Medical genetics

Numerical chromosome abnormalities

= Not 46 chromosomes

Common in zygotes, most result in early fetal loss

Trisomy/monosomy

• Change after the zygote = somatic mosaicism for numerical chromosome abnormalities

Analyse ex. with Karyotyping or QF-PCR

Structural chromosome abnormalities

Duplication, Inversions, Deletions, Insertion & Translocation

• Large -

• Small - structural rearrangements

Monogenic disease

One gene variant → one affected protein → one trait

• Most due to gene variants in, or close to, protein coding sequences

Main patterns of monogenic (mendelian) inheritance

Autos. dominant

Autos. recessive

X-linked recessive

X-linked dominant

Mitochondrial

Autosomal dominant inheritance

1 affected parent, both sexes affected

• Children have 50% risk of inherit the mutated allele

Autosomalt recessiv inheritance

Affected individuals have healthy parents, both sexes equally affected

• A couple with an affected child has a recurrence risk of 25% risk

X-linked recessive inheritance

Almost exclusively males affected

• Affected fathers transmit gene to all daughters

• Carrier women has a 25% risk of having affected boys

• Carrier females may have mild symptoms

X-linked dominant inheritance

Affects either sex, females more often than males

• Affected female children has 50% of being affected

• Affected males daughters has 100% risk of being affected, while non of the sons are.

X-chromosome inactivation in females

“Lyonisation” = Women inactivates one of two X

• Occurs randomly in each cell during early embryogenesis

Mitochondrial inheritance

Both sexes affected - only transmitted through females

• Variable symptoms within & between generations

Monogenic cancer-predisposition - “two-hit” model

A gene variant is inherted from one parent on 1 allele + a somatic variant is acquired sometime during life on the other allele in the vulnerable tissue

• Random mutation during life can lead to activation of an recessive gene → Cancer

Genetic heterogeneity

Different genes may, if mutated, cause the same phenotype

Alleleic heterogeneity

Different variants in a specific gene may cause variable phenotypes

Complications and variants in monogenic inheritance:

• Reduced penetrance ( or nonpenetrance)

• Late onset

• Variable expressivity

• New mutation

• Lyonisation

• Imprinting

Reduced penetrance

Penetrance of a character = the probability of who has the genotype will manifest the character

• Not all gene-carriers becomes affected - “skipping” generations

• E.g. breast cancer: 70% risk for the disease with associated genotype

  • Thus 30% chance of skipping

Late onset

A particularly important case of reduced penetrance is seen with late-onset diseases.

• Genetic conditions are not necessarily congenital (present at birth)

• The genotype is fixed at conception, but the phenotype may not manifest until adult life.

  • In such cases the penetrance is age related.

E.g. Huntington’s disease

Variable expressivity

Variable expression describes the frequent observation that affected individuals within a pedigree may show different degrees of severity or different features of the condition.

• Variable expression is especially a feature of dominant conditions.

• Variable symptoms, onset and progression among affected

New mutation

New mutations are individually rare.

However, in the context of serious dominant or X-linked diseases, they may appear to be very far from rare: they may account for a significant proportion of all cases.

• At least 80% of cases with achondroplasia (autosomal dominant) are new mutations.

Imprinting

An imprinting gene has one of its two copies silenced and the gene is expressed from only one chromosome.

• What allele is imprinted is dependent on its parental origin

• Impringing (“silencing”) occurs during oogenesis or spermatogenesis

Happens to compensate dosage, parentals silence to match

Genetics of complex (multifactorial) disease

Monogenic vs complex disease

Number of contributing gene variants and disease segregation in families:

• Monogenic disease = rare mutation

• Complex disease = many common gene variants with cumulative effect

Relative effect-size of gene variants vs. non-genetic factors:

• Monogenic disease = Impact of mutation in a single gene on disease phenotype

• Complex disease = Impact of variations in different genes on disease phenotype

Basis of human inter-individual genetic variation

SNPs

VNTRs/SSRs

CNV (copy number variation) and other types of structural variation

Epigenetic profilel

Alternative splicing

RNA editing

Structural variation in the genome

Deletion (CNV)

Duplication (CNV)

