine growth
retardation and stillbirth
 Factors: oral contraceptives, mutation (g.2021G>A) in prothrombin gene, methylene
tetrahydrofolate reductase (MTHFR) variant
Multifactorial Disease
Deep Vein Thrombosis – due to Factor V and prothrombin mutations
 Factors: trauma, orthopedic surgery, malignant disease, prolonged periods of
immobility, oral contraceptives, increased age
 Can develop to a pulmonary emboli  death
Hirschprung Disease (HSCR) – complete absence of the intrinsic
ganglion cells of the mesenteric and submucosal complexes of the
colon, preventing peristalsis and causing constipation and intestinal
obstruction
 Factors: RET gene at 10q11.2 of tyrosine kinase receptor, EDNRB gene at
13q22 of G-coupled protein endothelial receptor B, EDN3 gene at 20q13ligand (acting in parallel pathways to interact and develop ganglion cells)
Multifactorial Disease
Alzheimer’s Disease – neurogenerative disease that causes
dementia in individuals >65 years of age (1-2%),
characteristically exhibiting an accumulation of amyloid plaques
in the brain
Factors: associated with ApoE allele e4/e4 (19%), copper interaction
with the blood-brain barrier
At-risk: age, gender, family history, African-American or Caribbean
Hispanic ethnicity
L17
PRINCIPLES OF EPIGENETICS AND EPIGENETICS MECHANISMS
Epigenetics
Epigenetics – encompasses all
the heritable changes and
alterations of gene expression
(without changing the DNA
sequence)
 Stably transmitted from one cell
generation to next, forming “cellular
memory”
 Effects are reversible (through
reprogramming)
 Effects involve modifications (e.g.,
genomic imprinting, X-linker
inactivation, position effects)
Changes in regional chromatin
structure:
 Gene silencing – inaccessibility to the
condensed heterochromatin prevents
access to transcription factors,
preventing gene expression
 Position effect – permanent silencing
of a gene due to inversion or
translocation of a DNA sequence,
leaving it permanently condensed near
the centromere
Epigenetics
Epigenetic marker enzymes:
 “Writers” – add covalent chemical
marks to DNA or histones
 “Erasers” – remove covalent
chemical marks in DNA or histones
 “Readers” – effector proteins that
bind to interpret epigenetic markers
 Recruit additional proteins to create
different chromatic states:
 Compaction
 Chromatic remodeling – changes in
nucleosome spacing and structure, allowing
access to transcription factors
 Histone substitution (of non-standard histones)
Epigenetic markers:
 Patterns of methylation in cis-acting
regulatory elements of DNA strands
determine transcription (playing a
role in genomic imprinting and X-link
inactivation)
 Highly methylated regions exhibit no gene
expression
 Acetylation of histones in the
nucleosome triggers gene activation
 Others: ubiquination, sumoylation,
phosphorylation
Epigenome
Epigenome – total collection of epigenetic changes across a genome (cellspecific, varying with the current cell-state)
 Characteristic of individual cell types
 Plasticity (influenced by environment, development, aging)
 Relatively stable trans-generational transmission
 mCpG islands in DNA can change with the cell cycle
 Histone changes are more dynamic, changing within hours
Reprogrammable within differentiated cells:
 Somatic nuclear transfer (“Dolly” experiment): transplanting of a differentiated
nucleus into an oocyte cytoplasm allowed the development of a normal adult
 Induced pluripotent stem cells (iPSC): only requires 4 transcription factors to initiate
reprogramming from a differentiated fibroblast to a stem cell
Global effort: International Human Epigenetic Consortium (IHEC)
Epigenome
Sources of genetic changes that
can produce disease:
 Mutation in DNA
 Uniparental disomy
Epimutation – abnormal chromatin
formation at 1+ loci in the genome
 Primary epimutation – immediate
cause of disease (e.g., chemical or
environmental) through DNA
methylation or histone acetylation
 Secondary epimutation – controls
