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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5210_Библиотеки_им_академика_М_И_Перельмана
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Protein
Codon
230
G0 phase
(resting)
M
G
1
Semi-conservative
replication of DNA
G
2
Protein synthesis
U UUU UGG GG AACAA
mRNA
A
5' 3'
MET LYS PHE GLY
S
TranslationTranscription
STOP
Figure15.1 DNA replication, transcription and translation. BioRender/https://biorender.com/last
accessed December 02, 2023.
15.2 The Human Genome
15.2.1 Organisation ofHuman Genome
The smallest unit that enables the transmission of genetic characteristics to the next generation is
called a gene. In addition to individual genes, ‘gene families’, which have originated during the
evolution of the genome, encode homologous proteins and are formed by duplication or mutations
of ancestral genes. Non- functional copies of active genes are called ‘pseudogenes’, which are
formed by destructive mutations of the stop codon or frameshift type and are not transcribed.
Many gene products that are transcribed from specific genes but do not code for proteins play an
important role in regulating those that code for proteins. In addition to genes and pseudogenes,
there are many specialised regions in the organisational structure of the genome, such as noncoding RNAs, repetitive DNA sequences, intronic sequences, transposons and viral sequences.
Repetitive DNA regions can be divided into tandem and interspersed DNA. Tandem regions
(variable number of tandem repeats=VNTR or short tandem repeats=STR) are highly polymorphic regions and can be located in centromeric heterochromatin regions, telomeric regions, coding
or non- coding gene regions. Interspersed DNA repeats are retrotransposons (long interspersed
nuclear elements (LINEs), short interspersed nuclear elements (SINEs), long terminal repeats
(LTRs) and transposons, which account for up to 45% of the genome. Since transposons can independently translocate in the genome, they are important causes of mutations and some genetic
diseases. They damage the genome either by disrupting the function of genes in the regions where
they are integrated in the genome, by errors that occur during DNA repair in the regions they
leave, or by unequal crossing over(3, 4).
t.me/Dr_Mouayyad_AlbtousH
DNA double helix (2
Nucleosome (11
Chromatin fibre (30
Extended form of chromosome (300
Condensed form of chromosome (700nm)
Metaphase chromosome
Figure15.2 DNA packaging. BioRender/https://
biorender.com/last accessed December 02, 2023.
15.2 The Human Genome
nm)
nm)
nm)
nm)
231
The human genome is packaged during the cell cycle in order to be used most effectively. Histone
and non- histone proteins play an important role in these processes. The smallest unit (146–147
base pairs + 8 core histones) formed by two turns of the naked DNA molecule around core histones
(H2A, H2B, H3 and H4 × 2) is called a ‘nucleosome’ and is anchored by the histone protein H1.
Folding continues throughout the cell cycle to form solenoids, loops, chromatin and metaphase
chromosomes respectively (see Figure15.2).
15.2.2 Genome Variations
Permanent and heritable changes in the DNA sequence are called genetic variations. They are
called ‘polymorphisms’ if the MAF (minor allele frequency) observed in large population databases is greater than 1%, and ‘mutations’ if the MAF is less than 1%, especially if they are in coding
regions of the genome and are associated with Mendelian diseases.
Variations in the genome can occur at the gene, chromosome and genome levels (see
Figure 15.3). Genetic variations (silent, nonsense, missense, frameshift) can alter the genetic
code, gain or lose genetic material. Synonymous variations do not change the amino acid
sequence. Variations that cause a change in the amino acid sequence are called missense variation. Single- base substitutions that cause premature termination of the amino acid chain are
called ‘nonsense’ variations. There are also deletion or insertion variations which alter the reading
frame, called frameshift mutations.
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232
Figure15.3 Genetic variations.
BioRender/https://biorender.
com/last accessed December
02, 2023.
