genetics 3 Flashcards
Multifactorial Inheritance
Multifactorial inheritance is indicated when there is an increased risk to relatives, but there is no consistent pattern of inheritance within families. In this case, it is reasonable to assume multiple genes and environmental factors contribute to disease susceptibility. The genes that contribute to multifactorial disease susceptibility may or may not include the genes that cause the disease in patients showing Mendelian inheritance. To add to the complexity, the same disease may be the outcome of different multifactorial pathways.
Simple Mendelian
diseases are generally single-gene disorders characterized by inheritance patterns that follow Mendelian expectations and are discernable by examining pedigrees with multiple affected individuals. Reality is that many “simple Mendelian” diseases have characteristics not explained by the genotype at the causative locus. variable disease progression depending on other factors common. different alleles in same gene associated with varying levels of severity
Complex Traits
While inheritance of the disease may indeed follow such Mendelian patterns, it is often the case that the manifestation of the disease shows great variability among those with the same genetic risk factor(s). This variability in disease manifestation is likely due to many of the same characteristics that define complex traits. Aggregate (cluster) in families, Do not follow simple Mendelian mode of inheritance, Likely due to variants in multiple genes and non-genetic factors that may interact, No simple relationship between genetic variant and trait when looking at the population. Need to distinguish between clustering in families due to genetic factors and those due to shared environmental factors. Each measure of genetic contribution needs to be interpreted carefully, but as a group can provide compelling evidence for genetic contribution to trait.
Determining the Relative Contribution of Genetic and Environmental Variation
There are several epidemiological study designs that have been used to try to estimate the degree to which genetic variation is responsible for variation in who gets a particular disease or in a quantitative trait such as blood pressure. Need to distinguish between clustering in families due to genetic factors and those due to shared environmental factors. Twin, adoption, and immigration studies used. Each measure of genetic contribution needs to be interpreted carefully, but as a group can provide compelling evidence for genetic contribution to trait.
problem with family studies
One problem in family studies is that individuals who are genetically related often share a similar culture and environment.
Twin studies
provide a potential means of overcoming this problem. Monozygous (MZ) twins are identically matched for DNA sequence, age, and gender, and perhaps closely matched for environmental exposures. Dizygous (DZ) twins on average share 1/2 of their DNA sequences, but may be about as closely matched for other factors as are MZ twins. If it can be assumed that MZ and DZ twins are equally similar with respect to non-inherited factors, then twins can be used to get an estimate of the relative contribution of genetic vs. environmental variation to the trait. A much higher correlation among MZ compared to DZ twins for a quantitative trait suggests that genetic variation is relatively more important than variation in non-genetic factors. If twins raised together and assume same degree of similar environment then differences in concordance rate between mono- and dizygotic twins likely due to genetic factors.
Adoption Studies
Compare similarity between biological siblings raised apart and adoptive siblings. If biological sib 2 more concordant with biological sibling than adopted sibling, then have evidence for genetic component as opposed to environmental component.
Concordance rates
the presence of the same trait in both members of a pair of twins. However, the strict definition is the probability that a pair of individuals will both have a certain characteristic, given that one of the pair has the characteristic. For example, twins are concordant when both have or both lack a given trait. The ideal example of concordance is that of identical twins. Concordance rates are often used to compare MZ and DZ twins. A much higher concordance rate for disease in MZ than DZ twins suggests that genetic variation contributes to variation in risk more than variation in non-genetic factors
Heritability
The Heritability of a trait is the proportion of total variance in a trait that is due to variation in genes. A high heritability implies that differences among individuals with respect to a trait such as blood pressure in a population can be attributed to differences in the genetic make-up. The key to interpreting heritability estimates is to remember that we’re talking about and describing variation in BOTH genetic factors AND non-genetic factors. If one (alleles or environment) doesn’t demonstrate much variability, then it doesn’t have much potential to explain variability in a trait. Implication: A high heritability does not imply that non-genetic factors are not important. A low heritability does not imply that environment is not important.
Characteristics of Complex Traits
Complex traits demonstrate one or more of the following: Incomplete penetrance, Variable expressivity, Heterogeneity, and Presence of phenocopies
Incomplete penetrance
Some conditions are described as having reduced or incomplete penetrance. This means that clinical symptoms are not always present in individuals who have the disease-causing mutation. An example of an autosomal dominant condition showing incomplete penetrance is familial breast cancer due to mutations in the BRCA1 gene. Females with a mutation in this gene have an 80% lifetime risk of developing breast cancer. The penetrance of the condition is therefore 80%. , Common examples used to show degrees of penetrance are often highly penetrant. There are several reasons for this:Highly penetrant alleles, and highly heritable symptoms, are easier to demonstrate, because if the allele is present, the phenotype is generally expressed. Mendelian genetic concepts such as recessiveness, dominance, and co-dominance are fairly simple additions to this principle. And alleles which are highly penetrant are more likely to be noticed by clinicians and geneticists, and alleles for symptoms which are highly heritable are more likely to be inferred to exist, and then are more easily tracked down.
Variable expressivity
Variable expressivity occurs when a phenotype is expressed to a different degree among individuals with the same genotype. For example, individuals with the same allele for a gene involved in a quantitative trait like body height might have large variance (some are taller than others), making prediction of the phenotype from a particular genotype alone difficult. The expression of a phenotype may be modified by the effects of aging, other genetic loci, or environmental factors. Another example is neurofibromatosis, where patients with the same genetic mutation show different signs and symptoms of the disease.
Heterogeneity
the same disease or condition can be caused, or contributed to, by several factors. In the case of genetics, varying different genes or alleles. Example (allele): Cystic Fibrosis and Example (locus): Alzheimer Disease
Presence of phenocopies
Individuals who have the disease or trait for reasons that are not primarily genetic even though clinical presentation mimics the more genetic version. Example: Thalidomide-induced limb malformation vs. genetically-induced
Implications of Characteristics of Complex Traits
Because of the characteristics of complex inheritance, it is very difficult to predict whether or not individuals will develop a certain complex disease or trait, even when you may know something about their family history or their alleles at a certain locus. An example of this is that the proportion of individuals who develop Alzheimer Disease at certain ages according to their genotype at the APOE locus. This has implications for the informal genetic counseling that you may encounter in the clinical setting. These characteristics also make it difficult to identify the genetic variants that might contribute to disease. The role of non-genetic factors in contributing to variation in complex traits will vary from trait to trait and individual to individual. It’s very important to keep in mind that for some individuals, environment may play a large role, while in others their genetic make-up will play a much larger role in determining their disease risk and/or their physiologic characteristics.
Pharmacogenetics
The study of differences in drug response due to allelic variation in genes affecting drug metabolism, efficacy, and toxicity. The key conceptual elements here are that pharmacogenetics typically involves the study of just a few genes and these genes are selected based on a priori knowledge of their role(s) in drug metabolism.
Pharmacogenomics
the genomic approach to pharmacogenetics, is concerned with the assessment of common genetic variants in the aggregate for their impact on the outcome of drug therapy. Instead of analyzing individual genes and their variants according to what is known about how they influence pharmacokinetic and pharmacodynamic pathways, sets of alleles at a large number of polymorphic loci are being identified that distinguish patients who have responded adversely to what was considered a beneficial drug from those who had no adverse response.
Pharmacokinetics
The rate at which the body absorbs, transports, metabolizes, or excretes drugs or their metabolites. Genetic Examples: Cytochrome P450, glucuronyltransferase, thiopurine methyltransferase. pharmacokinetics is concerned with whether or how much drug reaches the target(s). Pharmacokinetics is broken down further into two basic ways that drugs are metabolized through biotransformations: Phase I (simplified): attach a polar group onto the compound to make it more soluble; usually a hydroxylation step and Phase II (simplified): attach a sugar/acetyl group to detoxify the drug and make it easier to excrete. Again, recognize that genetic variation in Pharmacokinetics/dynamics and/or Phase I/II metabolism that causes phenotypic variation in drug responses between humans becomes important clinically every time you write a prescription.
