HUMAN GENOME VARIATIONS

& EPIGENETICSAfiono Agung Prasetyo

HUMAN GENOME

VARIATIONS

Classes of genomic variation• Sequence variation:

– substitutions – insertions– deletions – indels

• Structural variation– 2 bp to 1000 bp– 1 kb to submicroscopic– Microscopic to subchromosomal– Whole chromosomal to whole genome

How to find genes and specific genetic variants responsible for diseases?

• Linkage analysis• Genetic markers: Positional cloning

• Linkage disequilibrium mapping• Genetic association analysis

COPY NUMBER VARIATION• DNA segments > 1 kb in size in which a comparison of two or more genomes reveals gains (by insertion or duplication) or losses (by deletions or null genotypes) of genomic copy number relative to a designated reference genome sequence

• copy number polymorphism is present when such variation is present in more than 1% of the reference or general population

CNV-Diet-Drugs Metabolism

• Duplication of the salivary amylase gene (AMY1)

• high starch diets have more AMY1 copies

• in populations with low starch diets there had been genetic drift and the locus had evolved neutrally

CNV-Diet-Drugs Metabolism• CYP genes encoding cytochrome P450 enzymes

• > two active functional copies of a specific CYP gene may show increased drug metabolism and absence of response at ordinary drug dosages, classified as ‘ultrarapid metabolizers’

• while ‘poor metabolizers’ lack functional enzyme due to defective or deleted genes.

• Lancet 2006: the death of an infant 13 days after birth who had lethal levels of morphine–Mother: a gene duplication of CYP2D6

CNV-multifactorial disease

• Psoriasis risk and β defensins copy number–each extra copy increased the relative risk of disease by 34%

• CCL3L1 and HIV-1–inhibits infection of cells by HIV-1 strains using the CC chemokine receptor 5 (CCR5)

–Most people: 1-6 copies of the CCL3L1 gene

–higher number of copies : risk was significantly reduced

SATELLITE DNA• Satellite DNA comprises very long arrays of tandem repeats typically 100 kb to several megabases in size, the repeat unit length varying between 5 and 171 bp.

• Minisatellite DNA arrays are of intermediate size and typically span between 100 bp and 20 kb, with each repeat unit between 6 and 100 bp in length.

• Microsatellite DNA comprises short arrays less than 100 bp in size, made up of simple tandem repeats 1–6 bp in length.

Functional Effects of VTR

• Modulates transcriptional regulation:–Alternative splicing–Genomic imprinting

• Alter the control of gene expression or the structure and function of the encoded protein

VTR-Diseases• Marker• Repeat instability has been associated with more than 20 diverse neurological disorders, including neurodegenerative and neuromuscular disease–CCTG repeats and dystrophia myotonica type 2

–ATTCT repeats and spinocerebellar ataxia type 10

–etc

EPIGENETICS

•Genetics: –Genes + their interactions–Only a few diseases can be explained by mutations in a single gene

•Genomics: –Genome

Genomics• Structural genomics: gene libraries, gene maps, complete sequencing of entire genomes, processing of sequencing data

• Comparative genomics: relationships between the genome & the phenotype

• Functional genomics: identify the biologic function of sequenced genes & the proteins they encoded

• Clinical genomics: genome & health• Pharmacogenomics

EPIGENETIC MECHANISMS

Epigenetic regulation

• DNA methylation• histone modifications• post - transcriptional alteration of gene expression based on RNA interference

• Epigenetic changes can occur on all building blocks of the nucleosome to ultimately constitute the epigenome:–post-translational histone modifications–incorporation of histone variants–remodelling of the DNA-histone interaction

–methylation of DNA–association with transcription (co)factors

–local changes in nucleosome density–changes in long-range chromatin interactions and compaction.

METHYLATION

NOTE:• Basic biological properties of DNA - segments such as gene density, replication timing and recombination are tightly linked to their guanine – cytosine (CG) content.

• 60 – 90 % of all CpGs are methylated in mammals

• Methylated C residues spontaneously deaminate to form T residues; hence CpG dinucleotides steadily mutate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency).

• Spontaneous deamination of unmethylated C residues gives rise to U residues, a mutation that is quickly recognized and repaired by the cell

• Unmethylated CpGs are often grouped in clusters called CpG islands, which are present in the 5' regulatory regions of many genes.

CpG islands• GC > 50% and with an observed CpG > 60%.

• In mammals: 200 – 3000 bp. • at or near the gene’s transcriptional start

• Promoters of tissue - specific genes that are situated within CpG islands are, normally, largely unmethylated

How cytosine methylation can regulate gene

expression?• 5 - methylcytosine can inhibit or hinder the association of some transcriptional factors with their cognate DNA recognition sequences

• methyl-CpG-binding proteins (MBPs) can bind to methylated cytosines mediating a repressive signal

• MBPs can interact with chromatin forming proteins modifying the surrounding chromatin, linking DNA methylation with chromatin modification

• Mostly, DNA methylation causes a repression of mRNA gene expression, however, when CpG methylation blocks a repressor binding site within a gene promoter, this may induce a transcriptional activation

• Hypomethylation, in general, is linked to chromosomal instability and loss of imprinting.

