The document provides an overview of molecular genetics concepts including DNA structure and packaging, transcription, translation, genetic variation, and types of polymorphisms and mutations. It discusses key discoveries such as Miescher discovering DNA, Levene proposing the nucleotide structure of DNA, and Watson and Crick deriving the double helix model. It describes DNA packaging into nucleosomes and chromatin and compaction into chromosomes. The processes of transcription, mRNA processing, alternative splicing, and translation are summarized. Different types of genetic variation such as SNPs, indels, and CNVs are defined as well as point mutations, insertions, deletions and other mutations.

1. PREPARED BY : IRSA IKHLAQ
SESSION : 2019-23
DEPARTMENT OF ZOOLOGY, GC SIALKOT

2. TOPIC :
MOLECULAR GENETICS MADE SIMPLE

3. • THE STRUCTURE OF DNAAND ITS PACKING
 The First Piece of the Puzzle: Miescher Discovers DNA
 DNA was first identified in the late 1860s by Swiss chemist Friedrich Miescher. Then,
in the decades following Miescher’s discovery, other scientist notably, Phoebus
Levene and Erwin Chargaff--carried out a series of research efforts that revealed
additional details about the DNA molecule.
 Due to its occurrence in the cells’ nuclei, he termed the novel substance “nuclein”—a
term still preserved in today’s name deoxyribonucleic acid.

4. • LAYING THE GROUNDWORK
 Levene Investigates the Structure of DNA
 Levene proposed that nucleic acids were composed of a series of nucleotides, and that
each nucleotide was in turn composed of just one of four nitrogen-containing bases, a
sugar molecule, and a phosphate group.
 Chargaff’s rule:
 The amount of adenine (A) is usually similar to the amount of thymine (T), and the
amount of guanine (G) usually approximates the amount of cytosine (C). In other
words, the total amount of purines (A + G) and the total amount of pyrimidines (C +
T) are usually nearly equal.

5. • LEVENE’S STRUCTURE OF DNA &CHARGAFF”S RULE

6. • WATSON AND CRICK’S DERIVATION
 Rosalind Franklin and Maurice Wilkins
contributed to Watson and Crick’s
derivation of the three-dimensional,
double-helical model for the structure of
DNA.
 DNA is in fact composed of a series of
nucleotides and that each nucleotide has
three components: a phosphate group;
or a deoxyribose (in the case of DNA)
sugar; a single nitrogen-containing base.

7. • DNA PACKAGING: NUCLEOSOMESAND
CHROMATIN
 The haploid human genome contains approximately 3 billion base pairs
of DNA packaged into 23 chromosomes, most cells in the body (except for female
ova and male sperm) are diploid, with 23 pairs of chromosomes. That makes a total of
6 billion base pairs of DNA per cell. Because each base pair is around 0.34
nanometers long, each diploid cell therefore contains about 2 meters of DNA [(0.34 ×
10-9) × (6 × 109)].it is estimated that the human body contains about 50 trillion cells—
which works out to 100 trillion meters of DNA per human.
 DNA, Histones, and Chromatin:
 Proteins compact chromosomal DNA into the microscopic space of the eukaryotic
nucleus. These proteins are called histones, and the resulting DNA-protein complex is
called chromatin.

8. • CHROMOSOMESARE COMPOSED OF DNA
TIGHTLY-WOUNDAROUND HISTONES

9. • THE NUCLEOSOME: THE UNIT OF CHROMATIN
 The basic repeating structural (and functional) unit of chromatin is the nucleosome, which
contains eight histone proteins and about 146 base pairs of DNA.
 Chromatin Is Coiled into Higher-Order Structures:
 The packaging of DNA into nucleosomes shortens the fiber length about sevenfold. In
other words, a piece of DNA that is 1 meter long will become a "string-of-beads"
chromatin fiber just 14 centimeters (about 6 inches) long. chromatin is further coiled
into an even shorter, thicker fiber, termed the "30-nanometer fiber," because it is
approximately 30 nanometers in diameter.
 Chromosomes Are Most Compacted During Metaphase:
 During the different phases of the cell cycle, the DNA varies in the extent of its
condensation. For example, during interphase the chromatin fibres are
organized into long loops, whereas in metaphase chromosomes, the DNA is
compacted to about 1/10,000 of its stretched out length

10. • TRANSCRIPTION
 Transcription is the first stage of the expression of genes into proteins. In
transcription, an mRNA (messenger RNA) intermediate is transcribed from one of the
strands of the DNA molecule.
 The RNA is called messenger RNA because it carries the "message," or genetic
information, from the DNA to the ribosomes, where the information is used to make
proteins.
 RNA and DNA use complementary coding where base pairs match up, similar to how
the strands of DNA bind to form a double helix.