epigenetic processes (e.g., deacetylase)
Disease - Chromatin Modifiers
Arise from a defect in “writers”: DNA methylases (DMNTs) and
chromatin modifiers (HDAC or HAT)
 Produces abnormal chromatin domains/loci across the genome, illiciting
phenotypic variability
Outcomes: incompatible with life, causing neurodevelopmental
diseases with intellectual disabilities
Rhett Syndrome – causes a rapid loss of language and motor skills
which eventually stabilizes (primarily in girls), leading to characteristic
handwringing movements with growth retardation, seizures, autistic
behaviours, and microencephaly
 Causal agent: loss of methyl-binding protein 2 (MeCP2)
Disease – Dysregulation of Heterochromatin
Heterochromatin: contains repetitive
DNA or silencer elements, expanding
across the entire chromosome, to
convert “open” chromatin to condensed
transcriptionally silent chromatin
(facilitated by communication between
nucleosomes)
 Silencing prevention: barrier elements that
protect genes from the surrounding
environment and nucleosome free areas
Facioscapulohumeral Dystrophy (FSHD):
 Variations leadings to toxic expression of
DUX4 transcription factors:
 FSHD1 – reduction in heterochromatin at
telomeric tandom microsatellite repeats at 4q35
 FSHD2 – creation of polyadenylation sites at the
end of tandem repeats (due to >DUX4 mutation)
Types of dysregulation:
 Heterochromatination of normally
expressed genes, leading to silencing of
active genes
 Position effect – silencing of a gene due to
inversion or translocation of a DNA sequence,
leaving it permanently condensed near the
centromere (in the heterochromatin region)
 Reduction of gene silencing and activation
of previously silent genes
 BRAC1 tumor suppressor protein – maintains
constitutive heterchromatin
 Mutations can cause a loss of heterochromatin
organization  mitotic recombination and genome
instability
Disease – Environmental Changes
Fluidity of epigenomic chromatin status is determined by
cytosine methylation patterns, histone modification patterns,
and nucleosome spacing
Environmental signals can significantly alter gene expression: Illumina
Infinium Human Methylation 450 Bead Chip
 Aging: marked by progressive inefficiency in cell, tissue, and organ function by
changing somatic cells of organs and stem cells that regenerate tissue
Disease – Genomic Instability
Universal characteristic of cancer cells: genomic instability (due to defects in
chromosome segregation and DNA repair deficiencies)
 Weakens the capacity to maintain genomic integrity, leading to chromosomal instability with
abnormal karytopes:
 Deletions – missing chromosomes
 Duplications or partial duplications – extra chromosomes
 Inversions and translocations – structural rearrangements
Epigenetic patterns help maintain genome stability by suppressing the excess
activity of transposons and maintaining the functions of centromeres and
telomeres
 Loss allows cells to revert to less differentiated states: prototype cancer allows for more
flexibility to adapt to the changing environment after reverting to a tumor-inducing
phenotype (through hypomethylation of highly repetitive DNA strands)
p53 mutated cells: show chromothripsis, shattering chromosomes into multitudes
of fragments before subsequent end-joining repair
 Allows proto-cancer cells to attain plasticity to escape normal growth controls
Disease – Development
Genomic imprinting – phenomenon where certain genes can be
expressed in a parent-of-origin manner
 At an imprinted diploid locus, there is unequal expression of the maternal or
paternal allele
 The parent specific imprinted locus “marks” are reprogrammed and susceptible
to errors
Uniparental disomy (UPD) – inheritance of 2 copies of chromosomes
from a single parent
Trans-regulation – a protein product acts at several chromosome
promoters to increase the activity (transcription) of multiple genes
Cis-regulation – a protein products acts on the same chromosome
