Human Genetics

1/21/2025

What is a genome?

• all genetic information contained in a cell

• 3gbp for a haploid cell (but we have 2 copies so really 6 GBP)

  • Males actually have 6.27 GBP

  • Females actually have 6.369 GBP

  • Because Y chromosome is shorter

Genes

• Gene: Basic physical and functional unit of heredity

  • Has to do something (encodes for a protein or other product like tRNA or rRNA)

• Knowing what parts of the genome are functional lets us understand genetic disease

• Humans have about the same number of PCGs as nematodes

  • Humans have many more mRNAs than protein-coding genes

    

• Eukaryotic gene structure

  

  • Pre-mRNA contains functional elements not present in mature mRNA

—Chromosomes

• 2 kinds (autosomes and sex chromosomes)

  • Distinguished because inherited diseases from sex chromosomes have different behaviors

• We have 22 autosomes and X and Y sex chromosomes

  • Each autosome comes in two different versions in all your somatic cells — so 44

  • Plus XX or XY

  • Plus Mitochondrial

  • So we have 47 chromosomes

• Chromosomes are numbered in order of length (longest to shortest)

• Karyotype: picture of chromosomes in an individual

  • Take cells, grow them, and treat them with chemicals that cause them to arrest at the condensation stage

    • Isolate the chromosomes and try to match them up

      

• Each chromatid is a single DNA molecule

  • Two identical chromatids make a chromosome

    

• Chromosomes in the cell cycle

  

  • Checkpoints ensure chromosomes are made

  • In beginning of S phase, chromosomes have centromeres but aren’t paired

  • In late S phase, there are two DNA double helices per chromosome

  • In M phase, sister chromatids separate to give two chromosomes that are distributed into two daughter cells — you need 2 sister chromatids to divide

• Chromosomes are critical to the accurate transmission and regulation of genetic information

• Chromosomes are fundamental to the generation of variation

• Incorrect meiosis, mitosis, or mutation can result in abnormal genetic composition

• Stained chromosomes can be visualized and studied for major changes in chromosome structure

Chromosome structure

• Types of chromosomes

  

  • The first chromosome has 2 sister chromatids, the second one only has 1 chromatid

  • Centromere can be used as a landmark to describe the location of genes

    • P arm (short) and Q arm (long)

    • Chromosome is never in the exact center

      

  • As the centromere goes to the end of the chromosome, the number of genes in the p arm goes down

    • Acrocentric chromosomes tend to have special functions (13, 14, 15, 21, 22)

• Homologous chromosomes: one came from mom, one came from dad.

  • NOT IDENTICAL

• Mitosis

  • Homologs do not pair

  • Sister chromatids separate during anaphase

  • The daughter cells are diploid (2n) like the parent cell

• Meiosis

  • Meiosis I: Homologs pair and separate — recombination happens here

  • Meiosis II: Sister chromatids separate

  • Reduction division: the parent cell is diploid (2n), the daughter cells are haploid (n)

• KNOW MEIOSIS AND MITOSIS

Meiosis I

• Metaphase I: Homologs pair (p-p, q-q all along the chromosome)

  • Homologous chromosomes are driven to pair up

  • Crossing over also happens here (non-sister chromatids exchange genes from one homolog to another). It happens on average once every meiosis for each arm for each homologous pair

    • Recombination happens in a sequence-specific manner

      

• Anaphase: recombined chromosomes get pulled apart

  

Meiosis II

• Chromatids are separated out so gametes have only 1 set of chromosomes

  

Spermatogenesis and oogenesis

Independent assortment

• Genetic variation can be generated via independent assortment

• Happens when homologs line up on the metaphase plate

• A chromosome does not influence B chromosome

  

• How are there so many combinations?

  

  • 2²³ different sperm, regardless of recombination

  • Recombination

  • Mutation

  • Incorrect meiosis

Numerical chromosome abnormalities

• Polyploidy: Extra whole sets of chromosomes (dispermy of fertilization of diploid cell)

• Aneuploidy: one or more chromosomes extra or missing (non-disjunction)

  • Disomy: two copies of a chromosome, the normal state for most human cells

  • Trisomy: 3 copies of a chromosome

  • Monosomy: one copy of a chromosome

  • Mosaicism: when two cell lines with different karyotypes exist in a single organism

• Mixoploidy: Two or more cell lineages mixed (mosaicism or chimerism)

• Non-disjunction:

  • Most aneuploidies result from meiosis I errors

    

    • You end up with a cell with no chromosome, and a cell with both homologous chromosomes

    • In the second division, two gametes end up with 2 chromosomes (one mom and one dad), and two end up with nothing

  • In meiosis II errors, two gametes end up normal, one ends up with 2 identical chromosomes (both from mom or dad), and one has nothing

Chromosome mutations

• There are commonly humans with translocations

• In reciprocal translocation, nothing is lost or gained as long as the breakpoint doesn’t disrupt the function

  • Produces 2 complete chromosomes, they just don’t look how they are meant to

• The drive for homologous chromosomes to pair is sequence-based

• When there is a translocation, pairing up gets weird

  • Normal and balanced translocation will lead to a normal zygote

    • Leads to translocation carrier

  • Duplicated and deficient = some areas are duplicated while others are missing

1/23/2025

Origins of polyploidy

• fertilization by 2 sperms

• doubling chromosomes but not dividing (endomitosis)

• euploid=normal

Fluorescence in situ hybridization (FISH)

• Good for quick chromosome count, detection of known/suspected deletions/deletions

• Quick turnaround (24-48 hrs)

• Limited bu probe availability

• Targeted

Microarray analysis

• A way to measure the quantity of a given sequence of DNA in a sample

• Can be used to detect full or partial aneuploidy including mosaicism

• Standard testing during initial work up for a person suspected to have a genetic syndromeX$

• Doesn’t work on translocation

  

Nomenclature

• 46, XY = 46 chromosomes, male (normal)

• 47, XY+21 = 47 chromosomes, male, trisomy 21 (downs syndrome)

• 46, XX, del(22)(q11.2) = 46 chromosomes, female, deletion on chromosome 22 at band q11.2 (digeorge syndrome)

  

Common aneuploidies

• Trisomy 21 (Downs syndrome)

• Trisomy 18 (Edward syndrome)

  • Characterized by small size, unusual ear shape, small jaw, heart defects, usually lethal

  • Partial or mosaic form may be milder

• Trisomy 13 (Patau syndrome)

• Monosomy X (turner syndrome)

• XXY syndrome (Klinefelter syndrome)

• Trisomy 16

• Triploidy

  • An extra copy of every chromosome

  • Most common polyploidy in humans

  • Lethal in non-mosaic state

  • Most commonly caused by diandry

Case studies

1. You are asked to evaluate Lucy, a 14 month-old girl with a history of

   microcephaly, dysmorphic facial features, and developmental delays. In reviewing her family history you learn that she has two siblings both of whom have intellectual disabilities

   • You order a microarray for Lucy and get the following result...

     • She has a 9.4 Mb gain on the p arm of chromosome 4 (partial trisomy) and a 6.8 Mb loss on the p arm of chromosome 8 (partial monosomy)

     • Seems like one of her parents had a translocation — she had a duplicated and deficient zygote

   • You then order a karyotype of the parents

     • Dad is 46, XY

     • Mother is 46, XX, t(4;8)(p16;p23)

       • Translocation in 4 and 8 at breakpoints 16 and 23 respectively

       • But she’s normal because she has all of her genes (translocation heterozygote)

       • Transfer of chromosomal regions between non-homologous chromosomes has an incidence of 1/500

         

• The couple wants to have another child. What are their options?

  • Reproductive options

    • In Vitro fertilization with preimplantation genetic diagnosis

    • Gamete donation

    • Adoption

    • No kids

  • Post conception options

    • ultrasound

    • Chorionic villus sampling

    • Amniocentesis

    • Cell-free fetal DNA analysis

Translocations

• Balanced: No gain or loss of genetic material at the translocation breakpoints

• Unbalanced: Some gain or loss of genetic at the translocation breakpoints;

  this may result in phenotypic anomalies

• Translocation Carrier: an individual who carries a balanced chromosome

  translocation; phenotypically normal (unless the breakpoints disrupt gene

  function)

• Origin of Translocations

  • Inherited: translocation carriers may pass the balanced (or unbalanced) form of a translocation onto their offspring

  • De Novo: a balanced or unbalanced translocation may arise through recombination in non-sister chromatids during (meiosis) or shortly after conception (mitosis)

• Autosomal Reciprocal Translocation:

  • The exchange of segments between two non-homologous (heterologous) autosomal chromosomes

  • Balanced carriers are generally unaffected

  • Miscarriage risk: 20-30% on average for female carriers (but this varies greatly by translocation

• Sex Chromosome Translocation:

  • The exchange of segments between one sex chromosome and another chromosome

  • The second chromosome may be an autosome or a sex chromosome (rare)

  • Generally causes infertility

• Robertsonian Translocation: the fusion of the long (q) arms of two acrocentric

  chromosomes (13, 14, 15, 21, 22); the short arms (p) are lost

  • Heterologous Robertsonian: A Robertsonian caused by the fusion of the q arms of two different acrocentric chromosomes

  • Homologous Robertsonian: A Robertsonian caused by the fusion of the q arms of two copies of the same chromosome

    
    

• Impact of translocations

  • Unbalanced translocations

    • Generally cause birth defects and intellectual disability

    • The larger the monosomic/trisomic segment the more severe the phenotype

  • Balanced translocation carriers

    • Generally healthy unless one of the breakpoints disrupts a gene

    • 20-30% risk for miscarriage with each pregnancy but may be as high as 50+% (general

      population risk is 15%)

    • The risk for miscarriage is generally higher if the translocation carrier is female

    • Males are at risk for oligospermia\

    • If a balanced de novo translocation is identified prenatally there is a 6% risk for an abnormal phenotype in the baby

IVF with PGD

• IVF: collection of sperm and egg samples allowing for fertilization outside the uterus, promising embryos are implanted in the mother

• PGT-A: testing done on embryos at the 8-cell stage, frequently via FISH, to look for imbalance, only balanced (normal or carrier) embryos are implanted

CVS and amniocentesis

• Amniocentesis: 1/300 wish of miscariage, tests amniotic fluid

• CVS: chronic villi, 1/100 risk of miscarriage

• FISH may be used to obtain a preliminary result. Microarray is fast replacing karyotyping as the standard of care for prenatal samples

• Cell-free fetal DNA analysis

  • Analysis of cell-free DNA in maternal circulation to assess for fetal aneuploidy

Case study 2

• You are asked to see Pamela, a 27 year old pregnant woman who is currently

  19 weeks. She had her fetal survey at her obstetrician’s office and the baby

  was noted to have short long bones, an absent nasal bone, and pelviectasis.

