Genetic Diseases miRNA-gene silencing. 1000 genes code for miRNAs, approximately 3% of the genome. Primary microRNA transcripts are processed within the nucleus to pre-miRNA. Pre-miRNA is transported to the cytoplasm via specific transporter Additionally modified by "dicer" to form mature dsmiRNA. miRNA gets unwound and incorporated into multiportein complex, RNA induced silencing complex (RISC). Base pairing between miRNA and target mRNA direct RISC to either cause mRNA cleavage or repress its translation. siRNAs function like miRNAs (RISC), except siRNA precursors are introduced into the cell. Hereditary: transmitted disorders in gametes through generations (familial). Congenital: "present at birth" some are not genetic (congenital syphillis).Mendelian Disorders (Diseases Caused by a single-gene defect) Autosomal dominant, recessive or X-linked. In some cases, both alleles of a gene pair may be expressed-codominance. Histocompatibility and blood group and polymorphism. Simple gene mutations may lead to many phenotypic effects (pleiotrophy), and conversely mutations at several genetic loci may produce the same trait (genetic heterogeneity). Marfan syndrome-gene encoding fibrillin. Retinal pigmentation.Transmission Patterns of Single-Gene Disorders Autosomal dominant disorders: Heterozygous state: Some patients have unaffected parents. Penetrance: some individuals inherit the mutant gene but are phenotypically normal. Expressivity: trait seen in all individuals carrying the mutant gene but is expressed differently among individuals. "Variable expressivity." 50% reduction in the normal gene product is associated with clinical symptoms. Two major categories: Proteins involved in regulation of complex metabolic pathways, often subject to feedback control. Familial hypercholesterolemia. Key structural proteins, such as collagen and cytoskeletal components of the red cell membrane. Dominant negative alleles impair the products of normal alleles.Autosomal Recessive Disorders1. Trait does not affect the parents.1. Siblings have on chance in four of being affected.1. If the mutant gene occurs with a low frequency, consanguineous marriage is likely.2. Expression of defect tends to be more uniform than in autosomal dominant disorders.2. Complete penetrance is common.2. Onset is frequently early in life.2. Many cases, enzymes are affected by the mutation.X-Linked Disorders All sex-linked disorders are X-linked. Typically recessive. Transmitted by heterozygous female carriers only to sons, who are hemizygous for X chromosome. Very few X-Linked diseases, and are much less common. Inheritance pattern is characterized by transmission of the disease to 50% of the sons and daughters of an unaffected heterozygous female.Diseases caused by mutations in structural proteins Marfan Syndrome: autosomal dominant disorder of connective tissue. Abnormalities affect fibrillin-major component of microfibrils found in ECM. Microfibrils serve as scaffolding for the deposition of elastin. Fibrillin 1-encoded by FBN1 gene (15q21), approximately 500 mutations have been found that lead to this disease. Heterozygotes have clinical symptoms, therefore, mutant fibrillin1 acts as a dominant negative. Skelton, eyes and cardiovascular system are principally manifested.Ehlers-Danlos Syndrome Defects in collagen synthesis and structure. Skin, ligaments, and joints. Skin is hyper-extensible and joints are hypermobile. Minor injuries produce gaping defects. Lack of tensile strength. Molecular basis: Deficiency of the enzyme lysyl hydroxylase: type I and III affected. Kyphoscoliosis EDS. Deficient synthesis of type III collagen resulting from mutations affecting the COL3A1 gene. Autosomal dominant, characterized by weakness of tissues rich in type III collagen (blood vessels, bowel wall). Defective conversion of procollagen type I to collagen. Results from a mutation in two type I collagen genes (COLA1 and COLA2) in arthrocholasia-type EDS.Diseases Caused by mutations in receptor proteins Familial hypercholesterolemia: Mong most common mendelian disorders. 