11. • TRANSCRIPTION

12. • DIFFERENCES IN TRANSCRIPTION
 There are significant differences in the process of transcription in prokaryotes versus
eukaryotes. In prokaryotes (bacteria), transcription occurs in the cytoplasm. In eukaryotes,
transcription occurs in the cell's nucleus. mRNA then moves to the cytoplasm for translation.
 DNA in prokaryotes is much more accessible to RNA polymerase than DNA in eukaryotes.
Eukaryotic DNA is wrapped around proteins called histones to form structures called
nucleosomes. Eukaryotic DNA is packed to form chromatin. While RNA polymerase interacts
directly with prokaryotic DNA, other proteins mediate the interaction between RNA
polymerase and DNA in eukaryotes.
 mRNA produced as a result of transcription is not modified in prokaryotic cells. Eukaryotic
cells modify mRNA by RNA splicing, 5' end capping, and addition of a polyA tail.

13. • STEPS OF TRANSCRIPTION
 Transcription can be broken into five stages:
pre-initiation, initiation, promoter clearance,
elongation, and termination.
 Pre-Initiation:
 The first step of transcription is called pre-
initiation. RNA polymerase and cofactors
(general transcription factors) bind to DNA and
unwind it, creating an initiation bubble. It's
similar in appearance to what you get when you
unwind strands of multi-ply yarn. This space
grants RNA polymerase access to a single
strand of the DNA molecule. Approximately 14
base pairs are exposed at a time.

14. • INITIATION
 The initiation of transcription in bacteria begins with the binding of RNA polymerase to
the promoter in DNA. Transcription initiation is more complex in eukaryotes, where a
group of proteins called transcription factors mediates the binding of RNA polymerase
and the initiation of transcription.
 Elongation:
 One strand of DNA serves as the template for RNA synthesis, but multiple rounds of
transcription may occur so that many copies of a gene can be produced.
 Promoter Clearance
 The next step of transcription is called promoter clearance or promoter escape. RNA
polymerase must clear the promoter once the first bond has been synthesized. The
promoter is a DNA sequence that signals which DNA strand is transcribed and the
direction transcription proceeds

15. • STEPS OF TRANSCRIPTION
Initiation: Elongation:

16. • TERMINATION
 Termination is the final step of transcription.
Termination results in the release of the newly
synthesized mRNA from the elongation complex. In
eukaryotes, the termination of transcription involves
cleavage of the transcript, followed by a process
called polyadenylation. In polyadenylation, a series
of adenine residues or poly(A) tail is added to the
new 3' end of the messenger RNA strand.
 pre-RNA and mRNA:
 After transcription, eukaryotic pre-mRNAs must
undergo several processing steps before they can be
translated

17. • PROCESSING OF MRNA
 The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling
factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not
specify the appropriate amino acids.
 5′ Capping:
 A cap is added to the 5′ end of the growing transcript by a phosphate linkage. This addition protects the
mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help
initiate translationby ribosomes.
 3′ Poly-A Tail:
 Once elongation is complete, an enzyme called poly-A polymerase adds a string of approximately 200 A
residue, called the poly-A (50–250 adenine molecules and a 70kDa protein) tail to the pre-mRNA.
This modification further protects the pre-mRNA from degradation and signals the export of the cellular
factors that the transcript needs to the cytoplasm.

18. • ALTERNATIVE SPLICING
 Alternative splicing is a molecular mechanism that modifies pre-mRNA constructs
prior to translation. This process can produce a diversity of mRNAs from a single
gene by arranging coding sequences (exons) from recently spliced RNA transcripts
into different combinations.
 Mechanisms of alternative splicing:
 Prior to RNA splicing, RNA polymerase II produces pre-mRNA transcripts by
transcribing gene sequences into a collection of non-coding introns and protein-
coding exons. When these pre-mRNA sequences undergo constitutive splicing, the
removal of introns is followed by the joining of exons in their DNA-corresponding
order

19. MECHANISMS OF ALTERNATIVE SPLICING

20. • TYPES OF SPLICING
 Exon skipping: This process involves the removal of
certain exons and their adjacent introns from mRNA
constructsprior to translation.
 Alternate 5’ or 3’ splicing: Alternative splicing can
also be mediated by the joining of exons at alternative
5’or 3’splice sites.
 Intron retention: This type happens when non-
coding portions of a gene are retained in the final
mRNA transcript.
 Importance of splicing:
 The mechanisms of alternative splicing help to
explain how one gene can be encoded into numerous
proteinswith various functions.