Gene
Silent
Missense
Nonsense
Frameshift
Variations
Chromosome
Deletion
Insertion
Translocation
Inversion
Genome
Euploidy
Triploidy(3n)
Tetraploidy(4n)
Aneuploidy
Trisomy
Monosomy
Chromosomal variations can be either numerical or structural. Structural chromosomal
variations are deletions, insertions, translocations, inversions (paracentric and pericentric) and
ring chromosomes. Numerical chromosomal variations are described as euploidy and aneuploidy;
any number of chromosomes that is an exact multiple of the number (n) in the haploid gamete is
called euploid (such as 3n=triploidy or 4n=tetraploidy) and is incompatible with life in humans.
If the chromosome number is not an exact multiple of the haploid number (such as 46,
XX, + 21=trisomy or 45, X=monosomy) it is called aneuploid(1–3, 5).
15.2.3 Gene Expression Regulation andEpigenetics
During the packaging of DNA into chromosomes, loosely (euchromatin) and tightly (heterochromatin) packed genomic regions are formed (see Figure 15.4). This organisation controls gene
expression and allows for epigenetic regulation. Euchromatin regions form structures that allow
the binding of transcription factors and enable the expression of genes located in these regions.
Euchromatin
Heterochromatin
Figure15.4 Euchromatin and heterochromatin
structure. BioRender/https://biorender.com/
last accessed December 02, 2023.
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15.2 The Human Genome
(a) Histone methylation
(b) Histone modifications
233
Me3
Figure15.5 Histone modifications. BioRender/https://biorender.com/last accessed
December 02, 2023.
Methyl group
Promoter
Target gene
However, heterochromatin structures are tightly packed and inactive regions for transcription.
Gene expression is regulated at multiple levels: at the DNA level by methylation, at the chromatin
level by histone modifications, chromatin remodelling and 3D genome organisation, and at the
post- transcriptional level by non- coding RNAs and RNA modifications(6).
Epigenetics can be thought of as the adaptation mechanism of DNA, which has a static structure, to dynamic environmental conditions. The study of heritable changes in gene function at the
cellular level that are not based on changes in DNA sequence. Epigenetic regulation includes DNA
methylation, histone modifications, chromatin remodelling, three- dimensional genome organisation and RNA interference mechanisms. Proteins involved in epigenetic mechanisms function in
three main groups: writers (histone acetyltransferases and histone methyltransferases), erasers
(histone deacetylases and lysine demethylases) and readers (bromodomains, chromodomains,
PHD fingers).
● DNA methylation is the addition of a methyl (CH
) group to the carbon at position five of
3
cytosine in a CpG dinucleotide and is catalysed by DNA methyltransferase enzymes (DNMT1,
DNMT3A and 3B). DNMT1 enzymes methylate the semi- methylated CpGs formed after repli-
cation. Demethylation is mediated by ten- eleven translocation (TET) proteins. The promoter
regions of genes are CpG (cytosine and guanine) rich regions and gene expression is regulated
by methylation/demethylation that occurs in these regions (see Figure15.5a).
● Another type of epigenetic regulation is histone modifications mediated by histone proteins. The
N- terminal ends of all nuclear histones and the C- terminal ends of H2A extend outside the
nucleosome and are modified by chromatin regulatory enzymes (acetyltransferases, methyl-
transferases, kinases, etc.). The N- terminal lysine, arginine and serine residues of histone pro-
teins undergo post- translational modifications. These modifications alter the chromatin
structure at the regions of interest and affect the ability to transcribe and are maintained during
cell divisions (epigenetic memory). Post- translational modifications of histones can lead to gene
activation (acetylation and phosphorylation), gene expression modulation (sumoylation, deami-
nation and proline isomerisation), or both (methylation and ubiquitination) (see Figure15.5b).