Pharmacodynamics
the response of the drug binding to its targets and downstream targets, such as receptors, enzymes, or metabolic pathways. Genetic Examples: Glucose-6-phosphate dehydrogenase, vitamin K epoxide complex. pharmacodynamics is concerned with what happens when the drug successfully reaches its target (note both phenomenon occur simultaneously in the race between drug effect (dynamics) and removal (kinetics).
cytochrome P450 (CYP450) genes
encode important enzymes that are very active in the liver and to a lesser extent in the epithelium of the small intestine. CYP450 enzymes metabolize a wide number of drugs. The CYP families (CYP1, CYP2, CYP3) are particularly active including six genes (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) that are involved in the Phase I metabolism of ~90% of all commonly used medications. CYP3A4 itself takes part in the metabolism of over 40% of all drugs used in clinical medicine. There is wide genetic polymorphic variation with phenotypic consequences in the CYP families of genes, which is why they are so important to prescribing physicians. Important Point: While most CYP genes are important in the rate of inactivation of a drug, in some cases the CYP gene(s) is required to activate a drug. The classic example of this is CYP2D6 activity being necessary to convert codeine (inactive, almost no analgesic effect) to morphine (active with a potent analgesic effect).
mechanism of CYP2D6
It is important since CYP2D6 is the principle Phase I metabolizer of ~70 drugs. Lots of mutations, several of which affect function are possible and the alleles are generally classified here as: Frameshift àà alter reading frame -> NO ACTIVITY. Splicing -> skip exons and/or alter reading frame -> NO ACTIVITY. Missense -> alter protein function -> usually REDUCED ACTIVITY. Copy number alleles -> increased gene copyalleles -> INCREASED (‘ULTRAFAST. Based on the combinations (since individuals have 2 alleles in their genotypes) have 3 major phenotypes: normal, poor (includes null alleles), and ultrarapid/ultrafast. Note: some papers will define 4 different phenotypes: poor, intermediate, extensive, and ultrarapid. Important Point: Not only can CYP variation be present and important within the human species as a whole, but ethnic population differences can also be noted.
CYP3A
Substrates: Cyclosporine. Inhibitors include: Ketoconazole, Grapefruit juice, Inducers include: Rifampin, [here more relevance of environmental factors than specific genotypes]
CYP2D6
Substrates: Tricyclic antidepressants and Codeine (activates), CPY2D6 is needed to activate codeine into morphine, inhibitors include Quinidine, Fluoxetine, Paroxetine
CYP2C9
detoxifies warfarin most active metabolite. A deficiency in this gene must guide dossage
N-acetyltransferase
It is an important phase II drug for Isoniazid for tuberculosis. If inactivation of this drug occurs too slow than peripheral neurophathy and bone marrow supression can occur. Those that metabolize it too fast, do not get the benefit of the drug.
TPMT
Drugs: 6-mercaptopurine, 6-thioguanine, if you give these children with ALL (leukemia) standard doses you will KILL the child due to immunosuppression
G6PD
Drugs: sulfonamide, dapsone, the mechanism is an x-linked enzyme, G6PD deficient individuals are susceptible to hemolytic anemia after drug exposures
VKORC1
Vitamin K is essential for blood clotting but must be enzymatically activated. This enzymatically activated form of vitamin K is a reduced form required for the carboxylation of glutamic acid residues in some blood-clotting proteins. The product of this gene encodes the enzyme that is responsible for reducing vitamin K 2,3-epoxide to the enzymatically activated form. Fatal bleeding can be caused by vitamin K deficiency and by the vitamin K antagonist warfarin, and it is the product of this gene that is sensitive to warfarin. In humans, mutations in this gene can be associated with deficiencies in vitamin-K-dependent clotting factors. In humans and rats it has also been associated with warfarin resistance different genotypes in confer different sensitivity ot warafin. Combining CPY2C9 and VKORC1 accounts for nearly half of the interindividual difference in warain dose requirment.
glucose-6-phosphate dehydrogenase deficiency
This condition mainly affects red blood cells. In affected individuals, a defect in an enzyme called glucose-6-phosphate dehydrogenase causes red blood cells to hemolysis. The most common medical problem associated with glucose-6-phosphate dehydrogenase deficiency is hemolytic anemia, which occurs when red blood cells are destroyed faster than the body can replace them. This type of anemia leads to paleness, yellowing of the skin and whites of the eyes (jaundice), dark urine, fatigue, shortness of breath, and a rapid heart rate. In people with glucose-6-dehydrogenase deficiency, hemolytic anemia is most often triggered by bacterial or viral infections or by certain drugs (such as some antibiotics and medications used to treat malaria). Hemolytic anemia can also occur after eating fava beans or inhaling pollen from fava plants (a reaction called favism). The G6PD gene provides instructions for making glucose-6-phosphate dehydrogenase. This enzyme is involved in the normal processing of carbohydrates. It also protects red blood cells from the effects of reactive oxygen species. Reactive oxygen species are byproducts of normal cellular functions. Chemical reactions involving glucose-6-phosphate dehydrogenase produce compounds that prevent reactive oxygen species from building up to toxic levels within red blood cells. As a result, reactive oxygen species can accumulate and damage red blood cells. Factors such as infections, oxidant drugs (sulfa), or ingesting fava beans can increase the levels of reactive oxygen species, causing red blood cells to be destroyed faster than the body can replace them. A reduction in the amount of red blood cells causes the signs and symptoms of hemolytic anemia. carriers of a G6PD mutation may be partially protected against malaria. This condition is inherited in an X-linked recessive pattern.
thiopurine s-methyltransferase deficiencey
Thiopurine methyltransferase methylates thiopurine compounds. Thiopurine drugs such as 6-mercaptopurine are used as chemotherapeutic agents and immunosuppressive drugs. Genetic polymorphisms that affect this enzymatic activity are correlated with variations in sensitivity and toxicity to such drugs within individuals. its role in the metabolism of the thiopurine drugs such as azathioprine, 6-mercaptopurine and 6-thioguanine. TPMT catalyzes the S-methylation of thiopurine drugs. Defects in the TPMT gene leads to decreased methylation and decreased inactivation of 6MP leading to enhanced bone marrow toxicity which may cause myelosuppression, anemia, bleeding tendency, leukopenia & infection.
cytochrome p450
a large family of enzymes that are heme containing proteins in the liver and in the and intestinal epithelium. Fe2+ allows them to accept electrons from electron donors such as NAPH and use them for catalyzing different reactions, mostly adding one molecule of O from O2 to a C,N, or S. For many drugs, CYP450 adds a hydoxyl group to the molecule (phase I). This provides a site to add a sugar or acetyl group to detoxify the drug and make it easier to excrete (phase II). Most CYPs function to inactivate drugs, but rarely are needed for activation.
UDP- glycosyltransferase
an enzyme that can glucuronidate many molecules including bilirubin extretion into bile and camptothecin (a chemotherapy drug). There is a lot of veriability in the metobilism and therefore risks to levels of toxicity.
cholinesterase
a family of enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation. An absence or mutation of the pseudocholinesterase enzyme leads to a medical condition known as pseudocholinesterase deficiency. This is a silent condition that manifests itself only when people that have the deficiency receive the muscle relaxants succinylcholine or mivacurium during a surgery. The effects are varied depending on the particular drug given. When anesthetists administer standard doses of these anesthetic drugs to a person with pseudocholinesterase deficiency, the patient experiences prolonged paralysis of the respiratory muscles, requiring an extended period of time during which the patient must be mechanically ventilated. Eventually the muscle-paralyzing effects of these drugs will wear off despite the deficiency of the pseudocholinesterase enzyme. If the patient is maintained on a mechanical respirator until normal breathing function returns, there is little risk of harm to the patient.
genotypic risk for adverse outcomes after cardiothoracic surgery
by combining patients genotype at loci involved in postoperative complications with other relavent information, you can obtain better risk analysis. Some of these loci inclue glycoproteins concerned with platlet affregation and the coagulation cascade. C-reactive protein and interleukin-6 helps judge the risk of stroke
CYP3A4
This family is responsible for breaking down 40% of all common drugs and there is less genetic variation in CYP3A4 than in other CYPs
nortriptyline metabolism
a tricyclic antidepressant. the greater the number of copies of functional CYP2D6 genes, the faster it is metabolized and the faster it leaves the patients system
Risk of Disease in Relatives
Can compare the frequency of disease in relatives of patients to see if higher than in general population. λs= risk of the disease in siblings of affected/ risk of disease in general population. Risk of disease in siblings of a type 1 diabetes patient is higher than risk in general population. Risk in general population ~ 0.4%. Risk to sibs of type 1 diabetes patient ~ 6%. lS ~ 15 for type 1 diabetes.
