Genetic Basics of Inherited Haematologic Disorders PDF

Genetic Basics of Some Inherited Haematologic Disorders Introductory Posting in Pathology & Pharmacology Delivered by: Dr. Adeyemi  Outline Introduction Overview of genetics Inherited disorders of red blood cell membrane Inherited disorder...

Genetic Basics of Some Inherited Haematologic Disorders Introductory Posting in Pathology & Pharmacology Delivered by: Dr. Adeyemi  Outline Introduction Overview of genetics Inherited disorders of red blood cell membrane Inherited disorders of red cell enzyme Inherited disorders of red blood cell haemoglobin Inherited bleeding disorders Conclusion  Introduction All the genetic information that makes up an organism is encoded in the DNA. This information is transcribed into mRNA, and then the triplet code of those mRNAs is translated into protein. Changes that affect the DNA or RNA sequence or its expression, either in the germline or acquired after birth, can cause haematologic disorders. These may be mutations that change the DNA sequence, including single base changes, deletions, insertions, and duplications, or they may be epigenetic changes that affect gene expression without any change in the DNA sequence  Introduction Inheritance patterns depend upon the biologic effect and chromosomal location of the mutation. Many of the haematologic diseases have a genetic basis. Understanding the genetics of a disorder is necessary for accurate genetic counseling.  Overview of Genetics All of the information required for the development of a complete adult organism is encoded in the DNA of a single cell—the zygote. This information, designated the genome, includes the data needed for the synthesis of all enzymes; all the plasma proteins, including the clotting factors, complement components, and the transport proteins; all the membrane proteins, including receptors; and all of the cytoskeletal proteins. The units of information into which the genome is organized are the genes, which are composed of sequences of DNA.  Overview of Genetics By serving as the blueprints of proteins in the body, genes ultimately influence all aspects of body structure and function. There are approximately 21,000 protein coding genes and an additional 10,000 genes that do not encode proteins but affect the regulation of genes. An error in one of these genes often leads to a recognizable genetic disease. To date, more than 20,000 genetic traits and diseases have been identified and cataloged.  Overview of Genetics DNA DNA has three basic components: – The pentose sugar molecule, deoxyribose – A phosphate molecule – Four types of nitrogenous bases Pyrimidines (single carbon-nitrogen rings): cytosine and thymine Purines (double carbon-nitrogen rings): adenine and guanine The four bases are commonly represented by their first letters: A, C, T, and G. Watson-Crick model of the DNA molecule.  Overview of Genetics DNA directs the synthesis of all the body’s proteins. Proteins are composed of one or more polypeptides (intermediate protein compounds), which are, in turn, composed of sequences of amino acids. The body contains 20 different types of amino acids, which are specified by the four nitrogenous bases. To specify (code for) 20 different amino acids with only four bases, different combinations of bases, occurring in groups of three, are used. These triplets of bases are known as codons. – Each codon specifies a single amino acid in a corresponding protein.  Overview of Genetics FROM GENES TO PROTEINS DNA is formed and replicated in the cell nucleus, but protein synthesis takes place in the cytoplasm. The DNA code is transported from nucleus to cytoplasm, and subsequent protein is formed through two basic processes: transcription and translation. These processes are mediated by RNA, which is chemically similar to DNA except that the sugar molecules ribose rather than deoxyribose, and uracil rather than thymine is one of the four bases. The other bases of RNA, as in DNA, are adenine, cytosine, and guanine. Uracil is structurally similar to thymine, so it also can pair with adenine. Whereas DNA usually occurs as a double strand, RNA usually occurs as a single strand. In transcription, RNA is synthesized from a DNA template, forming messenger RNA (mRNA).  