promoter to increase the activity (transcription) of the respective gene
Disease – Genomic Imprinting
Disease
Mechanisms
Chr. Loci
Genes
Prader-Willi
deletion, UPD, genomic imprinting
15q11/15q13
snoRNAs
Angelman
deletion, UPD, imprint defect, duplication
point mutation
15q11/
15q13
UBE3A
BeckwithWiedemann
imprint defect, UPD, 11p15.5 duplication,
translocation, point mutation
11p15.5
IGF2, CDKN1C
Silver-Russell
UPD, duplication translocation, inversion
epimutation
7p11.2/
11p15.5
several candidates in region/
biallelic expression of H19
and decrease of IGF2
Pseudohypoparathyroidism
UPD, imprint defect, point mutation
20q13.2
GNAS1
Disease – Trans-regulation
Disease
Gene
Effect
Rubinstein-Taybi
CREBBP/EP300
protein defect in co-activator of cAMP activity and
change in HAT activities
Rhett
MECP2
X-linked for females: activity binding to methyl CpGs
α-Thalassemia/
X-linked Mental Retardation
ATRX
Xq13: ATRX essential for survival of cortical neurons
ICF Syndrome
DNMT3B
loss of function mutations in methyl transferase
Disease – Cis-regulation
Disease
Gene
Effects
αδβ- and δβ-Thalassemia
deletion of locus control region (LCR)
leads to decreased globin expression
Fragile X
expansion of CCG repeat
abnormal methylation and silencing
of FMR1 causes a neurodegeneration
disorder
(premutation @ 60-200 repeats)
FSH Dystrophy
contraction of D4Z4 repeats
exposed less repressive chromatin
Multiple Cancers
MLH1 (mismatch DNA repair)
germline epimutation: abnormal
methylation of the promoter
L18
CLINICAL GENETICS: SINGLE GENE DISORDERS
1. Identify the clinical features of Huntington’s disease and distinguish its unique genetic
features.
2. Differentiate the genetic basis for inheritance of Myotonic Dystrophy.
3. Explain clinical features. Correlate the genotype-phenotype relationship in Myotonic
Dystrophy
4. Identify clinical features associated with Hereditary Motor and Sensory Neuropathy and
identify the genes involved and the functions of their protein products.
5. Identify clinical features associated with Neurofibromatosis. Distinguish between the genetics
of neurofibromatosis I (NF1) and Neurofibromatosis 2.
6. Identify clinical features associates with cystic fibrosis. Differentiate the biochemical and
molecular basis for manifestation of cystic fibrosis. Identify genes involved and correlate the
genotype-phenotype relationship.
7. Identify clinical features associated with Duchenne Muscular Dystrophy (DMD) and Becker
Muscular Dystrophy (BMB). Distinguish inheritance pattern of Duchenne Muscular Dystrophy
and the role of dytrophin gene mutations.
8. Identify clinical features associated with Hemophilia A and B.
9. Distinguish the inheritance of these diseases and current treatments
Hemoglobinopathies
Hemoglobin – 4 subunit globular protein that contains Fe2+ and
acts in O2-transport
Heme – Fe2+-containing porphyrin ring that binds O2, forming
oxyhemoglobin
 Deoxyhemoglobin (with Fe3+) does not bind O2
Adult Hb (HbA) – consists of 2 α and 2 β chains designated as Hb α2β2
Fetal Hb (HbF) – consists of 2 α and 2 γ chains designated as Hb α2γ2
 Possesses a higher affinity for O2, extracting from maternal circulation
Porphyrias – genetic or acquired disorders affecting the
enzymes of porphyrin or heme synthesis, causing neurological
dysfunction, photosensitivity, and cardiac arrhythmias
L19-L20
MOLECULAR BASIS OF CANCER
1. Describe the basis of three molecular mechanisms that can
predispose to cancer: Oncogenes (c-onc), Tumor Suppressor genes
(TSG), and DNA repair genes.
2. Describe the role of genetic mechanisms that underlie cancer
phenotypes: Germline mutations, Epigenetic signatures, and
miRNAs.
3. Understand how cellular processes are impacted and promoted by
neoplastic changes: Apoptosis, new angiogenesis, growth factors,
genetic instability, loss of cell adhesion, and loss of cell cycle
control.
4. What are heritable cancer syndromes and how are they identified?
Examples?