Heritability (h2)
Proportion of variance in trait that is due to genetic variation. If heretibility is less than 50% it is considered low. the proportion of observed differences on a trait among individuals of a population that are due to genetic differences. Factors including genetics, environment and random chance can all contribute to the variation between individuals in their observable characteristics (in their “phenotypes”). Heritability thus analyzes the relative contributions of differences in genetic and non-genetic factors to the total phenotypic variance in a population. For instance, some humans in a population are taller than others; heritability attempts to identify how much genetics play a role in part of the population being taller. Heritability is measured by estimating the relative contributions of genetic and non-genetic differences to the total phenotypic variation in a population. Heritability measures the fraction of phenotype variability that can be attributed to genetic variation. This is not the same as saying that this fraction of an individual phenotype is caused by genetics. In addition, heritability can change without any genetic change occurring, such as when the environment starts contributing to more variation.
HNF-1 alpha
is a transcription factor that is highly expressed in the liver and is involved in the regulation of the expression of several liver-specific genes. Changes in the HNF1A gene cause diabetes by lowering the amount of insulin that is produced by the pancreas. It allows insulin to be produced normally in childhood but the amount of insulin reduces as you get older. HNF1A is one of a group of familial types of diabetes called maturity onset diabetes of the young (MODY). HNF1A accounts for 70% of MODY cases.
allelic heterogeneity
Different alleles in the same gene result in same trait. Different alleles in the same gene result in different traits. Example: Cystic Fibrosis. Many alleles appear to have very similar clinical progression of disease. Alleles can be grouped into classes; severity of lung and pancreatic involvement depends on allele class
CFTR genotype
if you have a severe/severe genotype 85% present with pancreatic insufficiency while mild/mild or mild/severe only 15% of panctreatic insufficiency. Such genotype is also related to pulmonary function in CF patients, patients with mild mutations have generally better lung functions than those carrying severe mutations. But when we deal with particular patient from both groups it is very difficult to predict if they will have severe or mild lung disease. We can see some patients from the severe group that have relatively good pulmonary function and vice versa. We expect therefore that contribution of these other factors that we were talking about is much stronger for lung disease than for pancreas.
Locus Heterogeneity
Variants in different genes result in very similar clinical presentation. Classic example: Early onset Alzheimer disease (AD). Mutations in 3 different genes all result in identical clinical presentation of early-onset AD
phenocopy
environmentally caused phenotype that mimics the genetic version of the trait. Note that it can be argued that almost everything has some genetic component; intent here is that primary reason for the phenotype is not genetic. Example: Thalidomide-induced limb malformation vs. genetically-induced.
Type 2 Diabetes as an Example
Risk factors: increasing age, obesity, physical inactivity, family history, prior gestational diabetes, impaired glucose tolerance (IGT). Associated with: LDL cholesterol, triglycerides, blood pressure. Control often attained with diet, exercise. Insulin resistance generally thought to precede insulin deficiency, but both often present at time of diagnosis. Control of type 2 diabetes can usually be attained by maintenance of a regimented diet and exercise program, suggesting that environment plays an important role in the pathogenesis of the disease. AGE: most individuals not affected until later in life. However, it is clear that obesity is not the whole story . Estimates of concordance rates for MZ twins: About 2 times those for dizygotic twins - 35%-100%
Risk to 1st degree relatives of affected: ~ 3 to 4 times that of general population. Intermediate traits of blood glucose and insulin levels have heritability estimates as high as 50%
sensitivity
(also called the true positive rate, or the recall rate in some fields) measures the proportion of actual positives which are correctly identified as such (e.g. the percentage of sick people who are correctly identified as having the condition), and is complementary to the false negative rate. In medical diagnostics, test sensitivity is the ability of a test to correctly identify those with the disease (true positive rate), whereas test specificity is the ability of the test to correctly identify those without the disease (true negative rate). If 100 patients known to have a disease were tested, and 43 test positive, then the test has 43% sensitivity. If 100 with no disease are tested and 96 return a negative result, then the test has 96% specificity. Sensitivity and specificity are prevalence-independent test characteristics, as their values are intrinsic to the test and do not depend on the disease prevalence in the population of interest.[3] Positive and negative predictive values, but not sensitivity or specificity, are values influenced by the prevalence of disease in the population that is being tested.
specificity
sometimes called the true negative rate) measures the proportion of negatives which are correctly identified as such (e.g. the percentage of healthy people who are correctly identified as not having the condition), and is complementary to the false positive rate. A perfect predictor would be described as 100% sensitive (i.e. all sick are identified as sick) and 100% specific (i.e. all healthy are identified as healthy); however, theoretically any predictor will possess a minimum error bound known as the Bayes error rate.
pseudoautosomal region
are homologous sequences of nucleotides on the X and Y chromosomes. Pairing (synapsis) of the X and Y chromosomes and crossing over (recombination) between their pseudoautosomal regions appear to be necessary for the normal progression of male meiosis. Thus, those cells in which X-Y recombination does not occur will fail to complete meiosis. Structural and/or genetic dissimilarity (due to hybridization or mutation) between the pseudoautosomal regions of the X and Y chromosomes can disrupt pairing and recombination, and consequently cause male infertility. A high porportion of these genes are related to gonadal and genital development
testis determining factor
In the presence of this gene, the medullary tissue forms typical testes with seminiferous tubules and Leydig cells that, under the stimulation of chorionic gonadotropin from the glacenta, become capable of androgen secretion. The spematogonia, derived from the primordial germ cells by successive mitosis, line the walls of the seminiferous tubules, where they reside together with supporting setoli cells. In males, leydig cells of the fetal testes produce androgen which stimulates the mesonephric ducts to form male genital ducts. the sertoli cells produce a hormone that suppresses formation of the paramesonephric ducts
absence of y chromosome
if no y chromosome is present, the gonad begins to differentiate to form an ovary and the medulla regresses, and oogonia begin to develop within follicles. At about three months, oogonia enter meiosis I, but is arrested at dictyotene until ovulation occurs years later.
mesonephric ducts
a paired organ found in mammals including humans during embryogenesis. In a male, it develops into a system of connected organs between the efferent ducts of the testis and the prostate, namely the epididymis, the vas deferens, and the seminal vesicle. The prostate forms from the urogenital sinus and the efferent ducts form from the mesonephric tubules. For this it is critical that the ducts are exposed to testosterone during embryogenesis. Testosterone binds to and activates androgen receptor, affecting intracellular signals and modifying the expression of numerous genes. In the mature male, the function of this system is to store and mature sperm, and provide accessory semen fluid. In the female, with the absence of anti-Müllerian hormone secretion by the sertoli cells and subsequent Müllerian apoptosis, the Wolffian duct regresses, and inclusions may persist. The epoophoron and Skene’s glands may be present. Also, lateral to the wall of the vagina a Gartner’s duct or cyst could develop as a remnant.
paramesonephric ducts
are paired ducts of the embryo that run down the lateral sides of the urogenital ridge and terminate at the sinus tubercle in the primitive urogenital sinus. The development of the paramesonephric (Müllerian) ducts is controlled by the presence or absence of “AMH”, or Anti-Müllerian hormone (also known as “MIF” for “Müllerian-inhibiting factor”, or “MIH” for “Müllerian-inhibiting hormone”, or “APH” for Anti-Paramesonephric Hormone). In females, the paramesonephric ducts give rise to the uterine tubes, uterus, and upper portion of the vagina, while the mesonephric ducts degenerate due to the absence of male androgens. In contrast, the paramesonephric ducts begin to proliferate and differentiate in a cranial-caudal progression to form the aforementioned structures. During this time, the single-layered paramesonephric duct epithelium differentiates into other structures, ranging from the ciliated columnar epithelium in the uterine tube to stratified squamous epithelium in the vagina.
XX males
a rare sex chromosomal disorder. Usually, it is caused by unequal crossing over between X and Y chromosomes during meiosis in the father, which results in the X chromosome containing the normally-male SRY gene. When this X combines with a normal X from the mother during fertilization, the result is an XX male. Symptoms usually include small testes and subjects are invariably sterile. Individuals with this condition sometimes have feminine characteristics, with varying degrees of gynecomastia but with no intra-abdominal Müllerian tissue.
