Monday, 7 October 2013

Human genetics, part II

This post continues to define certain key concepts and themes in human genetics. Basics of genetics are not discussed here. The focus is on identifying genes causing diseases, mechanisms of cancer and the impact of genetic diversity to drugs. The previous post can be found here.


Pharmacogenetics studies how genes influence the efficacy and side-effects of drugs. It explains why different people react differently to medicines. Other related terms are pharmacogenomics (the interactions between drug and the genome), pharmacokinetics (metabolism of drugs) and pharmacodynamics (the interactions between drugs and their molecular targets).

Stages of drug metabolism
(c) Elsevier
The response to a drug can be continuous or discontinuous. In continuous response, the responses are normally distributed, because the response is multifactorial (depends on genetics and the environment). In discontinuous response the genotype of the user defines the  response, and there are only a few possible response types. Consider the stages of metabolism in the picture to the right. For example, homozyous recessive users (genotype aa) might follow the route distribution - breakdown - excretion, while homozygous dominant (AA) might immediately excrete the drug. For both response types the drug is ineffective. Heterozygotes (Aa) might metabolize the drug correctly. In this case, there are three possible responses to the drug.

Drug metabolism occurs mainly in the liver, where glucuronide conjugation and acetylation are the most common mechanisms of metabolising drugs. Metabolism is usually two-phased: First a polar group is added to the drug molecule to make it water-soluble. This is usually due to P450 enzyme, and includes hydroxylation or oxidation.Then the drug is metabolised into an excretable product. This phase often uses N-acetyltransferase (NAT) or glutathione S-transferase (GST).

Isoniazid is a drug used to treat tuberculosis. It absorbs quickly from the gut, and the level of isoniazid in the blood rises fast. The speed of metabolization depends on the enzyme NAT2, which has two alleles: fast acetylation and slow acetylation. Users homozygous for the slow acetylation metabolize isoniazid very slowly. For them the drug may cause liver damage and neural inflammation, because the toxic metabolites accumulate to the body. Certain genotypes concerning CYP2E1 and GSTM1 enzymes are also connected to slow metabolization of isoniazid and therefore hepatotoxicity. 50 % of West Europeans are homozygous slow acetylators.

Some hemolytic anemias such as favism are caused by deficiency of the enzyme G6PD, glucose-6-phosphate dehydrogenase. It also causes sensitivity against primaquine, a malaria drug. G6PD deficiency is inherited as X-linked recessive trait. If G6PD deficient people use primaquine, they develop black urine, jaundice and low hemoglobine due to destruction of red cells. The condition is not lethal. The deficiency is very common in Africa where malaria is common, which may support the claim that the deficiency protects against malaria.

Coumarin and other anti-blood-clotting drugs mut be very carefully dosed to each patient. There is extiensive variation between individual reactions to the drug, and a relatively high risk of severe bleeding complications if the dose is incorrect. Genes, ethnicity, the intake of vitamin K, diet, health and other drugs all have an impact on coumarin. Genetically the most important factors are polymorphism is CYP2C9 and VKORC1. The enzyme CYP2C9 has over 29 variants, and accounts for 15 % of all drug metabolism in the liver. Its most common variants are 20-70 % less effective enzymatically, and therefore need smaller dosages than fast metabolizers. VKORC1 is a vitamin K reductase, which reduces a by-product of blood-clotting factors into vitamin K. Vitamin K is essential in blood clotting. Warfarin, a drug similar to coumarin, inhibits VKORC1.

More on dosing blood clotting drugs can be found from

Ethical dilemmas associated with gene tests and prenatal screening

 Prenatal screening means the screening for inherited diseases in fetuses. It is a manner of genetic testing. Gene tests can also be done to humans of all ages to
  • detect carriers of inherited disorders
  • predict late-onset diseases, which cause symptoms only as the patient grows older
  • detect of increased risk of multifactorial diseases
  • detect of risk to adverse drug effects
  • diagnose cancer and determine most effective medication.
(c) McGraw-Hill
Prenatal screening is mostly based on detecting non-DNA markers, which detect metabolic abnormalities. There are several methods for prenatal screening, such as chrionic villus samples,amniocentesis (sample of the fetal liquid), blood sample of the umpibical cord and endoscope visualization of the fetus. These are invasive techniques, and pose a small risk to the mother or the fetus. Non-invasive techniques include blood samples from the mother or ultrasonography. Ultrasonography, also known as ultrasound, can detect trisomies (Down synrdome, Patau syndrome, Edwards syndrome) or Turner syndrome, where a female has only one X chromosome.