Cancer
Cancer – rapid and uncontrolled cellular proliferation (neoplasia) that produces a
malignant mass or tumor (neoplasm)
 Malignant – cell growth is no longer controlled by normal boundaries and is capable of
progressing and invading neighboring (and more distant) tissues
 Benign – noninvasive or metastatic cancers
Statistics: 2nd leading cause of death in the U.S., contributing to 20% of annual
deaths and 10% of medical care costs (in developed countries)
 Affects all ethnicities and genders regardless off age
Acting as a genetic disease:
 Gene dysfunction: initiates and drives cancerous changes in all tissues
 No 2 cancers will be the same, generating personalized medicine: Tyr Kinases are uniquely targeted by
candidate drugs
 Heritable cancer syndrome genes: linked to sporadic cancer development
 Analysis and documentation of common mutations in cancer tissue, creating a signature or
profile
 Genomic studies: identify changes associated with cancer
Nomenclature
Hereditary cancer syndrome – first inherited cancer (present in all cells) causing gene
mutations which is subsequently followed by somatic mutation at a later point in time
Sporadic cancer – a mutation occurring in a single cell that progresses to a tumor via
the accumulation of other mutations and clonal evolution
Oncogene (Onc) – a mutation of a proto-oncogene, stimulating cell division and
proliferation (neoplastic transformation)
 Proto-oncogene – a normal cellular protein coding gene that is involved in growth or cell
survival
Tumor suppressor genes (TSGs) – encode proteins that protect the transition of cells
through checkpoints (as gatekeepers) or protect cell division and integrity of the
genome (as caretakers)
 Gatekeepers: proteins regulators of mitosis and mediators of apoptosis
 Caretakers: proteins that detect and repair DNA mutations
 Loss of function: allows for the accumulation of deleterious mutations in proto-oncogenes and gatekeepers,
promoting the development of cancer
Cancer
Types:
 Sarcoma – originate in the mesenchymal tissues (e.g., bones, nervous system, muscle,
connective tissue)
 Carcinoma – originate in the epithelial tissues (e.g., GI tract, lung bronchi)
 Hemopoietic/Lymphoid – originate in the bone marrow, lymphatic system, and peripheral
blood
Tumor classification: site, tissue type, histological appearance, degree (or stage) of
malignancy
Cancer phenotypes: altered cell proliferation, invasiveness, apoptosis, generation of
new vasculature, increase in cellular signaling, genetic profile of “driver” mutations,
and increasing incidence with age
Multi-step process resulting from the dysregulation of genes  oncogenesis:
involved in cell growth and regulation, resistivity to cell apoptosis, and maintenance
of genetic stability
Oncogenesis
Carcinogenesis – the process that produces genetic mutations
induced by chemicals or physical agents
Initiation – irreversible genetic change in a single cell
 Activation of oncogenes or anti-apoptotic genes – dominant (requires a single
mutation)
 Mutations in tumor suppressor genes – recessive (requires mutation in both alleles)
Promotion – increased proliferative ability in the initiated cell through
clonal growth
Progression – accumulation of more genetic damage, allowing the cells
to develop a malignant phenotype
 Mechanism: proto-oncogene activation  progressive loss of tumor suppressor
genes  activation of anti-apoptotic genes  loss of pro-apoptotic gene expression
Oncogenesis
Tumorigenesis – driven by the accumulation of irreversible mutations (or
drivers) in key genes that encode functions for cell cycle regulation, cell
growth, and programmed cell death
 Require cooperativity: contributions from more than one defective oncogene,
allowing for uncontrolled cell division and growth (without regulation)
 Clonal evolution: each successive mutation in a cell confers a growth advantage,
outgrowing their “normal” neighboring cells
Tumor progression: accumulation of additional drivers after more
mutations (or epigenetic silencing of caretakers) that maintain
DNA/genome integrity or cytogenetic normality (along with altered
vascularization control, allowing for local clonal invasion or distant
metastasis)
K
K
Fearon-Vogelstein Adenoma-Carcinoma Model
