Poma Genetics

Medical Genetics For Modern Clinical Practice

• Molecular Medicine = DNA as it applies to medicine
• Genetics is a rapidly growing field
• having growing impact on all medical disciplines, and there is a growing need for the clinician to have an understanding of genetics
• all human variation is to some extent genetic – therefore all diseases have some genetic basis
• individual development depends on interplay of genes and environment
• possibility of treating or preventing clinical disease by manipulating environment of those with genetic predisposition/risk
• approx. 3% of all births have significant genetic disease or defect
• around 1 in 20 individuals either born with or develop a serious disease with a genetic component by age 25

Four Major Catergories of Genetically Determined Diseases:

1. Chromosomal
- entire chromosome involved e.g. the extra chromosome 21 in Trisomy 21 (Down Syndrome)

2. Single Gene Disorders
- just a single gene, rather than a whole chromosome, is defective e.g. B-thalassaemia, a defect in the B-globin gene of haemoglobin

3. Complex Genetic Diseases
- result from interaction of multiple genes
- also called polygenic/multifactorial diseases
- most common and least understood!
- major challenge for contemporary genetics
- e.g. diabetes mellitus, hypertension, schizophrenia, many congenital defects such as cleft lip, etc
- raises question/possibility of ‘personalised medicine’ – availability of karyotpying, when, for who and why should we analyse an individual’s genetic ‘fingerprint’?

4. Somatic Cell Genetic Disorders
- only in specific somatic cells (in contrast to others where the abnormality is in the DNA of all cells of the body, including germ cells)
- not transmitted to subsequent generations
- cancer paradigm – malignancy often develops as result of mutations in genes controlling cell growth (cancer can also have a hereditary component, however)

Genetic Data Online:
• www.ncbi.nlm.nih.gov – National Centre for Biotechnology Information
• www.bionews.org.uk – newsletter to keep up to date with advances in the field