XY females
a type of hypogonadism in a person whose karyotype is 46,XY. The person is externally female with streak gonads, and left untreated, will not experience puberty. Such gonads are typically surgically removed (as they have a significant risk of developing tumors) and a typical medical treatment would include hormone replacement therapy with female hormones. The first known step of sexual differentiation of a normal XY fetus is the development of testes. The early stages of testicular formation in the second month of gestation requires the action of several genes, of which one of the earliest and most important is SRY, the sex-determining region of the Y chromosome. Mutations of SRY account for many cases of Swyer syndrome. When such a gene is defective, the indifferent gonads fail to differentiate into testes in an XY (genetically male) fetus. Without testes, no testosterone or antimüllerian hormone (AMH) is produced. Without testosterone, the wolffian ducts fail to develop, so no internal male organs are formed. Also, the lack of testosterone means that no dihydrotestosterone is formed and consequently the external genitalia fail to virilize, resulting in normal female genitalia. Without AMH, the Müllerian ducts develop into normal internal female organs (uterus, fallopian tubes, cervix, vagina).
azoospermia factors
one of several proteins or their genes, which are coded from the AZF region on the human male Y chromosome.[1] Deletions in this region are associated with inability to produce sperm.
x inactivation
a process by which one of the two copies of the X chromosome present in female mammals is inactivated. The inactive X chromosome is silenced by its being packaged in such a way that it has a transcriptionally inactive structure called heterochromatin. As nearly all female mammals have two X chromosomes, X-inactivation prevents them from having twice as many X chromosome gene products as males, who only possess a single copy of the X chromosome (see dosage compensation). The choice of which X chromosome will be inactivated is random, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and its descendants in the organism. the choice of which x chromosome is to be inactivated is made early in development, thus females are mosaic. A fraction of the genes along the X chromosome escape inactivation on the Xi. The Xist gene is expressed at high levels on the Xi and is not expressed on the Xa.
XIST gene
it is contained in the x inactivation center in Xq13. it seems to be a key master regulatory locus for x inactivation and is only expressed from the allele on the inactive x.
non random x inactivation
unbalanced structural abnormalities of an x chromosome will cause that chromosome to be inactivated. Nonrandom inactivation is observed for most cases of x; autosome translocation. If the translocation is balanced, the noraml x chromosome is preferentially inactivated and the two parts of the translocated chromosome remain active so that the autosomal genes dont get inactivated. however, sometimes the break itself causes a mutation leading to an x-linked phenotype normaly only seen in males. in unbalanced offspring of unbalanced carriers, the translocated chromosome is inactivated to present partial trisomy.
X-linked intellectual disability
refers to forms of intellectual disability which are specifically associated with X-linked recessive inheritance. As with most X-linked disorders, males are more heavily affected than females. Females with one affected X chromosome and one normal X chromosome tend to have milder symptoms. Unlike many other types of intellectual disability, the genetics of these conditions are relatively well understood.[2][3] It has been estimated there are ~200 genes involved in this syndrome; of these ~100 have been identified.
hermaphroditism
Aside from having an ambiguous-looking external genitalia, true hermaphroditism in humans differs from pseudohermaphroditism in that the person’s karyotype has both XX and XY chromosome pairs (47XXY, 46XX/46XY, 46XX/47XXY or 45X/XY mosaic) and having both testicular and ovarian tissue. One possible pathophysiologic explanation of this rare phenomenon is a parthenogenetic division of a haploid ovum into two haploid ova. Upon fertilization of the two ova by two sperm cells (one carrying an X and the other carrying a Y chromosome), the two fertilized ova are then fused together resulting in a person having dual genitalial, gonadal (ovotestes) and genetic sex. Another common cause of hermaphroditism is the crossing over of the SRY from the Y chromosome to the X chromosome during meiosis. The SRY is then activated in only certain areas, causing development of testes in some areas by beginning a series of events starting with the upregulation of SOX9, and in other areas not being active (causing the growth of ovarian tissues). Thus, testicular and ovarian tissues will both be present in the same individual.
Loss-of-Function Mutations
Mechanisms: Caused by genetic mutations (deletions, insertions, or rearrangements) that eliminate (or reduce) the function of the protein. Of the four major mechanisms, this is the most common genetic mechanism leading to human genetic disease. In general, many metabolic diseases (enzyme defect in a metabolic pathway) are due to loss of function mutations and these loss-of-function mutations are often (but not exclusively) inherited as autosomal recessive diseases as both alleles need to be damaged before the phenotype develops. Included in the ‘loss of function category’ are also mutations that lead to reduction of protein function (either reduction of protein produced or a protein that functions less well/efficiently). the mutation may be in coding region or mutations affecting gene regulation or dosage.
Duchenne Muscular dystrophy
Complete loss of a protein: stop codon, frameshift, or deletion of multiple exons. DMD Xp21.2. Large deletions (multiple exons). Nonsense (stop) mutations / frameshift mutations à premature termination. (in-frame deletions -> milder Becker muscular dystrophy). Clinically: Boys with abnormal gait at 3-5 years, Calf pseudohypertrophy, Gower maneuver, Progressive involvement of respiratory muscles, Median age of death 18 years, Women may à cardiomyopathy. Large deletions (multiple exons)
Nonsense (stop) mutations / frameshift mutations -> premature termination, (in-frame deletions -> milder Becker muscular dystrophy). X-linked inheritance. Frameshift deletions = major loss-of-function mechanism in Duchenne Muscular Dystrophy. In-frame deletions (and also missense mutations) = major loss-of (i.e. reduction)-function in Becker Muscular dystrophy.
alpha-thalassemia
Reduction in amount of protein: deletion of copy. There can be transcription problems due to reduced or absent production of a globin mRNA because of deletions or mutations in regulatory or splice sites of a globin gene. There can also be translation issues due to nonfunctional or rapidly degraded mRNAs with nonsense or frameshift mutations
hereditary retinoblastoma
Somatic mutation leading to loss of tumor suppressor protein
Hereditary neuropathy with liability to pressure palsies (HNPP)
due to deletion of PMP22 gene leading to a phenotype where patients have temporary (usually reversible) neuropathy when pressure is applied to various nerves. Just as your arm may go to sleep if left in a certain position, these patients are more sensitive to pressure on nerves and their limbs can ‘go to sleep’ for longer periods of time (hours, days, to months). there can be transcriptional issues due to increased postnatal transcription of one or more γ-globin genes. Deletion of PMP22 gene = Loss-of-function à Hereditary Neuropathy with Liability to Pressure Palsies (HNPP). PMP22 protein is an integral membrane glycoprotein in nerves. Clinically: repeated focal pressure neuropathies (e.g. carpal tunnel syndrome and peroneal palsy with foot drop), First attack usually in 2nd-3rd decade, Recovery from acute neuropathy is often complete, Incomplete recovery -> mild disability, it is autosomal dominant
Osteogenesis imperfecta type I
Nonsense (stop) mutations / frameshift mutations in COL1A1ààpremature termination. Reduced amount of normal COL1A1 (collagen) protein causing a ‘milder’ form of osteogenesis imperfect. Clinically characterized by increased fractures, brittle bones, and blue sclera.
Gain-of-Function Mutations
Mechanisms: Caused by genetic mutations (often missense or sometimes promoter mutations) that enhance one or more normal functions of a protein (e.g. increased protein expression, increased half- life, decreased degradation, increased activity). Gain-of-function mutations caused by point mutations (missense mutations) often (but not always) show autosomal dominant inheritance patterns as carrying one mutant allele is sufficient to cause disease.
Hemoglobin Kempsey
(Beta hemoglobin gene, Asp99Asn missense mutation): leads to a hemoglobin molecule which has higher than normal oxygen affinity, and is less able to unload oxygen in the tissues. Beta hemoglobin gene: Asp99Asn missense mutation, which has Higher oxygen affinity. In normal hemoglobin binding of oxygen allowing for shift from tense (deoxygenated) to relaxed (oxygenated) form. 99Asn mutation prevents this shift. Hemoglobin remains ‘locked’ in the relaxed state (which has higher oxygen affinity) = Gain Of Function. Consequences: Hb Kempsey unloads less oxygen in tissues, Body ‘thinks’ it needs more oxygen -> makes more red blood cells-> polycythemia
Achondroplasia
FGFR3 Gly380Arg mutation increases the normal signaling though intracellular tyrosine kinase domain (essentially having the receptor constitutively in the ‘turned-on’ state). Skull growth abnormal: midface hypoplasia (otits, sleep apnea)…small cranial foramina (hydrocephalus, brainstem compression (10% of patients), increased frequency of hypotonia, quadriparesis, failure to thrive, central apnea, and sudden death). Between 3% and 7% of patients die unexpectedly during their first year of life because of brainstem compression (central apnea) or obstructive apnea. Gain-of-function mutations, Advanced paternal age, De novo mutation. Age at onset: prenatal, Rhizomelic short stature, Megalencephaly, Spinal cord compression, (trident hand). Achondroplasia is a form of short-limbed dwarfism. However, in achondroplasia the problem is not in forming cartilage but in converting it to bone (a process called ossification), particularly in the long bones of the arms and legs. Achondroplasia is similar to another skeletal disorder called hypochondroplasia, but the features of achondroplasia tend to be more severe. Health problems commonly associated with achondroplasia include episodes in which breathing slows or stops for short periods (apnea), obesity, and recurrent ear infections. In childhood, individuals with the condition usually develop a pronounced and permanent sway of the lower back (lordosis) and bowed legs. Some affected people also develop abnormal front-to-back curvature of the spine (kyphosis) and back pain. A potentially serious complication of achondroplasia is spinal stenosis, which is a narrowing of the spinal canal that can pinch (compress) the upper part of the spinal cord. Spinal stenosis is associated with pain, tingling, and weakness in the legs that can cause difficulty with walking. Another uncommon but serious complication of achondroplasia is hydrocephalus, which is a buildup of fluid in the brain in affected children that can lead to increased head size and related brain abnormalities.