DNA methyltransferases (DNMTs)

• Conduct DNA-methylation at position five of CpG-cytosines

• expressed in most dividing cells• Most DNMTs contain a sex-specific germline promoter which is activated at specific stages during gametogenesis.

• Genomic methylation patterns are largely erased during proliferation and migration of primordial germ cells and re – established in a sex - specific manner during gametogenesis, resulting in a high methylation of the genome.

• Close regulation of the DNMT genes during these stages and during early embryogenesis is needed

• After fertilization, a second phase of large epigenetic reprogramming takes place. Upon fertilization, a strong, presumably active DNA demethylation can be observed in the male pronucleus while the maternal genome is slowly and passively demethylated.

• Imprinting by methylation is maintained for both the paternal and the maternal genome. DNA - demethylation occurs until the morula stage, followed by de novo methylation

HISTONE MODIFICATIONS

HISTONE CODE?• Through alteration of gene expression and

destabilization of chromatin, histone modifications can have an impact on the risk of cancer

• acute promyelocytic leukemias: a chromosomal translocation leads to inappropriate HDACs activity

• gastric cancer, esophageal squamous cell carcinoma, and prostate cancer: an increased HADC1 expression

• Colon cancer: overexpression of HDAC2 causes a decreased expression of the APC (adenomatous polyposis coli) tumor suppressor gene

• in cancer of epithelial origin: an overexpression or mutation of HAT genes

• Some lines of lung, breast, and colorectal cancer have in common a mutation which inactivates a specific HAT

MICRO RNAs and siRNA

• small non – coding RNAs • Post transcriptionally regulate the expression of complementary mRNAs

• key controllers in cellular processes, including proliferation, differentiation and apoptosis

GENOMIC IMPRINTING

NOTE• Each autosomal gene is represented by two copies, or alleles, with one copy inherited from each parent at fertilisation.

• For the vast majority of autosomal genes, expression occurs from both alleles simultaneously.

• In mammals, <1% of genes are imprinted: gene expression occurs from only one allele.– The expressed allele is dependent upon its parental origin

• certain genes are expressed in a parent-of-origin-specific manner. It is an inheritance process independent of the classical Mendelian inheritance. Imprinted genes are either expressed only from the allele inherited from the mother (e.g. H19 or CDKN1C), or in other instances from the allele inherited from the father (e.g. IGF-2).

• involves methylation and histone modifications

Genomic Imprinting-example

• Prader-Willi syndrome & Angelman syndrome are associated with loss of the chromosomal region 15q11-13 (band 11 of the long arm of chromosome 15). – contains the paternally expressed genes (SNRPN and NDN) and the maternally expressed gene (UBE3A).

– Paternal inheritance: Prader-Willi syndrome (hypotonia, obesity, hypogonadism)

– Maternal inheritance: Angelman syndrome (epilepsy, tremors, smiling facial expression).

Genomic Imprinting-example

• NOEY2 (tumor suppressor gene?)– paternally expressed imprinted gene located on chromosome 1 in humans.

– if a person inherits both chromosomes from the mother, the gene will not be expressed and the individual is put at a greater risk for breast and ovarian cancer.

X-CHROMOSOME INACTIVATION (LYONIZATION)

The genes on the X & Y chromosomes

• Y chromosome (122 genes )– the instructions to make the baby develop as a male rather than a female

• X chromosome (1021 genes) – very important for growth and development; e.g. the genes that contain the instructions for a major protein in muscles (dystrophin), several proteins that control clotting in the blood and a number of genes involved in the development of intelligence.

• When these particular X chromosome genes are faulty, specific genetic conditions may result: haemophilia, Duchenne and Becker muscular dystrophy and fragile X syndrome–X-linked recessive inheritance

•Women who have the faulty gene copy as well as a ‘back up’ working gene copy on the other partner X chromosome copy, means that they are usually unaffected by these conditions

•Men have no other X chromosome to provide ‘back up’ so will usually be affected due to the faulty gene being expressed in the cells

• In order to adjust this potential imbalance between the genetic information in men and women, the cells have a system to ensure that only one copy of most of the X chromosome genes in a female cell is ‘active’. Most of the other genes on the other partner X chromosome copy are ‘inactivated’ or switched off. This mechanism ensures that only the genes located on the ‘active’ copy of the X chromosome are able to be used by the cell to direct the production of proteins.

• inactivated X chromosome: Barr body

• This system of inactivation in the body cells is usually random so that women’s bodies have a mixture of cells in regard to the inactivated X chromosome. Some cells will have the X chromosome switched off that came from their mother (an inactive maternal X chromosome); other cells will have the paternal X chromosome inactivated. The relative proportion of cells with an active maternal or paternal X chromosome varies from female to female (even between identical twins) because the process is usually random.

• X-inactivation only occurs in the somatic cells, since both X chromosomes need to be active in the egg cells for their normal development