Chromosomal disorders

• Human karyotypes: presentation of all the chromosome make up of an organism to detect gross changes of numerical or structural in chromosomes but not small deletion or point mutations.
- performed on any dividing cells such as bone cells, cancer cells, amniotic cells, chorionic villus, peripheral blood lymphocytes
 p arm: short segment of the chromosome
 q arm: long segment of the chromosome
 Centromere: part of the chromosomes that joins the two chromatids to each other and becomes attached to the spindle during mitosis and meiosis.
 Telomere: the end of a chromosomes which consists of repeated sequences of DNA that perform the function ensuring that each cycle of DNA replication is completed
 Types of chromosomes
- metacenteric: centromere roughly in the middle
- acrocentric: the centromere is much closer to one end
- submetacentric: intermediate between metacenteric and acrocentric
 Chromosomal abnormalities:
- numerical: aneuploidy
- structural: a break in one or more chromosomes resulting in overall loss or gain genetic material
- acquired
- constitutional
 Aneuploidy: the gain or loss of a whole chromosome. Trisomy is gain while monosomy is loss
- Occurrence: non-disjunctioning during meiosis I or II due failure of spindle formation or function and chromosomal pairs fail to segregate.
- mosaicism: production of two germlines as non-disjunctioning occurs post-fertilization giving a chimera.
- Trisomic disorder: 18 - edwards, 13 – patau
- Monosomic disorder: chromosome X – turners
 Reciprocal translocation: a two way exchange of material between two non-homologous chromsomes; if balance translocation then phenotype normal as no genetic material lost or gained. E.g. translocation of chromosome 11 material to 3
- carrier situation: if the derivative of chromosome 11 and 3 are inherited together to offspring, there is no loss in genetic material and the offspring is phenotypically normal
- non-carrier situation: when the derivative of chromosome 11 (shortened) are inherited with a normal chromosome 3, a loss of genetic material produced recurrent miscarriages, and birth of a dysmorphic baby and oligospermia in male carriers.
 Robertsonian translocation: translocation of the q arms between homologous or non-homologous acrocentric chromosomes only, e.g. chromosome 13, 14, 15, 21, 22. Patients with robertsonian translocation are phenotypically normal as the lost p arms are genetically insignificant.
 Inversion: intra-chromosomal structural rearrangement which involves two breaks on the same chromosomes and the segment rotates 180 degree and re-insert itself.
- pericentric: involves both arms
- paracentric: involves one arm
- Carriers of inversion are phenotypically normal with very low risk of having an abnormal offspring.
 Deletions: loss of chromosomal fragment
- terminal: single break at the terminal region and loss of the end fragment
- interstitial: two breaks in the same chromosomes and loss of the intervening fragment
- clinical cases: cri-du-chat syndrome with deletion of 5p15.1 and Prader-Willi syndrome interstitial deletion of 15q12
 Uniparental disomy: Inheritance of both chromosomes and parts of both chromosomes from one parent. This arises from meiotic error and subsequent monosomy or trisomy rescue. E.g. duplication of non-paired chromosome.
- isodisomy: result of monosomy rescue
- heterodisomy: result of trisomy rescue
 Molecular cytogenetic: methods of genetic examination
 Fluorescent In Situ Hybridization: using pre-designed piece of DNA called probe, labelled with a fluorochrome, the target DNA is denatured and reannealed with the probe DNA. If specific sequence complementary to the probe is present, the probe will bind and visualized under UV fluorescence.
 Range of specimens suitable for FISH:
- cultured cells
- uncultured cells
- bone marrow smears
- paraffin embedded tissues
- archival material
- any tissue with intact DNA
 Application of FISH:
- detection of micro-deletions
- painting of selected chromosomes with whole chromosome paints to identify marker chromosome
- rapid enumeration test on a selected set of chromosome
- detection of gene amplification, e.g. HER2 in breast cancer
- detection of specific gene rearrangement, e.g. chronic myeloid leukemia
 Comparative Genomic Hybridization: compares normal and target DNA by scanning the entire genome for overall gain or loss of DNA
 Microarray technology: CGH microarrays enable the simultaneous screening of hundred of DNA regions from the same individual spotted onto a microchip which allows the identification of DNA gain or losses from several region of the genome.
- Expression of microarrays using cDNA can screen the transcriptional activity of a particular gene or group of genes. This technology is still used at the research level but it is anticipated that it will be used diagnostically within the next decade.