  She will be meeting with you prior to her targeted fetal anatomy ultrasound

  to discuss the findings and her options. Her husband, Trevor, accompanies her

  today

  

• They opt for an amniocentesis

  • Result: 46, XY, rob(21;21),+21

  • Rob=robertsonian translocation (acrocentric, one chromosome is twice as long)

    • Leads to Down syndrome

• They then opt for karyotyping

  • Pamala is 45,XX,rob(21;21)

  • This means she has just 1, really long 21st chromosome

  • She will always have nondisjunction — can only make gametes with no chromosome 21 and ones with 2

    

1/28/2035

Mendelian genetics

• Key concepts

  • Inheritance of a characteristic follows mendel’s rules if its presence or absence is determined by the genotype at a single locus

  • Mendelian characteristics can be dominant, recessive, or co-dominant. A characteristic is dominant if evident in a heterozygote

  • Human Mendelian characters give pedigree patterns that are often recognizable

• Most characteristics are actually polygenic (multifactorial)

  

• Variable expressivity: a trait or disease can manifest differently among affected individuals

• Phenocopy: a phenotype that mimics the trait of interest but does not share the genetic etiology

• Disease alleles of genes are rare

  • Subject to certain conditions, allele frequencies and genotype frequencies are related by the Hardy-Weinberg formnalle=a

  • p+q=1

  • p²+2pq+q²=1

Inheritance patterns

• Autosomal recessive

  • Usually only one generation affected

  • Carriers are not affected

  • No association with sex

  • Children of a carrier couple are at 25% risk

    • Unaffected children have a 2/3 chance of being a carrier

  • Parents of a recessive child are obligate carriers

  • Consanguinity increases the risk for AR conditions

    • Fancy word for incest

  • Examples

    

• Autosomal dominant

  • Multiple successive generations (but may arise de novo)

  • NO association with sex

  • Children of an affected individual at 50% risk

  • Examples

    • Huntington’s disease

    • Treacher Collins syndrome

• X-linked

  • Multiple generations affected

  • Males tend to be more severely affected than females

  • Can be dominant or recessive but usually there is a broad spectrum

  • All daughters of affected males will be caries/affected

    • No male-male transmission

  • Each child of an affected female is at 50% risk

  • Examples

    

  • Pedigree is maybe a little unexpected — Mosaicism makes it so that women

    will sometimes express the diseased X

    • Which X (the paternal or maternal copy) is chosen for inactivation in an XX embryo is determined randomly and independently by each cell of the embryo, at the 10-2 cell stage

    • However, once a cell has chosen which X to inactivate, that X remains inactive in all its daughter cells

      

    • Barr bodies

      • XY males keep their single X active (no Barr body)

      • XX females inactivate one X in each cell (one barr body)

      • Females with turner syndrome (45,X) do not inactivate their X (no Barr body)

      • Males with Klinefelter syndrome (47,XXY) inactivate one X (one Barr body)

      • 47,XXX females inactivate two X chromosomes (two Barr bodies)

• Y-linked

  • Only present in Males

  • Only male-male transmission (females cannot be carriers)

  • All sons of affected male will be affected

  • Super rare

  • Example

    • Spermatogenic failure

• Mitochondrial

  • All affected children of an affected female will be affected (regardless of sex)

  • Affected fathers will not have affected children

  • Significant variation in severity with a tendency to get worse with each generation

  • Mitochondrial diseases caused by mutations in the nuclear genome follow an autosomal pattern of inheritance

    

  • Mitochondrial genes

    • MELAS

    • Leigh syndrome (also in nuclear genome)

  • Example: mitochondrial myopathy

  • Treatment

    • Mitochondrial replacement

Mendelian characteristics vs genes

• Locus heterogeneity: Sam clinical phenotype from mutations at any one of several different loci. Also called clinical heterogeneity

  

• Allelic heterogeneity: different mutations at the same locus result in the same clinical phenotype i.e. different mutations in the same gene may cause loss or gain of function leading to a specific mendelian disease

Mendelian complications

• Non-paternity

  • When the stated after is not the biological father

  • May be uncertain

  • Difficult to address with families

• Ascertainment bias

  

  • Not ¼ but 8/14

• Common recessive looks like a dominant

  • O is recessive but also really common

    

• Non-penetrance

  • Parent has condition but it doesn’t show up in the phenotype

• New mutations and mosaicism

Key concepts

• Gonadal mosaicism

  • When the gamete population of an individual is mosaic for an allele

  • Can cause an unaffected individual to have multiple children with an autosomal dominant condition

    

  • Chimerism

    

1/30/2025

Case study 1

• You are as ked to evaluate Alex, a 2 year old boy with a history of easy bleeding and bruising. As the family is very wealthy and influential you agree to make a house call. At the family home you meet Alex’s parents, Lexi and Nick, and his four older sisters. Lexi repor ts the following family history

  

• Hemophelia is x-linked, however about half of cases result from de novo mutation

• Deficiency of clotting factors 8 and 9, found at F8 and F09 genes

  • Locus heterogeneity

• You can test factor 8 and 9

  • Not able to diagnose 90% of female carriers

• Female carriers can be symptomatic and may be at-risk for bleeding especially during childbirth

Case study 2

• Maryann, a 45 year old woman, comes to yo ur clinic to request testing for a familial condition that causes dementia and involuntary movements . She reports that her father w as diagnosed at age 63. Her brother was also diagnosed 5 years ago

  

• Huntington is autosomal dominant and found on chromosome 4

  • Caused by a CAG repeat expansion in the HTT gene

  • 3 nucleotides are repeated over and over again — highly unstable

  • Normal alleles: 26 or fewer CAG repeats

  • Intermediate alleles: 27 – 35 CAG repeats; will not develop HD but

    may have children with HD due to anticipation

  • Reduced-penetrance alleles: 36 – 39 CAG repeats; some individuals

    in this range will never develop symptoms

  • Full-penetrance alleles: 40+ CAG repeats; 60+ CAG repeats may

    result in juvenile HD

  • Increasing repeat size is associated with earlier onset of symptoms

  • Easy to test for

• How did we figure out where the huntingons’s allele was?

  • Crossing over

  • Need a lot of families where the disease incidence is high

  • In one part of venezuela, there was a small founder population, and now the incidence rate is 1/125

Case study 3

• Omar and Nadia come to your clinic to discuss a family history of intellectual disability and dysmorphic facial features. Nadia is 28 weeks pregnant

  

  • Autosomal recessive

  • Consanguinity increases the chance for homozygosity by descent

  • Everyone has about 5-7 deleterious recessive alleles

  • Consanguinity increases the chance to have a child with an autosomal recessive condition

  • Increases the risk for birth defects that are not part of a

    known genetic syndrome

Case study 4

• Sally’s daughter, Claire, has been diag nosed with a rare, autosomal recessive condition called Random syndrome. Sally and Claire’s father are divorced. Sally and her new husband, Bernard, have just learned they are pregnant and are worried about the risk that their baby will also have Random syndrome.

  

• You know that that incidence of random syndrome is 1/14,400 but a

  carrier frequency has not been reported.

  Provide Sally and Bernard with a recurrence risk estimate for their baby.

• To calculate the risk to a pregnancy for an autosomal recessive disease you multiply

  the chance that each parent is a carrier by the ¼ chance that two carrier parents would both pass on the mutation (which is always ¼).

  • We know that Sally has to be a carrier

  • The incidence rate in the population is 1/14,400

  • 1×bernards chance of being a carrier×1/4

• Practical hardy-weinberg

  • The carrier frequency of an autosomal recessive disease in the general population can be determined from the disease incidence

    

  • So Bernards chance to be a carrier =2pq

    

  • Since p+q=1, and q is so small, we can assume that p=1

    

• Probability that the baby is a carrier = 1×1/60×1/4

  • 1 in 240

Ethnicity-based carrier screening

• Northern European Caucasian — cystic fibrosis

• French Canadian and Cajun — Tay Sachs disease

• Ashkenazi Jewish ancestry

  • Much of the US Jewish population

  • Evidence suggests that the Ashkenazi Jewish population bottlenecked and experienced founders effect

• Dor Yeshorim

  • International genetic screening system

  • Adults submit a blood sample tested for common autosomal recessive diseases

  • each participant receives an identification number but does not get the results

  • When two people are considering an engagement or when a rabbi is attempting to arrange a marriage, the identification numbers can be submitted to determine if the couple is compatible (not carriers for the same condition)

• Pan-ethnic carrier screening

  • Screening for many different genetic conditions regardless of ethnic background

2/4/2025

Birth Defects

• Birth defect = congenital anomalies

  • It can be major or minor

• 3-5% incidence rate

• Causees

  • Sporadic: usually isolated within a family, unknown etiology, not expected to be inherited

  • Chromosomal syndromes

  • Genetic syndromes

  • Teratogens

  • Positional

• Vocab

  • Isolated: not associated with other anomalies, etiology might be known, may be inherited

  • Syndrome: a well-known pattern of anomalies believed to have the same cause, often genetic/chromosomal but may be environmental

  • Sequence: a group of related anomalies believed to be caused by a single major anomaly that alters the development of other structures

Common major anomalies

• Heart defects

  • 1/200

  • Usually sporadic

• Neural tube defects (spina bifida)

  • 1/1000-1/200

  • Usually sporadic

  • May be environmental

• Club food

  • 1/1000-1/500

  • May be positional

• Pyloric stenosis

  • 1/500 males, 1/1000 females

• Clef lip

  • 1/1000-1/500

  • Usually sporadic by also associated with many genetic and environmental syndromes

• Gastroschisis

  • 1/10000

  • Usually sporadic, may be related to maternal age or smoking

Common minor anomalies

• 2,3-toe syndactyly

• Occular anomalies

  • Hypoterlorism/hypertelorism (eyes being close together or far apart

Syndrome vs sequence

• Syndrome: Achondroplasia

  • Dwarfism

  • Comes with short limbs, frontal bossing, low nasal bridge

  • FGFR3 gene mutation

• Sequence: Oligohydramnios sequence

  • Potters sequence

  • Limited lung development, joint conttractures

  • Low amniotic fluid causing fetal compression

  • Symptoms are all the result of one abnormality

Heirarchy!

