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Robbins & Contran Pathologic Basis of Disease - Chapter 5 Tue TOPIC: GENETIC DISORDERS LECTURER: Dr. Alconcel...

Robbins & Contran Pathologic Basis of Disease - Chapter 5 Tue TOPIC: GENETIC DISORDERS LECTURER: Dr. Alconcel Fri GENES AND HUMAN DISEASES 4 Categories of Human Genetic Disorders Disorders related to single gene mutations Mutations in Single follow the classic Mendelian pattern of inheritance Genes with Large high penetrance* Effects (Mendelian Disorders) structural or numerical alteration in the autosomes and sex chromosomes Chromosomal Disorder uncommon high penetrance Complex Multigenic caused by interactions between multiple variant forms of genes Disorders (polymorphisms) and environmental factors (Multigenic or more common Polygenic or low penetrance Multifactorial Disorders) caused by mutations in single genes, but they do not follow the Single-gene Disorders Mendelian pattern of inheritance with Nonclassic Patterns of Inheritance Examples: triplet-repeat mutations, mitochondrial DNA (mtDNA) mutations), genomic imprinting or gonadal mosaicism transmission *Penetrance - the presence of the mutation is associated with the disease in a large proportion of individuals. It is expressed in mathematical terms Ex. 50% penetrance indicates that 50% of those who carry the gene express the trait General Principles of the effects of Gene Mutations: Point mutations AKA Missense Mutations within coding Change in which a single base is substituted with a different base sequences including triplets of bases Divided in 2 categories: a. Conservative Missense: causes little change in the function of the protein b. Nonconservative Missense: replaces normal AA with a biochemically different one e.g Sickle cell mutation – CTC “glutamic acid”  CAC “valine” When point mutation prematurely terminates protein synthesis it is called Nonsense mutation e.g. β0-thalassemia – CAG “glutamine”  UAG “stop codon” (see figure below) MSU-GSC College of Medicine 1 Mutations within Mutations interfering with binding of transcription factors leading noncoding sequence to a reduction in or total lack of transcription May be caused by point mutations in introns Ex: thalassemias Deletions and Small deletions or insertions in the coding sequence insertions Effects on the Reading frame: a. Remains intact - when number of base pairs involved is three or a multiple of three e.g Cystic fibrosis (see figure below) b. Frameshift mutation - when number of affected coding bases is not a multiple of three e.g Single-base deletion at ABO locus (see figure below) Four-base Insertion in the hexosaminidase A gene (see figure below)  point mutations within introns may lead to defective splicing of intervening sequences Alterations in Coding genes can undergo structural variations result in aberrant protein-coding gain or loss of protein function such copy number changes such as genes other than amplifications, deletions, - translocations mutations Ex. Philadelphia chromosome (translocation) Alterations in Alterations in noncoding RNAs (ncRNAs) including microRNAs noncoding RNAs (miRNAs) and long noncoding RNAs (lncRNAs) Trinucleotide- amplification of a sequence of three nucleotides repeat mutations distinguishing feature of trinucleotide-repeat mutations is that they are dynamic where , the degree of amplification increases during gametogenesis almost all affected sequences share the nucleotides guanine (G) and cytosine (C ) Ex: fragile X syndrome (FXS) MSU-GSC College of Medicine 2 3 Major Categories of Genetic Disorders: 1. Disorders related to mutant genes of large effect 2. Disease with multifactorial inheritance 3. Chromosomal Disorders REMEMBER: Hereditary vs Familial vs Congenital  Hereditary disorders: derived from one’s parents and are transmitted in the germline through the generations and therefore are familial  Congenital: “born with” Note: Some congenital diseases are not genetic (e.g., congenital syphilis); and Not all genetic diseases are congenital (e.g Huntington disease) MENDELIAN DISORDERS Mendelian traits: described as dominant or recessive Note: Codominance – when both of the alleles of a gene pair contribute to the phenotype (e.g blood group antigens)  Pleiotropism – when a single mutant gene can lead to many end effects Example: sickle cell anemia  Genetic Heterogeneity - mutations at several genetic loci may produce the same trait Example: profound childhood deafness TRANSMISSION PATTERNS OF SINGLE-GENE DISORDERS Patterns of Single- Gene Disorders Description Examples Autosomal  Genotype: Aa/AA Dominant Disorders  Phenotype: Expressed (even if there’s only one affected gene)  At least one parent is affected  affect males and females equally, and both sexes can transmit the disorder  Enzyme proteins are not affected in autosomal dominant disorders; instead, receptors and structural proteins are involve  Clinical features vary in penetrance and expressivity, such as: a. Incomplete penetrance: individuals inherit the mutant gene but are phenotypically normal b. Variable expressivity: trait is seen in all individuals carrying the mutant gene but is expressed differently (e.g Neurofibromatosis type 1)  Age at onset is delayed  2 patterns for autosomal dominant diseases with deleterious mutations a. Diseases involved in regulation of complex metabolic pathways that are subject to feedback inhibition b. Key structural proteins, such as collagen and cytoskeletal elements of the red cell MSU-GSC College of Medicine 3  membrane Autosomal  Genotype: aa Recessive Disorders  Phenotype: Not usually expressed except if both recessive genes are inherited  enzyme proteins are frequently involved  males and females are affected equally  siblings may show the disease with one chance in four of having the trait  If the mutant gene occurs with a low frequency in the population, there is a strong likelihood that the affected individual is the product of a consanguineous marriage  expression of the defect tends to be more uniform with complete penetrance  Onset is frequently early in life  rarely detected clinically  Include almost all inborn errors of metabolism X--Linked Disorders  Genotype: depends more on the X chromosome (can be dominant or recessive)  Phenotype: Full expression, partial expression, or no expression of mutation  All sex-linked disorders are X- linked, and almost all are recessive  Males with mutations affecting the Y- linked genes are usually infertile  no Y-linked inheritance Types of X-Linked Disorders a. X-Linked Recessive Inheritance  An affected male does not transmit the disorder to his sons, but all daughters are carriers.  A heterozygous female  does not express the full phenotypic change unless there’s a random inactivation of one X chromosome  Sons of heterozygous women have one chance in two of receiving the mutant gene  Example: glucose-6-phosphate dehydrogenase (G6PD) deficiency b. X-linked dominant  caused by dominant disease- associated alleles on the X MSU-GSC College of Medicine 4  chromosome  affected heterozygous female  transmit it to half her sons and half her daughters  affected male parent + unaffected female parent  transmit to all his daughters but none of his sons  Examples: Vitamin D– resistant rickets and Alport syndrome BIOCHEMICAL AND MOLECULAR BASIS OF SINGLE-GENE (MENDELIAN) DISORDERS Mendelian disorders  result from alterations involving single genes.  