Mutations Mutations is any inherited alteration of genetic material. May cause disease or be subtle, silent substitutions that do not change amino acids. Missense mutation: A base pair change that alters a single amino acid Nonsense mutation: A base pair change that produces a premature stop codon. – Because they typically result in a complete loss of gene product, nonsense mutations usually produce a more-severe disease phenotype than do missense mutations. Frameshift mutation: Involves the insertion or deletion of one or more base pairs of the DNA molecule. These mutations change the entire “reading frame” of the DNA sequence because the deletion or insertion is not a multiple of three base pairs (bp; the number of base pairs in a codon). Splice-site mutations: Describe alterations of the DNA sequence at intron–exon boundaries. These result in a mature mRNA transcript that contains introns or lacks exons.  A functional classification of different types of mutations based on the level at which they modify gene action Transcription: Deletions, insertions, inversions, fusion genes, point mutations – Remove or inactivate gene or several genes in cluster – Involve major regulatory regions;-Promoters (DNA sequence for accurate initiation of transcription), Locus-control regions. Enhancers(increase promoter activity) – Trans-acting factors ,cap site (transcript of gene into RNA begin) Processing of mRNA: Point mutations, small deletions, insertions – Splicing (precise and efficient removal of RNA transcribed from introns) – Intron-exon junctions – Consensus sequences – Cryptic splice sites within introns and exons – Poly (A) addition site (polyadenylation at 3’ end)  Functional classification of mutations based on the level at which they modify gene. Translation: Nonsense or frameshift mutations, other point mutations, insertions – Initiation – Elongation – Termination Structure of gene products: Mis-sense mutations, deletions, insertions, frameshift, others – Processing – Function – Stability POINT MUTATION Frameshift Mutation  Mendelian Traits Traits caused by single genes are called mendelian traits. Each gene occupies a position along a chromosome known as a locus. The genes at a particular locus can take different forms (i.e., they can be composed of different nucleotide sequences) called alleles. At a given locus, an individual (diploid organism) has one allele whose origin is paternal and one whose origin is maternal. When the two alleles are identical, the individual is homozygous at that Locus; heterozygous, if non-identical. The composition of genes at a given locus is known as the genotype. The outward appearance of an individual, which is the result of both genotype and environment, is the phenotype.  Mendelian Traits A locus that has two or more alleles that each occurs with an appreciable frequency (classically defined as 1%) in a population is said to be polymorphic (or a polymorphism). – Single nucleotide polymorphisms (SNPs) – Copy number variants (CNVs): involve the presence or absence of larger pieces of DNA Balanced Polymorphisms – Occur when genetic variants, such as the alleles responsible for SCD, thalassaemia, or G6PD deficiency, reach polymorphic levels because the deleterious effects that they may have are counterbalanced by beneficial effects on survival, such as increased resistance to malaria.  Dominant and Recessive Traits Gregor Mendel established this concept. In dominant traits, such as von Willebrand disease or porphyria cutanea tarda type II, one copy of a disease- causing allele is sufficient for disease causation, so heterozygotes are typically affected. In recessive traits, such as β-thalassaemia, two copies of the disease-causing allele must be present, so the affected individual is a homozygote. A carrier is an individual who has a disease gene but is phenotypically normal.  TRANSMISSION OF GENETIC DISEASES The known single-gene diseases can be classified into four major modes of inheritance: – Autosomal dominant – Autosomal recessive – X-linked dominant – X-linked recessive The first two types involve genes known to occur on the 22 pairs of autosomes. The last two types occur on the X chromosome; very few disease- causing genes are found on the Y chromosome.  Pedigree for sickle cell disease. The double bar denotes a consanguineous mating. Because sickle cell disease is relatively common in some populations, most cases do not involves consanguinity.  TRANSMISSION OF GENETIC DISEASES PENETRANCE AND EXPRESSIVITY The penetrance of a trait is the percentage of individuals with a specific genotype who also exhibit the expected phenotype. Incomplete penetrance means that individuals who have the gene disease- causing genotype may not exhibit the disease phenotype at all, even though the genotype and the associated disease may be transmitted to the next generation. Penetrance can increase with age, and it can differ between the sexes. E.g haemochromatosis. Expressivity is the extent of variation in phenotype associated with a particular genotype. If the expressivity of a disease is variable, penetrance may be complete but the severity of the disease can vary greatly. Many haematologic conditions, including sickle cell disease and β- thalassemia, have variable expressivity.  