Prenatal Clinical Techniques in Genetics

• Prenatal testing: tests used during pregnancy to investigate the presence or probability of the fetus getting a genetic disorder
- diagnostic testing
- screening test
• Diagnostic testing: tests that determine if the baby has or will develop a particular problem or disorder due to changes in chromosome number or structure or specific gene mutations.
- Conditions detected may affect the child during post-natal period or not manifest until later childhood or adulthood.
• Screening tests: tests used to postulate if the baby has an increased risk of having a particular problem rather than actually diagnosing the disorder.
• Reasons for diagnostic testing: it is not possible to detect or diagnose every possible congenital or inherited disorder.
- family history of genetic disorder
- both parents carriers of the mutation
- one of the parent has a serious genetic disorder
- previous child suffering from serious problems
- family history of a condition that affects male members
- the mother is in her mid thirties or older
- parents exposed to chemical or environmental agents
- after a screening test that determined an increased risk for a particular disease of the mother
• Strategies for prenatal testing:
- screening tests: ultra sounds maternal serum analysis
- diagnostic tests: specific genetic analysis of cells from fetus to determine if the baby actually has the condition.
• Ultrasound:
- ultrasound can be performed any stage during pregnancy usually at 8 - 10 weeks
- at 18 weeks scan can pick up a range of physical problems
- non-invasive
- abnormality identified based on quality and experience of obstetrician
• Nuchal translucency ultrasound: a first trimester screening between 10-13 weeks in which the depth of the fluid in the space at the back of the baby's neck is measured with ultrasound. This measurement is used to calculate the risk or chance of the baby's having a constitutional chromosomal abnormality such as down syndrome.
- analysis of maternal serum taken at the same time as ultrasound increases accuracy of diagnosis
- not 100% specific or sensitive
- only provide risk of certain problems and further testing may be necessary.
- non invasive.
• Maternal serum testing: serum is extracted at around 15-17 weeks and different markers are analyzed involving alpha-fetoprotein, unconjugated estriol, and human chorionic gonadotropin. This is called triple test (three markers are tested)
- can pick up babies with neural tube defect such as spina bifida
- not all babies can be picked up
- non-invasive
• Chorionic villus sampling: diagnostic test where CVS cells from the developing planceta is taken and tested for cytogenetic abnormalities and single gene disorders.
- chromosomes or DNA from placenta is examined in the laboratory and test results taken about 1-2 weeks.
- invasive with transvaginal guiding probing and usage of ultrasound to extract villi. There is a 1% risk of miscarriage in addition to the background risk of miscarriage in the first three months.
• Aminiocentesis: diagnostic test using a fine needle to take a small amount of amniotic fluid around the baby. This occurs later in pregnancy where there are plenty amniotic fluid and done through the abdominal wall.
- cell is cultured for 4 -5 days
- baby's cell are removed from the fluid and the chromosome or DNA examined
- will provide a definitive answer for down syndrome
- invasive and less than 1% chance of miscarriage
• Pre-implantation Genetic diagnosis: diagnostic test used in cases of in vitro fertilization where DNA is extracted from a single cell removed from the blastocyst stage and analyzed. This is done prior to implantation to ensure there is no gene mutation or sex-linked disorders.
• Laboratory strategies: analysis of fetal cells obtained from either amniocentesis or VC biopsy. Depending on the situations, different strategies are employed. These include
- FISH: for abnormal chromosome number
- DNA analysis: PCR based where direct mutation analysis is used if gene and mutation are known in a family and indirect analysis if a linked or polymorphic maker to the unknown mutant gene is known febrile
• Importance of counseling:
- provide recent information about availability and use of tests prior to and during pregnancy
- provide an opportunity for informed decision to be made about utilization
- provide opportunity to discuss advantages and disadvantages of screening or diagnostic test
- provide guidance to procedures and action after test results, i.e. when a baby is found with a disorder

Mendelian Inheritance

• Autosommal dominant:
- Affected person have at least one affected parents
- Affects either sex
- Transmitted by either sex
• Lyonisation: female has 2 X chromosomes so one of them undergoes random inactivation. At times even in recessive X-linked disease, you can get inactivation of the normal gene and expression or the disorder
• Autosomal recessive inheritance:
- Affected people born to unaffected parents
- Affects either sex
- Increase incidence of parental consanguinity
• Non-penetrance: allele is inherited but no phenotypes, such as split foot/hand
• Hemizygous: the sex chromosome pattern for males with one X and one Y chromosome
• X-linked recessive: knights move pedigree pattern
- Affects mainly males
- Mothers carriers give birth to affected male
- No male to male transmission (X chromosome is not inherited from the dad)
• X-linked dominance:
- Affects either sex but more female than males
- Females have milder and more variable degree of effect due to lyonization
- Affected male pass to all his daughter but not son (help distinguish from autosomal dominance)
• Y-linked:
- Affects only males
- Affected father passes the disorder to all his son
- Affects son must have an affected father
• Complications:
- Common recessive conditions such as blood group O can give a false impression of dominance of other alleles
- New mutations or germline mosaicism including osteogenesis imperfecta
- Variable expression such as waardenburg syndrome in which a gene can cause several different effects such as hearing loss and white forelock
• Allelic heterogeneity: different types of mutation at the same locus, e.g. cystic fibrosis with more than 1000 mutation in the CFTR gene and can produce different phenotypes
• Locus heterogeneity: when mutation at different loci produce the same disease, i.e. spinocerebellar ataxia has more than 12 loci affected all autosomal dominantly inherited
• Hardy-weinberg equilibrium: helps predict genotypes and phenotypes in large populations involving a dominant and recessive allelic form.
- Theory state the proportion dominant alleles in population (p) and recessive alleles in population (q) must add up to 1. Thus, probability of say homozygous recessive will be then q2
- Application, helpful in clinical assessment as it can convert phenotypic frequency to heterozygote frequency.