1/500. Mutations in gene for LDL Receptor. Within hepatocytes, the LDL molecule is enzymatically degraded resulting in release of free cholesterol. Suppresses cholesterol synthesis by inhibiting the activity inhibiting the activity of 3-HMG-CoA reductase. Activates Acyl-CoA and ACAT which favors esterification and storage of excess cholesterol. Down regulates synthesis of cell surface LDL receptors, thus protecting cell from excessive accumulation of cholesterol. Monocytes and macrophages have receptors for chemically modified LDLs, amount catabolized is directly related to the plasma cholesterol level. Receptor problem causes LDL build up in plasma, as well as IDL build up. Reduced metabolism and increased biosynthesis. Marked increase in cholesterol traffic in the monocytes and macrophages leading to xanthomas and premature atherosclerosis. Hterozygous are usually asymptomatic until adult life. Homozygous-die of MI around age 15.Diseases caused by mutations in enzyme proteins Phenylketonuria: Autosomal recessive disorder Lack of phenylalanine hydroxylase leading to hyperphenylalaninemia and PKU. Affected infants at birth are normal, but within a few weeks develop a rising plasma phenylalanine level, which in some ways impairs brain development. BY 6 months, severe mental retardation. Inability to convert phenylalanine to tyrosine (phenylalanine hydroxylase). Approximately 400 mutant alleles of the phenylalanine hydroxylase gene. Only some cause PKU, others only result in partial deficiency.Galactosemia Autosomal recessive disorder of galactose metabolism. Lack of galactose-1-phosphate uridyltransferase, required to convert galactose to glucose. As a result, galactose-1-phosphate and galactitol accumulate in many tissues: liver, spleen, lens of the eye, kidney, and cerebral cortex. Liver, eyes, brain bear brunt of damage.Lysosomal Storage Diseases Inhibited lack of a lysosomal enzyme, catabolism of its substrate remains incomplete, leading to accumulation of the partially degraded insoluble metabolites within the lysosome. Autosomal recessive transmission. Commonly affects infants and young children. Storage of insoluble intermediates in the mononuclear phagocyte system, giving rise to hepatospleenomegaly. Frequent CNS involvement with associated neural damage. Cellular dysfunction, caused by storage of undigested material and cascade of secondary events triggered, for example, by macrophage activation and release of cytokines. Tay sachs disease: GM2 gangliosidoses: deficiency in hexosaminidase alpha subunit. Gangliosidases are characterized by accumulation of gangliosides, principally in the brain, as a result of a deficiency of a catabolic lysosomal enzyme. Mutation in and consequent deficiency of the alpha subunit of the enzyme hexosaminidase alpha which is necessary for the breakdown of GM2. Most mutations affect protein folding or intracellular transport. Storage of GM2 occurs in neurons, axons and glial cells. Affected cells appear swollen, possibly foamy. Whirling configuration within lysosomal/widespread CNS involvement and death 2-3 years. Unfolded proteins can trigger apoptosis (without chaperones).Niemann-Pick Disease, Types A and B Primary deficiency of acid sphingomyelin. A: Severe deficiency of sphingomyelinase. Breakdown of sphingomyelin into ceramide and PC is impaired, and excess sphingomyelin accumulates in all phagocytic cells and in the neurons. Spleen, liver, bone marrow, lymph nodes, and lungs, entire CNS, including splenic cord and ganglia. Affected areas become large and vacuolated. B: Organomegaly but no neurologic symptoms. Estimation of sphingomyelinase activity in the leukocytes or cultured fibroblasts can be used for diagnosis of suspected cases, as well as for detection of carriers. Prenatal diagnosis is possible by enzyme assays or DNA probe analysis. C: Distinct biochemical and molecular levels and is more common than types B and C. Mutations in NPC1 and NPC2, NPC1 is most common. Primarily due to defects in lipid transport. Heterogenous commonly presenting in childhood; ataxia, vertical supranuclear gaze palsy, dystonia, dysathria, and psychomoter regression. Guacher Disease Mutation in gene that encodes glucosylceramidase. 5 autosomal recessive variants. Marked deficiency in glucosylceramidase which normally cleaves glucse from glucosylceramide which leads to build up of glucosylceramide in phagocytes leading to the transformation to Gaucher cells. Wrinkled tissue looking cytoplasm. High levels of macrophage cytokines, IL2, IL6, TNF. Type I (non-neuronapathic form) accounts for 99%. Radiographic: have involvement/osteopenia, focal lytic lesions, and osteonecrosis. Hepatospleenomegaly of absence of CNS involvement.Mucopolysaccharidoses Defective degradation of mucopolysaccharides in various tissues. Part of ground substance, synthesized by fibroblasts. Mast is secreted, but a certain portion is broken down in lysosomes. Dermatan sulfate, Keratan sulfate, chondroitin sulfate. All MPs are autosomal recessive except Hunter's disease (x-linked). All result in coarse facial features, clouding of the cornea, joint stiffness, and mental