21. • TRANSLATION
 Translation is the process of converting mRNA into an amino acid chain. DNA stores the
information for proteins in its nucleotide sequence. The mature mRNA molecules can be
translated to proteins.
 This process takes place in the cytoplasm with the aid of ribosomes which are either
floating in the cytoplasm or chilling on the surface of the rough endoplasmic reticulum,
which are complexes of RNAs and proteins called ribonucleoproteins.
 The ribosomes are divided into two subunits: the smaller subunit binds to the mRNA,
while the larger subunit binds to the tRNA which carries the amino acids.
 The translation process ends with the stop codons UAA, UGA or UAG. The nascent
polypeptide chain is then released from the ribosome as a mature protein. In some cases,
the new polypeptide chain requires additional processing to make a mature protein

22. • STEPS OF TRANSLATION
1) Initiation:
 During initiation the small subunit attaches to the 5' end of mRNA. It then moves in the 5' → 3' direction.
The small subunit then reads the mRNA nucleotides in groups of three, called codons, until it runs into
the start codon which is always AUG
. 2) Elongation:
 In the elongation phase of translation, the tRNA with the correct corresponding anticodon will match
with the corresponding mRNA codon. A peptide bond, which is the type of bond that holds amino acids
together, The ribosome then shifts down moving in the 5' → 3' direction, making space for another tRNA
to match with its corresponding codon and thereby allowing another peptide bond to
form.Elongation always goes from the 5' end of the mRNAmolecule towards the 3' end.
3) Termination:
 The final phase of translation is termination. When the ribosome reaches a stop codon (UAG,
UAA, or UGA), a release factor will bind to the stop codon and cause the amino acid chain to
be released and the ribosome breaks off from the mRNA strand and the ribosome subunits to
separate.

23. • STEPS OF TRANSLATION (PROTEIN SYNTHESIS)

24. • GENETIC VARIATION
 Genetic variation refers to genetic difference between individuals within or between different populations.
These variations can be divided in polymorphisms and mutations.
 Polymorphism:
 Polymorphisms are defined as variants found in >1% of the general population. Due to their high
frequency they are considered unlikely to be causative of genetic disease. They can however, together
with other genetic and environmental factors, affect disease predisposition, disease progression or
response to treatments.
 Mutation:
 Mutations can be inherited from parents (germline mutations) or acquired over the life of an individual
(somatic mutations), the latter being the principal driver of human diseases like cancer.
 Germline mutations occur in the gametes. Mutations usually arise from unrepaired DNA damage,
replication errors, or mobile genetic elements.

25. • TYPES OF POLYMORPHISMS
 Three common types of polymorphisms are the
1. single nucleotide polymorphisms (SNPs)
2. small insertions/deletions (indels)
3. large-scale copy number polymorphisms (CNPs or CNVs).
 Single nucleotide polymorphisms:
 SNPs are single base changes that occur on average about every 1000 bases in the genome.
Most SNPs are neutral; yet 3–5% are thought to have a functional role, i.e. affect the phenotype
of the individual carrying them. Depending on their effect at the protein level.
 SNPs can be characterized as synonymous (coding for the same amino acid as the wild type
DNA sequence) or non-synonymous (coding for a different amino acid than the wild type DNA
sequence).

26. • SINGLE-NUCLEOTIDE POLYMORPHISMS
(SNPS)

27. • TYPES OF POLYMORPHISMS
 Small insertions/deletions (indels):
 Indels are small insertions or deletions ranging from 1 to 10,000 bp in length, although
the majority involves only a few nucleotides.
 They are considered the second most common form of variation in the human genome
following SNPs, with over 3 million short indels listed in public databases.
 Large-scale copy number polymorphisms (CNPs or CNVs).
 CNVs are variations in the number of copies of DNA regions. They can involve loss of
one or both copies of a region of DNA, or the presence of more than two copies of this
region. They can arise from DNA deletions, amplifications, inversions or insertions and
their size can range from 1 kb (1,000 bases) to several megabases

28. • COPY NUMBER VARIATIONS

29. • TYPES OF MUTATIONS
 Point mutations:
 Point mutationsin which a single nucleotide is changed for a different one.
 These are divided into :
 Missense mutations (meaning that when translated this DNA sequence leads to the incorporation of
a different amino acid into the produced protein,with possible implications in the protein function),
 Nonsense mutations (where the new nucleotide changes the sequence so that a ‘‘stop’’ codon is
formed earlier than in the normal sequence and therefore the producedprotein is truncated),
 Silent mutations (where the nucleotide change does not affect the amino acid in the corresponding
position of the producedprotein, and therefore the final protein productremains unaltered),
 Splice-site mutations (which affects the splice site invariant donor or acceptor dinucleotides (5’GT or
3’AG).