Huntington disease:

• Features: a neurodegenerative disorder, affecting the basal ganglia particularly
- Progressive motor disability involving both involuntary and voluntary movement
- Mental disturbances including cognitive decline and changes in personality.
- Family history consistent with autosomal inheritance
• Genetics: occurs on chromosome 4 and is the only gene associated with Huntington. The result is a trinucleotide CAG repeat expansion.
- Normal is 26 or fewer CAG repeats.
- Intermediate is 27-35 CAG repeats. Individual in this range does not develop HD but risking their child do. This is called mutable normal alleles and transformation factors include sex of transmitter (usually male), DNA sequence around CAG expansion and size of allele.
- Mutant gene is repeats over 36. Reduced penetrance are those just above 36 and have a chance of not developing HD while full penetrance alleles which is 40 or over is guaranteed development
• Mechanism of autosomal dominance pathology
- Gain of function of enzymes
- Lacking one functional allele so haploinsufficiency
- Abnormal protein produced with mutant allele
- Predisposing allele to cancer etc
In the case for Huntington, gene on chromosome 4 code for Huntington and CAG repeated caused polyglutamine expansion and a gain in function of neuronal cells. Linkage of glutamine with lysine residue of other protein creates aggregates that are insoluble and toxic.
• Genetic testing involved:
- Diagnosis of HD
- Predictive testing checks for HD mutation prior to onset of disease
- Prenatal involves checking for status of parents
• Method of clinical testing: targeted mutation analysis in which PCR amplify CAG region and electrophoresis (downside is contamination) or southern blot if sample does not amplify well due to juvenile onset of HD. The results should be interpreted in the context of family history
• Genotype-phenotype relationships: with increased penetrance and allelic expansion, the earlier the age of onset of HD, i.e. adults usually 36-55 while juvenile is over 60. Homozygotes exhibit an accelerated rate of disease progress than heterozygote though similar age of onset.
• Anticipation: phenomenon in which severity of disease increases with successive generations of inheritance. This is commonly paternal transmission with over 7CAG expansions per generation, possibly due to instability of spermatogenesis.
• Risk to family members: most individuals with HD have an affected parent. Due to anticipation, asymptomatic father may have intermediate allele or reduced penetrance. Sibling status depends on parents and the offspring of HD patients have 50% chance of inheritance and earlier age of onset
• Counseling:
- Testing does not provide definitive prediction of age-of-onset or severity but are useful
- Checks for possible impact of positive and negative results, whether its social, financial etc
- Long term follow up maybe arranged