alteration may lead to an abnormal protein or a reduction in the output of the gene product  virtually any type of protein may be affected ENZYME DEFECTS AND THEIR CONSEQUENCE 3 Major Consequences of an Enzyme Defect Accumulation of the Substrate A Metabolic Block and a Failure to inactivate a tissue- Decreased Amount of End damaging substrate Product  May also lead to an  If the end product is a  Uncontrolled substrates lead accumulation of one or both feedback inhibitor of the to continued tissue damage intermediates aside from the enzymes involved in the early well beyond necessary substrate reactions, tit may cause measures  Tissue injury may result if overproduction of  Example: deficiency of MSU-GSC College of Medicine 5  the precursor, the intermediates and their serum α1-antitrypsin intermediates, or the catabolic products which may products of alternative minor be toxic pathways are toxic in high  Example: Lesch-Nyhan concentrations syndrome  Example: Lysosomal storage diseases Defects in  Biologically active substances have to be actively transported across the cell Receptors and membrane, which may include receptor-mediated endocytosis. Ex: familial Transport hypercholesterolemia (FH) and cystic fibrosis Systems Alterations In Genetic defects resulting in alterations of nonenzyme proteins often have widespread Structure, secondary effects. For example: Function, or Defects in the structure of the globin molecule -> hemoglobinopathies Quantity of Defects in the amount of globin chains synthesized -> thalassemias Nonenzyme Defects in structural proteins Proteins ○ Defects in collagen -> osteogenesis imperfecta ○ Defects in spectrin -> hereditary spherocytosis ○ Defects in dystrophin -> muscular dystrophies  Pharmacogenetics - the study of how our genes affect the way we respond to Genetically medications Determined  Certain genetically determined enzyme deficiencies are unmasked only after Adverse exposure of the affected individual to certain drug where these genetic factors Reactions to have a major impact on drug sensitivity and adverse reactions. Drugs  Ex. G6PD deficiency + antimalarial drug primaquine = severe hemolytic anemia DISORDERS ASSOCIATED WITH DEFECTS IN STRUCTURAL PROTEINS MARFAN SYNDROME Description/  Connective tissues disorder manifested principally by changes in the skeleton, Etiology eyes, and cardiovascular system Prevalence  1 in 5000. Approximately 70% to 85% of cases are familial and transmitted by autosomal dominant inheritance and the remaining 15 to 30% from new mutations. Pathogenesis  Mutations of FBN1 and FBN 2 are different o Mutations of FBN1= Marfan syndrome; o Mutations of FBN2 = congenital contractural arachnodactyly  Caused by an inherited defect in an extracellular glycoprotein called fibrillin- 1 resulting to:  excessive TGF-β activation  causing inflammation which leads to: o Deleterious effect on vascular smooth muscle development o increases the activity of metalloproteases, causing loss of extracellular matrix  inhibit polymerization of fibrillin fibers (dominant negative effect) or reduces fibrillin content  weakens the connective tissue (haploinsufficiency) MSU-GSC College of Medicine 6  Note: Fibrillin is the major component of microfibrils found in the extracellular matrix where tropoelastin is deposited to form elastic fibers  Causes the cardiovascular and ocular symptoms Clinical Skeletal Abnormalities Features  unusually tall with exceptionally long extremities and long, tapering fingers and toes  Lax joint ligaments with possible double jointedness and hyperextension of the thumb back to the wrist  Skull is dolichocephalic (long-headed) with bossing of the frontal eminences and prominent supraorbital ridges  Spinal deformities including kyphosis, scoliosis, or rotation or slipping of the dorsal or lumbar vertebrae  Chest deformities such as pectus excavatum (deeply depressed sternum) or a pigeon breast deformity Ocular Abnormalities  Bilateral ectopia lentis/bilateral subluxation/bilateral dislocation (usually outward and upward) of the lens Cardiovascular System Abnormalities  Cardiovascular lesions with the most common being mitral valve prolapse (40- 50% of cases) and dilation of the ascending aorta  Mitral valve lesions  Floppy valve  Valvular lesions  Lengthening of the chordae tendineae  Mitral regurgitation  rupture of aortic dissections and cardiac failure leading to death EHLERS-DANLOS SYNDROMES (EDSs) Description/  A clinically and genetically heterogeneous group of disorders that result from Etiology some mutations in genes that encode collagen, enzymes that modify collagen, and less commonly other proteins present in the extracellular matrix Prevalence  1 in 5000 births Pathogenesis  Kyphoscoliosis type - autosomal recessive mutations in the PLOD1 gene encoding lysyl hydroxylase  Vascular type - genetically heterogeneous abnormalities of CL3A1 gene that encodes type III collagen  Arthrochalasia type - mutations that affect COL1A1 and COL1A2 abnormality in the conversion of type I procollagen to collagen  Dermatosparaxis type - mutations in the ADAMTS2 gene that encodes procollagen-N-peptidase  Classic type of EDS - 90% of cases, mutations in the genes for type V collagen (COL5A1 and COL5A2) MSU-GSC College of Medicine 7 Clinical Features DISORDERS ASSOCIATED WITH DEFECTS IN RECEPTOR PROTEINS REVIEW: NORMAL CHOLESTEROL METABOLISM Cholesterol  7% circulates in the plasma (LDL form)  Amount of plasma cholesterol is influenced by synthesis and catabolism  Liver plays a crucial role.  Derived from: o Diet o Endogenous synthesis  Dietary triglycerides and cholesterol are into chylomicrons in the intestinal mucosa → travel by gut lymphatics → blood  Enters the metabolic pool  Excreted as free cholesterol or as bile acids into the biliary tract Chylomicrons  Hydrolyzed by an endothelial lipoprotein lipase o In the capillaries of muscle and fat  Chylomicron remnants (rich in cholesterol) → delivered to the liver LDL receptor  Metabolizes ⅔ of the resultant LDL particles  Others are metabolized by a receptor for oxidized LDL (scavenger receptor)  Binds to apolipoproteins B-100 and E → involved in the transport of both LDL and IDL  75% are located on hepatocytes Endogenous Synthesis of Cholesterol and LDL 1. Begins in the liver 2. Secretion of triglycerides-rich very-low-density lipoprotein (VLDL) by the liver into the blood. 