Organelles

• Peroxisomes

  • Participate in energy metabolism

  • Replicate independently of the cell but do not have their own genome

  • Break down uric acid and other waste products

  • Lipid biosynthesis

  • Cause Zellweger syndrome

    • Autosomal recessive

    • 12 peroxin genes (locus, allelic, and clinical heterogeneity)

    • Low muscle tone, dysmorphic features, seizures, lover disease, usually die within the first year of life

Cellular structure

• Demyelinating diseases

  • Loss of the myelin sheath

  • Neurologic deficits due to failure to transmit signals across the neuron

  • Can be reversible

  • Causes:

    • B12 deficiency

    • Tay Sachs disease

    • Biral infection

    • MS

    • Alcholol

Skin Cells

• Epidermis, dermis, and hypodermis

• Protects against environment

• Sensory

• Fat storage

• Vitamin production

• Epidermolusis bullosa

  • Extreme skin fragility with blistering

  • Healing around the digits may lead to fusion of the fingers and toes

  • Mucosal tissues may also be involved

  • Teeth and nails fall out

embryology

• The zygote in each cell in very early stage vertebrate embryos can give rise to every type of adult cell

  • Stem cells

• 

• Fetal alcohol syndrome

  • Effect dependent upon exposure amount an timing

  • Changes to facial features

  • Intellectual disability

Case study

• Melody and Kevin have just given birth to their second child, a

  daughter named Fern. The family has recently moved

  to the area and Melody’s prenatal records have not yet been

  transferred. The couple shares with you that they were told that

  their baby would have cleft lip and palate and might have other

  concerns as well. You have been asked to evaluate the baby

  • MRI shows semi lobar holoprosencephaly

    

  • Holoprosencephaly

    • Abnormal development of the forebrain leading to incomplete separation of th cerebral hemispheres

2/5/2024

Genetic variation

• Genetic variation is measured at 2 levels

  • Within a population (among individuals)

    • Consider population history/current size

    • Big populations tend to have more diversity

    • Populations that have gone through a bottleneck tend to have less diversity

  • Among populations (of the same species)

    • Consider gene flow/connectivity

Types of genetic variation

• Chromosomal

  • Karyotypes describe what your chromosomes look like

  • Sex chromosomes vs autosomes

  • Haploid = n

  • Diploid = 2n

  • heterogametic = different sex chromosomes (XY)

  • homogametic = same sex chromosomes (XX)

  • Variation in chromosome number can be problematic for inter-breeding

    • horses: 2n=64, n=30

    • donkeys: 2n=62, n=31

    • mule: 2n=63, no n — infertile

      • one chromosome can’t pair up in meiosis

    • But things get weird! sometimes hybrids with odd numbers of chromosomes can reproduce

  • Chromosome variation is problematic during meiosis

    • Genetic information does not align during crossing over; interferes with gamete production

  • Chromosomes can fuse together, so you can have species within the same genus with very similar DNA sequences but different numbers of chromosomes

  • Polyploidy: Have different numbers of sets of chromosomes

  • Chromosomal rearrangements

    • Inversions

      • Chromosomal inversion separates annual and perennial ecotypes of monkey flower

      • Adaptive differentiation

    • Translocations

• Mitochondrial DNA

  • Useful for phylogenetic questions

  • Only maternally inherited

    • Eggs are big, sperm are small

    • There are many more mitochondria in the egg

  • It doesn’t vary much between individuals of the same family

    • Can be useful for separating groups

    • Whole genome acts as one locus

  • You can use mitochondrial DNA as a marker when you can’t with nuclear DNA bc you will always have more copies of the mitochondrial DNA

  • Used to look at differences between Ridley Sea Turtles across the world

  • Can also be used to look at differences among species (phylogenetic perspective)

• Chloroplast DNA

  • Useful for phylogenetic questions

  • Only maternally inherited

  • Chloroplast DNA variation resolved species relationships in the carrot/parsley family

  • Assaying variation in mitochondrial or chloroplast DNA

    • Sequence a gene region or the whole mitogenome (DNA sequencing)

    • Restriction enzyme analysis of a known polymorphism (genetic marker)

• Nuclear DNA

  • Biparental inheritance

  • Assay for nuclear DNA variation

    • DNA sequencing (whole genome sequencing)

    • Genetic marker approaches

      • Microsattelites:

        • Highly variable due to high mutation rate

        • Multiallelic

        • Typically neutral (not under selection)

        • Alleles differ by length of sequence

          • Made up of sequences of repeated blocks of DNA

• Genetic markers - microsatellites and SNPs

  • SNPs: Single Nucleotide Polymorphisms

    • Arise from point mutations

    • Bi-allelic

    • Abundant in the genome (millions)

    • Easily identifiable with modern genomic methods

2/6/2025

Twinning

• Monozygotic (identical) twins

  • Arise from one fertilized egg which splits into two embryos

  • Genetically identical at conception

  • Always same sex

  • Complications

    • Monochorionic diamniotic: twins that share a placenta but are in different amniotic sacs

      • Twin-to-twin transfusion system: Flow of blood from one twin to another causing severe stress on both babies systems

    • Monochorionic-monoamniotic: twins that share a placenta and are in the same amniotic sac

      • Can lead to cord entanglement or conjoined twins

    • Discordance for a genetic condition: de novo mutations can happen after the embryo splits, leading to one twin with a genetic disorder

• Dizygotic (fraternal) twins

  • Arise when two eggs are ovulated and fertilized

  • Genetic full siblings

2/13/2025

Cancer

• All cancers are genetic BUT cancer is very rarely inherited

• only 5-10 percent of all caners are caused by inherited mutations

• Mutations arise in somatic cells

Six features of cancer cells

1. Independence of external growth signals

2. Insensitivity to external anti-growth signals

3. Ability to avoid apoptosis and autophagy

4. Ability to replicate indefinitely

5. Ability of a mass of such cells to trigger angiogenesis and vascularize

6. Ability to invade tissues and establish secondary tumors

Important concepts

• We have several protective systems

  • Repair

  • Apoptosis

  • Immune response

• We need several mutations to convert normal cell to tumor cells

• Low probability except

  • Some mutations enhance cell division

  • Some mutations affect genome stability

  • Some mutations reduce immune response

Cancer mutations

• Multi-stage evolutionary process

• You need something to select for cancer expansion, or something that stops you from getting rid of the cancer cell

• Cell cycle dysregulation is the cardinal feature of cancer cells

  • In G1, RBP1 TP53 and CDKN2 play key roles

  • Associated with somatic and familial changes in tumor cells

Genes that result in acquired cancer cell

• Oncogenes — “gas pedal”

  • When they are normal, they promote cell proliferation

  • Gain of function mutations result in forms that are too active

    • 

    • Point mutations decrease activity increasing signal in pathway

      • GTP-RAS, MAP kinase pathway is on all the time

    • Rearrangements can activate oncogenes

      • Philadelphia chromosome: A balanced translocation 9;22 found in 90% of people with chronic myeloid leukemia — BCR-ABL1 chimeric tyrosine kinase fusion protein is always active.

      • In Burkitt lymphoma 8;14 translocation places MYc in transcriptionally active region - -MYC is upregulated under the influence of the normally highly expressed immunoglobulin genes IGH expresss

  • Discovered as part of retroviruses capable of transforming cells

    • Reverse transcribe DNA — form of duplication

    • Oncogenes turned out to be copies of normal cellular genes (proto-oncogenes).

    • 15% of cancers are caused by retroviral infection

• Tumor suppressor genes — “brake”

  • Normal activity limits cell proliferation, likks incorrigible cells, and maintains genome intgrity

  • loss-of-function mutations lose ability to control cell proliferation

    

  • Retinoblastoma is the paradigmatic TS gene and the two-hit hypothesis

    • You get a tumor when you get a second mutation

    • Inherited or sporadic mutation

    • Recessive but behaves like it’s dominant due to two hit hypothesis

      • Recessive at the cell level, dominant at the retina level

      • If you inherit the bad allele, you will get the sporadic mutation as well

        

    • Rb1 binds and inactivates E2F stopping progression into S phase

Mechanisms for loss of wildtype allele in RB

• Top of diagram is heritable heterozygous state. 2 markers flank the gene

  • A1 and A2 and B1 and B2 are different sizes

• N=normal. T=tumor

• Non-disjunction:

  • One chromosome with the RB gene, other chromosome is absent

  • Doesn’t have to be a mutation — this cell is homozygous for RB

• Non-disjunction+ duplication

  • Same as ^ Mutated chromosome is duplicated

• Mitotic recombination

  • Chromosome 2 has recombined and grabbed the part of chromosome 1 with RB

• Deletion: delete the part of the wild type chromosome with the allele

• Second mutation:

• Inactivation by methylation

LOH

• LOH= loss of heterozygosity

• Used as a marker to locate tumor suppressor genes

2/18/2025

Cancer facts

• 77% occurs in people over 55

• 5 year survival rate is 67%

Cervical cancer

• 12,000 women per year

• Caused by HPV

  • Strains 16 and 18 most likely to cause cervical cancer

  • Most common sexually transmitted virus in the US

• Virus invades epithelial cells, DNA is integrated into genomes

  

  • P53 is a tumor suppressor gene

• Prevention

  • Don’t have sex

  • Cervical cancer vaccine

The philidalpihia chromosome

• Chromosomal translocation that generates a kinase involved in the signal transduction cascade that is always on

• t(9;22)(q34;q11)

• Involved in leukemia

• Somatic (not germline) event

  • Cannot be inherited

• 3 clinically important isoforms associated with different cancer types

  • Acute lymphoblastic leukemia (ALL)

  • Chronic myeloid leukemia (CML)

  • Chronic neutrophilic leukemia (CNL)

• BCR-Abl fusion protein

  • Tyrosine kinase always on

  • Increased speed of cell division

  • Inhibits DNA repair (causing genomic instability)

• Cleevec

  • Treatment for the Philadelphia chromosome

  • Tyrosine kinase inhibitor — targets BCR-Abl protein

  • Doesn’t affect other tyrosine kinases

  • Only effective in Ph-positive CML

Inherited cancers

• 5-10% of cancer is inherited

• Young age of onset

• Organs affected bilaterally

• Multiple family members affected

• Multiple primary cancers

• Primary cancer: Arose as the result of a new event in the affected tissue

• Recurrent/Secondary cancer: The result of the incomplete eradication or metastatic of another cancer

Retinoblastoma

• Unilateral cases have an average onset of 24 months

• Bilateral cases have an average onset of 15 months

• Inherited retinoblastoma

  • RB1 gene

    • Tumor suppressor gene

    • Up to 8% will have a deletion that could also cause intellectual disability and birth defects

  • Autosomal dominant at the level of the organism, autosomal recessive at the level of the cell

  • 95% survival rate with treatment

  • Other cancers can also be associated

Breast/ovarian cancer

• Women have a 12% risk of breast cancer, 1.4% ovarian

• Inherited breast-ovarian cancer incidence: 1/800-1/2500

• 5-10% are caused by germline mutations (inherited) in BRACA1 and BRACA2

  

• BRCA

  • Expressed across many cell types

  • Mutations cause loss of gene function —> tomorrow suppressor genes

  • Suspected involvement in DNA repair

• Management

  • Climical breast exam

  • Mammography

  • Breast MRI

  • Ultrasound (ovarian)

  • Blood test (ovarian)