Class model for multi-stage progression of colorectal cancer:
Normal
colon cell
Mutation of APC
Loss of DCC
Mutation of K-ras
Hyperproliferation
Early
adenoma
Intermediate
adenoma
Late
adenoma
Accumulation other
genetic mutations
Loss of p53
DNA hypomethylation
Carcinoma
Metastasis
Phenotypes of Molecular Cancer
Immortality: indefinite proliferation due to protection over the
replication of telomeres, preventing chromosomal shortening, and
loss of growth control after the loss of tumor suppressor activity
Decreased dependence on growth factors for proliferation: selfgenerated production of tumor cell growth factors through autocrine
signaling
Loss of anchorage dependent growth/altered cell adhesion: allows
for metastasis (through proteolytic cleavage of the basement
membrane)
 Mechanism: detachment of tumor cells from the primary tumor, penetrating
through the basement membrane (by degrading the ECM)  migration to
foreign tissue sites through the blood and lymph
 Matrix metalloproteins (MPPs) – overexpressed in tumor cells
Phenotypes of Molecular Cancer
Loss of cell cycle control: multifactorial regulation of mitosis is controlled through
oncogenes and tumor suppressor genes, irreversibly committing the cell to divide
after an increase in mutations and resulting genome instability
 Cyclin-dependent kinase inhibitors (CKIs):
 Cip/Kip family: p21/Cip1/waf1/Sdi1, p21/Kip1, and p57/Kip2 – maintain a broad specificity, binding and
inactivating most of the cyclin/CDK complexes needed for cell cycle progression (and upon detection of
damaged DNA, p53 (stimulated by p21) halts the cell cycle)
 INK4 family: p16/INK4a, p15/INK4b, p18/INK4c, and p19/INK4d – regulate phosphorylation of Rb (a critical
cell cycle checkpoint)
Reduced sensitivity to apoptosis: overcomes promoters of apoptosis through
overexpression of Bcl-2 and mutation or suppression of Fas-R/CD95, inhibiting the
activity of the caspase family and associated p38 induced Bim/Bax interactions
 Phases: initiation phase after the activation of pathways from external signaling through death
receptor ligands (e.g., CD59—Fas-L)  decision and effector phase involving the disruption of
the mitochondrial membrane and release of cytochrome c into the cytoplasm (for protease
and nuclease activation)  degradation and execution phase
Phenotypes of Molecular Cancer
Increased genetic instability: confers a selective growth advantage through
translocation and associated rearrangements, acting as oncogenic drivers
 Chromosomal aneuploidy – gain or loss of 1+ chromosomes
 Chromosomal polyploidy – gain of extra sets of chromosomes
 Examples:
 Burkitt’s Lymphoma (t8,14) – translocation that results in transcriptional fusion protein, leading to the
expression of c-myc proto-oncogene expression
 Philadelphia Chromosome (CML) (t9,22) – translocation that splices c-Abl+BCR kinase + GAP, forming
consecutively active chimeric kinase) that drives cell proliferation)
 Gene amplification – multiple rounds of DNA replication at a single site (followed by FISH)
Angiogenesis: hypoxia inducible factor 1 (HIF1) interacts with vascular endothelial
growth factor (VEGF) (secreted by tumor cells) to stimulate neo-vascularization,
stimulating hyperpermeability in proliferating cancer cells
 Anti-tumor experiments target angiogenesis to reduce tumor growth
Oncogenes
Oncogenes – deregulated mutated derivatives of proto-oncogenes,
contributing to cell proliferation and decreased sensitivity to cellular
apoptosis, that confer a selective growth advantage
Classification based on cell location and activity:
 Growth factors and growth factor receptors: stimulate tumor cell growth
through self-generated autocrine signaling (and affect normal cells via
paracrine mechanisms)
 Membrane-associated G-proteins: Ras oncogenes function in cell
proliferation and survival, activating other oncogenes through constitutive
intracellular signaling to promote unregulated cell proliferation (through
kinase cascades)
 Serine-threonine protein kinases: after Ras recruitment, Raf activates MAPKs
to activate transcription factors (containing Elk-1) and protein kinase C (to
activate c-jun), acting as an intracellular signaling molecule
Oncogenes
 Cytoplasmic tyrosine kinases:
 SRC family – exhibits kinase activity (SH1) and promotes protein-to-protein interactions