3. The VLDL particle undergoes lipolysis and is converted to intermediate-density lipoprotein (IDL) (in the capillary endothelium of adipose tissue and muscle). Triglycerides is reduced 4. Cholestryl esters is enriched in IDL But IDL retains on its surface the VLDL- associated apolipoproteins B-100 and E. 5. Further metabolism of IDL occurs in two pathways: Most IDL particles are taken up by the liver through the LDL receptors Converted to cholesterol-rich LDL by a further loss of triglycerides and apolipoprotein E. 6. IDL is recycled to generate VLDL in liver cells MSU-GSC College of Medicine 8 LDL receptor pathway and regulation of cholesterol metabolism 3 regulatory functions of free intracellular cholesterol: 1. Suppression of cholesterol synthesis by inhibition of HMG-CoA reductase 2. Stimulating the storage of excess cholesterol as esters 3. Inhibition of synthesis of LDL receptors. Note:  PCSK9: causes intracellular degradation of LDL receptors in liver cells, reducing the level of LDL receptors on the cell membrane.  NPC1 and NPC2: are required for exit of cholesterol from lysosomes to cytoplasm.  Enzyme3-hydroxy-3-methylglutarylecoenzyme A reductase (HMG-CoA reductase): rate-limiting enzyme FAMILIAL HYPERCHOLESTEROLEMIA (FH) Description/  receptor disease Etiology  autosomal dominant disorder caused by mutations in the genes encoding the: 1. LDL receptor (85% cases) 2. ApoB protein (5% to 10% cases) 3. activating mutations of PCSK9 (1% to 2% cases)  Most common of mendelian disorders Prevalence  Heterozygous condition is 1 in 500  >20x prevalence in those with atherosclerotic cardiovascular disease Pathogenesis  Receptor abnormalities → loss of feedback control holds cholesterol synthesis in check → elevated levels of cholesterol → induced premature atherosclerosis → increase risk in myocardial infarction.  Results from mutation in the gene encoding the receptor for LDL or two genes that comprise its function. Details are elaborated below: b. Mutation in the LDLR gene  Heterozygote FH - possess 50% of normal number of high-affinity LDL receptor because of 1 normal gene - LDL catabolism by the receptor-dependent pathway is impaired  LDL in plasma ↑  Homozygote FH - No normal LDL receptors = LDL ↑ ↑ ↑ - Both the homozygotes and heterozygotes have increased synthesis of LDL → contributes to hypercholesterolemia  Impaired IDL transport. b. Mutation in Gene Encoding  Mutant ApoB reduces the binding of LDL molecules with LDL receptors. c. Activating mutation in the PCSK9 gene  reduces the number of LDL receptors on the cell surface because of their MSU-GSC College of Medicine 9  increased degradation during the recycling process. Classification Classifications of LDL Receptor Mutations of Disease  Class 1 mutations - lead to a complete failure of synthesis of LDL receptor protein (null allele)  Class II mutations - encode LDL receptor proteins that accumulate in the ER because their folding defects are impossible to transport to the Golgi complex  Class III mutations - affect the ApoB binding site of the receptor; the mutant LDL receptors reach the cell surface but fail to bind the LDL  Class IV mutations - encode LDL receptors are synthesized and transported to the cell surface efficiently. They bind LDL normally, but fail to localized in the coated pits, hence the bound LDL is not internalized  Class V mutations - encode LDL receptors are expressed on the cell surface, can bind LDL, and can be internalized; but the pH-dependent dissociation of the receptor and the bound LDL fails to occur. Receptors are trapped in endosomes and are degraded  Class VI mutations - fail the initial targeting of the LDL receptor to the basolateral membrane Clinical  Px develop hypercholesterolemia and xanthomas (fat build-up underneath the Features skin)  Heterozygote FH: ↑ Cholesterol, ↑ risk of atherosclerosis, CAD  Homozygote FH: ↑↑ ischemic heart disease DISORDERS ASSOCIATED WITH ENZYME DEFECTS REVIEW: LYSOSOME Lysosome Intracellular digestive system Contains a battery of hydrolytic enzymes that have two properties: 1. Function in the acidic milieu of the lysosomes 2. Constitute a category of secretory proteins that are for intracellular organelles. (requires processing within the Golgi apparatus) Lysosomal  Catalyze the breakdown of a variety of complex macromolecules enzyme  Large molecules derived from the metabolic turnover of intracellular organelles (autophagy) or acquired from outside of the cell by phagocytosis (heterophagy) Synthesis and intracellular transport of lysosomal enzymes Lysosomal enzymes or acid hydrolases, are synthesized in the ER and transported to Golgi apparatus. In the Golgi complex, they undergo various posttranslational modifications (i.e. the attachment of terminal mannose-6-phosphate groups to some of the oligosaccharide side chains) Phosphorylated mannose residues serve as an “address label” that is recognized by specific receptors found on the inner surface of the Golgi membrane MSU-GSC College of Medicine 10  LYSOSOMAL STORAGE DISEASES (LSD) OVERVIEW Description/ Involve in the etiology of several more common disease (e.g Parkinson’s disease) Etiology Lysosomes play a critical role in these diseases Abnormalities of lysosomal enzyme or proteins involved in: ○ Substrate degradation ○ Endosomal sorting ○ Lysosomal membrane integrity Distribution of the stored material and organs affected is determined by two interrelated factors: 1. The tissue where most of the material is degraded if found 2. The location where most of the degradation normally occurs For example, the brain is rich in gangliosides. So defective hydrolysis of gangliosides as occurs in GM1 and GM2 gangliosidoses, results primarily in accumulation within neurons LSD can be divided into categories based on the biochemical nature of the accumulated metabolites: ○ Glycogenoses ○ Sphingolipidoses (lipidoses) ○ Mucopolysaccharidoses (MPSs) ○ Mucolipidoses Prevalence 1 in 5000 in births Pathogenesis Two pathologic consequences of inherited deficiency of functional lysosomal enzyme: 1. Catabolism of the substrate of the missing enzyme remains incomplete Primary accumulation - leading to the accumulation of the partially degraded, insoluble metabolite within the lysosome. Stuffed with incomplete digested macromolecules Lysosomes become large and numerous to interfere with normal cell functions 2. Tight linkage between autophagy, mitochondrial functions, and lysosomes Mitophagy - autophagy is essential for turnover of mitochondria that serves as a quality control system where dysfunctional mitochondria are degraded. The rate of lysosomes process organelles delivered by autophagocytic vacoules is reduced → persistence of dysfunctional and leaky mitochondria with poor calcium-buffering capacity Damaged mitochondria generate free radicals and release molecules that trigger the intrinsic pathway of apoptosis. Impaired autophagy gives rise to secondary accumulation of MSU-GSC College of Medicine 11  autophagic substrates including ubiquitinated and aggregate-prone polypeptides such α-synuclein and Huntingtin protein Treatment 1. Enzyme replacement therapy 2. Substrate reduction therapy - if the substrate to be degraded by the lysosomal enzyme can be reduced, the residual enzyme activity may be sufficient to catabolize it and prevent accumulation 3. Molecular chaperone therapy - An exogenous competitive inhibitor of the enzyme can bind to the mutant enzyme and act as the folding template that assists proper folding of the enzyme and thus prevents its degradation. Hematopoietic stem cell transplants and gene therapy are also being evaluated in specific cases. MSU-GSC College of Medicine 12  TAY-SACHS DISEASE (GM2 Gangliosidosis: hexosaminidase a-Subunit Deficiency) Description/ A group of three lysosomal storage diseases caused by deficiency of the Etiology enzyme β-hexosaminidase resulting in an inability to catabolize GM2 gangliosides. There are two isoenzymes of β-hexosaminidase: ○ Hex A - consisting of two subunits, α and β ○ Hex B - a homodimer of β-subunits. Degradation of GM2 gangliosides requires three polypeptides encoded by three distinct genes: 1. HEXA (chromosome 15) - encodes the α-subunit of Hex A 2. HEXB (chromosome 5) - encodes the β-subunit of Hex A and Hex B 3. GM2A (chromosome 5) - encodes the activator of hexosaminidase Phenotypic effects are similar because they result from accumulation of GM2 gangliosides but different in enzyme effect.  