Colorectal cancer

• 5% risl

• Up to 33%B of colorectal cancers are caused by germline mutation but only 3% are highly penetrant

• Lynch syndrome: Caused by germline mismatch repair gene mutations

  • Immunohistochemistry (ICH): Tissue staining procedure, detects the presence or absence of proteins produced by mismatch repair genes

  • Microsatellite instability testing (MSI): Evaluation for instability in the size of microsatellite markers. More accurate than IHC

    

Li-Fraumeni syndrome

• “huntingtons disease of cancer”

• Incidence as high as 1/5000

• Caused by TP53 tumor suppressor gene

• Cuases many different cancer types

• 50% of affected individuals will have cancer diagnosed before 30 years old

Multiple endocrine neoplasia

• MEN1: Wermer’s syndrome

• MEN2A: Sipple syndrome

  • Incidence 1 in 35000

  • Autosomal dominant

  • RET gene, 10q11.2

    • Protooncogene

    • Regulates cellular environmental responses, promotes growth and division

    • Mutations over-activate the gene (tyrosine kinase is always active)

    • RET gene mutations also cause Hirschprung disease

• MEN2B:

Liquid biopsy

• Used to test fetal genome from mother’s blood

• Used for tumor diagnosis — tumors have genomic instability

2/25/2025 Analyzing genomes using technologies

testing

• Diagnostic testing: confirms or rules out specific genetic condition

• Predictive and pre-symptomatic testing: used to detect mutations associated with disorders that appear after birth but before symptoms (ex: BRCA)

• Carrier testing: used to identify individuals who carry one copy of a recessive condition

• Preimplantation genetic diagnosis (PGD): used in in vitro fertilization to identify embryos without genetic abnormalities for implantation and pregnancy

• Prenatal testing: Used to detect changes in a fetus’s genes or chromosomes before birth (amniocentesis or chorionic villus sampling)

• Newborn screening: Testing newborns for disorders (PKU or phenylketonuria)

Testing methods

• PCR: makes millions of copies of a target DNA sequence from very small sample and is the base technique for most genetic testing

  • Key techniques in most genetic tests

  • YOU WILL BE ASKED TO DESIGN A PCR EXPERIMENT ON THE EXAM

    

  • Directions are important (replication goes in 5’ to 3’ direction)

  • New bases get added to 3’OH

• Next generation DNA sequence: Sequencing of an individuals entire genome

• Microarrays (DNA chip technology: Analyze gene expression and detect SNPs. Basis of 23 and me

• Karyotyping: Visual examination of chromosome number and structure

• FISH: uses fluorescent probes to target specific sequences to assay for specific sequence

• Biochemical genetic testing: examines the amount or activity level of proteins that can indicate abnormalities

  

Types of variation

• SNPs

• Deletions and duplications

  • Indel: deletion and duplication <1000 bases

  • CNV: deletion and duplication >1000 bases

• Trinucleotide repeat expansion (TNR): increased number of trinucleotide repeats in a given area of a gene (Friedeich ataxia, Huntington

• Methylation: Addition of methyl groups to DNA can turn the gene off

• Mitochondrial DNA variants (MtDNA). Changes to the DNA in the mitochondria

Clinical genet tests

• Single gene testing: Single gene testing is done when there are symptoms of a

  specific condition or syndrome. (i.e. Duchene muscular dystrophy or sickle cell

  disease) or when there is a known genetic mutation in a family.

• Panel testing: Looks for changes in many genes in one test and are usually

  grouped in categories based on different kinds of medical concerns (i.e. low

  muscle tone, short stature, or epilepsy or risk of developing breast or colorectal

  (colon) cancer

• Large-scale genetic or genomic testing. There are two different kinds of large-

  scale genetic tests.

  • Microarrays and SNP arrays looks for INDELs and predetermined SNP

  • Whole Exome sequencing looks at all the expressed parts of genes in the DNA.

  • Whole Genome sequencing looks at all of a person’s DNA, not just the genes.

Large scale genotyping in humans

• Screening

  • DNA microarrays

  • Focused genome sequencing

    • SNP chips

    • PCR amplification

    • Hybridization capture

  • WGS

• Sxperimenting

  • Transcription profiling

    • RNA arrays, RNA seq, qPCR, ddPCR

  • Epigenetic analysis

  • Protein DNA interactions (ChIP-seq)

Human genetic variation

• Chromosome variation: typically limited to larger deletions, duplication. and translocations 5Ml>

• Segmental values (copied number variation): 1kb-5kb

  • Most but not all are associated with segmental duplications

  • Responsible for most differences (per base tail) between humans

• Single nucleotide polymorphisms (SNPs)

  • Base substitutions and small Indels

• COULD BE ON EXAM

• 

Microsatellites (SSRs)

• Several short repeated motifs

• Highly polymorphic

  • Good genetic marker

  • Good forensic tool

• Sometimes directly related to disease

  • Huntingtinon’s and fragile x

• Assay by PCR and length analysis

• Not all trinucleotides

  

  • Different lengths=different alleles

PCR

• How do we identify and detect a specific gene sequence in a genome?

• 2 big issues

  • There are a lot of other sequences in a genome that we’re not interested in detecting (specificity)

    • 1/4ⁿ is the probability of finding a sequence at random

  • The amount of DNA in samples we’re interested in is very small (amplification). Contamination is very dramatic

• Type of question that will be on the midterm

  

  • Mark the orientation of the forward and reverse primer

2/28/2025

• exam question: match red and blue lines with the PCR

Testing

• Microarrays are useful for detecting DNA deletions and duplications

Sequencing

• Traditional DNA sequencing/chain termination quequencing/sanger sequencing

  

  • Separate strands of different lengths by gel electrophoresis

  • Used dideoxyribonucleic acid — H on the 3’ carbon means it won’t bond with anything else

• Next generation DNA sequencing technologies

  • Much cheaper — don’t have to run gels

  • It is still expensive to analyze the genome

• Sequencing by Synthesis (Illumina)

  • Second generation sequencing

  • Goal is to monitor replication

    

  • P5 and P7 (red and green) are primers sites

  • Sequence on the inside surface of a flowcell

  • Primers are covalently attached to the glass

    
    

  • Needs to be amplified with PCR as well’

  • Bridge PCR

    

    • Just like normal PCR except primers are stuck to the glass

    • End up with clusters of identical DNA molecules

    

  • Need to get rid of color

    

  • Illumina stargazing

    

• PacBio

  • A bunch of wells in a plate

  • At the bottom of the wells there is a hole

  • Holes are such a size that we can look in but nothing will come out

  • Incorporating fluorescent nucleotides

  • Single molecule real time sequencing (SMRT)

    

• Nanopore sequencing

  • You have a protein hat unzips the DNA and pumps the sing strand through a pore in a membrane

  • Measure change in electrical activity across the membrane at every pore

  • Electrical activity changes for each nucleotide

  • Really long reads

  • MinION field sequencer

KNOW THE 4 SEQUENCING TECHNOLOGIES

Array based genotyping

• Arrays contain oligonucleotides that test the seuqnce of a sample

• Arrays target the test to a set of loci

• They function based on the discrimination of complementary base pairing or the ability to extend a base at a specific location

• Transcription profiling

  • Arrays can be used to measure the amount of RNA produced by a set of genes

  • Sequencing can be used to measure expression of a genome

Epigenetics

• Methylation — histones influence what portions of the genome are available for transcription

• bisulphite sequencing

  

  • If the C is not methylated, it switches it to be a U

  • Everywhere C that is remaining you know is methylated, so you can map these points

ChiP-seq

• Used to study protein DNA interactions

3/4/2025

Standard of care

• Standard of care: that which a minimally competent physician in the same field would do under similar circumstances.

• Not intended to replace the good judgement of clinicians

Cell free fetal DNA testing

• Can be used to detect gender as early as 8 weeks

  • Female is more susceptible to error

• Takes blood sample from mom

Karyotyping vs array

• Both detect aneuploidy and segmental variation

• Microarray can detect much smaller CNVs than karyotype

• Karyotype can detect balanced translocations (microarray can’t)

Case 1

• You are as ked to evaluate Sally, 2 years old, for a history of Tetralogy of Fallot, clubfoot (bilateral), failure to thrive, and recurrent infections. She is otherwise healthy and has shown normal development. Her parents report the following family history.

• Teralogy of fallot

  • Heart defect

  • Strongly associated with 22q11.2

  • 6% of isolated cases of ToF have 22q11.2 deletions

• 22q.11.2 deletion syndrome: digeorge syndrome

  • Incidence 1/4000

  • Can test using karyotyping or FISH or microarray, but microarray is standard of care

    

• For sally, the microarray identifies a 22q11.2 deletion

  

  • Next step is to offer testing for other family members via FISH

Case 2

• Betty is 19 weeks pregnant and has been referred to your practice. Her obstetrician is concerned because a recent ultrasound showed very small nasal bone, small cerebellum, choroid plexus cyst, and a single umbilical artery. Your ultrasound confirms the fetal findings. Betty and her husband, Luke, report that there is no significant family history

  • Small nasal mone — Down

  • Choroid plexus cyst — Trusomy 18

  • Single umbilical artery — could be anything

• Options

  

  • Microarray is best option

• A 5p15.2-p15.33 deletion is detected: Cri-du-chat (cat cr y) syndrome

  • 1/20000 -1/50000 incidence

  • Newborns make cat like cry

  • Microcephaly

  • Severe intellectual and motor disabilities

  • Dysmorphic facial features

• Next step: find a critical region

  • We know this is a spontaneous event

    

    • Look at where deletions actually span. Places critical region for cat-like cry phenotype in the 15.2 area

Case 3

• Edward is a 7 year old boy with a history of microcephaly, atrial septal defect, long palpebral fissures, large ears, polydactyly, intellectual disability, elevated liver enzymes, mild anemia, and several food allergies including eggs and

  wheat. He has already had an extensive genetic work -up which as been normal thus far. You are seeing Edward and his mother, Rhonda, for follow-up

• Tests already done

  • Karyotype: 46,XY

  • Fragile X syndrome: normal

  • MECP2 analysis (atypical Rett syndrome): normal

  • L1CAM analysis (X-linked hydrocephalus): normal

  • Williams syndrome: normal

  • Mitochondrial function studies : normal

  • Microarray: normal

• Options

  

  • Whole exome sequencing is the best option here

The Rominovs

• Determining who was in the mass grave

• Focused on mitochondrial DNA

  • We understand it’s the inheritance patterns

  • Evolves rapidly

• Mitochondrial DNA testing showed that Prince Philip shared a common female ancestor with Alexandra and three of her daughters from the first grave

  • Also showed that Duke of Fife and Princess Xenia shared a common female

    ancestor with Nicholas.

  • Nicholas’ remains showed a single point heteroplasmy in the mtDNA (16169 C/T) that was not seen in his maternal relatives (16169 T). This rare

    heteroplasmy was shared by his brother, Grand Duke Georgij Romanov.