retardation. Urinary secretion of accumulated mucopolysaccharides is often increased. Affects many organs; liver, spleen, heart and blood vessels.Glycogen Storage Diseases Hepatic type: deficiency in enzymes involved in glycogen metabolism is associated with two major clinical affects: Enlargement of the liver due to storage of glycogen and hypoglycemia due to a failure of glucose production. Von Gierke Disease (Type I glycogenesis)-lack of glucose-6-phosphatase. Myopathic type: muscle cramps after exercise, myoglobinuria, and failure of exercise to induce an elevation in blood lactate levels because of a block in glycolysis. McArdle Disease-deficiency of muscle phosphorylase. Type II Glycogenesis (Pompe disease)-deficiency of lysosomal acid maltase-deposition of glycogen in virtually every organ, but cardiomegaly is most prominent. Type IV-brancher glycogenosis is caused by deposition of an abnormal form of glycogen, with detrimental affects on the liver, heart, and muscles.Diseases caused by mutations in proteins that regulate cell growth Disorders with multifactorial inheritance Trait governed by the additive effect of two or more genes of small effect, conditioned by environmental, nongenetic influences. Risk of expressing a multifactorial disorder is conditioned by the number of mutant genes inherited. Rate of recurrence of the disorder is the same for all first degree relatives of the affected individual (2-7%). DM, hypertension, gout, schizophrenia, bipolar disorder, certain forms of congenital heart disease, and certain forms of some skeletal abnormalities.Cytogenic Disorders 1/200 50% of first trimester abortions. Can be due to alterations in the number or structure of chromosomes and may affect autosomes or sex chromosomes. Karyotype: photographic representation of a staiend meatphase spread in which the chromosomes are arranged in order of decreasing length. Allows certain identification of each chromosome as well as precise localization of structural changes. Nuemric abnormalities 2n=46 Polyploid (3n or 4n) casually results in a spontaneous abortion. Any number that is not an exact multiple of n is called aneuploid. Aneuploidy is caused by nondisjunction of a homologous pair of chromosomes at the first meiotic division, or a failure of sister chromatids to separate during the second meiotic division. Failure of pairing of homologous chromosomes followed by random assortment (anaphase lag) can also lead to aneuploidy. (n+1) or (n-1); if fertilize: 2n+1 or 2n-1. 2n+1 is trisomic 2n-1 is monosomy, which is incompatible with life (unless it is of sex chromosome). Mosaicism: presence of two or more populations of cells in the same individual. Postzygotic mitotic nondisjunction would result in the production of a trisomy and a monosomy daughter cell; the descendants of these cells would then produce a mosaic. Structural abnormalities: Result from chromosomal breakage-followed by a loss of rearrangement of material. P-short arm, q-long arm; each is divided into numbers, centromere out, fallowed by band numbers. Ex: 2q34; chromosome 2, long arm, region 3, band 4. Translocation: usually reciprocal, movement of a portion of one chromosome to another. Centric fusion type, or Robertsonian translocation: Break occurs close to centromere affecting the short arms of both chromosomes. Transfer of segments leads to one very large chromosome and one extremely small chromosome. Short fragments are lost and carrier has 45 chromosomes. Isochromosomes: result when the centromere divides horizontally rather than vertically. One of the two arms of the chromosome is lost, the remaining one gets duplicated resulting in a chromosome with two short arms or two long arms only. Deletion: loss of a protion of a chromosome. Inversion: two interstitial breaks in a chromosome, segment reunites after a complete turn around. Ring chromosome: variant of deletion, after loss of segments from each end of the chromosome, the arms unit to form a ring. Chromosomal disorders may be associated with obscene, excess or abnormal rearrangements. Imbalances of sex chromosomes are tolerated better than are similar imbalances of autosomes. Often produce subtle abnormalities, sometimes not detected at birth. Infertility is common, but only diagnosed at adolescence. Cytogenic Disorders Involving Autosomes Trisomy 21 (Down syndrome) 47 chromosomes Meiotic nondisjunction. Maternal age is a risk factor (1/25 if 45>yo) vs. (1/1550 in 20Angelman.Functions of Vitamin D Biologically active: 1,25 (OH)2 -D Steroid hormone Binds to a high-affinity nuclear receptor that in turn binds to regulatory