● One of the mechanisms of post- translational gene expression is non- coding RNAs, and they are
classified according to their length into short (>200 nt) (such as miRNA, snoRNA, piwiRNA)
and long (<200 nt) (such as XIST, H19, MALAT1) non- coding RNAs. These non- coding tran-
scripts have direct structural, regulatory and catalytic functions. They are generally involved
in gene- specific transcriptional regulation, epigenetic regulation (such as imprinting and
X- chromosome inactivation) and post- transcriptional regulation (such as mRNA splicing,
translation, RNA interference).
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Incidence at birth (per 1000)
multifactorial
gene
Chromosomal
234
15.3 The Basics ofInheritance
Genetic diseases are generally classified as multifactorial, chromosomal and single- gene disorders
(see Figure15.6). In multifactorial diseases, environmental influences act together with genetic
factors to predispose to the disease. Chromosomal disorders mostly occur de novo (trisomy, deletion, duplication) but can also be inherited (translocations). Non- Mendelian inheritance models
are also summarised below.
Figure15.6 Genetic disorders’ classification.
BioRender/https://biorender.com/last
accessedDecember 02, 2023.
Genetic
disorders
60%
Complex/
10%
Single
6%
15.3.1 Single- gene Disorders
Single- gene disorders are inherited diseases that follow a Mendelian pattern of inheritance (see
Figure15.7). They have a frequency of approximately 1in 100live births. A person is said to be
homozygous if he or she has a pair of identical alleles at a given locus; if the alleles are different,
the person is said to be heterozygous (a heterozygote or carrier). The term compound heterozygote
is used to describe a genotype in which mutations are present in two different regions of the same
gene. In the special case where a male has a mutant allele for a gene located on the X chromosome
and there is no other copy of the gene, he is neither homozygous nor heterozygous and is called
hemizygous. Single gene (or Mendelian) disorders are characterised by patterns of transmission
within families. To determine the mode of inheritance, a detailed family history is obtained, pedigrees of at least three generations are constructed and a detailed clinical evaluation is performed.
Mendelian inheritance models include autosomal dominant, autosomal recessive, X- linked dominant and X- linked recessive(1, 2, 7).
● In the autosomal dominant inheritance model, the trait/phenotype or disease occurs in heterozy-
gous form. More than half of Mendelian diseases have an autosomal dominant inheritance pattern. A single mutated allele is sufficient to disrupt cellular function and cause disease. Boys and
girls are equally affected, and each child has a 1in 2 (50%) chance of inheriting the disease.
Examples; Orofacial cleft- 1 (OMIM:119530), Dentinogenesis imperfecta (OMIM:125500),
Agnathia- otocephalycomplex (OMIM:202650), Amelogenesis imperfecta, type III (OMIM:130900).
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Autosomal recessive Autosomal dominant
X-linked recessive X-linked dominant
•
•
••
Figure15.7 Single gene inheritance models. Muge Sayitoglu.
235
● In the autosomal recessive inheritance model, both allelic copies must be mutated, in other
words, the affected person must be homozygous or compound heterozygous. In the autosomal
recessive inheritance model, both parents have to be carriers of one copy of the mutated gene.
Most recessive diseases are caused by loss of function mutations. Males and females are equally
affected and at conception, each child of parents who are both carriers have a 1in 4 (25%) chance
of being an unaffected noncarrier, a 2in 4 (50%) chance of being a carrier and a 1in 4 (25%)
chance of inheriting the disease. Consanguinity increases the likelihood that both parents may
be carriers of the same recessive gene mutation for a disease. Examples; Orofaciodigital syn-
drome VI (OMIM:277170), Orofaciodigital syndrome V (OMIM:174300), Dentin dysplasia, type
I (OMIM:125400), Periodontitis 1, juvenile (OMIM:170650) and Amelogenesis imperfecta, type
IC (OMIM:204650).