Insertion (CNV)

Inversion

Translocation

Complex rearrangements (CNV)

Copy number neutral loss of heterozygosity

Epigenetics

“The study of heritable changes in gene function that can not be explained by changes in DNA sequence”

The sum of genetic variation within the human body

Monozygotic (or identical) twins

• Fascinating experiment of nature and important resource in genetic and epidemiological research

• Two people who are clones from the same cell

3P

Preventive medicine

Personalized medicine

Precision medicine

Next generation sequencing

Upscaled snager sequencing - millions of molecules at the same time

• Sequencing by synthesis, is synthesizing the while strand from scratch

• Reversible dye terminators

• Solid phase support - bridge amplification

Solexa/Illumina sequencing

PCR bridge amplification

• Library preparation

• Cluster growth

• Sequencing

Reversible dye-terminators

DNA pol. incorporates adaptor oligonucleotides, each labeled with a different dye.

• Picture

• wash

• New oligonucleotide

Repeat

Bridge amplification

1. Adaptor binds covalently to flowcell flexible linker P5

   1. P5 adapter: common that the first read starts from this end

   2. P7 adapter: common the second read reads from this end

2. DNA synthesis → dsDNA denaturation → washing

3. ssDNA bending → annealing to P7 adapter

4. DNA synthesis → dsDNA denaturation → washing

5. ssDNA bending

osv…

= cluster of monoclonal DNA

RNA sequencing

Actually DNA sequencing but the RNA is converted to cDNA by reverse transcriptase.

• Don’t want to sequence RNA pga not as stable - tends to get hydrolysed

• Don’t want to just use reverse transcriptase pga unspecific

Input RNA is fragmented → fragmented RNA is converted to cDNA → by adding sequencing adapters its converted to DNA library

Chromatin organization

DNA is not randomly packaged in the nucleus, with the help of histones it is neatly condensed.

Cell-specific chromatin landscapes determine cell-selective glucocorticoid receptor occupancy

• Epig

Epigenetic ladscape

Determines the response to hormones/ligands pga it determines where the receptor can bind.

• Its a way of epigenomes to alter function in different organs in response to the same signal.

E.g. Cell-specific chromatin landscapes determine cell-selective glucocorticoid receptor occupancy

3 levels of epigenetic mechanisms

1. Packing of chromatin

2. Histon-modifications

3. DNA methylation

• 4-ish = spatial organisation

Local chromatin networks determine expression

E.g. spatial organisation as an epigenetic mechanism

• Cell changes in response to signals - genes alter between nuclear neighborhoods

  • Between silencing or expression

Classes of epigenetic enzymes

1. Writers

   1. Alters the sequence - epigenetic marks, e.g. by methylation

2. Readers

   1. Specifically bind (to docking sites) when the modifications have taken place

3. Erasers

   1. Need to be able to remove methylations osv.

Methylation of DNA

Usually on cytosine (C) and thereby creates a 5th base: 5-methyl-cytosine (5mc)

• 5mc influences chromatin structure and gene expression

• Occurs at CpG di-nucleotides

  • CpG occur in a low frequency in coding regions

  • Denser in promoter regions and are usually unmethylated = CpG islands

Epigenetics is partöy inherited

Methylation pattern can be maintained even if the DNA has been replicated

• Maintained by DNMT1 = main-methylation transferase

• Without it the pattern would be lost = passive demethylation

DNA methylation transferase family

Dnmt1 = main maintenance enzyme

Dnmt3 = more dynamic, establish the methylations

How is DNA-methylation removed?

Passive = replication without re-methylation

• Inhibit DNA transferare - interfere with the maintenance

Active = removing methylation

• Dnmt3 may act as demethylase

  • Depending on interaction with other proteins

• As a result from DNA repair - repaired seq. could be lacking the methylation

• Hydroxylation by Tet-enzymes => removes function

  • 5mC is hydroxylised

Reprogramming of DNA methylation

Egg and sperm carry DNA methylations patterns from parents, during embryogenesis enzymes that’s also carried by the parents removes majority of this pattern = de novo methylation, but will never reach a complete blank page - genomic imprinting

Genomic imprinting

Certain genes transfered during embryogenesis will be silenced/expressed

• While we inherit two copies of every gene (one from each parent), imprinted genes use only one active copy. The other copy is turned off.