DNA sequences, which induce transcription of genes coding for specific target proteins. Receptors are present on all nucleated cells, thus Vitamin-D has various biological activities beyond those involved in calcium and phosphorus homeostasis. Stimulates intestinal absorption of calcium and phosphorus. Collaborates with PTH in the mobilization of calcium from bone. Stimulates the PTH-dependent reabsorption of calcium in renal distal tubules. Effects of vitamin D on bone depend on the plasma levels of calcium. Deficiency states Rickets (children) and osteomalacia are skeletal diseases with worldwide distribution. May result from diets deficient in calcium and vitamin D, but probably more important is limited exposure to sunlight. Renal disorders cause decreased synthesis of 1,25-(OH)2-D or phosphate depletion and malabsorption. Elderly: milder forms of vitamin D deficiency: bone loss and hip fracture. Decreased vitamin D leads to hypocalcemia and increased PTH which leads to the activation of renal alpha1-hydroxylase thus increasing the amount of active vitamin D and calcium absorption. Calcium mobilized from bone. Decrease renal calcium secretion Increased renal secretion of phosphate. Vitamin D may be important for preventing demineralization of bones. Certain genetically determined variates of the Vitamin D receptor are associated with an accelerated loss of bone minerals with aging. Vitamin D toxicity. Prolonged exposure to normal sunlight does not produce an excess of vitamin D, but megadoses of orally administererd vitamin D can lead to hypervitaminosis. In children, may result in metastatic calcifications of soft tissues such as the kidney. Adults, bone pain and hypercalcemia. Toxic potential is so great that in sufficiently large doses, it is a potent rodenticide. Vitamin C Deficiency leads to scurvy. Bone diseases in growing children and by hemorrhages and healing defects in both children and adults. Function Accelerates hydroxylation and amidation. Activation of prolyl and lysyl hyroxylases form reactive precursors providing for hydroxylation of procollagen. Molecules that are not hydroxylated lack tensile strength. Deficiency also leads to suppression of the rate of synthesis of collage peptides. Vitamin C also scavenges free radicals and acts indirectly by regenerating the antioxidant form of vitamin E. Vitamin C toxicity Megaloading results in prompt excretion, but may cause uricoduria and increased absorption of iron, leading to iron overload. In large doses it does have antihistamine properties.Bleeding Disorders Tests: Bleeding time: time it takes for a standardized skin puncture to stop bleeding; measured in minutes. Provides a platelet response to limited vascular injury. 2-9 min. Platelet counts 150x10^3 to 450x10^3 cells/mm^3 PT: tests adequacy of the extrinsic and common coagulation pathways. Represents time needed to clot in the presence of an exogenously added source of tissue thromboplastin and calcium ions. Prolonged PT could rsult from a deficiency of Factor V, VII, or X, prothrombin, or fibrinogen. Partial Thromboplastin Time (PTT): Tests integrity of intrinsic and common clotting pathways. Time neede for plasma to clot in presence of kaolin, cephalin, and calcium is measured. Kaolin serves to activate the contact dependent factor XII, and cephalin substitutes for platelet phospholipids. Cephalin substitutes for phospholipids. Prolongation can be due to factors V, VIII, IX, X, XI, or XII, prothrombin or fibrinogen or an acquired inhibitor (typically and antibody). Abnormalities of Vessels Fragility-vitamin C deficiency, systemic amyloidosis, chronic glucocorticoid use, rare inherited condtions affection CTs, infectious and hypersensitivity related vasculitis. Spontaneous appearance of petechiae and ecchymosis in the skin and mucous membranes. Systemic conditions that activate or damage endothelial cells. If severe enough, such insults convert the vascular lining to a prothrombic surface that activates coagulation throughout the circulatory system. In such consumptive coagulopathies, platelets and coagulation factors are used up faster than they can be replaced, and the resulting deficiencies often lead to sever bleeding. Deficiencies of platelets: qualatative defects in platelet function: acquired (uremia) after aspirin ingestion, and in certain mycloproliferative disorders, or inherited, as in von Willdebrand. Easy bruising, nose bleeds, excessive bleeding from minor trauma and hemorrhage.Coagulation Disorders Congenital or acquired deficiencies of clotting factors. Most common: acquire coagulation factor