• But

  • The remains of only 3 children were discovered in the first grace, Alexei and one sister were missing

  • Found the second grave in 2007

• The second grave

  • Forensic anthropological analysis

    • The second grave contained two people

    • 15-19 year old female

    • 12-15 year old male

    • Ethnicity and height could not be determined for either victim

    • Silver fillings showed that at least one victim was an aristocrat

    • The grave was over 60 years old

  • DNA analysis

    • DNA was extracted from several bone sites (mostly long bones)

    • PCR was used to amplify and quantify (real-time PCR - qPCR) the

      extracted DNA

    • Multipex PCR was used to used to amplify and sequence the mt DNA

    • DNA from the Y chromosome was also amplified using PCR

  • Samples from the second grave matched the mtDNA studies from the first grave and from prince Philip

  • Genotyping results

    

    • Markers are microsatellites

    • Found using PCR — design primers to look for specific primers

  • Y-linked marker results

    

  • Conclusions

    • All of the Ramonov family were murdered on July 17, 1918 and buried in two separate mass graves

DNA forensics

• CODIS

  • CODIS: Combined DNA Index System

  • Operated by the FBI

  • Contains DNA profiles from convicted offenders and arrestees in states where collecting this data is legal

  • Contains DNA profiles found at crime scenes

  • Only works if everyone decides to use the same markers!!

• Process

  • Law enforcement agencies submit DNA profiles derived from samples

    taken as part of a criminal investigation

  • Sample DNA profile is compared to the CODIS database

  • If a match is found in the Convicted Of fender/Arrestee database, it is usually possible to obtain a warrant to collect a sample from the matched an individual for a reference sample

  • If a match is found in the Forensic Index the crime can be linked to

    one or more other crimes

• Accepted DNA profiles

  • STR (short tandem repeats): 20 loci

  • Y chromosome STR

  • Mitochondrial DNA (mtDNA)

  • Second 2 only ised to identify missing persons

• GEDmatch

  • Online tool

  • Users submit their raw DNA for analysis and comparisons with other users

  • Has been used by law enforcement to identify suspects and missing persons

  • Has a lot of data from both ancestry and 23andme

  • Open to everyone

The Golden State Killer

• Joseph James DeAngelo murdered at least 12 people

• Cold case for 44 years

• Arrested in 2016 at age 72

• No match for DNA sample in CODIS

• Comparison of DNA taken from a crime scene was submitted to GEDmatch and several partial matches were made

• Degree of relationship to the sample DNA was inferred from the amount of similarity between the sequences — people who he was related to

• This provided a narrower field of suspects
  

Brave new world

• Analysis of a DNA sample taken from a crime scene can be used to infer ethnicity and physical characteristics

• Modeling software was used to show the effects of age

Paternity testing

• Analysis of 10-20 markers

3/6/2025

Exam info

• modules 4 5 and 6

New genes come from gene duplication

• Mechanisms of gene duplication

  • Tandem gene duplication (direct or inverted) unequal crossing over (homologous chromosomes) or unequal sister chromatid exchange (same chromosome)

  • Duplicative transposition (typically retrotransposition)

  • Ancestral cell fusion (think endosymbiont theory)

  • Large-scale, sub genomic duplication (segmental duplication inter and intra chromosomal)

  • Whole genome duplication

• Protein coding genes beget protein coding genes

  

  • Involved in oxygen transfer

• We use phylogenetic trees to understand how genes evolve

  • Globins diverged in evolution

• Usually when there is a gene duplication, one becomes nonfunctional

  • Neofunctionalization is the process by which a gene acquires a new function after a gene duplication event.

    

  • Subfunctionalization is a neutral mutation process in which each paralog retains a subset of its original ancestral function

    

    • The part upstream of the gene

• Pseudogenes: genes that have been copied and are now not functional

  

  • Mutation in the stop codon or other critical region

  • In B, these are generated from reverse transcribed mRNA

    • Processed pseudogenes

    • You can tell if it’s processed by whether it has exons — if it’s processed, it will have already been spliced

  • Gene to make our own vitamin C is a pseudogene

    • Limited to primates and bats

    • This is why we need dietary vitamin C

Whole genome duplications

• How do we know these are whole genome duplications and not individual gene duplications

  • Genes that are similar to each other interact — form gene families

    

• X chromosome is completely conserved

  • Due to Barr bodies — silencing one x chromosomes

  • X genes on mice are probably in X genes in humans

Functional diversity of DNA in humans

• There are RNAs that control gene function and regulating transcription/translation

• There are probably 2x as many genes coding for RNAs as proteins

genome organization

• Transposons

  • Transposon repeats make up 40% of your genome

    

    • There are a lot of diseases associated with LINE-1 family elements

      

    • Alu family was one of the biggest barriers to sequencing the genome

    • Why do we have so many? A few ideas:

      • these are parasites — they care about their own replication and are successful because they can move around the genome and duplicate

      • DNA methylation evolved to silence these transposons

      • These are advantageous because they are a source of variation

Model organisms

• Used to understand facts of biology and evaluate potential therapies

• Comparing genome sequences provides genome-wide information on relatedness of DNA and protein sequences

• Used to infer function by orthology

  • harder with more evolutionary difference

• What makes a good model?

  • Easy to grow

  • Easy to manipulate

  • Not sympathetic

  • Closely related to humans

• Assigning function to human genes

  • We cant experiment on humans

  • Model organisms are great for experiment advancing our understanding how a protein functions

  • We can identify the same protein in human genomes and assign the function determined in the model

  • 50% of human gene functions come from yeast

• Dogs are good for understanding genes that code for morphological and behavioral differences between breeds

  • Chondrodysplasia: a collection of diseases that can affect a person's stature

  • Studied in dogs

  • All small breeds have FGF4 retrogene on CFA12

Genes across species

• Odine’s curse: congenital central hypoventilation syndrome

  • Affects the autonomic nervous system

  • Hypoventilation — children with this disease need a thrach to survive

  • Hirschprung disease

  • Caused by a mutation in the PHOX2B gene (4p12), only gene associated with it

  • Autosomal dominant

  • Homeobox gene family

    • Made up of 235 genes and 65 pseudogenes

    • transcription factors

      • Homeodomain: DNA binding region (helix-turn-helix)

    • Expression of these genes during development follows same patterns in model organisms

    • Can also lead to mixed up limbs and in model organisms

    • In people, they can impact development of digits

• Extra SMNs — case study

  

  • SMA

    • SMN1 gene, 5q13.2

    • 95-98% are homozygous for a deletion or truncation of exon 7

    • 62% of SMN1 genes are composed of repetitive elements

    • Autosomal recessive

    • Degeneration of motor neurons

  • 

• Evolution of the SMN gene

  • Mice and rats only have one SMN gene → the inverted duplication arose after humans diverged from mice

Sickle cell anemia

• 20A→ T mutation HBB gene — 11p15.4

• Autosomal recessive

• Sickled cells clog small blood vessels

• important in malaria resistance

betal globin gene family

Alpha globin gene family

Apert syndrome

• Apert syndrome is caused by mutations in FGFR2 gene

• Craniosyntosis, midface hypoplasia, syndactyly of hands and feet, fusion of cervical vertebrae

• There are 7 other syndromes caused by the same mutations

• Most mutations are missence mutations

• FGFR2 shows alternative splicing. A known Alu insert in exon IIIc inappropriately promotes splicing to form FGFR2

Lil bub

• 

• homozygous deletion of exon 8 of the TNFRSF11A gene

• Encodes RANK protein

3/13/2025

Types of variation

• SNPs: Changes to a single nucleotide within the DNA

  sequence, such as single base substitutions, insertions, or deletion

• Deletions + duplications: Missing or extra genomic information. Both very small (single nucleotides) and very large (whole chromosomes)

  • Indels

  • CNVs

• Trinucleotide repeat expansion: increased number of trinucleotide repeats (three specific nucleotides) in a given area of a gene (e.g., Friedreich ataxia, Huntington’s)

• Methylation: The addition of methyl groups to DNA can turn the activity of a gene off. Changes to the methylation pattern can cause disease (e.g., imprinting disorders such as Angelman syndrome). These changes do not change the DNA sequence.

• Mitochondrial DNA variants: Changes to the DNA in the mitochondria can include single nucleotide variants and deletion/duplications (e.g., mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes or MELAS).

Nomenclature

• There is a standard way to communicate human genetic variation necessary to make use of that data

• Each variant has a unique identifier (e.g. rs212570 rs = “reference SNP”)

• These variants can be described using a set of common conventions. (e.g. g.1162G>A or p.F508del).

• g-genomic, c-cDNA, r-rRNA, p-protein

Molecular mechanism of pathology

• Loss-of-function mutations: gene products have no or reduced function.

  • Can display haplo-insufficiency

  • Allelic homogeneity (all mutations with same phenotype)

• Gain-of-function mutations: gene product does something positively abnormal.

  • Often due to abnormal product interactions, increased expression or expression in the wrong place.

• Pathogenic: Variant has been shown to disrupt gene function in the lab, is strongly associated with disease in humans, and does not appear in the general population

• Likely pathogenic: Variant is predicted to disrupt gene function is seen in some individuals with disease, and does not appear in the general population

• Variant of uncertain significant (VOUS): Evidence for variant pathogenicity is conflicting or absent

• Likely benign: Variant is not predicted to disrupt gene function and has not been seen in individuals with disease.

• Benign: Variant has been shown not to disrupt gene function and is present in high frequency in the general population

Funtional consequences of a variant

• Missense mutation

• Frameshifts

• Nonsense mutations

• Mutations that affect splicing

• Mutations that affect expression

• Short tandem repeats

Central dogma

Missense mutation

• SNP that leads to change in the protein

Nonsense mutation

• Mutation that gives rise to a stop codon

Dynamic mutations

• SHort tandem-repeat polymorphic loci become unstable above threshold size

• Many but not all are pathogenic

• Can display anticipation increased severity or lower age of onset in successive generations

Case study

• It is 1968 and George and Lorraine asked you to

  evaluate their two oldest children, Dave and Linda.

  They report that Dave has always been a “sickly” child

  and he was not able to attend kindergarten due to his

  frequent respiratory infections. Linda is also often ill

  but her parents are mostly concerned about her size; she

  is less than the 5th percentile for height and weight.

  George and Lorraine also have a 4 month old son,

  Marty. Linda tells you that Marty is a healthy boy but

  she thinks it is very odd that she always tastes salt when

  she kisses him. George is of Irish descent and Linda is

  of French and English descent

  

Kabuki syndrome

• KMT2D (12q13.12) and KDM6A (Xp11.2) genes

• Characteristic facial features

  • Arched eyebrows

  • Long eyelashes

  • Long palpebral fissures

  • Flat tip of nose

  • Large earlobes

• Intellectual disability

• Siezures

• Microencephaly

• Hypotonia

• Autosomal dominant inheritance

• Finding the gene

  • Whole-exome sequencing of 10 individuals with Kabuki syndrome (unrelated) with varied ethnic backgrounds

  • Why was it so hard to find the gene?