Alzheimer disease in Trisomy 21
Patients with an extra copy of chromosome 21 have 3 total copies of the APP (21q21) leading to increased production of APP protein which contributes to early-onset Alzheimer disease in this patient population.
Charcot-Marie-Tooth disease type 1A
due to duplication of PMP22 gene (see HNPP) which leads to elevated PMP22 protein. Duplication of PMP22 gene = Gain-of-function à Charcot Marie Tooth Syndrome type IA (CMT1A). PMP22 protein is an integral membrane glycoprotein in nerves. Clinically: Demyelinating motor and sensory neuropathy, Often presents in lower extremities with weakness and muscle atrophy and mild sensory loss, Progressive; typical patterns on nerve conduction studies, is autosomal dominant
Novel Property Mutations
Mechanisms: Caused by genetic mutations (often missense) that confer a novel property on the protein, without necessarily altering its normal functions. Although the introduction of a novel property has sometimes been advantageous from an evolutionary standpoint, the majority of such changes result in a novel protein property that reduces fitness (i.e. can lead to disease). (relatively uncommon)
Sickle cell anemia
the Glu6Val mutation of the beta globin gene results in a hemoglobin molecule which transports oxygen essentially normally. However under low oxygen states the Val residue leads to polymerization of hemoglobin into long protein-fibers which deform and restrict the normally flexible red blood cells
Huntington disease
A triplet repeat disorder where by expansion of CAG repeat ‘triplets’ in the gene increase the number of glutamine residues (the CAG codon codes for glutamine). Increased polyglutamine residues above a certain threshold leads to a novel toxic effect on the huntingtin protein. Expansion occurs in paternal allele
Ectopic or Heterochronic Expression Mutations
(relatively uncommon; seen in cancers) Mechanisms: Caused by genetic mutations that alter regulatory regions of a gene and alter either the timing (wrong time = heterochronic) or location (wrong place = ectopic) of expression. Cancers: a gene that is normally silent (for example in differentiated cells) is abnormally expressed (i.e. turned back ‘on’) and leads to abnormal proliferation. Oncogenes are examples of this situation.
Hereditary persistence of fetal hemoglobin
the normal switch from fetal to adult hemoglobin does not occur and fetal hemoglobin, which has a higher affinity for oxygen, remains expressed beyond infancy. One mechanism for this is deletion of several genes in the beta hemoglobin locus, but retention of the fetal (γ) gene such that the γ-globin gene continues to be expressed because the β-globin gene is missing and the normal switch from γ to β cannot occur
The 8 Steps at Which Mutations Can Disrupt the Production of a Normal Protein
transcription, translation, polypeptide folding, posttranslational modification, assembly of monomers into a holomeric protein, subcelular localization of the polypeptide or the holomer, cofactor of prosthetic group binding to the polypeptide, function of a correctly folded assembled and localized protein produced in normal amounts
hemoglobinopathies
More than 70 hemoglobinopathies are due to abnormal hemoglobins with amino acid substitutions or deletions that lead to unstable globins that are prematurely degraded, e.g., Hb Hammersmith
I-cell disease
a lysosomal storage disease that is due to a failure to add a phosphate group to mannose residues of lysosomal enzymes. The mannose 6- phosphate residues are required to target the enzymes to lysosomes.
osteogenesis imperfecta
in some types, an amino acid substitution in a procollagen chain impairs the assembly of a normal collagen triple helix
Familial hypercholesterolemia
a genetic disorder characterized by high cholesterol levels, specifically very high levels of low-density lipoprotein (LDL, “bad cholesterol”), in the blood and early cardiovascular disease. in the carboxyl terminus of the LDL receptor, that impair the localization of the receptor to clathrin- coated pits, preventing the internalization of the receptor and its subsequent recycling to the cell surface
homocystinuria
an inherited disorder of the metabolism of the amino acid methionine, often involving cystathionine beta synthase. It is an inherited autosomal recessive trait. The enzyme cannot function due to poor or absent binding of the cofactor (pyridoxal phosphate) to the cystathionine synthase apoenzyme
Hb Kempsey
caused by an impaired subunit interaction locks hemoglobin into its high oxygen affinity state, otherwise this protein is normal
Unstable Repeat Sequences
In general mutations are relatively stable from generation to generation for most disorders. An exception to this can be found in the ‘unstable repeat expansion’ disorders. These genes contain tri, or tetra-nucleotide repeats which are believed to make the genes susceptible to slipped mispairing during DNA replication. The consequence of this is that the repeat numbers for each allele are prone to change from parent to offspring. An expansion of repeat numbers beyond certain thresholds can lead to clinical disease. Huntington disease, a progressive neurodegenerative condition is the classic example of this class of disease.
Huntington disease
a neurodegenerative genetic disorder that affects muscle coordination and leads to cognitive decline and behavioral symptoms. All humans have two copies of the Huntingtin gene (HTT), which codes for the protein Huntingtin (Htt). Part of this gene is a repeated section called a trinucleotide repeat, which varies in length between individuals and may change length between generations. If the repeat is present in a healthy gene, a dynamic mutation may increase the repeat count and result in a defective gene. When the length of this repeated section reaches a certain threshold, it produces an altered form of the protein, called mutant Huntingtin protein (mHtt). The differing functions of these proteins are the cause of pathological changes which in turn cause the disease symptoms. The Huntington’s disease mutation is genetically dominant and almost fully penetrant. It is not inherited according to sex, but the length of the repeated section of the gene and hence its severity can be influenced by the sex of the affected parent. HTT contains a sequence of three DNA bases—cytosine-adenine-guanine (CAG)—repeated multiple times (i.e. … CAGCAGCAG …), known as a trinucleotide repeat. CAG is the 3-letter genetic code (codon) for the amino acid glutamine, so a series of them results in the production of a chain of glutamine known as a polyglutamine tract (or polyQ tract), and the repeated part of the gene, the PolyQ region. Generally, people have fewer than 36 repeated glutamines in the polyQ region which results in production of the cytoplasmic protein Huntingtin. However, a sequence of 36 or more glutamines results in the production of a protein which has different characteristics. This altered form, called mHtt (mutant Htt), increases the decay rate of certain types of neurons. Regions of the brain have differing amounts and reliance on these type of neurons, and are affected accordingly. Generally, the number of CAG repeats is related to how much this process is affected, and accounts for about 60% of the variation of the age of the onset of symptoms. The remaining variation is attributed to environment and other genes that modify the mechanism of HD. 36–39 repeats result in a reduced-penetrance form of the disease, with a much later onset and slower progression of symptoms.