● X- linked dominant or recessive inheritance patterns are distinguished by the phenotype in
heterozygous females. A male with a mutant allele at an X- linked locus is hemizygous for that
allele, while females can be either homozygous or heterozygous for either the wild- type or
mutant allele. In X- linked recessive inheritance, a trait or disease is caused by a mutation of a
gene on the X chromosome. Males show the phenotype because they have one X chromo-
some, while females usually do not show the phenotype because they have two X chromo-
somes. Diseases are transmitted from carrier females to their sons but affected males do not
inherit the disease in subsequent generations. If the mother is a carrier, there is a 50% chance
that her daughters will also be carriers, while her sons have a 50% chance of being affected.
X- linked dominant inheritance is a rare condition in which both males and females are
affected. Due to X inactivation, affected males show the phenotype more severely. Affected
females have a 50% chance of passing the disease on to their sons and daughters. Examples;
Orofaciodigital syndrome I (OMIM 277170) and Amelogenesis imperfecta, type 1E
(OMIM:301200).
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236
15.3.2 Chromosomal Disorders
The chromosomes, called autosomes, are identical in males and females, and the two pairs of sex
chromosomes (XX in females and XY in males) are called gonosomes. A karyotype is a representation of an individual’s chromosomes, indicating their number and morphology on a graph.
Structural or numerical chromosomal abnormalities can affect somatic or germ cells and can occur
at different stages of life. Structural chromosomal abnormalities can occur spontaneously or as a
result of radiation, carcinogens and viral infections. They can be balanced variations without
genomic loss (such as translocations) or unbalanced variations with genomic loss (such as deletions, duplications, inversions)(1–3).
Chromosomal imbalances resulting in the addition or loss of genes in chromosomal diseases are
expected to have a specific phenotypic effect determined by the dosage of genes on the excess or
missing chromosomal segments. Depending on the size of the affected area, 1000 of genes and
therefore many systems and organs can be involved in the phenotypes.
Chromosomal mutations can affect autosomes or sex chromosomes.
● Monosomies (the presence of a single copy of a chromosome) that affect autosomes are incom-
patible with life, but partial monosomies (such as 5q deletions and 22q deletions) lead to pathogenic phenotypes. Autosomal trisomies (the presence of three copies of a chromosome) are
incompatible with life except for three: trisomy 21, 18 and 13. These trisomies have rather distinct phenotypes, but each of them is characterised by mental retardation and multiple congenital anomalies and for prenatal screening, trisomies of these three chromosomes are screened in
high- risk mothers.
● X- and Y- chromosome aneuploidies are disorders that occur in 1in 400 to 500live births and
cause less severe phenotypes than autosomal aneuploidies due to inactivation of the X chromosome and low gene content on the Y chromosome. The most common sex chromosome defects
in live- born infants and fetuses are trisomic types (XXY, XXX and XYY). Monosomy for X (Turner
syndrome) is less common in live- born babies and is the most common chromosomal abnormality reported in spontaneous abortions. Sex chromosome abnormalities most commonly affect
sexual development and cause infertility in affected females and males.
15.3.3 Complex Inheritance
Complex or multifactorial inherited diseases affect a large proportion of the population and occur
under the influence of genes and environmental factors. Phenotypes are influenced by more than
one gene and although each gene individually segregates as a Mendelian type of inheritance, the
diseases or phenotypes do not fit the Mendelian type of inheritance. Asthma, diabetes, hypertension, spectrum disorders, coronary artery disease are common diseases with genetic and environmental components.
They usually occur later in life and have a relatively low risk of recurrence. In this context, the
concepts of incidence (the number of new cases per population at risk in a given time period) and
prevalence (the proportion of cases in the population at a given time) are important for understanding risk assessment in families. Phenotypes with complex characteristics can be assessed
quantitatively or qualitatively.
The quantitative approach is usually based on the distribution and frequency of a complex disease within a family or using concordant and discordant phenotypes in twin studies. Quantitative
trait loci (QTL) are controlled by many genetic loci and show varying degrees of dominance; some
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QTLs have stronger effects than others (major effect and minor effect genes, respectively). Linkage
studies and genome- wide association studies (GWAS) are used to locate the genes that influence
quantitative traits.
