Penetrance and Expressivity in Genetic Disorders

Human Genome Organisation

• Dark bands represent DNA regions that have a low density of G-C base pairs and a generally low density of exons and genes.
• Pale bands represent DNA regions with a high density of G-C base pairs, as well as a generally high density of exons and genes.
• Centromeric heterochromatin is illustrated by red blocks.
• Other constitutive heterochromatin is shown as blue blocks.
• Large amounts of noncentromeric heterochromatin are present on the Y chromosome, the short arms of acrocentric chromosomes (13, 14, 15, 21, and 22), and chromosomes 1, 9, and 16.
• Numbers to the left of the image represent individual chromosome bands, according to the nomenclature of chromosome banding.
• Only 2% of the genome is transcribed.

Noncoding RNA

• Tiny noncoding RNAs are smaller than 35 nucleotides in length.
• Cytoplasmic microRNAs (miRNAs) are 20-22 nucleotides long.
• Piwi protein interacting RNAs (piRNAs) are present.
• PiRNAs function in germ cells to reduce excessive activity of transposons.
• MicroRNAs (miRNAs) are single-stranded regulatory RNAs that down-regulate expression of target genes by base pairing to complementary sequences in their transcripts.
• miRNA genes undergo transcription and cleavage in the nucleus, producing a stem-loop RNA which is then exported to the cytoplasm and cleaved asymmetrically.
• The passenger strand is cleaved and degraded, leaving the guide strand as a mature single-stranded miRNA.
• They are typically 20–22 nucleotides long.
• Correct base pairing is important for the 'seed' sequence, which covers the first eight or so nucleotides from the 5' end of the miRNA.
• Some mismatches are tolerated when the remaining part of the miRNA pairs up.
• A typical human miRNA binds to and regulates transcripts produced by hundreds of different genes, due to limited miRNA length and tolerance of some base mismatches.
• It is thought at least 50% of protein-coded genes are regulated by miRNAs.
• Individual mRNA often has multiple miRNA-binding sites.
• As an example, the mRNA from the human PTEN tumor suppressor gene has binding sites in the 3' untranslated region for miRNAs belonging to seven miRNA families.
• There are hundreds of miRNA families.
• Most have tissue-specific expression.
• Many are important in early development.
• Important regulators exist in a whole range of different cellular and tissue functions.
• Long non-coding RNAs (lncRNAs) are greater than 200 bases long.
• They are transcribed, but do not code for protein.
• They are implicated in cellular functions, gene transcription, and regulation.
• There are 50,000-60,000 IncRNAs annotated.
• Antisense RNA is transcribed using the sense strand, is not subject to cleavage or splicing, and binds to complementary sense RNA for down-regulation.
• Long regulatory RNAs can be capped, spliced or polyadenylated and are formed from primary transcripts, regulating neighboring or other genes.
• Some long non-coding RNAs are not non-coding, but code for tiny peptides (<100 amino acids) with essential functions.
• Micropeptides were missed due to arbitrary cutoffs such as ORF size of 300 nucleotides, AUG start codon usage, and sequence conservation.
• They lack an N-terminal signal, which means they freely roam in the cytoplasm.
• They are highly conserved for up to 550 million years, present in both prokaryotes and eukaryotes.
• They can act autocrine, endocrine or paracrine manner. -Some are shown to be critical for muscle and cardiac functions.
• A pseudogene is a DNA sequence that shows a high degree of sequence homology to a non-allelic functional gene but is itself nonfunctional.
• It may not make a protein like its closely related homolog, but may make a functional non-coding RNA.

Unprocessed pseudogenes

• Arises from a gene copy made at the level of genomic DNA.
• For example, after tandem gene duplication.
• Has copies of all exons and introns of the parental gene, along with neighboring regulatory sequences like the promoter.A protein-coding gene transcribed, introns are excised from the transcript yielding mRNA.
• Acquisition of deleterious mutations can lead to gene inactivation ('silencing') and subsequent decay, as well as sometimes instability.
• Substantial amounts of the DNA sequence can be lost, leaving just a fragment of the parental gene.
• Often found in the immediate chromosomal vicinity of the parental functional gene, but can be transposed to other locations.