• Haemochromatosis: autosomal recessive disorder in which iron overload due to increased absorption in iron causes organs and tissue to be damaged over time. Onset age around 40
- 1 in 10 caucasians are carriers and 1 in 400 is homozygous
- Male onset early than female due to no menstruation
- Tiredness and lethargy
- Organ damage can lead to other diseases such as cirrhosis (liver), diabetes (pancreas), cardiomyopathy (heart) etc
• Diagnosis and histological features:
- Raised iron saturation and serum ferrtin
- Increased iron in liver biopsy (blue staining or dry weight) and risk of hepatocellular carcinoma
- Fibrosis or cirrhosis of liver
• Management of haemochromatosis:
- Assessment to observe extend of damage of iron overload, status of other family members
- Venesection to deplete iron sources (remove blood)
• Genetics: familial pattern linked to the HFE gene on chromosome 6. The gene codes for a transmembrane protein, when homozygous for the point mutation on the gene from cysteine to tyrosine at amino acids 282 (C282Y), disorder occurs. 90% of haemochromatosis is affected by this while 5% is mutation at H63D (histine to aspartate).
- Non HFE mutation include transferrin receptors mutation, ferroportin gene (export of iron) etc and juvenile haemochromatosis is produced by haemojuvelin gene.
• Variable penetrance: not all sufferers will develop the disease due to environmental modifiers, e.g. growth, pregnancy, alcohol or viral hepatitis. Other genetic factors affecting include hepcidin
• Impact of HFE gene discovery
- Haemochromatosis diagnosis is now based on abnormal iron studies (check for increase iron, ferritin etc) and HFE gene testing while liver biopsy is no longer needed for most cases (only used if HFE gene test is negative and there is concern about cirrhosis – provide prognostic information)
- Allows screening of family members
• Detection of mutation (HFE analysis): change in base pair from TGC to TAC creates a new restriction site thus produce one more fragment than normal when cut with Rsa I. Electrophoresis reveals results
• Clinical issues:
- Screening criteria is met due to late onset of disease and allows early effective intervention but there is a problem of understanding the true penetrance and risks with screening with haemochromatosis
- Personal right to comply and understand reason for screening
- Can test for partner


• Haemophilia: x-linked recessive clotting disorder due to deficiency of either factor VIII or factor IX. Clinical features include recurrent and spontaneous bleeds into joints, muscles, soft tissue and CNS and long term damage to affected joints.
- lyonization can act as a clinical tool to pick up carriers
- staining of the barr body to reveal sex of offspring and risks
- affects only 1 in 5000 patient but have extreme implications for social, work, education, family in both short term and long term
• Haemophilia management:
- Replacement of coagulation factors and pain relief, but problem of infection of blood products such as HIV etc
- Resting of affected joint
• Genetics: mutations of the gene for factor VIII and IX through either deletions, insertions or rearrangement of the gene or point mutations. Therefore is genetic heterogeneous
- This causes complications to genetic diagnosis as mutation must be classified in order to examine and also severity of the condition may vary
• Diagnosis:
- Pedigree analysis: examining the family tree to give probabilities of offspring getting haemophilia. This is effective in conjunction with ultrasounds to determine the sex. Key purpose is to determine odds or chances of the status of the female, carriers or not carriers.
- Phenotype studies: measurement of the phenotype which in this case is the level of clotting enzymes in the blood. Normal factor VIII and IX levels are 50-150 umole/L . A patient with clotting levels in the normal range but very close to lower limit is possible to be carriers. This technique is also able to diagnose prenatal cases, i.e. fetal blood sampling. Problems with method include variability of factor VIII level due to exercise, stress oestrogen and late diagnosis for prenatal is difficult.
• Genetic diagnosis: DNA analysis much more effective. Sources can be obtained from blood, skin, chorionic villus or amniotic fluid and then can be analyzed with two method:
- Direct mutation analysis: used when mutation responsible for the condition is known and so don’t require other family members. For some haemophilia case, it is a 20.7kb deletion of the factor VIII gene and so electrophoresis will reveal an abnormal shorter band. Point mutation can gain/lose new restriction sites or direct sequencing of the gene.
- Linkage analysis: important when the method of mutation is not known. Using RFLP or VNTR that located near the mutant gene can be used as markers. Using electrophoresis, if a variable DNA segment found in the affected parent is also found in the child, then child may also have the mutant gene as the two are inherited together. Problem with this method include need for family study, unable to apply to fathers, non-definitive (only very high possibility due to recombination)
• Restriction fragment length polymorphism: intron segment of the chromosomes that varies in fragment length when cut with a restriction enzyme)
• Variable number tandem repeats: intron DNA segment that have different number of sequence repeating in different people
• Advantage of genetic diagnosis: accurate, precise and allow preimplantation diagnosis for in vitro fertilization, involving fetal sexing and blood sampling so to select the non-affected embryos.
• Therapy: recombinant coagulation factors that is safe but expensive and gene therapy