General features of multifactorial inheritance are;
● Most affected children have normal parents
● The risk of recurrence increases with the number of affected children in the family
● The risk of recurrence increases with the severity of the defect; a more severely affected parent
is more likely to give birth to an affected child
● Consanguineous marriage slightly increases the risk of an affected child
● The risk for affected relatives decreases very rapidly with the degree of consanguinity.
15.3.4 Non- classical Inheritance
In addition to the classical inheritance models mentioned above, some situations in which a single
gene is inherited but outside the rules of Mendelian inheritance can be said to be non- classical or
non- Mendelian inheritance (see Figure15.8).
Mosaicism: the coexistence of cells with different genetic characteristics derived from a single
zygote in the same organism. One of the best examples of this is X inactivation in females; due
to random X inactivation, they are mosaic in terms of the X chromosomes they possess (some of
paternal and some of maternal origin). Mosaicism is the result of genetic variations that occur
either prenatally or postnatally in a single cell, which then develops clonally through postzygotic divisions. Rarely, it can occur in the gonads (germline mosaicism) and is known to be a
complicating factor in genetic counselling (see Figure15.8a)(8, 9).
Mitochondrial Inheritance: Mutations that occur in mitochondrial DNA (mtDNA, <17 Kbp) are
transmitted to subsequent generations through maternal inheritance. Cells contain hundreds
of mtDNA molecules in their mitochondria, which change according to their energy needs.
Mature oocytes contain more than 100,000mtDNA, and each cell division replicates a large
number of copies of mtDNA in each mitochondrion and randomly transfers them to newly
synthesised mitochondria.
237
Homoplasmy is the state in which all mutant or wild- type mtDNA is present in the daughter
cells that result from cell division. The transfer of a mixed composition of mutant and wild- type
mtDNA into new cells is called heteroplasmy. In maternal inheritance, the mother passes the
disease to her sons and daughters via the mitochondria in the cytoplasm of her oocytes, but only
sick daughters can transmit the disease to the next generation. The phenotype of the disease varies
according to the homoplasmic/heteroplasmic status (see Figure15.8b)(10, 11).
Genomic Imprinting: A condition in which the expression of genes in different alleles varies
according to the sex of the parent from whom they are inherited. Different diseases/phenotypes
result from mutated alleles inherited from either the mother or the father. Imprinting is essential for normal embryonic development. It occurs during gametogenesis just before fertilisation
and marks certain genes as originating from the mother or father. After conception, imprinting
controls gene expression within the imprinted region in some or all of the embryo’s somatic
tissues. The imprinted state persists postnatally throughout the somatic tissues of the embryo
(see Figure15.8c)(12, 13).
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Gene
Gene
expression
(a) Mosaicism (b) Mitochondrial inheritance
238
(c) Genomic imprinting
Father
A
B
Gene A
Gene B
Figure15.8 Non- classical inheritance models. BioRender/https://biorender.com/last accessed
December 02, 2023.
Mother
X
Paternal
(d) Uniparental disomy (UPD)
UPD-heterodisomy UPD-trisomic rescue UPD-isodisomy Normal
Uniparental Disomy: refers to the condition in which both copies of a specific chromosome are
inherited only from the mother or only from the father. If both homologues of the parental pair
are present, the condition is called heterodisomy; if one of the parental homologues is present as
a pair, the condition is called isodisomy (see Figure15.8d)(9, 14).
Dynamic Mutations: In classical models of inheritance, when a mutation occurs, it is stably
inherited over generations. It can be described as unstable repeat expansions of regions that
are polymorphic between individuals in the general population and repeats of three (such as
CAG or CCG) or more nucleotides in tandem create an expansion within the gene. There are
two important terms in this case: anticipation and premutation. Anticipation is the occurrence
of phenotypes/diseases earlier and more severely between generations. Premutation refers to
the number of repeats that are higher than normal but not enough to cause disease and are
likely to be inherited as full mutations. Premutation carriers are phenotypically normal but
prone to disease.