Processed pseudogenes

• Introns are excised from the transcript to yielding mRNA.
• The mRNA is naturally converted into an antisense single-stranded cDNA using a cellular reverse transcriptase function.
• If this occurs in the germ line and the transcript integrates randomly into the genome: it is incapable of expression (it will lack a promoter) and will degenerate into a retropseudogene, after acquiring deleterious mutations (red asterisks, bottom left).
• If the transcript inserts next to an endogenous promoter, it may be expressed, expressed gene may be useful and be preserved as functional retrogene (bottom right).

Mitochondrial DNA

• It is a circular 16.6 kb genome.
• It has 24 RNA genes, with 2 genes making the 12S and 16S rRNAs.
• There are 22 genes making tRNAs.
• It has 13 protein-coding genes that make a few components of the oxidative phosphorylation system.
• Large intergenic region at the 12 o'clock position has important regulatory sequences.
• Two promoters, PH and PL, transcribe the heavy and light strands respectively in opposite directions (clockwise and counterclockwise).
• This generates large multigenic transcripts from each strand that are subsequently cleaved.
• Paternal mtDNA is destroyed in the very early embryo, hence the matrilineal inheritance.
• Each cell has several hundred to thousands copies of mitochondria.
• A human egg cell contains more than 100,000 mtDNA molecules.

Heteroplasmy

• It describes the bottleneck effect in the primary oocyte stage.

Mitochondrial DNA diseases

• Mitochondrial genome is more prone to mutations due to its exposure to ROS.
• Muscle and brain are the most affected from mitochondrial diseases due to their high-energy demands.
• MtDNA disorders are transmitted by females only.

Human Genome Repeats

• Around 30% of the human genome are composed of tandem repeats, where 2 or more nucleotides are repeated tandemly.
• They include satellite, microsatellite and minisatellite repeats.
• They contain various kinds of transposon sequences which are broadly classified into two groups.
• Class I (RNA) transposons are comprised of retrotransposons that transpose through an RNA intermediate, all contain reverse transcriptase.
• Long terminal repeat (LTR) retrotransposons.
• Non-LTR transposons.
• Class II (DNA) transposons contain transposase, and they do not use RNA as an intermediate
• LTR acts like a promoter.
• The gag gene encodes structural proteins that form the virus-like particle (VLP), inside which reverse transcription takes place.
• The pol gene encodes several enzymatic functions, including a protease that cleaves the Pol polyprotein, a reverse transcriptase (RT) that copies the retrotransposon's RNA into cDNA, and an integrase that integrates the cDNA into the genome. The env encodes envelope genes to produce viral particles - absent in human LTRs.
• Full length LINES are 6-8 kb long and can encode a RT, lines are truncated to nearly 1 kb long.
• There is around 80-100 copies of LINEs that are transposing in human genome.
• Full length SINs are around 100-300 bases, belonging to Alu repeat family with dispersed around our genome.
• They are primate specific and seem to have evolved from cDNA copies of 7SL RNA.
• They are often transcribed but no coding potential.
• ALU can transpose but need LINE for RT.
• Class II transposons are DNA transposons and have no RNA intermediate.
• The transposase catalyses cut and paste of the DNA.
• They are flanked by short repeats (yellow triangles).
• LINE1 (L1) repeats can be transcribed but their weak polyA signal might not be sufficient for the stop and transcription may go on.
• Since the L1 is close to the gene end, the polyA signal of the gene is adopted.
• The new RNA is reverse transcribed to DNA and inserted to another gene, the exon of the previous gene carried together.

Origins of DNA Sequence Variation

• Mutation describes both the process that produces altered DNA sequences and the outcome of that change.
• They may contribute to normal or disease phenotype.
• They may have no obvious phenotype.
• Mutations originate as a result of changes in the DNA not corrected by DNA repair systems.

Types of Human Genetic Variation

• Changes that do not affect the DNA content (number of nucleotides); copy number is the same.
• Balanced translocations.
• Inversions.
• Changes that affect the DNA content: change in copy number.
• Abnormal chromosome segregation.
• Deletion or insertion of a single nucleotide.
• Altered numbers of specific short oligonucleotide sequences to megabase lengths of DNA.