    • Locus heterogeneity

    • Failure of WES to find the mutations in the targeted exome

    • Causative mutations are outside the targeted exome

  • How was this addressed?

    • Use less strict criteria

    • Found 16 varients shared in at least 7 patients

    • MLL2 (KMT2D) — 7 cases had mutations

      • Loss of function found in 9

  • What is MLL2

    • FOund in drosophila nad mouse

    • Involved with histones and chromatin regulation

    • MLL2 provides instructions for making an enzyme called lysine-specific methyltransferase 2D

    • Loss of MLL2 is an embryonic lethal in mice

What does finding genes do for patients?

• Allows for earlier diagnosis and treatment

• Prenatal testing

• Confirmation of diagnosis

• BEtter understanding of molecular mechanisms

Genome-wide association studies (GWAS)

• Identify genetic variants (Haplotypes) associated with a trait or illness.

• Mainly use SNP Chip technologies like 23 and me rather than whole genome sequencing.

• GWAS typically involve large numbers of individuals – GIANT consortium use 5.4 million human data sets

Haplotype

• A set of DNA variations, or polymorphisms, that tend to be inherited

  together. A haplotype can refer to a combination of alleles or to a set of single nucleotide polymorphisms (SNPs) found on the same chromosome.

• They are transmitted throughout the pedigree as a block without recombination.

• What determines the size of a haplotype?

  – Age of the segment

  – Local rate and pattern of recombination

  

Mapping genes conferring susceptibility to complex diseases

• Major genetic effects on human health are complex (many

  genes).

• An approach to finding susceptibility factors (GWAS) finds statistically significant associations between ancient haplotypes and disease.

• Results are somewhat disappointing

  – Common disease-common variant hypothesis

• Lots of variants with small effects

  – Mutation selection hypothesis

• Variants with significant effect lost be selection

  

• Problems with GWAS

  • Variants must be old and able to persist for many

    generations to be associated with a haplotype shared by

    many.

  • Variants with strong effects are removed from the

    population by selection but replaced by new mutations

    in different haplotypes.

  • May be different strong mutations in genes from same

    pathway making them untraceable by linkage because

    genes for similar process are not clustered

  • Variants with small effects require lots of cases to find

    statistical significance.

Four basic gene therapy approaches

• Gene replacement: the delivery of a functional gene to replace a non-working gene

• Gene silencing: inactivation of a mutated gene that has become toxic to cells

• Gene addition: over expression of a “foreign” or exogenous gene to impact cellular function

• Gene editing: a permanent manipulation of a gene in a patient’s genome.

SEE SLIDES ——- 23 and ME

CRISPR

• Stands for clustered regularly interspaced short palindromic repeats

• CRISPR will go to any gene that you want

  • Can knock the gene out by making it make errors (NHEJ)

  • Can give it a new DNA sequence to repair it (HDR)

• Bacterial immune system

• In vivo

  • injecting gene therapy into individual and target it

• Ex vivo

  • Culture the cells, do gene therapy on the cells, and infuse the cells back in the patient

Gene delivery vehicles

• For CRISPR: usually use viruses

  • Adenovial

  • Retroviral

  • Lentiviral

  • Adeno-assocaited viral

• Viral vector components

  • The protein capsid defines the vector’s tissue or cell tropism and antigen

    recognition

  • The transgene of interest, which when expressed in cells, serves to confer a desired effect

  • The“regulatory cassette” that controls expression of the transgene as an episome or as a chromosomal integrate

Dont need to memorize below

3/25/2025 — pharmacogenomics

HIstory/evolution of adaptations to xenobiotics

• Substances that are foreign to the body or an ecological system

• The great oxygenation event (GOE) 2.45 BYA nearly wiped out obligate anaerobes probably gave rise to the enzyme families like P450

  • Genetic solution (adaptation) to xenobiotic toxin (oxygen)

• The great Animal Plant Wars last 800 Million years

  

• Example: possums vs eucalyptus

  • Eucalyptus is a potential food source for possums and koalas

  • Eucalyptus expresses terpines (toxic)

  • Possums feeding on Eucalyptus express >50 more CYP450 increasing metabolism and clearance of terpines by order of magnitude

Evolution of drug metabolism as a science

• Morphine was discovered by Freidrich Wilhelm Adam Serturner

  (1783-1841), a 21-year-old pharmacist’s assistant. He wondered

  about the medicinal properties of opium, which was widely used by

  18th-century physicians

• Began transformation of pharmaceutical chemistry from alchemistry

• Richard Tecwyn Williams

  • 1942, worked on the metabolism on TNT with regard to toxicity

    in munitions workers; due to the war he assembled teams to work on metabolism of sulfonamides, benzene, aniline, acetanilide, phenacetin, and stilbesterol. Established the role of metabolism in drug action

How do drugs work

• Pro drug: compound that you take that you have to metabolize for it to be active. The metabolites from your system produce the bioactive compounds

• Active drug: The drug you are taking is in the active form, but it has to get where you want it to go before it metabolizes

  

• Biotransformation of drugs into active metabolites

  

• Organs of drug metabolism

  • Liver — most common

  • Kidneys

  • Muscle tissue

  • Intestinal wall

  • Lungs

  • Skin

  • Blood

Main ways of biotransformation of drugs

• Dont need to memorize

• We have a whole bunch of genes that make proteins (enzymes) that metabolize drugs

Pharmacogenomics

• Pharmacogenomics: study of how an individuals entire genetic makeup determines the body’s response to drugs

• Core concept: because so many interactions occur between a drug and proteins within the patient, many genes and many different genetic polymorphisms can affect a person’s response to a drug

• Knowing someone’s genotype should help inform drug therapies

Optimizing drug therapies

• On average, a drug will be effective in only about 50% of patients who take it

• Trial and error is used until the correct drug is found for a patient

  • This is a waste of time and can be dangerous

• Pharmacogenomics increases the efficacy of drugs by focusing drugs on subpopulations of patients who will benefit

• Personalized pharmacogenomics is already widely practiced in diagnosis and treatment of cancer

• It is important to understand each patient’s genetic profile to select an appropriate treatment — particularly those newer treatments based on molecular characteristics of tumors

• HER-2

  • Personalized medicine was successfully used in the HER-2 gene and the use of the drug Herceptin in breast cancer

  • The human epidermal growth factor receptor 2 gene is located on chromosome 17 and codes for transmembrane tyrosine kinase receptor protein called HER-2

  • In about 25 percent of invasive cancers, the HER-2 gene is amplified and the protein is overexpressed on the cell surface.

  • In some breast cancers, the HER-2 gene may have as many as 100 copies per cell.

  • This amplification is associated with increased tumor invasiveness, metastasis, and cell proliferation as well as poorer patient prognosis

  • HER-2 is an oncogene

  • THerapy

    • Using recombinant DNA technology, Genentech Corporation in California developed a monoclonal antibody known as trastuzumab (or Herceptin®).

    • When bound to the receptor, Herceptin appears to inhibit the signaling capability of HER-2 and may also flag the HER-2 expressing cell for destruction by the patient’s immune system.

    • In cancer cells that overexpress HER-2, Herceptin treatment causes cell-cycle arrests, and in some cases, death of cancer cells.

  • Optimizing therapy

    • Herceptin only acts on breast cancer cells that have

      amplified HER-2 genes; therefore it is important to

      know the HER-2 phenotype of each cancer.

    • Herceptin has potentially serious side-effects;

      therefore its use must be limited to those who could

      benefit from the treatment.

    • The treatment is directed toward “HER2 Positive”

      tumors.

Reducing adverse drug reactions

• Every year, about 2 million people in the US experience serious side-effects from pharmaceutical drugs

• The costs associated with these adverse drug reactions (ADRs) are estimated to be $136 billion annually.

• Sequencing variations ina large number of genes can affect drug responsiveness

• Cytochrime P450 families of enzymes are particularly significant and are encoded by 57 genes

  • Superfamily of heme enzymes. Can catalyze different reaction types, mainly hydroxylation

  • Human: 18 families, 43 subfamilies, 57 sequenced genes

  • can be induced and inhibited

  • Occur in most tissues (except muscles and blood)

  • Exhibit genetic polymorphism (atypical biotransformation

  • Nomenclature:

    

Important human liver drug CYP450s

Drugs and grapefruit juice

• Grapefruit juice interacts with CYP450s

• Can block enzyme that degrades drug in drugs that are active when you take it — can lead to overdose

• Can block transporter that brings drug to body’s cells, leads to too little drug

LEFT OFF HERE IN FLASHCARDS

Reducing adverse drug reatctions

• People with some cytochrome P450 gene variants metabolize and eliminate drugs slowly, which can lead to accumulations of the drug and overdose

• In contrast, other people have variants that cause

  drugs to be eliminated quickly, leading to reduced

  effectiveness.

• Example:

  • There are more than 70 variant alleles of the gene CYP2D6 which encodes the debrisoquine hydroxylase enzyme. This enzyme is involved in the metabolism of approximately 25 percent of all pharmaceutical drugs, including diazepam, acetaminophen, clozapine, beta blockers, tamoxifen, and codeine

• Another example of pharmacogenomics in personalized medicine is that of the CYP2C9 and VKORC1 genes and the drug warfarin.

  • Warfarin (Coumadin) is an anticoagulant drug prescribed to prevent blood clots after surgery and to aid people with cardiovascular disease who are prone to blood clots.

  • Warfarin inhibits vitamin-K-dependent synthesis of several clotting factors.

  • If the dosage of warfarin is too high, the patient may experience serious hemorrhaging; if it is too low, the patient may develop life-threatening blood clots.

    • 20 percent of patients are hospitalized during

      their first six months of treatment due to warfarin side-effects.

  • Two single-nucleotide mutations in CYP2C9 lead to reduced elimination of warfarin and increased risk of hemorrhage.

    • Patients who are heterozygous or homozygous for some alleles of CYP2C9 require a 10–90 percent lower dose of warfarin.

  • The FDA has recommended the use of CYP2C9 and VKORC1 genetic tests to predict the likelihood that a patient may have adverse effects to warfarin.

  • It is estimated that the use of warfarin genetic tests could prevent 17,000 strokes and 85,000 serious hemorrhages per year.

Acetaminophen pathway

• APA is an active drug. SOme metabolites are hepatotoxic

• Acetaminophen belongs to a class of drugs called analgesics (pain relievers) and antipyretics (fever reducers). The exact mechanism of action of acetaminophen may reduce the production of prostaglandins that cause inflammation and swelling.