Ethical dilemmas arise from several factors. One is the clinical validity: how trustworthy the test is? Ultrasonography images and test results may show unclear results, subtle anomalies of unknown significance or offer mild indications of a disease. In which case should these findings be concidered strong enough to decide whether to keep or abort the fetus? Laws may dictate that abortion is either illegal or legal only under certain conditions. Invasive tests also increase the risk of miscarriage. Lastly, how to estimate the risk of anxiety if the test results are inconclusive, or a sick child is born despite negative test results?

There is also a possibility that the result is erroneous, but it can be easily checked by taking another test.

Fundamental ethical principles are
  • autonomy: the respect for privacy and absolute confidentiality. The patient is informed of the risks and validity of the offered test, and other options available.
  • beneficence: the test is done for the best interest of the patient. 
  • non-maleficence: the principle of not doing harm.
  • justice: fairness, equity of access and opportunity. This is especially important when concerning countries without government-supported healthcare, where all mothers should have equal opportunities to participate to gene tests.

Gene therapy

Gene therapy is the deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposes. Currently gene therapy is still only experimental, and tried mostly on cancers. No major breakthroughs have been achieved.
Gene therapy can be either done on germline cells or autosomal cells. Germline mutations are permanent and inherited, due to which they are banned in many countries. No current trials use germline therapy. Autosomal cell therapies aim to modifying specific cells or tissues of the patient.

There are a variety of techniques used for gene therapy:
– Delivery of synthetic or recombinant nucleic acids into humans
– Genetically modified vectors (viruses or plasmids). This is the most used technique, covering over 60 % of all gene therapy trials. The usability of a given virus depends on the amount of foreign DNA it can carry, how it interacts with the host genome and how the transgene is expressed.
– Genetically modified stem cells
– Oncolytic viruses, which target only cancel cells, proliferate inside it and kill the cell after releasing new virusparticles to another cancer cells.
– Nucleic acids associated with delivery vehicles
– Naked nucleic acids
– Antisense techniques
– Genetic vaccines
– RNA interference
– Xenotransplantation of animal cells (but not solid organs)

For somatic cell therapy, the techniques can be divided into four methods. In gene supplementation a working copy of a gene is inserted to its target cells to treat loss-of-function conditions.Gene replacement replaces a mutant gene, and targeted inhibition silences certain genes. Lastly, gene therapy can be used for targeted killing of specific cells. In all cases the target gene can be inserted either directly to the patient cells (in vivo), or by growing and infection the patient's own cells with the target gene in a cell culture before injecting them to the patient (ex vivo).

All the previous techniques can be used to treat cancer. Supplementation can restore tumor suppressor gene function. Inactivation can silence an oncogene, and manipulation of tumor cells can lead to apoptosis. Cells can also be made more antigenic, and thus promote the immune system to target the cancer cells. Cell killing can be done by using oncolytic viruses, which targets certain cells. Another method is the suicide gene therapy. In suicide gene therapy the cells are modified to turn a safe substance into a toxic one. For example, a retrovirus is modified to infect cells with herpes virus kinase. The virus is injected directly to the tumor, where it infects only dividing cells. When the cells have been infected, they are treated with a drug, which the kinase metabolizes into a toxic substance. The infected and drug-treated cells die. 

Problems in gene therapy arise from several factors. Most serious problems are
  • temporary effects: to be effective, some therapies need constant injections or "rounds" of gene therapy.
  • immune response: patient's body may recognize the engineered cells and destroy them. Since the immune response is stronger in later infections, the patient may become immune to gene therapy.
  • usage of viral vectors carries a risk of viral infections and mutated viruses in addition to possible toxicity and immune responses.
  • multigene disorders cannot at the moment be treated with gene therapy at all. It is suitable only for monogenic diseases.

Ups and downs of gene therapy (c) Elsevier

RNA-therapy is a form of genetic therapy which uses RNA to affect genes and genetic expression. One example is the antisense nucleotide therapy used to treat Duchenne muscular dystrophy.