Single nucleotide variants (SNP or SNV)

• Reference allele: base found in the reference genome.
• Alternative allele: any other base other than the reference allele found at a locus.
• Protein-coding mutation in LMNA gene cause Progeria or accelerated aging and can be lethal.
• A major allele in one population can be a minor allele in another population.
• Major allele can identify an individual- around 300 SNVs can help determine their ethnicity.
• Minor Allele Frequency: the frequency of the second most common allele that occurs in a population.
• The minor allele is not always the alternative allele. -Major and minor is defined on the basis of frequency and reference one is on the basis of reference- nothing to do with frequency. -If a DNA variant is common the population, the DNA variation is described as single nucleotide variant or polymorphism (SNV or SNP).
• DNA variants that have frequencies less than 1% are rare variants. -The reference allele can be different than the major allele and a SNV has no effect on its detection.
• Does not mean that rare variants are difficult to sequence or if its rare in genome or smth.
• Just defines the frequency of a variant in a population, not difficulty in sequencing or being found in the genome.

Copy number variants (CNVs) and Imprecise Cuts

• Always affects more than one base.
• CNV Inversion: copy number is same, change in directionality of a segment, affects inversion and duplication which impacts cancer.
• Indels are strictly speaking copy number changes that are (arbitrarily selected) less than 100 bases.

Imprecise cut between CNVs and indels

• There are DNA variants that differ by the presence or absence of one or few nucleotides: insertion/deletion variation or polymorphism (indel).
• It is hard to sequence.
• Copy number changes are those that span longer than 100 bases
• The frequency of indels is one-tenth of SNVs.
• Short size indels are more common than long size indels.
• There are as little as 1-10 long indel sequences.

Aneuploidy, Polyploidy and Uniparental Disomy

• Having a normal chromosome set is euploidy.
• Aneuploidy is missing a chromosome.
• Nondisjunction Mechanism: paired chromosomes fail to segregate, or sister chromatids fail to disjoin at either meiosis II or mitosis.
• If non-disjunction occurs in mitosis, the individual becomes mosaic.
• The stages known as Anaphase lag are lethal.
• Polyploidy describes the gain of complete chromosomes.
• Triploidy has rare conditions such as as 3% of pregnancies produce a triploid embryo through two sperm fertilizing a single egg.
• The rare lethal occurence Tetraploidy as well.
• Uniparental Disomy: A zygote develops in which both copies of one chromosome originated either from mother or father , which can disrupt genomic imprinting.

Structural Abnormalities

•Involve Double stranded breaks and are Commonly • The abnormal chromosomes with one centromere can go through with success. • Those which lack (acentric) or have two centromeres (dicentric) will not segregate stably during mitosis, hence be eventually lost.

Translocations

• Derivative chromosome- Translocating a region of one chromosome to another chromosome and creating a new chromosome.
• Chromosomal territories: chromosomes 4, 13 and 18 tend to translocate more often due to chromosomal proximity: chromsomal territories.
• Robertsoninan translocation: chromosomes 13, 14, 15, 21 and 22 are acrocentric chromosomes, (the centromere is close to a telomere) and have short arms containing 30-40 large tandem repeats for rRNAs.
• They congregate at the nucleolus to produce ribosomal RNAs.
• During this close association, breaks in the short arms causes translocations or fusions.
• The sequences are vey similar, so the DNA repair machinery cannot distinguish them.

DNA Methylation

• Modification, not mutation and Cytosine is a base in DNA (C).
• Gene expression is correlated with "open" chromatin, which has more DNA space.
• "C" is always followed by "G" in CpG.

DNA methylation

• DNA methylation involves adding a methyl group to certain cytosine residues, forming 5-methylcytosine (5-meC).
• Cytosines that are methylated occur within palindromic sequence that is the CG dinucleotide.

Underrepresentation of CpG dinucleotides

• 5-methylcytosine becomes thymidine give CpGs erode in time in a Deamination reaction.
• The 41% of genome is made up of G-C base pairs, giving individual base frequencies of 20.5% each for G and C; the expected frequency of the CG dinucleotide is ~ 4.2%, the observed CG frequency is around 1%.