• The process relies on chemical tags like DNA methylation added during egg or sperm formation.

• Old marks are erased during germ cell development and reset.

Histone modifications

= Epigenetic modifications

N-terminal tails modified by: acetylation, methylation, phosphorylation & ubiquitination

• Important modifications for gene regulation

  • In the promoter = a lot of histone acetylation

Variation in histone modification marks certain features

• Enhancer vs promoter - different methylation

Epigenetic modifications altering transcriptional activity

Modified lysine: Binding sites for regulatory proteins

Epigenetic repression:

• Methylated C by methylation transferase → opens chromatin → activation

• Histone acetylation → closes chromatin → repression

Does epigenome change during life?

Slightly due to environmental influence, ex:

• behavioral changes and long-term memory - requires histone acetylation

Epigenetic changes can affect behavior and phenotype

Locus

The specific physical location of a gene or other DNA sequence on a chromosome, like a genetic street adress.

Allele

One of two or more variants of a locus

Genetic marker

A DNA sequence with known physical location on a chromosome

• STR microsatellites

  • PCR based linkage analysis

• SNVs/SNPs

  • Hybridization array

  • Large scale sequencing

Genetic mapping

The process of establishing the locations of loci on the chromosomes and the distance between them

• Creating a genetic map

Recombination

Chromosomal crossover to recombination between paired chromosomes inherited from each of ones parents

• The more recombination → loci further away

Genetic distance

The distance between two loci, based on recombination fraction, measured in cM (1cM = 0.01 recombinants).

• Translates recombination events into genetic distances, based on recombination fraction

Linkage analysis

Linkage = The tendency of genes or other DNA sequences at specific loci to be inherited together as a consequence of their physical proximity on a single chromosome.

• LOD score: likelihood of linkade (theta < 0.5) / likelihood the loci are unlinked (theta = 0.5).

Linkage analysis:

• Mathematical model of the extent to which a given marker tends to be co-transmitted with a disease through families

• Mapping a disease to a specific chromosome region

• Recombination events narrow the region

Linkage analysis works when:

Inheritance is mendelian

Variants in one gene (homogeneity)

Childhood onset of disease

• Helthy individuals are truly healthy

Linkage analysis weaknesses:

Comples disorders - hard to get correct linkage for more than 1 gene

Heterogeneity

Lack of informative families

Large families needed

Genetic association

= Is the co-ocurrence of two or more traits in a population of individuals more often than can be explained by chance.

• Can also be a marker allele and phenotype

Association analysis

Can be family- or population-based

• Linkage methods are always family-based.

Family-based association design

Mendelian traits can be used when families are small but numerous

• Case-parents trios

• Parents used as controls

• Measure association of genetic markers in nuclear families by transmission from parent to offspring.

Population-based design

Mostly used in population genetics (complex traits)

• Risk alleles for traits

To be able to find genetic variants, there must be a genetic component in the disorder.

• Adoption studies…

Linkage disequilibrium

LD = the non-random association of alleles at different genetic loci in a population.

• Case-control study:

  • Susceptibility alleles may be in LD with the marker alleles that are over-represented in cases; protective alleles may be in LB with those that are over-represented in controls.

Genome Wide Association

Population-based case-control analysis has long been employed in candidate gene studies.

• Identify genes with biologically plausible role in disease pathology

• Genotype SNPs in these genes and test for association with disease status.

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GW SNP arrays: Homozygosity mapping

Locus homozygousity = two identical inherited alleles

GW SNP arrays: Autozygosity mapping

= means homozygosity for markers identical by descent, inherited from a recent common ancestor

• In the same family - find homozygotic regions

Large scale sequencing

Large scale sequencing - data analysis

Alignment → variant calling and annotation → filtering and predictions → candidate variants

Disease inheritance probability

Mutations can arise:

Spontaneously

• DNA replication, DNA repairs, recombination, etc.