Genetic anticipation
an important (and testable) concept that describes the clinical observation of disease severity worsening in subsequent generations. Genetic anticipation is explained by the mechanism of tri/tetra nucleotide repeat number expansions occurring from parent to offspring. The offspring inheriting an expanded disease allele is more likely to present earlier and progress faster. There are three principal pathogenic mechanisms for this to occur: 1) Expansion of noncoding repeats and loss of function, consequences are impaired transcription, mutant RNA is not made therefore mutant protein is not made. examples include fragile x and friedreich ataxia 2) Expansion of noncoding repeats conferring novel properties. RNA has novel property (abnormal RNA binds and soaks up RNA-binding proteins -> affects other gene products), Mutant RNA is made but Mutant protein not made. examples include Myotonic dystrophy types 1 and 2, Fragile X-associated tremor/ataxia syndrome (FXTAS) 3) Expansion of codons in exons. this causes novel property on expressed protein and both mutant RNA and protein is made which is toxic. Examples include Huntington disease and spinocerebellar ataxias. Most of these disorders have neurological consequences and are autosomal dominant in their inheritance pattern. Exceptions are Fragile X (X-linked), Friedreich ataxia (autosomal recessive), and some spinocerebellar ataxias (most are autosomal dominant, but recessive forms also reported)
Characteristics of Autosomal Recessive (AR) Disorders
Phenotype expressed only in people who have two mutant alleles of the same gene. Both parents of an affected child are obligated carriers of the disease-causing allele(s). Men and women are usually equally affected. Horizontal pedigree (affected individuals are usually siblings). Carriers are usually undetected, thus the birth of the first affected child is usually unexpected.
The recurrence risk is 1 in 4 (25%) for each unborn child of the same couple. The probability of an unaffected sibling being a carrier is 2/3. The majority of mutant allele(s) are present in carriers instead of patients. Sometimes with a higher frequency within people of a small group (high-risk group). Increased incidence of parental consanguinity for a child affected by a rare AR disorder.
Compound heterozygote
one who carries two different mutant alleles of the same gene.
Phenylketonuria (PKU) Phenotype
Microcephaly and profound mental retardation if untreated during infancy. Neurobehavioral symptoms such as seizure, tremor, and gait disorders are common. High phenylalanine and low tyrosine levels in the plasma because the conversion from Phe to Tyr is impaired. High levels of phenylalanine metabolites in urine and sweat gives a characteristic “mousy” odor.
Phenylketonuria (PKU) Frequency
Disease frequency is 1/10,000 births (q2 = 1/10,000, q = 1%) among individuals of Northern European ancestry. Carrier frequency is about 1/50 (2pq ≈ 2q = 2%).
Phenylketonuria (PKU) Biochemical defects
PKU is an inborn defect of phenylalanine metabolism. Most PKU cases are caused by defects in the PAH gene encoding phenylalanine hydroxylase, a liver enzyme that catalyzes the conversion of Phe to Tyr using molecular oxygen and a cofactor tetrahydrobiopterin (BH4). A small fraction of PKU patients (1~3%) have normal PAH but are defective in genes that are needed for the synthesis or regeneration of BH4, the cofactor of PAH. BH4 is also the cofactor for two other enzymes, tyrosine hydroxylase and tryptophan hydroxylase, both of which synthesize monoamine neurotransmitters. The high phenylalanine level in PKU damages the developing central nervous system in early childhood and interferes with the function of the mature brain, although the mechanism of damage is unclear. BH4-deficient PKU patients have problems caused by both hyperphenylalaninemia and neurotransmitter imbalance.
Phenylketonuria (PKU) Molecular basis
The PAH gene is at chromosome 12q22-24. Most mutations in PAH are partial or complete loss-of-function alleles. PAH gene exhibits high allelic heterogeneity; over 400 alleles have been identified. Most PKU patients are compound heterozygotes (i.e. having two different mutant alleles of the PAH gene). Severity of phenotype varies and probably reflects compound heterozygosity.
Phenylketonuria (PKU) Newborn screening
Guthrie test (aka Guthrie bacterial inhibition assay) for PKU was developed in the mid- 1960s. This test is based on the findings that thienylalanine inhibits the growth of the bacterium Bacillus subtilis and that such inhibition can be overcome by a high level of phenylalanine in the blood sample of a PKU baby. Mass Spectrometry is the current method of choice for many newborn screenings including PKU. A mass spectrometer simultaneously sorts many molecules in a blood specimen by weight (mass) and size, and also measures the quantity of each molecule. Phe/tyr ratio is also a good measure of PKU because phe is convereted to tyr by PAH, therefore without PAH the ratio is high. Timing of Test: The sensitivity of PKU screening is influenced by the age of the newborn when the blood sample is obtained. Phenylalanine level is typically normal in PKU babies at birth because of normal PAH in maternal supply and increases progressively with the initiation of protein feedings during the first days of life. Early detection and treatment is crucial to prevent irreversible damage to the developing brain. However, if tested too early (within 1-2 days of birth), some affected children can be missed. Newborns are tested first after birth and then again at their first pediatrician’s visit days later.
Phenylketonuria (PKU) Treatment
When treated early with low-phenylalanine diet, the mental retardation can be prevented. Phenylalanine is an essential amino acid and thus cannot be eliminated from the diet. The low-phenylalanine diet should be maintained throughout childhood and school years, and preferably the patient’s whole life. BH4-deficient PKU patients are treated with oral BH4, low-phenylalanine diet, and supplements (L-dopa and 5-hydroxytryptophan etc) to balance neurotransmitter levels.
Maternal PKU
It is important to maintain PKU women on a low-phenylalanine diet throughout their child- bearing years. PKU mothers who are not on a low-phenylalanine diet have a markedly increased risk of miscarriage and giving birth to children with congenital malformations, mental retardation, and growth impairment, irrespective of the genotypes of the children. These defects are caused by elevated phenylalanine levels in the maternal circulation during fetal development and thus termed maternal PKU.
α1-Antitrypsin Deficiency (ATD) Phenotypes
ATD patients have a 20-fold increased risk of developing emphysema, with more severe symptoms among smokers. This disorder is late-onset, especially in non-smokers, but 80- 90% of deficient individuals will eventually develop disease symptoms. Many patients also develop liver cirrhosis and have increased risk of liver carcinoma due to the accumulation of a misfolded α1-AT mutant protein in the liver.
α1-Antitrypsin Deficiency (ATD) Frequency
α1-antitrypsin deficiency is a common genetic disorder among Northern European Caucasians. Disease frequency is 1/2,500, carrier frequency ~1/25. The most common normal (wild-type) allele, the M allele, occurs with a frequency of 95%; thus 90% (0.952 =0.9025) of white Europeans have M/M genotype. Most ATD diseases are associated with two mutant alleles, the Z and S alleles. Individuals with Z/Z genotype have only 10-15% of normal α1-AT activity and account for most cases of the disease. Individuals with S/S genotype have 50-60% of normal α1-AT activity and usually do not express disease symptoms. Z/S compound heterozygotes have 30-35 % of normal α1-AT activity and may develop emphysema.
α1-Antitrypsin Deficiency (ATD) Biochemical defects
α1-antitrypsin (ATT or SERPINA1) is made in the liver and secreted into plasma. SERPINA1 is a member of serpins (serine protease inhibitor), which are suicide substrates that bind and inhibit specific serine proteases. The main target of SERPINA1 is elastase, which is released by neutrophils in the lung. When left unchecked, elastase can destroy the connective tissue proteins (particularly elastin) of the lung, causing alveolar wall damage and emphysema.
α1-Antitrypsin Deficiency (ATD) Molecular basis
The SERPINA1 gene is on chromosome 14 (14q32.13). There are ~20 different mutant alleles, although the Z & S alleles account for most of the disease cases. The Z allele (Glu342Lys) encodes a misfolded protein that aggregates in the endoplasmic reticulum (ER) of liver cells, causing damage to the liver in addition to the lung. The S allele (Glu264Val) expresses an unstable protein that is less effective.
α1-Antitrypsin Deficiency (ATD) Screening
Sequence specific oligonucleotide probes can be used to distinguish the M, Z and S alleles in a target population and provide accurate prenatal diagnosis.
α1-Antitrypsin Deficiency (ATD) Environmental factors (Ecogenetics)
Smoking accelerates the onset of emphysema in ATD patients. Tobacco smoke damages the lung, prompting the body to send more neutrophils to the lung for protection. More neutrophils release more elastase, causing more severe lung damage.
α1-Antitrypsin Deficiency (ATD) Treatment
Two approaches of delivering human SERPINA1 to the pulmonary epithelium are being studied: intravenous infusion and aerosol inhalation.
Tay-Sachs disease (GM2 gangliosidosis type I) Phenotypes
T-S is an inherited disorder that progressively destroys neurons in the brain and spinal cord. The most common form of T-S is an early-onset, fatal disorder apparent in infancy. T-S infants appear normal until the age of 3-6 months, when early symptoms such as muscle weakness, decreased attentiveness, and increased startle response appear. As the disease progresses, the T-S children experience symptoms of neurodegeneration including seizures, vision and hearing loss, diminishing mental function, and paralysis. An eye abnormality called “cherry-red spot” is a characteristic of T-S. Children of T-S usually live only till 3-4 years of age.