Prader Willi and Angelman Sydrome

• Chromosome 15q11-q13: the genes affecting the Prader Willi and the Angelman syndrome are located in this region of chromosome 15. On the paternal chromosome the PW is active and AS is inactive while on the maternal chromosome, PW is inactive and AS is active.
- Pathogensis of AS or PD is due to either the loss of the active gene or the loss of the imprinted inactive gene.
• Prader Willi syndrome: in about 75% of the cases is due to deletion of the paternal loci while 25% is due to uniparental disomy of the maternal chromosome (i.e. no paternal loci), and in less than 5% due to mutation of the imprinting center (i.e. no imprinted PW on maternal)
- Features: failure to thrive in
- infancy and childhood with loss of muscle tone, small hands and feet, obesity and short stature and skin hypopigmentation. Cases are sporadic and can reoccur with imprinting mutations.
• Angelman syndrome: 60% of cases have deletion of maternal loci and 5% with paternal UPD (loss maternal loci). A few % have mutation in the PW/AS imprinting center (no imprinted AS on paternal). Cases are sporadic with imprinting mutation and other undefined mutations.
- Features: open-mouthed expression, mental retardation, ataxia, hypotonia, absent of speech and skin hypopigmentation as well.
• Genetic imprinting: the restriction of either the paternal or the maternal allele in somatic diploid cells of embryos and adults. This process is epigenetic which means it causes heritable change in DNA expression without changing the DNA sequence itself and operates at the transcriptional level (methylation). Hence causes different expression from genetically identical alleles, thus produce alteration in pedigree patterns.
• Features of imprinting:
- Imprinting can vary between tissues and during different times of the development, e.g. for female it occurs during oocyte maturation and for male prior to meiosis to generate primary spermatocyte and later in the embryo, demethylation removes imprinting.
- Imprinting occurs with methylation of cytosine residues in CpG dinucleotides (cytosine right next to a guanine connected by a phosphate) of promoter regions and causes transcriptional silencing. The methylation can be carried through cell division by methytransferase activity
- Methylation is reversed if the methyltransferase is inhibited or sequestered.
• CpG islands: region of DNA longer than 200 bases with majority of the bases G and C. They are located in promoter regions of genes and regulate transcription when methylated.
• Confirmation of imprinting: individual must be heterozygous for sequence of the target gene, i.e. two different alleles with two different sequences. If imprinting occurs only one loci will be expressed and the mRNA in the cell can be analyzed to determine which allele was expressed. This can then be traced back to the parents and find the origin of the allele.
• Imprinting disorders:
- Beckwith-Wiedemann chromosome: disorder on chromosome 11p15 producing pre and postnatal overgrowth due to lack of imprinting. There is also increase tumour predisposition.
- Wilm’s tumour: loss of imprinting causes excessive IGF2 of the paternal gene to be expressed, leading to deficiency of the maternally growth suppressor (CDKN1C expression).
- Familial paraganglioma
• Mitochondrial DNA: double stranded circular DNA found only in the mitochondria of female. It has 15569 nucleotides encoding 13 proteion, 22 tRNA and 2 rRNA. Mutation rate is 10 times than of normal nuclear DNA due to lack of protective histones or repair mechanism.
• Mitochondria DNA mutation: proteins that participate in the respiratory chain are encoded by both DNA and mtDNA. There are 5 complexes located in the inner mitochondrial membrane with the function of ATP production, apoptosis, production of ROS and cellular oxidation/reduction. Thus mtDNA mutation creates energy disorder affecting organs that have high energy requirement.
- Chronic progressive external ophthalmoplegia: slow paralysis of the extraocular muscle commencing with ptosis (drooping of upper eyelid)
- Kearns-Sayre syndrome:

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