Mutagens induced

• Endogenous mutagens

• Exogenous mutagens

Spontaneous mutation

DNA strand is damaged → induces DNA repair mechanism → cell goes into senescence, apoptosis or uncontrolled cell division (cancer)

Spontaneous errors

DNA replication errors:

• Base mismatches

• Short tandem repeats (STRs)

Chromosome segregation:

• Altered numbers of whole DNA molecules

  • Germ line = embryonic lethality or congenital disorder

  • Somatic cells = cancer

Recombination errors

• Deletions/insertions/inversions when misaligning

Mutagens induced errors

Chemical DNA damage:

• Endogenous = spontaneous damage within cells

  • Hydrolytic damage: depurination/depyrimidination/deamination

  • Oxidative damage: cellular metabolism generates electrophilic molecules/ions e.g. ROS

  • Aberrant DNA methylation

• Exogenous = damage induced by external sources

  • Radiation:

    • Ionizing radiation = generates ROS breaking DNA strands

    • Non-ionizing UV radiation = causes covalent bonding between adjacent pyrimidines on a DNA strand

  • Harmful chemicals:

    • Interact with cellular molecules to generate ROS

Four classes of chemical (mutagen-induced) damages to DNA

• Strand breakage

• Base deletion

• Base modification

• Base crosslinking: may block DNA replication or transcription: triggering cell death!

DNA repair

DNA repair on single strand:

• Base mismatch repair

• Base-excision repair (BER)

• Nucleotide-excision repair (NER)

DNA repair on both strands:

• Homologous recombination (HR) - mediated DNA repair

• Nonhomologous end joining (NHEJ)

Base-excision repair (BER)

Single strand DNA repair - repairing chemically modified single bases

• Modified base is taken away (deaminated, alkylated…)

• Gap filled by DNA polymerase and DNA ligase

Base mismatch repair

Single strand DNA repair

• Corrects errors in DNA replication, often caused by replication slippage

  • Endonuclease → Exonuclease → olymerase → ligase

Nucleotide-excision repair (NER)

Single strand DNA repair - repair of bulky, helix-distorting DNA lesions

• Damaged site is opened out → DNA is cleaved some distance away on either side of the lesion → generating an oligonucleotide of about 30 nucleotides containing the damaged site → resynthesis of DNA

Non-homologous end-joining (NHEJ)

Repair of DNA damage on both strands

• Broken ends are quickly fused together, special protein binds and recruit a special DNA ligase

Homologous recombination-mediated repair

Repair of DNA damage on both strands

• Highly accurate; uses undamaged sister chromatid as template for repair.

Hardy-Weinberg distribution assumptions

No selection

No mutation

No migration

Large population

Random mating

Recombination

Crossing over during meiosis

• Occurs in the early stages of meiosis

• Usually not random - hotspots

• Increases genetic variation in offspring

Linkage disequilibrium (LD)

The non-random association of alleles at different loci

Haplotype

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

Haplotype blocks

Are created by correlated SNPs inherited together

The boundaries between blocks are usually recombination spots

• Can differ between populations and often do

Changing allele frequencies

Allele frequencies are subject to change over time because of a number of factors:

• Mutation

  • somatic or germline mutations

• Drift

• Selection

Factors that determine if mutations persist/increase in frequency

Genetic drift: Neutral mutations

• Noticeable affect/difference in big or small populations

• A long-term small effective pop. size will reduce the genetic diversity

  • Bottlenecks/Founder effect

Mutation:

Selection:

• Works on phenotype and only indirectly on the genotype

• Most phenotypes do not depend on a single genotype but a combination of:

  • Epigenetic

  • Environmental factors

  • Multiple genotypes at numerous loci

Cancer as an example of a disorder of our genome

Cancer is a genetic disease of predominantly somatic cells

• Consistent with twin studies showing a low level of heritability

At least 5% of patients inherit a predisposing mutation to develop cancer

Cancer is a multi-step process

Model for development of colon cancer:

1. Normal epitelium → loss of mutation of the APC gene

2. Hyper-proliferative epithelium → DNA hypomethylation

3. Early adenoma → activation of KRAS

4. Intermediate adenoma → Loss or mutation of the SMAD4 gene

5. Late adenoma → Loss or mutation of the p53 gene

6. Carcinoma

Two principally different genetic mechanisms in cancer development

Oncogene activation (gain of abnormal function):

• Normal cell → translocation between two chromosomes → cancer cell → tumor progression

Inactivation of tumor suppressor gene (loss of normal function):

• Normal cell → mutation in a tumor suppressor gene leads to a predisposed cell → second mutation → cancer cell → tumor progression

p53 and retinoblastoma pathway

= The two most critical tumor-suppressor pathways in human cells.