More Facts about CpG Islands

• The CpGs are not simply random points but clusters throughout the genome
• CpG islands:sequence ranges where value the O/E is greater than 0.6 and the GC content is greater than 50% for 200 base window, which is mostly associated with vicinity of transcriptional start sites. -These CpG islands might not methylated everywhere throughout our body tissues.

Further information on Aberrant DNA Methylation

• Cells use S-adenosylmethionine (SAM) as a methyl donor for methylation.
• Sometimes SAM can inappropriately methylate DNA to produce harmful bases and Each day, around 300-600 adenines are converted to 3-metyladenine, a cytotoxic base that distorts the DH , crucial DNA-protein interactions..

Sources for the Origins of Genomic Variation

• Normally cellular metabolism generates strongly electrophilic (highly reactive) molecules or ions
• The Reactive oxygen species, in result of reduction of oxygen, have roles in signalling pathways, mostly in mitochondria, causing breaking of sugar bonds in DNA strand, potentially attacks DNA bases, and block the polymerases

Other causes of Genetic Mutation

• External Mutagens are caused by Ionising radiation(harmful chemicals), such as UV radiation covalent bonding between adjacent pyrimidines (C and T) pyrimidines on a DNA strand.
• Cigarette smoke and automobile fumes are large aromatic hydrocarbons that can bind to DNA and distort the double helic.

Error in DNA replication leads to Proofreading by DNA polymerase

-In the context that billions of nucleotides must be accurate when they are replicated.

• During the insertion by wrong base pairings induces the DNA repair.
• It is then reparied the own DNA polymerase via exonuclease activity for the DNA mismatching.
• If proofreading fails DNA mismatch repair system is activated.
• A repair is needed to counteract DNA mutations by different sensors that trigger the for DNA repair pathway, or the cell cycles are arrested. -The double stranded breaks:The break can inactivate genes.
• Homologous recombination (HR) mediated DNA repair requires an intact DNA strand using a template to guide repair in mitosis and during cell division

DNA repair

• Nonhomologous end joining (NHEJ): No template strand is necessary since the broken ends are simply fused together in the DNA.

Mutagenic Sequences in the genome

• The GC-rich sequences are methylated and prone to spontaneous deamination
• The Microsatellite contains short tandem repeats (STRs) causing slippage and indel formation (3% of the genome)
• The Meiotic recombination and Late replicating are prone with more chance mutations

Population and Human Variation

• On average each day there are thousands of DNA lesion occuring in the cells.
• Due to Popultaion bottlenexk there the human population was around 10,000, but humans are better at preventing genetic heterogenuity to to recent and rare occurance and more de novo mutations.
• These variations vary on their position. -Single nucleotide variation has > 75% of genomes

  • 350 Million is common SNV and about (8,000 novel variants (Discussion point)

• Personal geno sequence each possess on average on Single nucleotide variation per 1 kb and >2000 CNV
• Carry 120 gene-inactivating variants

Variation Patterns

• Single nucleotide variations are not homogenuously distributed
  • Different DNA regions have different mutation rates
  • The Alternative nucleotides have been marked on ancestors to chromosomes, in present day populations. -Human segments also share Chromosome Segments
  • These related patterns of Ancestral (common ancestor and within previous four generations )

-For reference the Follicular variant is very similar for the chimpanzee or gorilla with high allele frequency, due to is a result of changes refer to changes from that common sequence

• There is high variation that contains two amino acids (modern people and Neanderthols) -Many variations in cognitive skills

Lecture 3 Topics

• Each nucleated cell contains the same genome, achieving expressions through gene regulation.
• Epigenetic pathways help to ensure this regulation.
• Cis regulatory elements function in gene regulation that is limited to the single DNA molecule on which it resides.
• Trans regulatory elements are free to migrate by diffusion to regulate expression of both alleles, and often regulate different genes.
• Promotors, always on 5' end of the end of the gene, have canonic TATA boxes for promoter elemements
• Other promoter elemements lack the DPE, BRE. -Enhancers, regulated by specific motif of transcriptive region, are DNA elements that modulate the genes over genome long distances.
• Their activity is often orientation independent -The action is clusters of focused high affinity for the DNA in promoters, which lead to increased or decreased the rate of transcription
• Silencers are which regulatory sequences lowers the rate of initiation of the transcription element or the processerity for the RNA Polymerase. -The RNA Polymerase II assembles with basal and activator factors together in the cell, but can synthesize with some promoter but cannot recognise the TFs, because transcription does not act like other promoters.
• Mediator complex complex makes the interaction between the promoter and the enhancer in a specific way. -It communicates regulators DNA factors for the transcription of RNA Polymerase and regualtes the gene expression due to presence and absense.
• The high range of 5 to 10Mb are interactions are dependent on cell type.
  • These interacttions are either the rate for the burts frequency, or they are long rare events