results from mutations in the α-subunit locus on chromosome 15 that cause a severe deficiency of hexosaminidase A. Prevalence  Among Jews, particularly among those of Eastern European (Ashkenazic) origin  a carrier rate of 1 in 30 Morphology  Hexosaminidase A is absent from virtually all the tissues, so GM2 ganglioside accumulates in many tissues (e.g., heart, liver, spleen, nervous system)  But the involvement of neurons in the central and autonomic nervous systems and retina dominates the clinical picture ○ On histologic examination, the neurons are ballooned with cytoplasmic vacuoles, each representing a markedly distended lysosome filled with gangliosides  Cytoplasmic inclusions can be seen with electron microscope. ○ the most prominent being whorled configurations within lysosomes composed of onion-skin layers of membranes ○ progressive destruction of neurons ○ proliferation of microglia ○ accumulation of complex lipids in phagocytes within the brain substance ○ A cherry-red spot thus appears in the macula, representing accentuation of the normal color of the macular choroid contrasted with the pallor produced by the swollen ganglion cells in the remainder of the retina Ganglion cells in Tay-Sachs disease. (A) Under the light microscope, a large neuron has obvious lipid vacuolation. (B) A portion of a neuron under the electron microscope shows prominent lysosomes with whorled configurations. Part of the nucleus is shown above. Clinical Feature Affected infants begin to manifest signs and symptoms at age 6 months but appear normal at birth. MSU-GSC College of Medicine 13  There is motor and mental deterioration, resulting in motor incoordination and intellectual disability → leading to muscular flaccidity, blindness, and increasing dementia. In the early course of the disease, the characteristic (not pathognomonic) cherry-red spot appears in the macula of the eye in most patients. Over 1-2 years a complete vegetative state is reached, followed by death at the age of 2-3 years Antenatal diagnosis and carrier detection are possible by enzyme assays and DNA-based analysis. NIEMANN-PICK DISEASE TYPES A and B Description/  two related disorders that are characterized by lysosomal accumulation of Etiology sphingomyelin due to an inherited deficiency of sphingomyelinase.  Patients usually survive into adulthood  Autosomal recessive  Heterozygotes who inherit the mutant allele from the mother can develop Niemann-Pick disease Prevalence  common in Ashkenazi Jews Pathogenesis  Enzyme deficiency blocks the degradation of the lipid, resulting in its progressive accumulation within lysosomes Morphology  Innumerable small vacuoles of relatively uniform size are created, imparting foaminess to the cytoplasm,  Vacuoles are engorged secondary lysosomes that often contain zebra bodies - Zebra bodies - membranous cytoplasmic bodies resembling concentric lamellated myelin figures  Vacuolation and ballooning of neurons - constitute the dominant histologic change that, in time leads to cell death and loss of brain substance.  A retinal cherry-red spot is also seen Classification of  Type A Disease - severe infantile form with extensive neurologic involvement, marked visceral accumulations of sphingomyelin. - progressive wasting and early death within the first 3 years of life  Type B - Organomegaly but generally no central nervous system involvement. MSU-GSC College of Medicine 14 Clinical  Lymph nodes are markedly enlarged throughout the body Features NIEMANN-PICK DISEASE TYPE C Description/  more common than types A and B combined Etiology  Mutations in two related genes, NPC1 and NPC2  Due to a primary defect in nonenzymatic lipid transport. ○ NPC1 is membrane-bound ○ NPC2 is soluble ○ Both are involved in the transport of free cholesterol from the lysosomes to the cytoplasm.  clinically heterogeneous Prevalence  NPC1 mutations responsible for 95% of cases Pathogenesis  a primary defect in nonenzymatic lipid transport  Clinical course is marked by:  Ataxia  Vertical supranuclear gaze palsy  Dystonia, dysarthria  Psychomotor regression. Clinical  Present as hydrops fetalis and stillbirth, as neonatal hepatitis, or, most Features commonly, as a chronic form characterized by progressive neurologic damage GAUCHER DISEASE Clinical Type I Features first appear in adult life (splenomegaly and bone involvement) Common: pancytopenia or thrombocytopenia secondary to hypersplenism Progressive but compatible with long life Type II and III CNS dysfunction, convulsions, progressive mental deterioration Diagnosis: ○ Homozygotes: measurement of glucocerebrosidase activity in PBS (peripheral blood smear) or in extracts of cultured skin fibroblast ○ Heterozygotes: detection of mutations (more than 150 mutations cause Gaucher disease, single genetic test is not possible), and molecular test (if causative mutation is known) Treatment Replacement therapy with recombinant enzymes Transfer of normal glucocerebrosidase gene in hematopoietic stem cell Substrate reduction therapy with inhibitor of glucosylceramide synthetase (under evaluation) MUCOPOLYSACCHARIDOSES (MPS) Etiology Group of closely related syndromes that result from genetically determined deficiencies of enzymes involved in degradation of mucopolysaccharides (glycosaminoglycans) Mucopolysaccharides are long-chain complex carbs linked with proteins to form proteoglycans (abundant in ECM, joint fluid, CT) Accumulation of glycosaminoglycans (dermatan sulfate, heparan sulfate, keratan sulfate, chondroitin sulfate) 11 enzymes degrade these molecules; if not cleaved, chains accumulate within MSU-GSC College of Medicine 15  lysosomes Pathogenesis All 11 MPSs are inherited as autosomal recessive, except Hunter syndrome (X- linked recessive) Characterized by coarse facial features, clouding of cornea, joint stiffness, intellectual disability Hepatosplenomegaly, skeletal deformities, valvular lesions, subendothelial arterial deposits in coronary artery, and brain lesions ○ Coronary subendothelial lesions → myocardial ischemia ○ Cause of death: myocardial infarction and cardiac decompensation MPS I: deficiency of α-L-iduronidase