30. • TYPES OF MUTATIONS
 Insertions:
 Insertions in which one or more nucleotides are inserted in the normal DNA sequence,
therefore disrupting it. This can have a moderate or severe effect on the corresponding
mutant protein product.
 Deletions:
 Deletions in which one or more nucleotides are deleted from the normal DNA
sequence. As in the case of insertions this can lead to minor (e.g. single amino acid
changes) or major protein defects.

31. • TYPES OF MUTATIONS
 Amplifications
 Amplifications leading to multiple copies of chromosomal regions and consequently to an increased number of copies
of the genes located within them and increased levels of the corresponding proteins.
 Inversions
 Inversions involving the reversal of the orientation of a DNA segment, with variable implications for the protein
product.
 Translocations
 Translocations where regions from non-homologous chromosomes are Loss-of-function mutations can be associated
with haploinsufficiency, a common occurrence in the molecular cardiomyopathy setting.
 Haploinsufficiency occurs when the gene product of one of the two alleles in an individual is lost due to a DNA deletion
or to instability/degradation of the mutant protein. Other terms used to describe the effect of a mutation on the fitness of
the carrier are: harmful or deleterious mutations (decreases the fitness of the carrier), beneficial or advantageous
mutations.

32. • TYPES OF MUTATIONS

33. • DISTINGUISHING POLYMORPHISM FROM
MUTATION IN GENES
 Distinguishing Polymorphism from mutation in genes:
 A gene is said to be polymorphic if more than one allele occupies that gene's locus within a
population. In addition to having more than one allele at a specific locus, each allele must also
occur in the population at a rate of at least 1% to generally be considered polymorphic.
 Mutation” and “polymorphism”: earlier definition
 The uniform and unequivocal description of sequence variants in human DNA and protein
sequences (mutations, polymorphisms) were initiated by two papers published in 1993. In this
context, any rare change in the nucleotide sequence, usually but not always with a disease
causing attribute, is termed a “mutation”. This change in the nucleotide sequence may or may
not cause phenotypic changes.

34. • DIFFERENCE BETWEEN GENE
POLYMORPHISM AND MUTATIONS
 A rule of thumb that is sometimes used is to classify genetic variants that occur below 1% allele
frequency as mutations rather than polymorphisms. However, since polymorphisms may occur at low
allele frequency, this is not a reliableway to tell new mutations from polymorphisms.
 Identification:
 Polymorphisms can be identified in the laboratory using a variety of methods. Many methods employ
PCR to amplify the sequence of a gene. Once amplified, polymorphisms and mutations in the sequence
can be detected by DNA sequencing, either directly or after screening for variation with a method such
as single strand conformation polymorphism analysis.
 Genes which controlhair colourare polymorphic.
 Gene polymorphisms can occur in any region of the genome. The majority of polymorphisms are silent,
meaning they do not alter the function or expression of a gene. Some polymorphisms are visible. For
example, in dogs the E locus can have any of five different alleles, known as E, Em, Eg, Eh, and
e.Varying combinationsof these allelescontribute to the pigmentation and patternsseen in dog coats.

35. • DISEASE DUE TO VARIANT OF A GENE
 A polymorphic variant of a gene can lead to the abnormal expression or to the
production of an abnormal form of the protein; this abnormality may cause or be
associated with disease.
 For example, a polymorphic variant of the gene encoding the enzyme CYP4A11, in
which thymidine replaces cytosine at the gene's nucleotide 8590 position encodes a
CYP4A11 protein that substitutes phenylalanine with serine at the protein's amino
acid position 434. This variant protein has reduced enzyme activity in metabolizing
arachidonic acid to the blood pressure-regulating eicosanoid, 20-
hydroxyeicosatetraenoic acid. A study has shown that humans bearing this variant in
one or both of their CYP4A11 genes have an increased incidence of hypertension,
ischemic stroke, and coronary artery disease.

36. DISEASE DUE TO MUTATION OF GENE

37. • MODE OF INHERITANCE—CLINICALAND GENETIC
HETEROGENEITY
 Once a mutation has been directly associated with a pathological phenotype a number of
additional parameters need to be evaluated in order to maximize its value in the clinical
setting.
 .The categorization gonosomal or autosomal depends on whether the mutations are
located on either of the sex chromosomes or not. For example, a mutation on the Y
chromosome will only affect males.
 In hypertrophic cardio myopathy (HCM) a number of cases have been reported with
homozygosity for the pathogenic mutation.
 For example, in Egyptian HCM cohort, none of the mutation-positive patients were
homozygous for the mutation detected (data not published) which might be explained
either by the rarity of its occurrence in the specific cohort or due to technical limitations
in the mutation screening method.