Tay-Sachs disease (GM2 gangliosidosis type I) Frequency
The Ashkenazic Jewish population is at 100-fold higher risk for T-S (~1/3,600) than the general population (~1/360,000). Other high-risk groups for T-S are certain French- Canadian communities of Quebec, the Old Order Amish community in Pennsylvania, and the Cajun population of Louisiana.
Tay-Sachs disease (GM2 gangliosidosis type I) Biochemical defects
T-S is a lysosomal storage disease. Inability to degrade GM2 ganglioside results in up to 300-fold accumulation of this sphingolipid inside swollen lysosomes in neurons of the central nervous system. A defective hexosaminidase A (HexA) needed for in metabolizing GM2 is responsible for T-S. HexA is a heterodimer of αβ, which are encoded by the HEXA and HEXB genes, respectively. Although HexA is a ubiquitous enzyme, the impact of T-S is primarily in the brain where most of GM2 ganglioside is synthesized. Sandhoff disease (GM2 gangliosidosis type II) presents the same neurological symptoms as T-S. Sandhoff disease patients have defects in both Hexosaminidase A and Hexosaminidase B (HexB); Hex B is a homodimer of ββ. T-S is caused by a defective α subunit; only HexA activity is affected. Sandhoff disease is caused by a defective β subunit; both HexA and HexB activities are affected. The α subunit gene HEXA and the β subunit gene HEXB reside on chromosomes #15 and #5, respectively. AB-variant of Tay-Sachs is a rare form of T-S in which both HexA and HexB are normal but GM2 accumulates due to a defect in the GM2 activator protein (GM2-AP), which facilitates interaction between the lipid substrate and the HexA enzyme (α subunit) within the cell. Note: HexA and HexB refer to the two enzymes; HEXA and HEXB refer to two genes encoding the α and β subunit, respectively.
Tay-Sachs disease (GM2 gangliosidosis type I) Molecular basis
Over 100 HEXA mutations are known. The most common mutant allele (~80%) in the Ashkenazi Jewish population is a 4 bp insertion in exon 11 of HEXA, causing a frameshift and a premature stop codon in the coding sequence of the gene (i.e. it’s a null allele).
Tay-Sachs disease (GM2 gangliosidosis type I) Screening
Enzymatic activity assay: both HexA and HexB enzymes are present in the serum. Their activities can be distinguished in such assays because only HexA is inactivated by heat. Carrier Screening: primarily among Ashkenazi Jewish population, the enzyme test has 97% accuracy because carriers have lower HexA enzyme levels in the blood. Prenatal screening: the enzyme test can also be performed on cultured amniotic fluid cells to detect T-S fetus when both parents are known to be carriers. Notably, this screening has reduced the number of T-S cases by about 95% over the past 30 years. DNA testing: The tests currently available can detect about 95% of carriers in the Ashkenazi Jewish population and about 60% of carriers among non-Jewish individuals. Therefore, some carriers will be missed by DNA test alone.
Other forms of Tay-Sachs disease (GM2 gangliosidosis type I)
Juvenile and adult-onset forms of T-S are very rare with milder symptoms appearing in childhood, adolescence, or adulthood; the patients have decreased HexA activity.
Hemoglobin Structure
The major form of adult hemoglobin (HbA) is a α2β2 tetramer of two α- and two β-globin chains. Each globin contains one heme group with a covalently linked iron that binds oxygen, so one HbA can simultaneously bind four oxygen molecules.
Chromosomal Localization of Globin Genes
All of α and α-like genes are in the α-cluster on chromosome 16, while all of β and β-like genes are in the β-cluster on chromosome 11. All of these globin genes are likely to have arisen from a common ancestral gene via gene duplication events. There are two copies of α in the α-cluster but only one copy of β in the β-cluster. The genes in each cluster have the same 5’-to-3’ transcriptional orientation. At the molecular level the promoter and enhancer regions of these genes are very similar. α-cluster: zeta-alpha2-alpha1 (ζ-α2-α1). β-cluster: epsilon-gammaG-gammaA-delta-beta (ε-γG-γA-δ-β). Pseudogene: resembles a gene but makes no protein. ψζ, ψα, and ψβ are all pseudogenes. The 5’-to-3’ spatial order of genes within each cluster coincides with the temporal order of their expression during development. The sequential expression of these genes during development is under the regulation of the Locus Control Region (LCR), which is located at the most upstream region of each cluster. It is currently thought that the distance between the LCR and a particular globin gene affects its expression.
Locus Control Region (LCR),
are defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number-dependent manner at ectopic chromatin sites. The concept derives from the idea that developmental and cell lineage-specific regulation of gene expression relies not only on gene-proximal elements such as promoters, enhancers, and silencers, but also on long-range interactions of various cis-regulatory elements and dynamic chromatin alterations. It is currently thought that the distance between the LCR and a particular globin gene affects its expression. The LCR presumably makes physical contact with the promoter and/or negative regulatory regions via specific transcriptional factors to influence gene expression by opening up the chromosome to TF through a DNase 1 hypersensitivity site. the LCR along with associated DNA binding proteins, interacts with genes of the locus to form a nuclear compartment called the active chromatic hub, where beta globin gene expression takes place. This hub moves from the 5’ gene (ε-globin) to the δ and β globin genes in adults. Deletions of the entire LCR of the beta cluster cause beta-thalassemias, a condition in which zero β-globin synthesis leads to precipitation of the α-globin chains.
Hemoglobin Expression and Development
Adults have two Hb forms (made in the bone marrow): major form: HbA = α2β2 (97%), minor form : HbA2 = α2δ2 (2%), Note that δ level is much lower than β because δ has a weaker promoter. More Hb forms are present during embryonic and fetal development Embryonic hemoglobins (made in the yolk sac): ζ2ε2 = Hb Gower I α2ε2 = Hb Gower II ζ2γ2 = Hb Portland, Fetal hemoglobins (made in the liver): α2γ2 = HbF. Globin Switching: turn-off of ζ and ε, turn-on of α and γ during early embryogenesis, turn-offofγ,turn-on of β and δ around the time of birth. Significance of Globin Switching: HbF has higher affinity for O2 at low pO2 than HbA. Thus, HbF in fetal blood is better suited to bind O2 at the placenta (lower pO2) than HbA, which binds O2 at the lung (higher pO2).
Genetic Variants of Hemoglobin
~ 600 variants of hemoglobin. About half of them are clinically significant. The some different variants of hemoglobin include structural, thalassemias, and hereditary persistence of fetal hemoglobin
Structural Variants (qualitative hemoglobinopathies)
Mutations that alter the globin polypeptide properties without affecting its synthesis. Most structural variants are found in the β-globin chain. These mutations may alter O2 binding such as HbKemsey (too tight) and HbKansas (too weak), cause heme loss and denaturation of Hb, or make Hb less soluble such as HbS and HbC. Some can lead to serious red blood cell (RBC) diseases.
Thalassemias (quantitative hemoglobinopathies)
Disorders of imbalanced globin levels resulted from markedly reduced or no synthesis of one globin type. Can be caused by virtually all types of genetic mutations including deletions, missense and nonsense mutations, and defective transcriptional control. Note: A functional Hb tetramer is composed of two α (or α-like) chains and two β (or β-like) chains. Homotetramers (e.g. α4, β4, γ4) are poor O2 carriers and precipitate inside the RBC. Thalassemias are caused by an imbalance in the relative levels of the α and β globin chains, which leads to the precipitation of the globin in excess and decreases the life span of the RBC.
Hereditary Persistence of Fetal Hemoglobin (HPFH)
HPFH is a group of clinically benign conditions that impair the perinatal switch from γ- to β-globin synthesis, leading to continued high-level production of HbF in adults. HPFH may alleviate the severity of certain hemoglobinopathies and thalassemias, and is selected for in populations with a high prevalence of these conditions (which in turn are often selected for in areas where malaria is endemic). Thus, it has been found to affect Americans of African and Greek descent.
Sickle cell anemia (HbSS)
Most common among people of African origin, where carrier frequency is ~10%.it is a severe autosomal recessive hemolytic condition characterized by the red blood cells becoming grossly abnormal in shape under conditions of low o2 tension. Single base mutation at codon#6 in the β-globin gene changes glutamate to valine. HbS is 80% less soluble than HbA when not bound to O2, and polymerizes into long fibers that distort the RBC into a characteristic sickle shape. These sickled cells become lodged in the micro-capillaries and further exacerbate the sickling crisis.