Why do we need to understand how genes function?

• Different mutations can cause the same disorder

• Knowing what specific mutations a person has can direct effective treatment

• This may be the most useful outcome of personal genomics

• Ex cystic fibrosis is caused by thousands of genes. It is useful to know which gene it is caused by

  • CFTR: cystic fibrosis transmembrane

  • CF is caused by dysfunctional CFTR

  • Leads to infection, inflammation, and airway damage

  • We usually treat this by treating the symptoms (breaking up the mucus, antimicrobials, etc.)

  • We would like to be able to treat the genetic defect more directly

    

Critical concepts for the future of pharmacogenomics

Phenylalanine hydroxylase deficiency (PKU)

• PAH gene, 12q23.2

• Autosomal recessive

• Deficiency of phenylalanine hydroxylase

• Inability to process phenylalanine

• Characteristics (untreated)

  • Severe intellectual disability

  • Epilepsy

  • Microcephaly

    

• Treatment

  • Restriction of dietary Phe at least through adolescence

  • Supplementation with Phe-free formula

• Can avoid negative consequences based on genetic test

Rh disease

• Antigens are genetically inherited

  

• Rehus factor (Rh)

  • D antigen

  • If you have the gene for Rh, you are Rh positive (dominant)

• AB+ = universal acceptor

• O- = universal donor

  

• Rh incompatibility

  • Transfusion of Rh positive blood to an Rh negative recipient will produce a transfusion reaction

  • Hemolytic disease of the newborn

    • Rh- mother, Rh+ father

    • Mother becomes sensitized to the Rh positive blood type during delivery

      • All subsequent pregnancies are at risk if the fetus is Rh positive — once sensitized, the mother can’t be desensitized

    • The immune responded will be more severe with each pregnancy

      

    • You need to know the fetus’s genotype for the treatment to be affected

Single gene disorders

• Fabry disease

  • GLA gene, Xq22.1

    • Alpha-galactosidase A enzyme

    • Active in lysosomes, break sown Gl-3

    • Buildup of GL-3 is toxic to the body

  • X-linked

  • Episodes of severe extremity pain

  • Angiokeratomas

  • Eye anomalies

  • Heart disease

  • Stroke

  • Males more severely affected

  • Treatment

    • enzyme replacement therapy (fabrazyme)

      • IV infusion given every 2 weeks, may take 4-5 hours

      • Proven to reduce Gl-3 buildup, but limited change in overall lifespan

      • Most people have adverse side effects

    • Galafold

      • Not enzyme replacement

      • Binds to misfolded protein and refolds it

3/27/2025

What is epigenetics

• epigenetics: the study of changes in gene activity that do not involve changes to the DNA sequence itself, but still get passed on when the cells divide — and sometimes across generations. These changes affect how genes are turned on or off

• Mechanisms include

  • Methylation

  • Histone modification

  • Non-coding RNAs

• First coined by Conrad Waddington in the 1940s

Key concepts

• DNA methylation

  • The addition of a methyl group (-CH3) to cytosine bases, usually at CpG sites

  • Often silences gene expression by preventing transcription factors from binding or recruiting repressive proteins

• Histone Modification

  • Post-translational modifications (acetylation, methylation, phosphorylation) of histone proteins around which DNA is wrapped.

  • Function: alters chromatin structure to make DNA more or less accessible to the transcription machinery

• Non-coding RNAs (ncRNAs)

  • Small and long RNAs that don’t code for proteins but regulate gene expression epigenetically

  • Can recruit chromatin-modifying complexes or directly interfere with mRNA stability are translation

• Epigenetic inheritance

  • The transmission of epigenetic marks (like DNA methylation) across generations without altering DNA sequence

  • May help adapt offspring to environmental conditions experienced by parents

Analogy: the genome as a library

Chromatin

• Chromatin structure is regulated in part by histones

• Euchromatin: loosely-packaged chromatin that is actively available for transcription

• Heterochromatin: tightly-packaged chromatin that is not expressed; has a tendency to spread

  • Constituative

    • Chromatin structure is stable during the cell cycle

    • Largely composed of repeats, few genes

  • Facultative

    • Reversible heterochromatin, can be used to regular gene expression

      • Ex: the X chromosome in females is regulated via the formation of facultative heterochromatin (X-inactivation forming Barr bodies)

• Methods for chromatin modification

  • Histone-modifying enzymes

    • Enzymes attach or remove chemical groups from histones

    • Non-histone proteins bind to the chemical groups to regulate gene expression

    • Ex: methylation

  • ATP—dependent remodeling complexes

    • Alter the location of nucleosomes

    • Causes formation or disassembly of nucleosomes

    • Actively transcribed DNA regions tend to have fewer nucleosomes

  • These mechanisms can interact

Histone octamer/nucleosome

• Nomenclature

  • 

Proteins that manage and interpret post-translational modifications (PTMS) on histones

• Writers: add modifications

  • These enzymes catalyze the addition of chemical groups (e.g. methyl, acetyl, phosphate) to histone tails

• Erasers: Remove modifications

  • These enzymes remove the modifications effectively, reversing the writer’s work

• Readers: recognize and bind modifications

  • These proteins or domains recognize specific histone marks and help recruit other proteins or complexes to regulate chromatin and gene expression

DNA methylation

• Adding methyl groups by DNA methyltransferases to cytosine molecules that are immediately upstream of a guanine molecule (CpG sequences)

• Methylation of repeat sequences is common; represses transcription; inappropriate methylation has been linked to some cancers and other diseases

  

Epigenetics

• Lasting changes in gene expression that are not caused by changes to the DNA sequence

• DNA methylation is a simple example

X-inactivation

• X-inactivation patterns are stably transmitted from a daughter cell to its daughter cells

• Not transmitted from parent to child

  

• The X-inactivation center (XIC)

  • Xq13

  • Codes for XIST (X-inactivation specific transcript)

    • Produces a non-coding RNA
      Recruits other proteins to inactivate the X-chromosome

    • In the case of an X-autosome translocation, only the X chromosome material containing XIC deactivates, neighboring autosome material does not deactivate

    • Not all genes are inactive on the inactive X-chromosome

  • Repressed on the active X

• Skewed X-inactivation

• Duchenne muscular dystrophy

  • Dystrophin gene at Xp21 (1/4 of probands have de novo mutation)

  • Progressive loss of muscle function (ultimately including breathing and heart muscles

  • Lose ability to walk around age 9

  • Usually die in late teans or early 20s

  • Female “carriers”

    • Skewed X-inactivation is associated with the female phenotype

    • small proportion have muscle weakness and heart disease

• Rett syndrome

  • MECP2 gene at Xq28 (usually de novo)

  • Onset at 6-18 months in females. Lethal in males

  • Microcephaly and seizures

  • Rapid loss of developmental skills which stabilizes at 2-3 years old

  • highly skewed X-inactivation may result in asymptomatic women

• Atypical Rett syndrome and affected alleles

  • Severe neonatal encephalopathy

    • Most common presentation in surviving males with MECP2 mutations (rare in females)

  • Features

    • Seizures

    • Microcephaly

    • Abnormal muscle tone

    • Breathing difficulty

    • Lethal by age 2

  • Mildly affected females and makes with classic Rett syndrome have been reported

    

• MECP2

  • Most mutations are de novo

  • Involved in neuron maturation

  • Expression varies across development and tissue type (differential splicing leads to brain-specific transcripts)

  • MECP2 protein binds to methylated CpG dinucleotides which can either activate or repress transcription

  • MECP2 mutations result in failure of gene silencing

Imprinting

• Imprinting: gene expression is determined by the parent of origin. Imprinted genes are deactivated

• Each imprinted region is likely to contain clusters of genes that are differently imprinted and some which are not imprinted at all

• May be tissue-specific or developmental-stage specific

• Regulated via methylation which is erased and reset shortly after conception

Chromosome 15

• Prader-willi syndrome imprinting center (PWS-IC)

  • Function: controls the paternal expression of several genes in the region

  • Normally active on the parental allele

  • Regulates genes like SNRPN, SNORD116, and others that are paternally expressed

  • Mechanism: PWS-IC is required for establishing and maintaining the unmethylated state of the paternal allele during gametogenesis, allowing transcription of these genes

• Angelman syndrome imprinting center (AS-IC)

  • Function: indirectly regulates the maternal expression of UBE3A by controlling methylation of the PWS-IC

  • Normally active on the maternal allele

  • Regulates imprinting and silencing of the paternal-type expression pattern in the maternal allele. Ensures UBE3A is expressed only in the maternal allele in neurons

  • Mechanism: THe AS-IC initiates methylation of the PWS-IC on the maternal allele during oogenesis, silencing ghte paternal-only genes there, and allowing expressing of maternal-specific genes

• Summary:

  

• Uniparental disomy (UPD)

  • When two copies of the same chromosome are inherited from one parent

  • If the chromosome involved in the UPD is imprinted then this may cause an abnormal phenotype

  • Often arise via trisomy rescue

    • When trisomy mosaicism is noted it may be important to test for UPD

    

• Angelman syndrome (AS)

  • Normal pregnancy and birth

  • Happy affect with inappropriate laughter, extremely social

  • Motor delays and severe intellectual disability

    • Abnormal gait

    • No speech

  • Seizures

  • Disrupted sleep

  • Normal lifespan

  • Maternal allele usually active!!!!

    

• Prader-willi syndrome (PWS)

  • Motor developmental delay

  • Mild intellectual disability

  • Short stature

  • Infancy

    • Hypotonia

    • Poor appetite

    • Failure to thrive

  • Baby

    • Normal appetite and feeding

    • Normal growth

  • Childhood

    • Hyperphagia

    • Slow metabolism

    • Obesity

  • Parternal allele usually active!!!!