Diagnostic tool: ⬆ urinary excretion of accumulated mucopolysaccharide Morphology Generally found in mononuclear phagocytic cells, endothelial cells, intimal smooth muscle cells, fibroblast, also in neurons Common sites of involvement: spleen, liver, bone marrow, lymph nodes, blood vessels, heart Microscopic: distended with apparent clearing of cytoplasm to create “balloon cells” EM: swollen lysosomes contain finely granular periodic acid-Schiff + material CNS involvement: lysosomes in neurons are replaced by lamellated zebra bodies (similar in Niemann-Pick) Clinical Hurler Syndrome (MPS I-H) Features α-L-iduronidase deficiency Children: normal at birth, develop hepatosplenomegaly at 6-24 months, growth retardation, coarse facial features, skeletal deformity, and death by age 6-10y.o. Due to cardiovascular complications Hunter Syndrome (MPS II) X-linked Absence of corneal clouding, milder clinical course GLYCOGEN STORAGE DISEASE Description/  Hereditary deficiency of one of the enzymes involved in the synthesis or Etiology sequential degradation of glycogen Pathogenesis  Divided into 3 major subgroups: a. Hepatic forms  ↓ liver enzymes  ↑ glycogen in the liver + hypoglycemia  e.g von Gierke disease, or type I glycogenosis b. Myopathic forms  ↓ Glycolytic enzymes glycogen storage occurs in the muscles  impaired energy production  muscle weakness  e.g McArdle disease: lack of muscle phosphorylase gives rise to storage in skeletal muscles and cramps after exercise c. Glycogen storage diseases associated (1) deficiency of acid alpha-glucosidase (acid maltase) and (2) lack of branching enzyme MSU-GSC College of Medicine 16 MSU-GSC College of Medicine 17 GLYCOGEN STORAGE DISEASE Mnemonics: “Victorious People Can Always Make History Today” “Villainous President Called And Molested Her” TYPE & EPONYM ENZYME DEFICIENCY INVOLVED TISSUES MAJOR CLINICAL FEATURES 0 Glycogen synthetase Liver I von Gierke Glucose 6-phosphate Liver, kidney, Hepatomegaly, hyperlipidemia, lactic intestines acidosis, severe fasting hypoglycemia II Pompe Lysosomal acid α-1,4 Most tissues Cardiomegaly, muscle weakness, death in glucosidase(acid maltase) infancy and adult III Cori Debranching enzyme Liver, muscle, Hepatomegaly, muscle weakness, fasting WBCs hypoglycemia IV Anderson Branching enzyme Most tissue Portal cirrhosis, death in infancy V McArdle Muscle phosphorylase Muscle Pain and stiffness after excretion, myoglobinuria VI Hers Liver phosphorylase Liver, WBCs Hepatomegaly, mild fasting hypoglycemia VII Tarui Muscle Muscle Pain and stiffness on exertion phosphofructokinase VIII Adenylate kinase Liver, brain Spasticity, decerebration, high urinary catecholamines, death in infancy IX Phosphorylase kinase Liver Hepatomegaly, occasional fasting hypoglycemia Lifted from: Burtis, Ashwood, Bruns Tietz Textbook of Clinical Chemistry and Molecular Diagnostics © 2016, Elsevier. COMPLEX MULTIGENIC DISORDERS  Complex Disorders - result from the collective inheritance of many polymorphisms - For example, of the 20 to 30 genes implicated in type 1 diabetes, 6 or 7 are most important, and a few HLA alleles contribute more than 50% of the risk  Polymorphisms are common to multiple diseases of the same type, while others are disease specific - Example: Autoimmune diseases CHROMOSOMAL DISORDERS  Normal Karyotype - human somatic cells contain 46 chromosomes  22 homologous pairs of autosomes  2 sex chromosomes, XX in the female and XY in the male - short arm of a chromosome is designated p (for petit) - long arm is referred to as q (the next letter of the alphabet)  BANDED KARYOTYPE– each arm of the chromosome is divided into 2 or more regions bordered by prominent bands. Regions are numbered (e.g., 1, 2, 3) from the centromere outward MSU-GSC College of Medicine 18 STRUCTURAL ABNORMALITIES OF CHROMOSOMES aberrations underlying cytogenetic disorders may take the form of an abnormal number of chromosomes or alterations in the structure of one or more chromosomes Terms to Remember: EUPLOID– any exact multiple of haploid number of chromosomes (23) ANEUPLOIDY–error occurs in meiosis or mitosis and a cell acquires a chromosome complement not an exact multiple of 23 caused by nondisjunction and anaphase lag. NONDISJUNCTION–occurs during gametogenesis, the gametes formed have either an extra chromosome (n + 1) or one less chromosome (n − 1) ANAPHASE LAG– one homologous chromosome in meiosis or one chromatid in mitosis lags behind and is left out of the cell nucleus. Result is one normal cell and one cell with monosomy MOSAICISM  Mitotic error in early development give rise to 2 or more cells with different chromosomal complement in same individual  result from mitotic errors during the cleavage of the fertilized ovum or in somatic cells.  Autosomal mosaicism seems to be much less common than that involving the sex chromosomes DELETION  Loss of portion of a chromosome  Single break may delete a terminal segment.  2 interstitial breaks, with the union of proximal and distal segment, result in loss of internal segment.  Terminal deletions result from a single break in a chromosome arm, producing a fragment with no centromere, which is then lost at the next cell division MSU-GSC College of Medicine 19 INVERSION  2 interstitial breaks in a chromosome with reincorporation of the inverted, intervening segment  Inversion involving only one arm of the chromosome is known as paracentric.  Breaks are on opposite sides of the centromere– pericentric RING  A variant of deletion. After loss of segments from each chromosome, arms CHROMOSOME unite to form a ring.  Produced when a break occurs at both ends of a chromosome with fusion of the damaged ends  Do not behave normally in meiosis or mitosis and usually result in serious consequences ISOCHROMOSOMES  results when one arm of a chromosome is lost and the remaining arm is duplicated, resulting in a chromosome consisting of two short arms only or of two long arm  has morphologically identical genetic information in both arms  MOST COMMON present in live births involves the long arm of the X chromosome and is designated i(X)(q10). CYTOGENETIC DISORDERS INVOVLING AUTOSOMES TRISOMY 21 (DOWN SYNDROME) Description/ Most common of the chromosomal disorders; major cause of intellectual Etiology disability.  Have mild phenotypic changes and may even have normal or near-normal intelligence. Prevalence US incidence in newborns: 1 in 700 MSU-GSC College of Medicine 20   Approx. 95% of affected individuals have trisomy 21; chromosome count is 47 Etiology Usually caused by meiotic nondisjunction Characterized by extra copy of chromosome 21  Maternal age has a strong influence on the incidence of trisomy 21. Pathogenesis Meiotic nondisjunction of chromosome 21 that occurs in the ovum. Majority of protein coding genes mapped to chromosome 21 are overexpressed. Included is the gene for amyloid-beta precursor protein (APP).  