The principle of antisense oligonucleotide therapy for Duchenne muscular dystrophy

Dystrophin in a DMD patient and
patients treated with AON.
(c) Garland science
Duchenne muscular dystrophy, DMD, is a genetic disorder of muscle weakness and degeneration caused by lack of dystrophin in the muscle tissue. DMD occurs in 1 of 3500 boys, and first symptoms occur in early childhood. Most patients die as young adults due to degeneration of breating and cardiac muscles. Dystrophin can be seen in tissue samples as a strong border around muscle cells. In DMD patients the stains show very little or no dystrophin, and the muscle cells are without real form or support.
A milder version of DMD is the Becker syndrome, where the patients have a normal life expectancy. Becker syndrome patients have a partially functioning dystrophin, which can be smaller in size than normal, and/or have reduced abundance. The difference of DMD and Becker is caused by a genetic mutation. In DMD, the dystrophin protein has an out-of-frame deletion, which leads to an altered reading frame and eventually to a truncate and unstable protein. Becker patients have an in-frame deletion, which leads to a shorter but partly functional protein.

The antisense oligoribonucleotide (AON) therapy targets at fixing the broken reading frame. The deletion between exons 48 and 52 is severe, because the exons are not compatible. Therefore the spliceosome stops at the exon 48, unable to continue to 52. The gene therapy aims at bridging this cap. The antisense oligoribonucleotides are tailored to hybridize to exon 51 and hide it from the spliceosome. The exons 48 and 52 are compatible, so the spliceosome continues and runs to the end of the protein. The final product is a shortened protein, which is however party functional. Gene therapy does not cure DMD - it merely makes the disease milder and increases the lifespan of the patients.

Currently gene therapy has been tested on 150 antisense oligonucleotides. The deletion of exons 49-50 is just one possible mutation causing DMD, and other mutations need their own oligonucleotides. First clinical studies have been performed on three AON therapy methods to hide the exon 51. One of these requires weekly abdominal injections.

Examples of genetic diseases

Disease nameCauseInheritanceSymptoms
Hutchinson-Gilford Progreria Syndromede novo point mutations in LMNA gene in 1q22-Growth failure, loss of body fat and hair, aged-looking skin, stiffness of joints, hip dislocation, generalized atherosclerosis, cardiovascular (heart) disease and stroke
Huntington diseaseCAG-repeat expansion in 4p16.3 ADMovement abnormality, involuntary movements, memory impairment, dementia
Spinal and bulbar muscular atrophyExpansion of a CAG-repeat in androgen receptorX-linkedMuscle wasting, weakness, contractions, swallowing difficulties
Myotonic dystrophyCTG-repeat expansion in 3'UTR or CCTG-repeat expansion in ZNF9 (leads to gain-of-function RNA)
Progressive muscle weakness and myotonia. DM1 allele causes a more severe disease than DM2
Fragile-XCGG-repeat in promoter region of FMR1 (suppresses transcription due to hypermethylation -> slow development of cerebral neurons)X-linkedLearning difficulties, typical facial features (large ears, high forehead, long face)
Facioscapulohumeral muscular dystrophy (FSHD)Shortened megasatellite repeat in 4q35 leading to hypomethylation and cascading effects (deregulation of several muscle genes)ADStriking asymmetry of muscle involment from side to side, unbalance between right and left side muscles
AchondroplasiaDe novo mutations in FGFR3 gene in 4p16.3, usually a missense mutation Gly380ArgADShort stature (120-130cm), characteristic facial features
Cartilage-hair hypoplasia (CHH)Mutation in RMRP (insertion which prevents transcription or SNP), leads to lack of RNAse required for cell growth-Short-limbed dwarfism (100-140 cm), sparse hair, immunodeficencies
Diastrophic dysplasia (DTD)SNP mutation in SLC26A2, leads to undersulphation of cartilage matrix-Short stature (100-160 cm), deformities of joints, hip dysplasia, hand deformities
Tibial muscular dystrophyHeterozygous deletion-insertion in titin geneADMild weakness of tibial muscles (calves)
Down syndromeTrisomy 21 (47, +21). Can be due to translocation (t14;21) or t(21;21) or mosaicism. Extra chromosome often from motherHypotonia, mental retardation, characteristic facial features, adult height of ~150 cm. Lifetime expectancy of 50-60 years.
Patau syndromeTrisomy 13 (47,+13). Can be due to translocation (t13;14)-Central nervous system malformations, heart defects, growth retardation, cleft lip and palate. Most die as newborns.
Edwards syndromeTrisomy 18-Malformations in many organs, elfin features, mental deficiency. Most die within a week from birth.
Klinefelter syndrome (47, XXY)Extra X chromosome(s) in males. Can also be (48, XXXY) or (49, XXXXY).-Taller than average, long lower limbs. All patients are infertile.
Turner syndrome (45, XLack of chromosome X in women-Short stature, infertility, no puberty.
XXX femalesMore severe symptoms if more than one extra X chromosome-Possible mild reduction in intellectual skills
HermafroditismPaternal X-chromosome has Y-chromosome sequences; Chimerism of XX and XY cell lines; androgen insensitivity in males; adrenal hypoplasia in females-Possibly taller stature