TRANSMISSION OF GENETIC DISEASES X-LINKED INHERITANCE Some genetic conditions are caused by mutations in genes located on the sex chromosomes, and that mode of inheritance is termed sex linked. Only a few diseases are known to be inherited as X- linked dominant or Y chromosome traits  Examples Of Haematologic Diseases Caused By Different Types of Mutations Transmission Pattern of Single-Gene Disorder Examples Autosomal Dominant disorders Hereditary spherocytosis, von Willebrand disease Autosomal Recessive Disorders Sickle cell anaemia, thalassaemia, haemachromatosis X-linked Recessive Disorders Haemophilia A and B, chronic granulomatous disease, Glucose 6-phosphate dehydrogenase deficiency, aggamaglobulinaemia, Wiskott- Aldrich syndrome  INHERITED DISORDERS OF RED CELL MEMBRANE Hereditary Spherocytosis Spherocytic cells have increased osmotic fragility of RBC Shape –results from weak anchoring of the cell membrane to the cytoskeletal proteins. Loss of more lipid than protein in the form of lipid vesicles – surface area is reduced. Autosomal dominant inheritance. Mutation in the genes of β spectrin band. Mutation in genes for ankyrin and band 3 protein. May present in childhood clinical course varies from mild to severe anaemia, jaundice and splenomegaly.  INHERITED DISORDERS OF RED CELL ENZYME GLYCOGEN 6-PHOSPHATE DEHYDROGENASE ENZYME (G6PD) G6PD is an enzyme whose synthesis is inherited in an x-linked manner. The deficiency leads to abnormality of glucose metabolism characterized by failure of generation of NADPH involved in the production of GSH which protect the red cell against oxidative stress with attendant haemolysis.  Biology And Molecular Aspect of G6PD Deficiency The gene for G6PD is located on x-chromosome at xq28 The gene spans approx 20kb with 13 exons and 12 introns The gene is transcribed as a monomer of 514 amino acids to produce equilibrium of dimers and tetramers The mutation affects enzyme stability leading to rapid decline in its activities Vast majority of the mutation are missense  Complete inactivation of the enzyme is incompatible with life Clinical consequences are virtually confined to rbc G6PD activity decreases significantly as rbc ages with a half-life of about 60days Reticulocytes have 5x higher enzyme activity than the oldest rbc subpopulation  G6PD VARIANTS Specific G6PD alleles are associated with G6PD variants with different enzyme levels and, thus, different degrees of clinical disease severity. The variation in G6PD levels accounts for differences in sensitivity to oxidants. G6PD B: World wide – is the wild type of allele (normal variant) G6PDA: African Variant (Asparagine replaced by Aspartic acid) G6PDA-: Deficient (Decreased stability in vivo) A and A – have common nucleotide substitution at Nucleotide 376 – hence the rapid movement on electrophoresis A- above plus mutation at nucleotide 202 this accounts for its in vivo instability  More than 400 variants have been defined and the exact mutation determined Most are sporadic but some occur at high frequency Variants divided into 3 categories based on the type of haemolysis that they cause – Acute intermitent haemolytic anaemia – Congenital non-spherocytic haemolytic anaemia – Chronic haemolytic anaemia No obvious risk of haemolysis eg A+  G6PD – Product of structural gene located on the X chromosome. Near the tip of the long arm of the X chromosome (band Xq 28) genetic disease is closely linked to gene for – Haemophilia A (factor VIII) – Mental retardation – Protan and Deutan Colour blindness – Adrenal leukodystrophy. – No association with Haemophilia B – Duchene muscular dystrophy Inherited Disorders of Red Cell Haemoglobin Haemoglobinopathies –Are a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassaemias) or the synthesis of a structurally abnormal haemoglobin (HbS) Haemoglobin gene variants are haemoglobinopathies  Types of Globin Abnormalities Synthetic (Quantitative) abnormalities Thalassaemias arising from reduced synthesis Thalassaemia syndromes are sub-classified based on the gene involved. α and β thalassaemias are further sub-divided into α+, β+ or αo, βo depending on whether some (+) or no (o) globin protein is produced Structural (Qualitative) abnormalities Homozygous or double heterozygous abnormal globins e.g. sickle cell syndromes  Haemoglobin Hb; - 68 000 Consists of two pairs of unlike globin chains (i.e., α plus β, δ, or γ) In healthy adults, ∼ 95% of the Hb is Hb A (α2β2) with small amounts (1000 mutations causing haemoglobinopathies have