Epigenetics and Chromatin

• It is the study of of heritable changes occur within the DNA sequence.
• The methylation that cytocines in CpG context and cell specific,is important in genomic imprinting and for retrotransposon silencing.
• The chromatin is has Histone Octamer with and has Nucleosome that wrapps or alters the modification of the Histone tails( DNA)
• Different variants of histone molecules wrap DNA for stability changes by modifying new sites
• With that, the Epigenetic has been modified to alter transcription of which include, acetylation (Ac), methylation (Me), phosphorylation (P), ubiquitination, all which effect chromatin packaging, and may be located with a TATA box
• The chromatin packaging unit involves mapping histone with ChIP-seq and DNA binding proteins with formaldehyde cross linked cells.
• Non coding RNAs in epigenetic regulation
  • Around 20% of the non coding RNAs regulate structure and architecture of the chromatin regulatory ncRNAs, trans act like a signals for the transcripted genes.

Lecture 4 Topics Cont.

• LncRNA Recruit, to introduce modifications to the Histone during transcriptions, in cell division

Chromatin Packaging and Transcription

• The local action is set by Chromatin, so that action occurs next to the transcription unit.
• This is done by the "Tail of histones",
• "Heterochromatin
• "Euchromatin
• :This is an area of high chromosome
• *"Transcriptions occure in burst-in and out."
• Inactivate and Active and
• It is important. It can be mapped via Hi Cap and Chromosome Conformation.
• The is also the Polycomb repressive complex that occurs in Chromosomes.
• Therefore a way in which structure, function,and genetic information are carried to a variety of different cell functions. and can be on a nucleosome -Positional Effect: Is element can be activated depending on its surrounding chromatin Genomic Imprinting: for some genes, there is a defined parent of origin and with parthenogenesis
• • a subset of is expressed only
• -The methylations are imprinted in different regions

Epigenetics and Genome/Gene Function

• The long process involves the expression of cell-type specific functions within the transcription regulatory system, and then these interactions are expressed from there by different TFs.
• That means that the "structure" of the genome at local scale is a powerful way to set the tone of what genes and elements are to be transcribed, and what genes and elements are to not to be transcribed.

A quick way to summarize the key points

• That gene control of transcription is related to the heritability of genetics, and structure, function,regulation, and a lot of the factors surrounding these functions.
• • In many humans genome the "disorders" are very apparent which cause "disease
• (and are affected by the environment)

Monogenic diseases

• In cases of Single gene it that caused by different mutations to one genes:
  • In which all mutations have an effect, leading to the loss or gain of genes.
• -Some are inherited and some are not and there many different disorders (Mendelian inheritance patterns): "Dominant,recessive,and allolactic which can change the gene.
• • "Also if inherited they are insensitive to the genome"
• -There are known examples of many genes which each has a role in embryonic or of placenta growth that give insights to the development in of humans

Causes of non-Monogemetic (rare) diseases.

• Noncoding genic mutations: :There'r -There must some activation of "cryptic splice" sites:

• where the "Spilce acceptor is "in the Intron", and creates a 'framshift" and there will be no proteins

• -Structural aberrations, and allolactic disorders These include: Pathogenic exchanges that lead to nonallelic homologous recombination, or over or under transcription that has sideffects

Explore the concepts of penetrance and variable expressivity in genetic disorders through multiple-choice questions. Understand how these phenomena affect the expression of inherited traits, focusing on factors influencing phenotype manifestation. Questions cover probability, mutation inheritance, and the genetic basis of diseases like tuberous sclerosis.