    

• Recurring risks (for siblings)

  

  • Most cases are not inherited

• Imprinting and inhertance

  

  • Most cases aren’t inherited, but when they are, we can study the pattern

  • Need to understand what is happening on the maternal and paternal chromosomes

    

  • The paternal imprinting center is unmethylated, the maternal one is methylated

  • Paternal genes are not expressed (suppressed expression)

  • So this disease can be passed from mother to children, but not father to children

• Key players in imprinting

  • 

• What happens on paternal allele

  • Ice is unmethylated

  • Expression of SNRPN and UBE3A-ATS

  • UBE3A-ATS silences maternal genes in Cis

• What happens on maternal allele

  • Ice is methylated —> silenced

  • UBE3A is expressed

Imprinting on a genome-wide scale

• Molar pregnancy

  • Complete hydatidiform mole

    • Diploid conception with only paternal DNA present

    • No fetus is present

    • Always ends in a miscarriage

  • Partial hydatidiform mole

    • Triploid conception (69,XXX)

    • Usually caused by dispermy

    • Very abnormal placenta

    • Usually causes miscarriage, but rarely may result in a living child

  • Not all diandric triploidies cause PHM

    

• Molar pregnancies are classic examples of imprinting disorders

  • Complete moles have only paternal chromosomes (uniparental disomy), leading to overexpression of paternally expressed genes

  • Lack of maternal contribution leads to loss of maternal-imprinted gene expression, disrupting normal embryonic development

Genome-wide uniparental disomy/diploidy (GWUPD)

• Case history

  • Baby girl born at 36 weeks (normal size)

  • Biliary duct cyst removed at 2 days of age

  • Umbilical hernia repaired at 2 months age

  • Kidney cyst noted but resolved spontaneously

  • Hearing loss diagnosed at 3 months of age

  • Cochlear implant at 2 years of age

    • GJB2 and SLC26A4 gene Sanger sequencing

      negative

    • Homozygosity for novel V413I variant in the ESRRB

      gene found (Sanger sequencing) – likely benign

  • Height and weight remained consistent and in the normal range through age 11 years

  • Mild leg length discrepancy noted

• Additional genetic testing for hearing loss

  • Paternity confirmed using STR markers on chromosomes

  • Sanger sequencing of the exons of ESRRB done for family members

  • Whole genome SNP array fone for whole family

• Results

  • V413I variant in the ESRRB gene found in the girl (homozygous), her brother (heterozygous) and her father (heterozygous) but NOT her mother

  • Paternity testing showed the girl to be homozygous at all sites for a paternal marker; no maternal markers were found

  • SNP array confirms genome-wide homozygosity for paternal markers

  • Follow-up testing og multiple tissue types showed biallelic inheritance in fibroblasts but primarily paternal inheritance in blood and saliva

  • Likely shows somatic mosaicism

• What is the likely cause of her hearing loss?

  • Homozygosity for mutation in a gene causing an autosomal recessive form of hearing loss

• What other conditions might she be at increased risk for?

  • Other individuals with similar GWUPD generally develop cancer

Hongerwinter

• Lots of people were exposed to very low caloric intake

• The effect from their environment affected their offspring

  

• IFG2 gene — insulin-like growth factor II

  • 11p15.5

  • Prenatally expressed

  • Regulates cellular development

  • Maternally imprinted (paternally expressed) except in the liver (bi-allelic)

  • May be associated with Wims tumor and/or Beckwith-Wiedemann syndrome

  • DMR: differentially methylated region

• Study population: hunger winter family study

  • 600 individuals conceived during the hunger winter

  • Measureed methylation of IGF2 DMR

  • Compared i=people with early prenatal exposure to those with late prenatal exposure

• Famine during early gestation associated with hypomethylation of the IDF2 DMR

• Early embryonic/fetal development may be a sensitive period for developing appropriate epigenetic markers

NASA twin study

• Identical twins Mark and Scott

  • Scott spent one year in space

  • Mark stayed on earth

• Study conclusions

  • Physiological effects of spaceflight

    • Stress caused by oxygen deprivation

    • Increased inflammation

    • Nutrient shift

  • Genetic effect of spaceflight

    • Telomeres lengthened while in space but returned to normal within two days of Scott’s return to Earth

    • Epigenetic regulation was altered during spaceflight which may highlight candidate genes related to cellular stress responses (all but 7% of these returned to normal shortly after his return

• Central dogma revisited

  • One gene can have many promoters altering how and when

    the gene is expressed

  • Alternative splicing can allow one gene to produce several

    different protein

  • Post-transcriptional RNA editing alters the RNA transcript so

    that the mRNA has a different sequence than the DNA

    template

  • Environmental factors may modify epigenetic markers to

    cause lasting changes to DNA

4/8/25 — recorded lecture, need to watch

put notes in flashcards!

4/10/25

• took most notes in flashcards!

Cystic fibrosis

• caused by mutations in the CFTR gene disrupt flow of chloride ions,

• Cholera

  • vibrio cholerae hyperactivates the CFTR ion channels

  • Increased ion flow leads to more watery secretions producing the intense watery diarrhea associated with cholera

Microbiome

• Environmental alterations can produce changes in microbiome composition

• Composition changes over course of lifetime

4/15/2025 — also in flashcards

Case of the cranky baby

• Length of pregnancy: 41 weeks

• Delivery by C-section

• Feeding: normal progression of feeding strategies

• One sibling

• Incidence of infections

  • 

• Ear infections

  • Tubes that drain the inner ear build up, causes pressure

  • Can be treated with antibiotics

  • 3+ infections warrants referral to an ENT

• C.difficile

  • Most expensive healthcare associated infection

  • Contagious

  • Related to antibiotic use

  • Normally suppressed by native bacteria

  • High recurrence rate following treatment

  • Possible mechanism

    • Intestinal bacteria normally regulate bile

      acids

    • Antibiotic-related loss of native bacteria may

      lead to an increase in the products of bile

      acid metabolism (deoxycholate)

    • Deoxycholate promotes the growth of C. difficile

Case study — microbes and obesity

• things to consider in a fecal donor

  • BMI: you can’t donate for fecal transplantation if your BMI is too high

  • Microbiome is so tied to weight

Review

Know

• types of mutations

  • mutations in places that disrupt splicing — cause insertions in reading frame

  • base pair changes

• Types of variation

  • Methylation

  • TNR expansion

  • SNPs

  • Deletions/duplications

  • mtDNA

• Nomenclature

  • p.F508 — most common deletion in CF

• Pathogenic vs benign and everything in between

• CF: everything in the slide picture

  • Caused by thousands of independent mutations

  • Shortened proteins

• Sickle cell

  • Caused by missence mutation

• Kabuki syndrome

  • KMT2D (12q13.12) and KDM6A (Xp11.2)

• GWAS

4/24/2025

Evolution basis

• Multi-regional model (Thorn and Wolpoff

  • Modern HUmans evolved in multiple regions from Homo erectus

  • Based on fossil evidence linking ancient and modern populations

• Out of Africa model

  • Modern humans evolved in Africa and dispersed recently

  • Based on genetic evidence

Major events in the study of human evolution

1. Discovery of DNA structure

2. Development of molecular clock hypothesis

   • Proposed that mutations accumulate at a roughly constant rate. Enabled estimation of divergence times between species

3. Mitochondrial Eve and Out-of-Africa hypothesis

   • Analyzed mitochondrial DNA from global populations and concluded that all modern humans share a common maternal ancestor who lifed in Africa

4. Y-chromosome Adam

   • Studies Y-chromosome variation revealed a common paternal ancestor, supporting African origin of humans

5. Completion of the human genome project

   • enabled comparative studies with other hominins and primates, revealing what makes humans unique

6. Sequencing of Neanderthal genome

   • Demonstrated interbreeding between Neanderthals and modern humans

7. Advances in Ancient DNA techniques

8. Populartion genomics and modern human diversity

9. CHISPR and functional genomics

Mitochonrial eve

• Most recent common female ancestor from which all modern humans descended

• Comparative analysis of mtDNA variation suggests that a single woman who lived ~200,000 years ago is the ancestor of all humans alive today

• Showed that all lineages recently trace back to Africa

Y-chromosome Adam

• Most recent common male ancestor from which all modern humans descended

• Comparative analysis of Y chromosome variation suggests that a single man who lived 180-200,000 years ago

1000 genomes project

• Catalogue of human genetic variation across many population'

• Sequenced the genomes of 2504 people across 26 populations

  • Low coverage WGS

  • Deep exome sequencing

  • Dense microarray analysis

• Lacks full representation of some global regions

How does 23 and me work?

• Genotyping your DNA

• Comparing to reference panels

• Statistical algorithms

• Output: your ethnicity estimate

• Important caveats

  • Estimates evolve

  • Geographic labels are fuzzy

  • Not suitable for detailed ethnic identification

  • More accurate for recent ancestry

Neanderthals

• First discovered in 1856

• Neanderthals left Africa 500,000 years ago and migrated to Europe and Asia before modern humans

• Largely limited to Europe

• Went extinct 30,000 ya

• Complex societies

Neanderthal DNA

• Alleles contribute to behaviors

  • Chronotype

  • Tendency towards loneliness and isolation

  • Frequency of lack of enthusiasm and disconnect

  • Smoking statuesnyt

• Neanderthal dessert: area of a genome without neanderthal DNA

• We think that humans had complex language, but neanderthals didn’t

• FOXP2

  • 7q31.1

  • Regulates the brain’s control of the lips, mouth, and tongue

  • Critical for normal language development

  • FOXP2-related speech nd language disorder

    • Apraxia of speech

Sickle cell anemia

• 5 haplotypes associated with hemoglobin S muation

ATA

• 2003 skeleton found in Chile

• 6 inches big

• Mitochondrial B2 haplotype matched to South American populations

• bone age suggested 6-8 years of age.

• 2 X chromosomes - predicted to be female

• Multiple SNVs found associated with skeletal phenotypes

April 29 2025

Ethical issues in human genetics

• Pace of tech change is faster than society’s ability to comprehend and adapt

• Genotyping and disease prediction is misunderstood

• Porential benefits are being missed

• New tools of genetic modification, forensic testing, etc. are already here

Testing

• A test can have implication for other family members

  • A person has, at some level, the right to not know

• Is there a duty of care?

  • If you are a practitioner and you know that someone has a disorder, do you have to tell them?

Huntington disease

• No cure or effective treatment

• Genetic testing is available and extremely accurate

• 75% of individuals at HD risk choose not to get tested

• Key features that lead to ethical dilemma

  • autosomal dominant (50% risk to offspring)

  • anticipation (getting worse or appearing earlier in future generations) in some cases

  • No cure

  • Testing deeply affects identity

  • Disclosure to at-risk relatives

Most genetic tests have serious limitations

• Most have limited predictive power

• Might know that the condition will exist but not how severe it will be

• Might find a disease that they weren’t expecting to find

Potential for discrimination

• Particularly with presymptomatic testing

• Laws protecting us are limited and subject to change

Testing and reproductive choices

• Children are a major issue for many people

• The effect of parents’ genetic status complicated things

• Termination vs preparation are major decisions with very strong social and cultural ramifications

Direct to consumer testing

• Limited state and federal regulation

• Questionable clinical value

• not all insurance concerns are addressed

• privacy policies not backed by laws

• no regulation of genetic variants that can be included in risk assessments

• increases global health inequality

Inappropriate genetic testing

• Sex selection

• Paternity

  • Generally requires establishing legal chain of custody to be used in court in cases of

    • custody

    • child support

    • inheritance claims

    • immigration

    • adoption

  • Forensic testing generally done a pre-existing samples

Forensic DNA databases

• CODIS

  • uses only DNA from people with convictions

• Genetic genealogy (gedmatch)

  • Works better than CODIS

  • Open database

  • More likely to get a match to a family member

• Appropriate uses

• Potential for error