Saturday, 5 October 2013

Human genetics, part I

Human genetics is the study of human genome and individual genes. It includes many different aspects and views, such as pharmacogenetics and -genomics (the study of interactions between genes and drugs), gene therapy and mapping out disease susceptibility genes. In this post I summarize some topics of human genetics.

The human genome

Humans have genetic material, DNA and RNA, in two places: in the nucleus and mitochondria of cells.

Mitochondrial genome is always inherited from the mother. It is 16,6 kb long, and consists of one circular (plasmid) DNA module. It can be divided to light and heavy strains as opposed to the leading and lagging strand of the nuclear genome. Mitochondrial genome therefore has no chromosomes. Each cell has several, even thousands, copies of the plasmid, but the copy number varies in cell types. The mitochondrial genome has only 13 genes, is mostly free of binding proteins and has very little repetitive sequences. It is thus very dense in genes: it has ~1 gene in 0,45 kb of DNA. It also has no introns, and over 66 % of the DNA codes a protein. There is no recombination in the mitochondrial genome.

Mitochondrial genome has even a small strand of triple strand DNA, called the 7S DNA. Like all plasmids, the mitochondrial genome has an ORI-segment (origin of replication), but also promoters for both the light and the heavy strand. Over 98 % of the mitochondrial genome is highly conserved. It also has a different amount of stop codons and amino acid coding codons than the nuclear genome.

Human mitochondrial genome and genes. (c) NCBI

The nuclear genome is what traditionally is understood by a "genome". It is located in the nucleus as chromosomes, of which humans have 23 (the haploid number). The nuclear genome is 3,1 Gb in size - that's 250000 times larger than the mitochondrial genome! The nuclear genome is packaged around histone proteins. It has plenty of repetitive sequences, untranslated regions and introns. Approximately half of the nuclear genome is repetitive sequences such as microsatellites. Recombinations are common in nuclear genome, and it is inherited chromosomally: Mendelian inheritance for X and autosomes, paternal inheritance for Y chromosome.

The nuclear genome has over 20 000 protein-coding genes at a density of 1 gene / 120 kb. Only 1 % of the genome codes proteins. Single genes vary in size, the smallest being few hundred basepairs and the longest 2,4 Mb. One gene is transcribed from one strand, unlike in mitochondrial genome where by swapping between light and heavy strands one sequence can be translated differently. Circa 6 % is highly conservated, ~45 % transposons and the rest is poorly conserved for other reasons.

Both genomes participate in some important parthways in the human body. Both code subunits for the oxidative phosphorylation system, with nuclear genome providing 80 and mitochondrial genome 13 subunits. They also both participate in building protein synthesis complexes: mitochondrial genome is solely responsible for producing the rRNA and tRNA, while nuclear genome produces only ribosomal proteins.

# of genes and sizes of human chromosomes.
(c) Wikipedia

Repetitive sequences and variation in the human genome

Repetitive sequences are common in the human genome, and nearly 50 % of the nuclear genome is repetitive. Microsatellites, minisatellites and the telomeres at the end of chromosomes are examples of repetitive sequences which are often used in genetic studies. There are also repetitive sequences within genes. The repetition number can cause illnesses and diseases due to a malformed protein or the gathering of long mRNA chains in the cell. Illnesses caused by insertion leading to the lengthening of a repetitive sequence are Huntington disease, fragile X and myotonic dystrophy.