been documented – Only a limited number are associated with disease states and clinical significance People who inherit combinations of haemoglobins S, C, E, D Punjab, β thalassaemia, or α zero (α0) thalassaemia may have a serious haemoglobin disorder Each at-risk ethnic group has its own combination of common Hb variants and thalassaemia mutations  Inheritance of Thalassaemias The thalassemia syndromes and some of the Hb variants are inherited as autosomal recessive conditions. Very rarely, β thalassaemia demonstrates an autosomal dominant inheritance pattern. Some Hb Variants also have an autosomal dominant inheritance pattern Genetic classification of the Thalassaemias and Related Disorders α Thalassaemia α0 α+ Deletion (- α) Non-deletion (αT) β Thalassaemia β0 β+ Variants with unusually high level of HbF or A2 Normal HbA2 ‘Silent’ δβ Thalassaemia (δβ)+ (δβ)0 (Aγδβ)0 Classification of the Thalassaemias and Related Disorders – γ Thalassaemia – δ Thalassaemia Δ0 δ+ – εγδβ Thalassaemia – Hereditary persistence of fetal haemoglobin Deletion (δβ)0 Non-deletion Linked to β-globin-gene cluster – Gγβ+ – Aγβ+ – Unliked to β- globin-gene cluster  Alpha Thalassaemia This is caused by defective synthesis of the α chain with resultant excess of γ and β chains. It is usually caused by gene deletion or mutation in the termination codon (Hb Constant Spring) Classification of Alpha Thalassaemia  Clinical Classification of Alpha Thalassaemia Hydrop foetalis: This is associated with the deletion of all four α genes (α0/ α0) leading to the production of Hb Barts, Hb H, and Hb Portland. Hb H disease: This results from deletion of three α genes (α0/ α+), and is associated with detection of Hb H, moderate anaemia, marked poikilocytosis, anisocytosis, microcytosis, and splenomegaly. – Diagnosis is by detection of Hb H. Treatment is with Folic Acid, avoidance of oxidant drugs, and if necessary splenectomy.  Clinical Classification of Alpha Thalassaemia Deletion of two α genes (α0/α or α+/ α+): This is associated with mild anaemia, osmotic fragility curve typical of thalassaemia. – Diagnosis is the finding of Hb H inclusion in 0.1 to 0.3% of red blood cells, low MCV, low MCH, and increased red cell count. α-thalassaemia trait: This is associated with mild condition, 1-2% Hb Barts production and resembles thalassaemia minor.  β Thalassaemia Beta thalassaemia result from defective synthesis of the β globin chain. It may be due to: – single point mutation in the β globin gene – fusion genes – cross over between δ and β genes. Rarely, the β gene may be deleted. The most common-thalassemia mutations: – IVS1-5 (G➝C), codon 15 (G➝A), codon 26 (G➝A), codon 30 (G➝C), and codon 41/42 (−TCTT). – These accounted for 85% in 80 -thalassemic alleles deciphered from 56 patients, including-thalassemia major and carriers  Classification of β Thalassaemia Thalassaemia major Thalassaemia minor Thalassaemia Intermedia  β-Thalassaemia Major Usually resulting from homozygous genetic abnormality, with survival ranging from 1 year to the 3rd decade. Causes: – Deletion of the β globin gene – Defective production of mRNA – Defective processing of high molecular weight RNA – Unstable RNA – Unreadable RNA  Pathophysiology Excessive production of globin chains without complement leads to accumulation of free chains which are not incorporated into tetramers and so remain dimers. – Dimers dissociate into monomers which are unstable resulting in formation of precipitates referred to as Heinz bodies. There is ineffective erythropoiesis and haemolysis. Compensatory formation of Hb F, a high affinity haemoglobin results in tissue hypoxia and marrow hyperplasia.  Clinical Features Presentation is in the first four years of life with severe anaemia, hepatosplenomegaly Extension of the marrow space to create the ‘hair-on-end’ appearance Multiple fractures Diseases Resulting from Structural Haemoglobin Variants Haemolytic anaemia – HbS, HbC – Unstable haemoglobins Hereditary polycythaemia – High-oxygen-affinity haemoglobins Hereditary cyanosis – M haemoglobins – Low-oxygen-affinity variants Thalassaemia phenotype – Highly unstable haemoglobins – Chain-termination haemoglobin variants – Fusion-chain haemoglobin variants  Structural Haemoglobin Variants-Mutations 90% of haemoglobin variant have single amino acid substitution in α, β, δ, γ chains, embryonic variant unknown 10% are abnormal HbS with 2 substitutions in the same globin chain, deletions or insertions, N-terminal or C-terminal elongations, or hybrid globins. Majority (-75%) of structural variants described are due to mutations in in α or β chains of the major adult component, HbA. Most important are S,C, O Arab and E A few structural variants are synthesized ineffectively and so have a thalassemic phenotype  Structural Variant Prevalence HbS - Sub-Saharan Africa, parts of the Mediterranean , Middle east and India HbC – mainly in West Africa HbE – 15-30% in Cambodia, Thailand, parts of China, Vietnam.  