38. • MUTATION SCREENING
 Mutation screening by de naturing
high performance liquid
chromatography (dHPLC) using
WAVE, Transgenomics. dHPLC can
be used as initial mutation screening
method, being dependent on hetero
duplex (wild type-mutant)
formation, and variant profiles from
the wild pattern are subsequently
sequenced.
 Note however, that dHPLC is not
capable of detecting homozygosity.

39. CONTINUED…..
 The phenomena of variable expressivity (variations
in a phenotype among individuals carrying a
particular genotype) and epistasis (one gene is
modified by one or several other genes, e.g.
modifier genes) can lead to a range of pathological
characteristics despite the presence of the same
mutation. These parameters, potentially in
combination with environmental factors, can often
lead to significant clinical heterogeneity in most
inherited CVDs, between unrelated individuals as
well as family members carrying the same
mutation.

40. CONTINUED….
 For example, in HCM the presence of multiple pathogenic mutations could be
included amongst the risk stratification criteria. Multiple mutations have been
observed in about 5% of HCM patients and they are usually associated with higher
septal thickness and worse clinical outcomes, such as heart failure and sudden death.
 Double heterozygosity is commonly detected in the β Myosin heavy chain (MYH7)
and Myosin binding protein C (MyBPC) genes.
 Compound heterozygosity in MyBPC however, leading to the absence of a normal
protein, has been reported to results in neonatal death in two independent cases, where
the parents were each heterozygous for one of the mutations. Similarly, to HCM,
double heterozygosity has been reported in other CVDs such as long QT, with a
similar frequency of 5%.

41. • SIGNIFICANCE OF GENETIC TESTING IN CARDIOLOGY
 In clinical practise, genetic testing can serve 3 main purposes:
 To determine the mode of inheritance of the specific disease in the specific family and identify if there is risk for
other family members.
 To organise the clinical assessment of unaffected family members through genetic testingTo identify if there is ri
sk for other family members.
 Predictive genetic testing:
 To identify those who are at risk for the disease and should be treatedregular cardiac monitoring (mutation carrie
rs) and those who do not mutation non-carriers.
 Identification of distinct genotype–phenotype correlations:
 Genetic screening:
 If patient is valuable, then Initially, provided the genotype–
phenotype relationship, at the diagnostic/prognostic/therapeutic level associations were formed.
 These associations vary greatly between individuals.
cardiovascular diseases, various genes, and various mutations.
 It is important to note, however, that genetic testing in the cardiovascular field is still in its early stages.

42. • IMPORTANCE OF PRE-SYMPTOMATIC GENETIC TESTING
 For the proband's family members,ranges from ensuring that carriers of unaffected mutations receive regular
clinical follow-up to
prophylactic treatment (where available) to reassure that clinically'suspicious' findings are not present.
 Negative Genetic Testing:
 Negative genetic test result in the proband's death.
 Family members cannot rule out the presence of disease in general, because a large number of people have it.
 Pathological Cardiovascular Phenotype:
 By chance, a family member may be a carrier,
a distinct gene mutationAn HCM positive family's pedigree from the BA HCM Study.
 The sister's HCM diagnosis was ruled out.
 However, the proband's symptom-free and echo-clear son.
 At the age of 12 years, he tested positive for the mutation and was given a pre-
symptomatic diagnosis of HCM.
 Symbols in white represent unaffected individuals, while those in black have HCM based on clinical or genet
ic evidence.
.

43. • GENETIC TESTING
 For long QT syndrome and catecholaminergic polymorphic ventricular tachychardia, and occasionally in
high risk HCM families, in which preventive measures or prophylactic therapy is advisable for
asymptomatic mutation positive family members, genetic testing should be undertaken in early childhood,
i.e. regardless of age.
 On the other hand, for late-onset and/or reduced penetrance diseases, it is reasonable to proceed with
clinical monitoring as needed during childhood, leaving the genetic testing option open for when the
individual reaches adulthood
 When a child has already presented with a CVD, the use of genetic testing is complementary to all other
clinical tests, and especially valuable for identifying other family members at risk, since childhood-onset
cases, even when presumed as sporadic, can often have a genetic aetiology.
 Example:
 Hypertrophyhas a genetic basis and bridging the cardiovascular and genetic cause.

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