Mitochondria are abnormal both morphologically and functionally in several tissues. Clinical Features Flat facial profile, oblique palpebral fissures, and epicanthic folds that are evident at birth. OTHER TRISOMIES Trisomy 18 (Edwards syndrome) and 13 (Patau syndrome) are common. They share several karyotypic and clinical features with trisomy 21 Most cases result from meiotic nondisjunction Carry a complete extra copy of chromosome 13 or 18. In contrast to trisomy 21, malformations are more severe and wide ranging Infants rarely survive beyond the first year of life CHROMOSOME 22q11.2 DELETION SYNDROME Description Encompasses a spectrum of disorders that result from a small deletion of band q11.2 on the long arm of chromosome 22. Prevalence Fairly common, occurring in as many as 1 in 4000 births Etiology Result from a small deletion of band q11.2 on the long arm of chromosome 22. MSU-GSC College of Medicine 21 Pathogenesis Approximately 30 candidate genes have been mapped to the deleted region. TBX1, a T-box transcription factor is expressed in the pharyngeal mesenchyme and endodermal pouch from which facial structures, thymus, and parathyroid are derived. targets of TBX1 include PAX9, a gene that controls the development of the palate, parathyroids, and thymus. Morphology Deletion of genes at chromosomal locus 22q11.2 gives rise to malformations affecting the face, heart, thymus, and parathyroids. Clinical Features Congenital heart defects, abnormalities of the palate, facial dysmorphism, developmental delay, and variable degrees of T-cell immunodeficiency and hypocalcemia. Above clinical features also represent 2 different disorders: DiGeorge syndrome and velocardiofacial syndrome. In a very small number of cases there is a deletion of 10p13-14. and velocardiofacial syndrome and in a number of cases, there is a deletion of 10p13-14. DiGeorge syndrome – thymic hypoplasia, with resultant T-cell immunodeficiency parathyroid hypoplasia giving rise to hypocalcemia, a variety of cardiac malformations affecting the outflow tract, and mild facial anomalies. CYTOGENETIC DISORDERS INVOLVING SEX CHROMOSOMES Genetic diseases involving sex chromosomes are more common than autosomal aberration Imbalances of ex chromosomes are better tolerated than autosome imbalances Factors peculiar to sex chromosomes: ○ Lyonization or inactivation of all but one X chromosome ○ Modest amount of genetic material carried by Y chromosome Regardless of the number of X chromosome, the presence of a single Y chromosome determines the male sex SRY (sex-determining region Y gene) located on distal short arm MSY (male-specific Y region) harbors 75 protein coding gene Y chromosome deletions are associated with azoospermia Features common to all sex chromosome disorders: Cause subtle, chronic problems relating to sexual development and fertility Difficult to diagnose at birth; first recognized at puberty The greater the number of X chromosomes (male and female), the greater the likelihood of intellectual disability LYON HYPOTHESIS Idea of X-inactivation by Mary Lyon (1961) ○ Only one of the X chromosomes is generally active ○ The other X chromosome (either maternal or paternal origin) undergoes heteropyknosis→ inactive ○ This inactivation occurs at random among all cells of the blastocyst on or about day 5.5 of embryonic life ○ This inactivated X chromosome persists in all the cells derived from each precursor cell Females have the same dosage of X-linked active genes as males (inactivated maternal/ paternal X chromosome) Inactive X chromosome is seen in interphase nucleus as darkly staining small mass in contact with the nuclear membrane (Barr body or X chromatin) Molecular basis of X inactivation involves the gene XIST whose product is lncRNA retained KLINEFELTER SYNDROME Etiology Male hypogonadism due to ○ Uneven dosage compensation during X inactivation → overexpression of X chromosome Short-stature HomeobOX (SHOX) gene: growth-related gene that maps on the pseudoautosomal region of Xp is not subject to X inactivation MSU-GSC College of Medicine 22  ○ Androgen receptor gene maps to the X chromosome and contains highly polymorphic CAG repeats Shorter CAG repeats are more sensitive to androgens X chromosome bearing androgen receptor allele with the shortest CAG repeat is inactivated In XXY males with low testosterone levels, expression of androgen receptors with long CAG repeats exacerbates the hypogonadism → low testosterone levels Occurs when two or more X chromosomes and one or more Y chromosomes 47,XXY (90%), 46,XY/ 47,XXY (15%), 47,iXq,Y Incidence: 1 in 660 live male births Comorbidity: ○ Type 2 diabetes/ ⬆ insulin resistance ○ Congenital heart disease (mitral valve prolapse, atrial and ventricular septal defects) ○ Osteoporosis and fractures due to sex hormonal imbalance ○ Extragonodal germ cell tumors (mediastinal teratomas) ○ Breast cancer and autoimmune dse (SLE) Other Genetic cause of reduced spermatogenesis and male infertility Information ○ Testicular tubules are atrophied and replaced by pink, hyaline, collagenous ghosts ○ Leydig cells appear prominent ○ ⬆ FSH, ⬇ testosterone, ⬆ plasma estradiol Ratio of estrogens and testosterone determines the degree of feminization Clinical Features Distinct body habitus with increase in length between the sole and pubic bone (appearance of elongated body) Eunuchoid body habitus with abnormally long legs Small atropic testes often associated with small penis Lack of secondary male characteristics (deep voice, beard, male distribution of pubic hair) Gynecomastia Average to low cognitive abilities with modest deficit in verbal skills TURNER SYNDROME Etiology Complete of partial monosomy of X chromosome Hypogonadism in phenotypic females ○ Missing an entire X chromosome 45,X (57%) ○ Structural abnormalities (14%) 46,X,i(X)(q10) isochromosome of the long arm → loss of short arm 46,X,r(X) deletion of portions of both long and short arms 46X,del(Xq) or 46X,del(Xp) deletion of portions of short or long arm ○ Mosaics (29%) – almost normal appearance with primary amenorrhea 45,X/ 46,XX 45,X/ 46,XY (5-10%; high risk for gonadoblastoma) 45,X/ 47,XXX 45,X/ 46,X,i(X)(q10) Incidence: 1 in 2000 live-born females Comorbidity/ Risks: ○ Congenital heart disease (25-50%) ○ Left-sided cardiovascular abnormality (preductal coarctation of aorta, bicuspid aortic valve) ○ Aortic dilation (30%) ○ Autoantibodies against thyroid gland → hypothyroidism ○ Glucose intolerance MSU-GSC College of Medicine 23  ○ Obesity ○ Nonalcoholic fatty liver disease ○ Insulin resistance (due to growth hormone therapy for short stature) 80% of cases the X chromosome is maternal origin, indicating an abnormality in paternal gametogenesis Fetal ovaries develop normally early during the first 18 weeks of gestation, but the absence of the second X chromosome leads to an accelerated loss of oocytes ○ Ovaries are reduced to atrophic fibrous strands, devoid of ova and follicles (streak ovaries) ○ “Menopause occurs before menarche” Somatic features are determined by genes on short arm ○ Haploinsufficiency of SHOX give rise to short stature Fertility and menstruation are determined by genes on long arm Clinical Features Edema of the dorsum of the hand and foot due to lymph stasis during infancy Swelling of the nape of the neck (distended