There are three types of errors in unstable repeat expansion; Class 1: preRNA transcription in the
affected gene can be disrupted, resulting in loss of protein function (examples: Fragile X syndrome
and Friedreich’s ataxia), Class 2: can confer new properties to the RNA (examples: Myotonic dystrophies 1 and 2, Fragile X- associated tremor/ataxia syndrome) and Class 3: disorders caused by a
codon (such as CAG for glutamine) repeat expansion that confers new properties to the affected
protein (examples: Huntington’s disease, spinocerebellar ataxia)(15, 16).
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15.4 Milestones ofGenome Studies
The human genome refers to the entire genetic code of a human being and includes coding and
non- coding regions. Deciphering the human genome sequence is one of the greatest achievements
of the scientific community. Reference human genome sequences are invaluable for detecting
genomic variants and understanding the origin of the human species. The complete sequencing of
the genome took 32 years and the journey is summarised in this chapter.
The Human Genome Project (HGP): The HGP aimed to decipher sequences of the human genome
as well as a set of model organism’s genomes. The second goal of the HGP was to store the
genome data in databases, develop data analysis tools and address ethical, legal and social
issues (ELSI) that may arise during and after the project. The HGP launched in 1990with an
international collaborative effort and took 13 years to complete ~92% of the first genome
sequence (~3 billion base pairs) with 99.9% accuracy(17, 18).
The reference genome sequence was generated from blood samples of multiple individuals and
made publicly available. The HGP used Sanger sequencing technology, which can only sequence one
strand at a time. Due to technical limitations, highly repetitive regions (centromeres and telomeres)
could not be sequenced. Before HGP, the human genome was estimated to contain ~100,000 genes.
However, when the project was completed, it was shown that the human genome contains approximately 20,000–25,000 protein- coding genes and that only 1.5% of the genome encodes proteins, while
the rest consists of RNA genes, regulatory sequences, introns and other intergenic regions.
We learnt that genes are not evenly distributed along chromosomes, but are instead distributed
in clusters, with some chromosome regions carrying more genes and others carrying fewer. The
identification of polymorphic regions in the genome, especially single nucleotide base variations
(SNPs), has led to the development of several revolutionary methods for further computational
tools and the creation of larger projects such as HapMap, ENCODE and 1000 genomes(3, 17, 19).
239
International HapMap Project: International HapMap Project launched in 2001 and aimed to
develop a haplotype map of the human genome for defining genes and genetic variations in
health and disease. The HapMap is an important tool for researchers and all data are freely available to the public through the single nucleotide polymorphism database (dbSNP)(20).
The Encyclopedia of DNA Elements (ENCODE) Project: The ENCODE Project started in 2003 and
aimed to identify functional elements in the human and mouse genomes. All ENCODE data is
shared in databases publicly. Today, ENCODE provides a broad data resource including RNA
transcription, chromatin structure and modifications, DNA methylation, chromatin looping
and occupancy by RNA- binding proteins and transcription factors(21).
The International 1000 Genomes Project: The international 1000 Genomes Project launched in 2008
to develop a catalogue of human common genetic variations freely available. Initially, 1092individuals from 14 populations were sequenced by whole genome and exome sequencing, and each
passing year the number of populations and volunteers is increasing(22, 23).
The Telomere- to- Telomere (T2T) Consortium: After 32 years of initiation of the HGP, the T2T con-
sortium announced that the gaps were filled and sequence of human genome was complete in
2022. Researchers used long- read sequencing technology to decipher repeated rich parts of the
human genome. Cost of generating the first human genome was ~$1 billion, but nowadays, it
takes less than a day to sequence a person’s entire genome and costs less than $1000. The complete blueprint will lead to better understanding of genomic variations and their contribution to
disease development(24–26).
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