deficiencies. Vitamin K is essential for prothrombin synthesis, and clotting factors VII, IX, and X and its deficiency causes a severe coagulation defect. Parenchymal diseases of the liver are common causes of complex hemorrhagic diseases. Hereditary deficiencies have been identified for each factor. Hemophelia A: factor VIII Hemophelia B: factor IX Both are x-linked. Most are autosomal, but are rare except for HA, HB and VW. Deficiencies of Factor VIII-vWF Complex Hemophelia A and von Willebrand disease Qualitative or quantitative deficeincies of factor VIII-vWF complex. vWF-in plasma with VIII, platelet granules, endothelial cells, unusual cytoplasmic vesicles called Weibel-palade bodies, subendothelium, where it binds to collagen. Subendothelial vWF binds to platelets, facilitating the adheison of platelets to damaged blood vessel walls. Tests: Ristocetin which binds platelets and promotes the itneraction between vWF and platelet membrane glycoprotein Ib. Creates interplatelet "bridges" that lead to the formation of platelet clumps, an event that can be measured easily. vWF is produced by both megakaryocytes and endothelial cells. Von Willebrand Disease Marked by spontaneous bleeding from mucous membranes, excessive bleeding from wounds, menorrhagia, and a prolonged bleeding time in the presence of a normal platelet count. Platelet defects. Type I-autosomal dominant characterized by a reduced quantity of circulating vWF. Deficiency causes a secondary decrease in factor VII, but not to levels that are clinically significant. Factor VIII Deficiency Hemophelia A is the most common hereditary disease associated with serious bleeding. X-linked recessive disorder. Hemarthroses (bleeding into joints) leads to progressive deformities that can be crippling. Petechiae are commonly absent. Prolonged PTT. Factor IX Deficiency X-linked disorder that is indistinguishable clinically from hemophelia A, but is less common. PTT is prolonged, and bleeding time is normal.Diagnosis of Genetic Diseases Aberrations in genetic material may be in the germ line (present in each cell) or somatic (restricted to certain tissue types or lesions). Karyotype analysis of chromosome G-banding remains the classic approach for identifying changes at the chromosomal level. Resolution is fairly low, though. Major limitation of karyotyping is that it is applicable only to cells that are dividing or can be induced to divide in vitro. FISH: Fluorescence in Situ Hybridization FISH utilizes DNA probes that recognize sequences specific to chromosomal regions. Usual size of FISH probe is the order of ~1 megabase (1x10^6 nt), this defines the limit of resolution of this technique for identifying chromosomal changes. Such probes are labeled with fluorescent dyes and applied to metaphase spreads or interphase nuclei. Probe binds to its complementary sequence on the chromosome and thus labels the specific chromosomal region that can be visualized under a fluorescent microscope. Ability of FISH to circumvent the need for dividing cells is invaluable when a rapid diagnosis is warranted. Amniocentesis cells Chorionic villus biopsy Umbilical cord blood Peripheral blood lymphocytes Archival tissue sections Used for numeric abnormalities of chromosomes, for the demonstration of subtle micro-deletions or complex translocations not detectable by routine karyotyping. Analysis of gene amplification, and for mapping newly isolated genes of interest to their chromosomal loci. Comparative Genomic Hybridization (CGH): Global strategy to detect chromosomal abnormalities without prior knowledge of what aberrations may be the root of disease. CGH, the test DNA and a reference DNA are labeled with two different fluorescent dyes (Cy5 and Cy3). The two specimens are then hybridized together. If the contributions of both samples are equal for a given chromosomal region, then all regions of the genome will fluoresce yellow due to an equal admixture of green and red dyes. If the test sample shows an excess of DNA at any given chromosomal region, there will be a corresponding excess of signal from the dye with which this sample was labeled. Reverse is true in the event of a deletion, with an excess of the signal used for labeling the reference sample. CGH still lacks the ability to detect submicroscopic alterations. Array-based CGH has been developed, wherein short segments of genomic DNA are "spotted" on a solid matrix, usually a glass slide. Representations of the human genome at regularly spaced intervals, and usually cover all 22 autosomes and the X chromosome. Steps following are similar to CGH.Molecular