There are several factors causing variation in the human genome: mutations, genetic drift, selection and migration. Mutations can be classified to substitutions, deletions and insertions.

  • Substitutions are either synonymous (or silent), where the final amino acid chain of the protein does not change, or nonsynonymous, where the amino acid chain is changed. Non-synonymous mutations can alter the amino acid at the point of the mutation, cause a stop codon or lead to aberrant splicing or altered gene expression (mutations in promotor sequences).
  • Deletions are either multiples of three or not. Multiples of three lead to deletions of whole amino acids. Other deletions disrupt the reading frame, causing premature termination or loss of function of the gene product.
  • Insertions are like deletions: multiplies of three add amino acids, while non-multiples disrupt the reading frame. Insertions can also be insertions of entire genes or amplification, where the gene stability, function or expression is altered.

Cancer: inheritance and proto-oncogenes

Cancer is a genetic disease of somatic cells. Therefore all forms of cancer are genetic, some more than others. Environmental factors, such as lifestyle, are important factors in developing cancer. 37 % of cancers in the USA are estimated to be caused by diet high in fats, while ~30 % of cancers are caused by tobacco.

Cancer can never be inherited. Only genes which may or may not increase susceptibility to certain types of cancers, are hereditary. Cancer development depends on a few types of genes:
  • proto-oncogenes: normal genes, which may develop into oncogenes if activated
  • oncogenes: mutated forms of proto-oncogenes. Important in cell growth and differentiation. Can be classified to viral oncogenes and cellular oncogenes. Both types can be
    • growth factors
    • growth factor receptors
    • intracellular signal transduction factors
    • DNA-binding nuclear proteins
    • Cell cycle factors
  • Tumor suppressor genes: inhibit and control cell proliferation
  • DNA repair genes: repair faulty and broken DNA, and can fix mutations. In cancer the gene repairs are not working for example due to mutation. Mutations in repair genes can be inherited, which may cause leukemia, solid tumors and other types of cancer.
Proto-oncogenes can be activated in many ways. Duplication is common, and many cancer genes have multiple copies of normal oncogenes. Point mutations can activate oncogenes of the RAS family, which mediate cell signaling. Translocations can create novel chimeric genes. Leukemia is most often caused by a chimera called the Philadelphia chromosome t(9;22).  Ph1 does not answer to common cell function controls. Chimeras are also common in hematologic (to do with liver) tumors and sarcomas.Oncogene can also be translocated to an transcriptionally active region, and the oncogene becomes overexpressed.

For example, colorectal cancer can be caused by hereditary conditions such as familial adenomatous polyposis (FAP) and hereditary non-popyposis colorectal cancer (HNPCC). FAP is an autosomal dominant disorder, where patients develop several polyps in their large intestine. In addition they have germline mutations in a tumor suppresson gene on chromosome 5, which causes the adenomas. HNPCC, also known as Lynch syndrome, is autosomal dominant disorder. It is caused by mutations in DNA repair genes.

Breast cancer can also be due to hereditary conditions. 15- 20 % of breast cancers in Western Europe are in women who have a family history of breast cancer. The susceptibility genes are BRCA1 and BRCA2, which are found in 30-50 % of all breast cancer families. Both are DNA repair genes. A heterozygous carrier can have 85 % chance of developing breast cancer. 

Methylation of DNA is a normal way of the body to increase or decrease the activity of a single gene or the whole genome. Methylation silences the gene, because a methylated gene cannot be transcribed. In hypomethylation the gene is inadequately methylated, and normally silent genes are expressed. Imprinting is also lost is the imprinted gene is hypomethylated. Reversely, hypermethylation silences a normally active gene. Cancel cell genomes are often hypomethylated. Genes can be silenced by methylation, mutation or both.