lymphatic channels → cystic hygroma As they develop, swellings subside and leave bilateral neck webbing and persistent looseness of skin on the back of the neck At puberty, there is failure to develop normal secondary sex characteristics (infantile genitalia, inadequate breast development, little pubic hair) Short stature (rarely exceeding 150cm) Amenorrhea True hermaphrodite: presence of both ovarian and testicular tissue Pseudohermaphrodite: disagreement between the phenotypic and gonodal sex (female pseudohermaphrodite has ovaries but male external genitalia) Genetic sex: presence of absence of Y chromosome Gonodal sex: histologic characteristics of gonads Ductal sex: presence of derivatives of the mullerian or wolffian ducts Phenotypic/ genital sex: appearance of external genitalia Treatment Growth hormone and estradiol MSU-GSC College of Medicine 24 SINGLE-GENE DISORDERS WITH NONCLASSIC INHERITANCE  Does not follow classic Mendelian principles, classified into four (4) categories: o Trinucleotide-repeat mutations o Mitochondrial genes mutations o Genomic imprinting o Gonadal mosaicism TRINUCLEOTIDE  Expansion of trinucleotide repeats  causes neurodegenerative disorders -REPEAT  Fragile X Syndrome (FXS) caused by trinucleotide repeats  landmark in MUTATIONS human genetics  Causative mutations: expansion of a stretch of trinucleotides that share nucleotides G and C  Associated with unstable tetranucleotides, pentanucleotides, & hexanucleotides which is fundamental mechanism for neuromuscular disease.  Inclination to expand depends on the sex of the transmitting parent: o FXS: expands during oogenesis o Huntington disease: Spermatogenesis  3 key mechanisms which unstable repeats cause disease: o Loss of function (affected gene): via transcription silencing, location at the NON-CODING part o Toxic gain of function: alterations of protein structure, i.e. Huntington or spinocerebellar ataxias, location at the CODING PART of the gene o Toxic gain of function mediated by RNA: i.e. Fragile X-associated tremor/ataxia syndrome, same in FXS, location is at the NON- CODING part of the gene MSU-GSC College of Medicine 25  FRAGILE X SYNDROME (FXS) Description Most common genetic cause of intellectual disability in males & overall second (2nd) most common cause after Down syndrome Prevalence Male frequency: 1 in 1550 - Carrier males:  Approx 20% of males who carry a fragile X mutation and are clinically normal  Called: Normal transmitting males Female frequency: 1 in 8000 - Affected females:  30% to 50% are affected (i.e., intellectual disability etc) Etiology Cause: TRINUCLEOTIDE EXPANSION MUTATION in the familial mental retardation 1 (FMR1) gene FMR1: present in Fragile X-associated tremor/ataxia syndrome and fragile X- associated primary ovarian insufficiency Fragile site: constriction in the long arm of X chromosome Pathogenesis Anticipation: ○ FXS worsens with each successive generation (i.e. grandsons and great grandsons[through daughters]) Xq27.3: locale that is responsible for the disease 6 to 55 CGG repeats: Normal population 55 to 200 CGG repeats: Normal transmitting males & carrier females 200 to 4000 repeats (Full mutation): for patients of FXS Process of further amplification of CGG repeats seen in premutations: ○ Carrier males: transmits repeats in with small changes in repeat number ○ Carrier female: High probability of dramatic amplification of the repeats leading to intellectual disability in most male offsprings and 50% of female offsprings MSU-GSC College of Medicine 26  ○ Thus: Oogenesis permutations can be converted to mutations by triple- repeat amplification This explains the unusual inheritance pattern: likelihood of intellectual disability being higher in grandson than in brothers of transmitting males. Fragile X mental retardation protein (FMRP) ○ It is the product of FMR1 gene ○ When the FMR1 gene is suppressed, this results in the absence of FMRP causing phenotypical changes ○ FMRP is most abundant in the brain and testis ○ It selectively binds mRNAs associated with polysomes and regulate their intracellular transport to dendrites ○ It is a translation regulator; which suppresses protein synthesis form the bound mRNAs in response to signaling through group I metabolic glutamate receptors (mGlu-R) ○ Reduction in FMRP in FXS = increased translation of the bound mRNAs at synapses ->imbalance in production of proteins at synapses - > loss of synaptic plasticity (which is essential for learning and memory) PCR: diagnostic test for detection of repeats Clinical  Male symptoms: Features o Intellectual disability o Large mandible, large everted ears, & large testicles (macro-orchidism; observed in 90% postpubertal male) o Hyperextensible joints o High arched palate o Mitral valve prolapse o Epilepsy (30%) o Aggressive behavior (90%) o Autism spectrum disorder o Anxiety/Hyperactivity disorder (50 to 75%)  Risk of phenotypic effects: o Depends on the position of the individual in the pedigree o Example: 9% risk (intellectual disability) for brothers of transmitting males, 40% risk for grandsons of transmitting males FRAGILE X- ASSOCIATED TREMOR/ ATAXIA SYNDROME AND FRAGILE X-ASSOCIATED PRIMARY OVARIAN FAILURE  FMR1 GENE causes these 2 disorders through a distinct mechanism involving toxic gain of function  Fragile X-associated primary ovarian failure: o approx 20% of females carrying the permutation (carrier females) have ovarian failure ( before the age of 40) o Manifests menstrual irregularities and decreased fertility o FSH levels are elevated o Antimullerian hormone levels are decreased (both of these are markers of declining ovarian fxn) MSU-GSC College of Medicine 27  o Develop menopause 5 years earlier  Fragile x-associated tremor/ataxia syndrome: o Approx 50% of permutation-carrying males (transmitting males) exhibit a progressive neurodegenerative syndrome starting in their sixth decade. o Intention tremors & cerebellar ataxia -> parkinsonism  How do permutations cause disease? o FMR1 gene continues to be transcribed instead of being methylated or silenced o CGG-containing FMR1 mRNAs are “toxic” o Intranuclear inclusions are formed in both CNS & PNS o FMR1 mRNA have been detected in granulosa cells and ovarian stromal cells  Read on these key concepts: MUTATIONS IN MITOCHONDRIAL GENES - LEBER HEREDITARY OPTIC NEUROPATHY  mtDNA is derived entirely from the ovum (maternal inheritance)  This is due to the presence of numerous mitochondria in the ova as opposed to spermatozoa  37 genes are contained in Human mtDNA. 22 transcribed in transfer RNA & 2 into ribosomal RNA; the remaining 13 genes are in respiratory chain enzymes  Mutations of these genes have deleterious effects on CNS, skeletal muscle, cardiac muscle, liver, and kidneys. (due to its effect on oxidative phosphorylation)  Each mitochondrion contains thousands of copies of mtDNAs, where individuals may have both wild-type and mutant mtDNA, this is called heteroplasmy  Threshold effect : minimum number of mutant mtDNA before oxidative phosphorylation gives rise to disease.  