Structural variant- HbS Sickle Hb (HbS)-Point mutation Nucleotide sequence of beta globin gene at β6 is GTG instead of GAG, beta globin mRNA form of beta globin gene at β6 is GUG instead of GAG – Insertion of valine instead of glutamic acid DeoxyHbS undergoes structural change resulting in sickled erythrocyte  Haplotype Residing within a cluster of beta-like /alpha genes ,the beta/alpha globin gene are in linkage disequilibrium with multiple nearby polymorphic sites. Different combinations can be identified by restriction endonuclease cleavage which thereby defines discrete beta –locus haplotype. betaS gene tend to reside on one of several distinct chromosomal patterns referred to as Senegal, Benin, Bantu, Cameroon, Arab-India haplotype. Haplotype analysis provide information on the chromosomal background on which mutation has occurred e.g identical mutation on different haplotype may be associated with small differences in clinical phenotype indicating that modifying sequence element such as Hb F production. Useful in population genetics and Prenatal diagnosis  HbC HbC- beta globin mRNA is AAG instead of GAG at β6 resulting in insertion of lysine (β6 glu –>lys) HbC –less soluble and lower O2 affinity than HbA tendency to crystal formation Homozygote state –mild anaemia associated with moderate splenomegaly. Not symptomatic except during stress such as pregnancy  HbD, O-Arab HbD Punjab/Los Angeles, Ibadan: Glutamine (CAG) replaces glutamic acid (GAG) at position β121 (β glu –>gln) HbO Arab: Lysine (AAG) replaces glutamic acid at the same site (β121 glu –>lys). Hb O Arab and Hb D Punjab - Clinical manifestations are minimal. The major clinical impact of these hemoglobinopathies results from co-inheritance with Hb S These abnormal haemoglobins as well as Hbs Lepore, Boston, when inherited along with the gene for HbS result in clinically significant sickle cell disease.  HbE HbE – Mild thalassaemia and abnormal Hb. Results from the substitution of lysine for glutamic acid at position β26 (β26 glu –>lys).(GAG-> AAG) This is mildly unstable and susceptible to oxidant damage Homozygote exhibit microcytosis but asymptomatic Compound heterozygote with β- thal resemble β- thal intermedia or major but more prone to infection and pulmonary hypertension that homozygous β - thal  Prognosis Clinical features of haemoglobinopathies and factors that modify the clinical features, clinical course, complications and treatment of the disorders Modifies by acquired, environmental and hereditary (heterozygote/homozygote) factors  Inherited Bleeding Disorders Haemophilia A - Genetics FVIII gene is 186kb Located near the tip of the long arm of the X chromosome Xq2.6 region – Has 26exons and 25 introns – It codes for a 2351 amino acids protein which is produced in the liver Post translational removal of 19AA must occur to give the 2332AA mature plasma protein The FVIII protein has a triplicated region A1,A2,A3, a duplicated region C1,C2 & a heavily glycosylated B domain Inheritance of Haemophilia Inheritance of Haemophilia  Genetics of Haemophilia Over 142 mutations have been described Missense Nonsense Frameshift splicing Deletions – large or small Insertions flip tip inversion particularly produce very severe disease Hemarthrosis (acute)  von Willebrand Disease von Willebrand factor – Synthesis in endothelium and megakaryocytes – Forms large multimer – Carrier of factor VIII – Anchors platelets to subendothelium – Bridge between platelets Inheritance - autosomal dominant Incidence - 1/10,000 Clinical features - mucocutaneous bleeding  Hereditary haemochromatosis Is a group of diseases in which there is excessive absorption of iron from the GIT leading to iron overload of the parenchymal cells of the liver, endocrine organs and, in severe cases, the heart. Most patients are homozygous for a missense mutation in the HFE gene which leads to insertion of a tyrosine residue rather than cysteine in the mature protein (C282Y). HFE is involved in hepcidin synthesis and therefore hereditary haemochromatosis caused by HFE mutation is due to low serum hepcidin levels  CONCLUSION The detection of mutations that cause a variety of diseases is now possible and has become a routine method for the diagnosis of some disorders. Inheritance patterns depend upon the biologic effect and chromosomal location of the mutation. Understanding the genetics of a disorder is necessary for accurate