Chemical carcinogenesis

Chemical carcinogenesis is a system where chemicals cause cancer. It was discovered in 1770s, when chimey sweeps were often diagnosed with scrotal cancer. Some key terms in chemical carcinogenesis are
  • Genotoxicity: toxicity to the genome (A substance can be genotoxic)
  • Mutagen: chemical or physical agents inducing alterations in DNA
  • Carcinogen: cancer-causing agent
  • Co-carcinogen; chemicals that cause cancer only in specific combinations
  • Genotoxic carcinogen: DNA reactive agents (mutagens) or agents acting on the chromosome level (causing aneuploidy)
  • Non-genotoxic carcinogen: does not affect DNA but promotes growth in other ways (hormones, some organic compounds)
Chemical carcinogenesis is a multistep process involving initiation, promotion, malignant conversion, and progression. In initiation, a carcinogen causes an irreversible mutation. In promotion non-genotoxic agents cause the expansion of the initiated, mutated cells. This step requires many faulty mechanisms to be effective: cell to cell communication must be hindered, apoptosis must be blocked and other forms of cell proliferation inhibition must fail too. Promotion leads to conversion, where the cells become a malignant cell population. This step requires tumor suppresson genes to be inactivated and oncogenes to be amplified. Last step is the progression, where the cells continue to divide, eventually leading to metastasis.

Well known and unfortunately widely used chemical carcinogens are tobacco, alcohol, pesticides and certain types of fibers and dusts (e.g. asbestos and coal). Many substances become carcinogenic only after metabolization in human cells. For example, some smokers have overactive activator enzymes in their lungs, and most if not all precarcinogens in tobacco are metabolized to actual carcinogens. Smokers, whose activator is lazier, accumulate much less carcinogens from the same about of precarcinogens. Same kind of mechanism is in the kidneys, where a carcinogen eliminating enzyme can either be very effective or very passive. Examples of such enzymes are N-acetyltransferase (NAT) and glutathione S-transferases (GSTs).

Cytochrome P450 genes, also known was CYP, catalyze reactions where polycyclic aromatic hydrocarbons (PAH-compounds) are metabolized to carcinogens. Humans have 57 CYP-genes. Some are expressed in the lungs, some in the kidneys. Different CYP-genes have been scientifically proven to be connected with lung cancer.

Example: asbestos
- Asbestos is inhaled when working with asbestos without adequate protective gear
- Asbestos fibers penetrate lung epithelium, causing inflammation and tissue damage
- Free radicals are produced due to prolonged contact between asbestos and mesothelial cells, and due to prolonged exposure to inflammatory cells.
- Reactive oxygen causes DNA damage. Antioxidants can hinder this reaction, but cannot entirely prevent it.
- Asbestos fibres in the lung become covered in iron and calcium. Macrophages ingest asbestos fibres, whereupon growth factors are released, and fibroblasts coat the macrophages with collagen.  

Molecular genetics of Hutchinson-Gilford progeria syndrome

(c) Georgia Gwinnett College
Hutchinson-Gilford progeria syndrome or HGPS for short is a disease causing premature ageing in children. The children are born healthy, but begin to age rapidly when they reach 18-24 months of age. The disease causes growth failure, heart issues, loss of hair, loss of body fat, aged-looking skin and stiffness of joints. The childred die of atherosclerosis around 13 years of age. Progeria is very rare, 100 patients have been diagnozed within as many years.

The disease is caused by a mutation in lamin-forming gene LMNA. Most cases are new mutations, meaning that the parents are not carriers of the disease and the susceptibility cannot be estimated. 90 % of the patients carry the same single-point mutation, which leads to aberrant splicing and a lack of 50 amino acids in lamin A.

Due to the aberrant splicing the protein lamin A is not postprocessed correctly. A farnesyl group is added to end of lamin A in a cell. In a normal cell, this farnesyl group is soon removed, and the final protein, callen lamin A, is formed. In progeria-infected patients the farnesyl group remains, leading to instability of the protein (which is called progerin). The instability is what causes the premature aging, since lamin A is very important factor in maintaining cell integrity and structure.

Progeria is related to normal ageing. Each chromosome has long telomeres at the ends. Each division in normal cell shortens the telomeres, until they begin to "wear out". Shortened telomeres induce the production on progerin - the very same protein which in progeria-patients induces premature ageing, and for the same reason (aberrant splicing). In other words, progeria patients experience all of the symptoms and die of old age when ~13 years old.

Progeria cannot be cured. The progress of the disease has been hindered by farnesyltransferase inhibitors (FTIs). The current status of the research is available at