Diagnosis of Genetic Disorders Many genetic diseases are caused by alterations at the nucleotide level that cannot be detected by FISH or even CGH. It is possible to diagnose on the molecular level, inherited diseases; Remarkably sensitive. PCR DNA-based tests are not dependent on a gene product that may be produced only in certain specialized cells or expression of a gene that may occur late in life. Because the defective gene responsible for inherited genetic disorders is present in germline samples, every post-zygotic cell caries the mutation. Prenatal diagnosis of genetic diseases is possible due to the few numbers of cells required for molecular techniques. Two distinct molecular diagnosis of single-gene diseases: direct detection of DNA mutations and indirect detection based on linkage of the disease gene with surrogate markers in the genome. Direct Detection of DNA Mutations by PCR Analysis: PCR analysis RT-PCR Prerequisite for direct detection is that the sequence of the normal gene must be known. To detect the mutant gene, two primers that bind to the 3' and 5' ends of the normal sequence are designed. DNA can be sequence to obtain a readout of the order of nucleotides, and by comparison with a normal sequence. Mutations can be identified. Ready availability of automated sequencers has made the previously laborious task of manual sequencing obsolete, and thousands of base pairs of genomic DNA can now be sequenced in a matter of hours. Gene chips have become available that can be used for sequencing genes or portions of genes. Short sequences of DNA (oligonucleotides) that are complementary to the wild-type sequence and to known mutations are "tiled" adjacent to each other on the gene chip, and the DNA sample to be tested is hybridized to the array. Before hybridization the sample is labeled with fluorescent dyes. The hybridization will be strongest at the oligonucleotide that is complementary to wild-type sequence if no mutations are present. The presence of a mutation will cause hybridization to occur at the complementary mutant oligonucleotide. DNA can also be digested with restriction enzymes, if the specific mutation is known to affect a restriction site, then the amplified DNA can be digested. Mutation affects, the mutant and normal alleles give rise to products of different sizes. These would appear as different bands on agarose gel electrophoresis. Considerably lower in throughput than automated or array-based sequencing, but remains useful for molecular diagnostics in instances when the casual mutation always occurs at an invariant nucleotide position. PCR is helpful when a mutation is associated with deletions or expansions. Fragile X-FMR1 gene (trinucleotide repeats). Linkage Analysis Direct diagnosos of mutations is possible only if the gene resonsible for a genetic disorder is known and its sequence has been identified. Several deseases that have a genetic basis, including some common disorders, direct genetic diagnosis is not possible, either because the causal gene has not been identified or because the disease is multifactorial and no single gene is involved. Surrogate markers in the genome, also known as marker loci, must be used to localize the chromosomal regions of interest, based on their linkage to one or more putative disease-causing genes. Linkage analysis deals with assessing these marker loci in family members exhibiting the disease or trait of interest, with the assumption that marker loci very close to the disease allele, are transmitted through pedigrees. With time it becomes possible to define a "disease haplotype" based on a panel of marker loci all of which co-segregate with the putative disease allele. Eventually, linkage analysis facilitates localization and cloning of the disease allele. Marker loci is a naturally occurring variation in DNA sequences known as polymorphisms. Occur at a frequency of approximately one nucleotide in every~1000-base pair stretch. SNP SNPs are found throughout the genome. Serve both as a physical landmark within the genome and as a genetic marker whose transmission can be followed from parent to child. SNPs can be used in linkage analysis for identifying haplotypes associated with disease, leading to gene discovery and mapping.Indications for Genetic Analysis Prenatal and postnatal analysis. Prenatal genetic analysis should be offered to all patients who are at risk of having cytogenetically abnormal progeny. Can be performed on cells obtained by amniocentesis, on chorionic villus biopsy material, or on umbilical cord blood. Important indications: Mother of advanced age (