Mitochondria and their DNA are randomly distributed to daughter cells during cell division. With this, expression of disorder resulting from mutations in mtDNA is variable  Leber hereditary optic neuropathy: prototype of disease associated with mitochondrial inheritance (rare) o Neurodegenerative disease that manifests with:  Progressive bilateral loss of central vision  Manifests between ages 15-35, that eventually leads to blindness  Cardiac conduction defects & minor neurologic manifestations have also been observed MSU-GSC College of Medicine 28  GENOMIC IMPRINTING POINTS TO  Imprinting: differences that exists paternal and maternal allele REMEMBER  Maternal imprinting: transcriptional silencing of the maternal allele  Paternal imprinting: inactivation of the paternal allele  This process occurs in the ovum or sperm, before fertilization  Best illustrated by two (2) uncommon) genetic disorders: Prader-Willi syndrome Angelman syndrome  Intellectual disability, short stature,  Intellectual disability, hypotonia, profound hyperphagia, microcephaly, ataxic gait, obesity, small hands and feet, and seizure, and inappropriate hypogonadism. laughter  60% to 75%: deletion of q12 in long arm  Referred to as “happy of chromosome 15, del(15)(q11.2q13) puppets”  In all cases the deletion affects paternally derived chromosome 15  3 mechanisms involved in genomic imprinting: o Deletions : maternal 15q12 is imprinted (silenced) thus functional alleles are provided by the paternal chromosome, when these are lost as a result of deletion, the person develops Prader-Willi syndrome; if the same process happens with the paternal chromosome, only the maternally derived allele is active, deletion of this allele gives rise to Angelman syndrome. (deletion accounts for 70% of cases) o Uniparental disomy: Inheritance of both chromosomes (two maternal copies of chromosome 15, since Prader-Willi syndrome px have this) from one parent. Same goes for px with Angelman syndrome with two copies of paternal chromosome 15. (second most common mechanism, around 20% to 25% of cases) o Defective imprinting: Occurs in a small minority of patients (1% to 4%). Some patients with Prader-Willi syndrome, the paternal chromosome carries maternal imprint, conversely Angelman syndrome px, the maternal chromosome carries paternal imprint  Angelman syndrome affected gene: UBE3A, a ubiquitin ligase. MSU-GSC College of Medicine 29  o Absence of this gene inhibits synapse formation and synaptic plasticity (expressed from both alleles most tissue)  Prader-Willi syndrome series of genes affected: 15q11.2-q133 interval o Loss of SNORP family of genes that encode small nucleolar RNAs is believed to contribute to the condition.  Read on these Key concepts: GONADAL  Some autosomal disorders (such as osteogenesis imperfecta) phenotypically MOSAICISM have normal parents that have more than one affected child  Violates the laws on Mendelian inheritance  Responsible for unusual pedigrees  Mutation that occurs postzygotically during early embryonic development  Mutation affects only cells that from the gonads, the gametes that carry the mutation, but somatic cells are normal.  Parents with gonadal mosaicism can transmit to the offspring through their mutated gametes  Since progenitor cells of the gametes carry the mutation, it can affect multiple offsprings. MOLECULAR GENETIC DIAGNOSIS  Emerged in thr half of the lattee 20th century in the form of labor-intensive, low throughput methods such as conventional karyotyping (Down Syndrome) and DNA-based assays such as Southern blotting (Huntington’s Disease) Genetic Markers Constitutional Somatic Present in every cell of the affected person Restricted to specific tissue types or lesions MSU-GSC College of Medicine 30 Example: CFTR mutation in a patient with Example: mutation in RAS in a variety of human cystic fibrosis cancers DIAGNOSTIC METHODS AND INDICATIONS FOR TESTING Laboratory  Focus on the sensitivity, specificity, accuracy and reproducibility of considerations different methods o Standard cystic fibrosis testing for pathogenic mutations in CFTR has a sensitivity of 94% in Ashkenazi Jews, but identifies less than 50% of affected patients in Asian populations  BUT, there are no pathogenic mutations are identified in approximately 10% of patients with classic cystic fibrosis → NGS assays miss certain genetic disorders (e.g. kilobase scale deletions and gene arrangement) Indications for analysis  Possible indications for prenatal testing: of inherited genetic o Advanced maternal age alterations o A parent known to carry a unbalanced chromosomal rearrangement → increases the frequency of abnormal chromosome segregation during meiosis and the risk of aneuploidy in the fertilized ovum o Fetal anomalies observed on ultrasound o Routine maternal blood screening indicating an increased risk of Down syndrome or another trisomy  Prenatal testing may also be considered for fetuses at known risk for Mendelian disorders (e.g., cystic fibrosis, spinal muscular atrophy) o At present it is usually performed using amniocentesis, chorionic villus biopsy or umbilical cord blood  Following birth, testing is ideally done as soon as the possibility of constitutional genetic disease arises (commonly performed on peripheral blood DNA). These are the indications o Multiple congenital anomalies o Suspicion of metabolic syndrome o Unexplained intellectual disability, and/or developmental delay o Suspected aneuploidy(e.g., Down syndrome) or other syndromic chromosomal abnormality (e.g., deletions, inversions) o Suspected monogenic disease  Possible indications for older patients o Inherited cancer syndromes (triggered by either family history or an unusual cancer presentation) o Atypical mild monogenic diseases (e.g., attenuated cystic fibrosis) o Neurodegenerative disorders (e.g., familial Alzheimer disease, Huntington disease) Indications for analysis  Common indications: of acquired genetic o Diagnosis and management of cancer alterations  Detection of tumor-specific mutations and cytogenetic alterations (e.g., BCR-ABL fusion genes → chronic myeloid leukemia[CML]) o Clonality as a indicator of a neoplastic condition o Identification of specific genetic alterations (e.g., amplification of HER2 [official gene name ERBB2] in breast cancer OR mutation of EGFR B [official gene name ERBB1] in lung cancer) MSU-GSC College of Medicine 31  o Treatment efficacy (e.g., minimal residual disease detection in patients with CML by quantitative PCR for BCR-ABL) o Drug-resistant secondary mutations in malignancies treated with therapies that target specific proteins (e.g., mutated EGFR) o Diagnosis and management of infectious disease  microorganism -specific genetic material for definitive diagnosis (eg., HIV, mycobacteria, HPV, herpes virus in CNS)  Genetic alterations in the genomes of microbes that are associated with drug resistance  Treatment efficacy (e.g., assessment of viral loads in HIV, EBV and HCV) PCR and Detection of DNA Sequence Alterations  PCR analysis → synthesis of short DNA fragments from a DNA template  PCR uses heat-stable DNA polymerases and thermal cycling, the target DNA (