Working together to regulate cell division, the DNA damage response, and programmed cell death (apoptosis).

P53: Triggers cell-cycle arrest in response to stress or DNA damage

Retinoblastoma (RB): Controls cell cycle entry from the G1 phase into the S phase, halting cell proliferation when activated.

RB inactivation

Retinoblastoma - tumor suppressor inactivation (inactivation of RB1 from chromosome 13) leading to familial tumor syndrome, autosomal dominant inheritance.

• Two-hit hypothesis

Two-hit hypothesis

= Both copies (alleles) of a tumor suppressor gene must be damaged or inactivated for cancer to develop. LOH = loss of heterozygosity

• Hereditary form: Individual inheriting the first mutated allele (germline mutation), only one extra somatic mutation (second hit) is needed.

• Sporadic form: Individual born with two healthy alleles, both hits must happen spontaneously in somatic cells over lifetime

BRCA1 inactivation

Ex. of inactivation of tumor suppressor gene on chromosome 17, leading to breast/ovarian cancer.

• Because failed DNA repair due to LOH, or epigenetic silencing (chemical changes like promoter hypermethylation block the gene from working even if the DNA sequence is normal)

• Can be inherited = autosomal dominant inheritance

Loss of heterozygosity (LOH)

Cancer starts when the second, healthy copy is accidentally lost or damaged in a cell over time.

• The only thing hindering cancer formation is the heterozygosity (one healthy allele)

• Loosing it = becoming homozygous for the mutated alleles

Genome of cancer cells is unstable

Gene level: microsatellite instability, e.g. point mutations

Sub-chromosomal level: usually missing large chromosome parts, e.g. deletions

Chromosome level: Aneuploidy, extra or missing whole chromosomes

Cancer cells accumulate various mutations during the disease development and progression (is a multistep process).

Detection of Sub-chromosomal level instability

Ex. by using microsatellite markers

• Compare the microsatellites between maternal and paternal chromosomes - combine and see what’s missing

• By identifying LOH or abnormal inheritance patterns of short tandem repeat sequences via PCR amplification and fragment sizing.

Cancer genes

Oncogenes & tumor suppressor genes

Oncogenes

= Mutated gene that can push cells to grow and divide to fast and cause cancer - activating mutations.

• Often fusion proteins

• The tumor cells are heterozygous for oncogenic mutation

• Seldom cause inherited dominant cancer syndromes.

Tumor suppressor genes

Genes are expressed in most tissues, although tumors arise only in selected organs.

Cause most inherited tumor syndromes; retinoblastoma

Tumor cells usually homozygous for inactivating mutations (double hit)

Deletions are commonly causing inactivation.

Familial cancer

Caused by heterozygous inherited mutations - autosomal dominant heritage

• Clinical characteristics:

  • Early onset

  • Multiple (bilateral) tumors

  • Associated forms (cancer syndromes)

Loss of Y (LOY)

Males live shorter than females - LOY is most likely the biggest reason.

LOY is the most common post-zygotic (somatic acquired) mutation

Hematopoietic LOY is associated with mortality and risk for numerous diseases.

• All-cause mortality and cancer

• Smoking causes LOY

• Alzheimer’s disease

LOY is an aneuploidy - Removing the entire chromosome

Pleiotropic effects of LOY - single mutation involved in pathogenesis of many diseases

• Y chromosome not only sex determining, important for immune system

How to recognize a macromolecule

Using affinity reagents for specific binding:

• For DNA and RNA: complementary, base pairing probes

• For proteins: Probes must be selected

Decoding the identity via the sequence of building blocks:

• For DNA and RNA: NGS records the sequence of nucleotides in individual molecules.

• For proteins: mass spectrometry identifies aa. sequences of individual molecules.