Hemoglobin C disease (HbCC)
A milder form of hemolytic anemia than sickle cell anemia. Caused by a single base mutation at codon#6 of the β-globin gene, changing glutamate to lysine. HbC is less soluble than HbA and tends to form crystals, reducing the deformability of RBC.
HbS or HbC trait
Both sickle cell anemia and hemoglobin CC disease are of autosomal recessive inheritance. Sickle cell trait (HbS trait) or hemoglobin C trait (HbC trait) describes the conditions expressed in individuals who are heterozygous of HbS/HbA and HbC/HbA, respectively. They are clinically normal except when under severe low pO2 stress.
Hemoglobin SC disease
Compound heterozygotes (βS/βC) have a milder anemia than sickle cell disease.
HbS Diagnosis Using RFLP
A recognition site (CCTNAGG) of the restriction enzyme MstII is destroyed in exon 1 by the A-to-T change in the βS mutant allele. The normal allele βA gives 1.15 kb + 0.20 kb fragments, whereas the βS mutant allele give one1.35 kb fragment. Note: MstII can distinguish between βA and βS but not between βA and βC.
α -Thalassemias
Mostly caused by deletions of one or both copies of the α-globin gene in the α-cluster. Thus, γ- and β-globin are in excess. Affects the formation of both fetal and adult hemoglobins.
α-thal-1 allele (- -)
Common in Southeast Asia. Caused by deletion of both copies of α-globin genes in the α- cluster. Homozygous state (- -/- -) results in “hydrops fetalis” (stillborn). Most fetal hemoglobin is γ4 (Hb Bart’s) although there is enough ζ2γ2 (Hb Portland) to sustain fetal development. Heterozygotes (αα/–) have mild anemia, a.k.a. α-thalassemia-1 trait.
α-thal-2 allele (α -)
Common in Africa, Mediterranean, and Asia. Deletion of one of the two α-globin genes in the α-cluster. 50% decrease in α-globin synthesis. No disease phenotype in heterozygote (αα/α-, silent carrier). Mild anemia in homozygotes (α-/α-), a.k.a. α-thalassemia-2 trait.
α-thal-1/α-thal-2 (α -/- -)
Compound heterozygous individuals with only 25% of normal α-globin level. Severe anemia. a.k.a. HbH disease. About 5-30% of their hemoglobin is β4 (HbH), which precipitates.
β -Thalassemias
Similar to alpha thalasemia, beta can cause hypochromic, microcytic anemia and the imbalance in globin synthesis leads to precipitation of the excess alpha chains, which in turn leads to damage of the red cell membrane. In contrast, the onset is not until a few months after birth, when beta globin normally replaces γ as the major non alpha chain. It is also usually due to single base pair substitutions rather than deletions (as in alpha). Show a wide range of severity and can be cause by virtually every possible type of mutation in the β-globin gene. High allelic heterogeneity means most patients with β-thalassemia are compound heterozygotes carrying two different mutant alleles of the β-globin gene. Categorization of β-thalassemias: Based on clinical conditions- thalassemia major vs thalassemia minor, Based on the molecular nature of the mutation – simple β-thalassemia vs complex thalassemia, Based on the biochemical nature of the diseases -β+-thalassemia vsβ0-thalassemia,
Thalassemia major
Characterized by severe anemia, in which most RBCs are destroyed before being released into the circulation. Thinning bone cortex, enlarged liver and spleen resulted from massive effort of blood production at these sites. Treat temporarily with blood transfusions; however, iron accumulation from repeated transfusion leads to organ failure. Iron chelation therapy (e.g. desferrioxamine) is used to reduce the complications of iron overload.
Thalassemia minor
Clinically normal, carriers of one β-thalassemia allele.
Simple β-thalassemia
Caused by mutations or deletions that impair the production of the β-globin chain alone. Other genes in the β-globin cluster unaffected. These mechanisms include promoter mutants, RNA splicing (in 5’ GT or 3’ AG), capping or tailling mutants, and frameshift or nonsense mutations
Complex thalassemia
Caused by large deletions that remove the β-globin gene plus other genes in the β-cluster, or the LCR. Note that some deletions within β-cluster cause HPFH instead of thalassemia
β+-thalassemia
Most common form of β-thalassemia (90% of the cases). Some β-globin is made so that some HbA is present. Decrease in β-globin synthesis can be caused by mutations affecting transcription, RNA processing, or protein stability.
β0-thalassemia
Zero β-globin synthesis so that no HbA is present. Caused by deletion of the β-globin gene, nonsense or frameshift mutations at the 5’ of the coding region that lead to an early stop codon, or mutations that result in no RNA synthesis. [Hb] is ~5% of normal level (of which 95% is α2γ2 with 5% α2δ2), which is not enough for good survival.
δβ0-thalassemia
No δ or β synthesis due to deletion of the coding sequences for both δ- and β-globin. Milder clinical phenotype than β0-thalassemia because the remaining γ gene(s) is still active after birth instead of switching off as would normally occur. Therefore, HbF (α2γ2) compensates for the absence of HbA (α2β2), about 5-18% of normal level of total hemoglobin production. Note: People with deletions that remove the δ and β genes (γ is intact) may have either δβ0- thalassemia or HPFH, depending on the range of the deletion.
HPFH (hereditary persistent fetal hemoglobin)
No δ or β synthesis because of deletions of both genes. Increased γ-globin expression caused by either of the two following mechanisms: (1) extended deletion of additional downstream sequences, which likely brings a cis-acting enhancer element closer to the γ-globin gene, or (2) mutations in the promoter region of one of the two γ-globin genes that destroy the binding site of a repressor, thereby relieving postnatal repression of γ. HPFH individuals are disease free, since adequate levels of γ chains are still made due to the disruption of the perinatal globin switch from γ to β. 100% of hemoglobin is HbF (α2γ2), which is about 17-35% of normal level of total hemoglobin production. HPFH individuals have higher HbF (17-35%) level than δβ0-thalassemia individuals (5-18%). Understanding the mechanism of HPFH may make it possible to express HbF at high levels postnatally to treat patients of β-thalassemia and sickle cell anemia.
heterochronic expression
The heterochronic expression of genes controls the timing of developmental events. heterochrony is defined as a developmental change in the timing of events, leading to changes in size and shape. There are two main components, namely (i) the onset and offset of a particular process, and (ii) the rate at which the process operates.
ectopic expression
the expression of a gene in an abnormal place in an organism. This can be caused by a disease, or it can be artificially produced as a way to help determine what the function of that gene is.
Erythropoiesis
the process which produces red blood cells (erythrocytes). the termporal switch of globin synthesis (moving down the chromosome in th same transcriptional orientation), is accompanied by chanes in the major site of synthesis. In the early fetus, erythropoiesis takes place in the mesodermal cells of the yolk sac. HbF (α2γ2) is the main oxygen transport protein in the human fetus. By the third or fourth month, erythropoiesis moves to the liver. After seven months, erythropoiesis occurs in the bone marrow. Beta chains are only synthesized in high levels only near the time of birth. δ synthesis continues after birth but never accounts for more than about 2% of adult hemoglobin. On the alpha chain the order is ζ, Ψ (both are psuedogenes), than α (all in fetal development).
Hemoglobin A (HbA)
also known as adult hemoglobin or α2β2, is the most common human hemoglobin tetramer, comprising over 97% of the total red blood cell hemoglobin. It consists of two alpha chains and two beta chains
Hemoglobin A2 (HbA2)
a normal variant of hemoglobin A that consists of two alpha and two delta chains (α2δ2) and is found at low levels in normal human blood. Hemoglobin A2 may be increased in beta thalassemia or in people who are heterozygous for the beta thalassemia gene.
Fetal hemoglobin
(also hemoglobin F, HbF, or α2γ2) is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus and persists in the newborn until roughly 6 months old. Functionally, fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother’s bloodstream.
gene dosage and ontogeny in globin genes
a single mutation in beta affects 50% of beta chains whereas a single alpha chain muation affects only 25% of the alpha chain. Howerver, the beta globin mutation has no prenatal consequence because gamma globin is the beta like chain in HB F, which constitutes three quarters of the total hemoglobin at term. alpha globin mutation can cause sevre phenotypes both prenataly and after birth.