(SH2/SH3), generating constitutive signaling
 Bcr-Abl (chimeric fusion of t9,22 in Philadelphia chromosome) – exhibits kinase activity (cAbl) and activates GTPase (Bcr), promoting unregulated cell growth (through Ras signaling)
 reduces GF dependence, alters cell adhesion properties, and enhances cell viability
 Nuclear proteins:
 Transcription factors: Burkitt’s Lymphoma (t8,14) moves the Ig heavy chain (IgH) promoter
driving c-myc (regulates by binding to E-box sequences) expression, inducing uncontrolled
cell proliferation (as an oncogenic switch for oncogenes and tumor suppressor APC)
 Tumor suppressor APC – mediates the degredation of β-catenin which, in turn, acts as a transcription
factor for Tcf, activating c-myc expression
 Telomerases – synthesize the hexamer (TTAGGG) that caps chromosomal ends (telomeres),
allowing tumor cells to proliferate indefinitely (due to a mutation in the telomerase gene)
 Cytoplasmic proteins engaged in cell survival: increased expression of Bcl-2
oncogene disrupts apoptosis
Tumor Suppressor Genes (TSGs)
“Loss of function” mutations: point or small insertions/deletions
(must lose heterozygosity to induce tumor development)
Gatekeepers – directly involved in cell growth and death
Caretakers – promote genetic stability through DNA repair, increasing the
mutation rate of DNA and accelerating tumor formation
Loss of Rb protein (recessive)  deregulation of cell cycle and
uncontrolled cell division, continually activating target genes
Normal mechanism: dephosphorylation of Rb allows for binding to E2F
transcription factor, preventing transcription of E2F transcription target
Tumor Suppressor Genes (TSGs)
p53 tumor suppressor gene – transcription factor that promotes
DNA repair and apoptosis while inhibiting growth (for genetic
stability)
 Loss of function  tumorigenesis: mutation of p53 gene (in sporadic tumors
and Li-Fraumeni Syndrome), vital transforming antigens (by SV40 T antigen),
and cytoplasmic sequestration (preventing entrance into the nucleus to
activate target genes)
Adenomous Polyposis Coli (APC) gene: initial mutation in early colon
carcinogenesis, leading to the activation of c-myc (can be inherited)
Phosphatase and Tensin Homologue (PTEN): first identified in
glioblastoma multiforma (aggressive brain tumor), inhibiting cell
cycle progression and inducing apoptosis (as a negative regulator of
Akt activation, opposing VEGF)
Translocations
Chromosomal translocation can rearrange the genome and activate
“new” promoter/enhancer elements, dysregulating normal gene
expression
Chronic Myelogenous Leukemia (CML) – Philadelphia chromosome
t(9,22), moving Abl to the “break point cluster” Bcr, resulting in
chimeric fusion (Bcr-Abl) that enhances Abl kinase activity
 Drug target: imatinib – inhibits kinase activity
Burkitt’s Lymphoma – B cell-derived tumor of the jaw (in African
children) due to t(8,14), positioning Ig heavy chain (IgH) enhancer to
drive defunct c-myc expression  uncontrolled cell growth and
proliferation
Follicular B cell Lymphoma t(14,18) – IgH promotion of translocated
Bcl-2, increasing the B cell population and inhibiting apoptosis
Heritable Cancers
Li-Fraumeni: p53 gene mutation (70%)
Breast and/or Ovarian Cancer: BRCA1 (DNA repair and genomic stability
as a cell cycle checkpoint), BRCA2, RAD51C (DNA repair), and RAD51D
mutations (10-15%)
Lynch Syndrome: MLH1 and MLH2 mutations (DNA repair), causing
colorectal cancer
Bloom’s Syndrome: BLM (a recQ helicase) mutation, causing short stature
and increased light sensitivity
Autosomal recessive:
 Xeroderm Pigmentosum (XP): XPC mutation (DNA repair), causing increased light
sensitivity and subsequent skin cancer
 Franconi’s Anemia (FA): FRACB (DNA repair) mutation (in Ashkenazi Jews and South
Africans), causing acute myelogenous leukemia and subsequent bone marrow failure
Others
miRNA – post-transcriptionally
degrade or inhibit the expression of
target mRNA, acting as gene
regulators and tumor suppressors
 Functions: cell proliferation and
apoptosis
Carcinogenesis:
 Planar chemicals – integrate into DNA,
forming adducts that introduce
mutations when replicated
 E.g., vinyl chloride, dimethylnitrosamine
(DMN), benzopyrene, UV light,
benzene, 3-MCA, nitrogen mustards