Genetics in haematology2012

• 1. Prepared by:Col (Dr) Amir Muhriz AbdulLatiff M.D (USM) Mpath(Haematology)Senior Lecturer andHaematologistMedical Faculty UiTM 1

2. LEARNING OBJECTIVES1. Describe gene & chromosome abnormalities in haemoglobinopathies, leukaemias, (Philadelphia chromosome and other related chromosomal abnormalities) & bleeding disorders2. Interpret the chromosome and gene nomenclature in relevant diseases.3. Relate the importance of genetic studies in the classification and management of disease.4. List common test used in the detection of genetic abnormalities in haematology5. Discuss the importance of carrier detection in haematological diseases. 2 3. Contents 1. Important definitions 2. Chromosomal and molecular abnormalitiesina. Haemoglobinopathy and thalassaemiab. Leukaemiac. Bleeding disorders 3. Importance of genetic and chromosomalstudies in haematology 4. common test used in the detection of geneticabnormalities in haematology 5. Importance of carrier detection inhaematological diseases 11/27/20123 4. Important definitionGenetics:The branch of science concerned with the means andconsequences of transmission and generation of thecomponents of biological inheritance.(Stedman, 26th ed)Molecular genetics: The study of the molecular constitution of genes andchromosomes (World English Dictionary)Chromosomes:any of several threadlike bodies, consisting of chromatin, that carry the genes in a linear order: the human species has 23 pairs, designated 1 to 22 in order of decreasing size and X and Y for the female and male sex chromosomes respectively.(dictionary.com)4 5. Importance of genetics The only way to understand hereditarydiseases What we are begins with our genetic heritageand is modified by our environment andexperiences Our genetic heritage determines susceptibilityto multifactorial diseases such as: HypertensionDiabetes Vascular diseaseCancer OsteoarthritisAutoimmune diseases Critical to developing new disease treatments Statins tPA EPO 6. The scope of genetics The human genome has been sequenced! Approximately 35,000 genes, most of which encode a protein, in a haploid genome of 3 X9 10 base pairs Only about 1.5 % of the DNA actually encodes functional genes All living organisms are remarkably similar at the genetic level Same genetic code About 50 % of genes comparable between us and plants All nucleated somatic cells have a complete set of genes Only a small fraction of genes are active in a single cell Enables cloning 7. The burden of Mendelian (single gene)disorders Although individually rare, genetic diseasescollectively constitute a major health problem About 5 - 8 % of admissions to a pediatric hospitaland about 1 % of admissions to an adult hospitalare for Mendelian disorders 9 % of pediatric deaths are due to Mendeliandisorders About 1- 2 % of the population has a Mendeliandisorder Most Mendelian disorders are apparent bychildhood Life span is reduced in about 60 % of thesedisorders Each person is estimated to have 1 - 5 lethal 8. From cellular to molecular biology 9. Genetic terminology Gene: The fundamental unit of heredity. About 35,000 in the human genome. A typical gene:53enhancer promoter repressorintronexon 5 UTR 3 UTR AAAAAAAAAmRNA coding domain (ORF) 10. Exons, introns and alternative splicing Most genes have introns Alternative splicing is common Many alternate proteins can be generatedfrom a single gene, each of which can have aunique function 11. Transcription and translation Transcription: Generation of an RNA copy of asingle gene Translation: Synthesis of a protein using themRNA as a template 12. Important definitions Alleles: Alternative forms of a gene that can bedistinguished by their alternate phenotypic effects orby molecular differences; a single allele for each locusis inherited separately from each parent Autosome: One of chromosomes 1 - 22 Dominant allele: An allele whose phenotype isdetectable (even if only weakly) in a single dose orcopy Recessive allele: An allele whose phenotype isapparent only in the homozygous or hemizygousstate. Heterozygous: Having a normal allele on onechromosome and a mutant allele on the other 13. More terms to know Hemizygous: Having half the number of alleles(e.g. males are hemizygous for all X chromosomegenes) Expressivity: The severity or intensity of thephenotype of an allele. Penetrance: The degree to which a geneexpresses any observable phenotype Locus (pl. loci): The position on a chromosome of agene or other chromosome marker; also, the DNAat that position. Proband: The first affected individual who comes toclinical genetic evaluation. Indicated by an arrow onthe pedigree diagram. 14. Major mutation types Single base substitutions that cause prematuretermination of protein synthesis, change of aminoacid, suppress termination of protein translation, alterlevel of gene expression, or alter patterns of mRNAsplicing Translocations, that bring disparate genes orchromosome segments together Deletions of a few nucleotides up to long stretches ofDNA Insertions and duplications of nucleotides up to longstretches of DNA Many different mutations can occur within a givengene, although it appears that genes have differentdegrees of mutability Different mutations affecting a gene can result indistinct clinical syndromes 15. Types of mutations Point mutations: Change of the normalbase to another Possible consequences: Silent mutation: No consequence Missense mutation: changes the codon to one encoding a different amino acid Nonsense mutation: Changes codon from one encoding an amino acid to a stop codon Splice site alteration: can abolish or create a splice site Regulatory region mutation: Can result in net increased or decreased gene expression 16. Location, location, location 17. Small mutations can have subtle or drasticeffects In frame deletion of one codo: No frameshift Deletion of one base: Frameshift Out of frame deletion ofthree bases:Frameshift 18. Clues that suggest a Mendeliandisease Positive family history Characteristic syndrome Unusual syndrome (e.g. progressive neurologic deterioration, multiple organ system abnormalities, intermittent neurologic symptoms) at any age Common syndrome at unusually early age Lack of environmental or other primary cause of symptoms and signs 19. Taking a family history Inquire about the health of each family memberthrough second degree relatives(grandparents, first cousins) Pay special attention to any signs or symptomsrelated to your patients condition in relatives Inquire about causes of any deaths, includingany stillbirths or early deaths,institutionalizations Obtain medical (and death) records of relativesas well as of proband Inquire about any possible consanguinity Recognize that false paternity does occur 20. Some essential nomenclature for chromosomal notationsComma (, )Separates chromosome numbers, sex chromosomes andchromosome abnormalities.del Deletion denotes both terminal and interstitial deletioninv Inversion of a part of a chromosomeMinus ( -)Chromosome loss, monosomyp Short arm of chromosomeq Long arm of a chromosomePhPhiladelphia chromosomet translocation 11/27/201220 21. Pedigree symbols 22. Constitutional karyotypeThere is a role of constitutional karyotype that predispose to malignancy.e.g.Down syndrome, trisomy 21- increased risk of developing acuteleukaemia.Klinefelters syndrome 47, XXY.Blooms syndromeFanconis anaemia.11/27/2012 22 23. Chromosomal and molecularabnormalities in leukaemiaDiseasegenesCorrespondin Method ofg proteinactivationAML t(8;21) (q22;q22)ETOAML-1 fusionAML, M3 t(15,17) (q22;q21) PMLPML-RARAfusionAML M4inv (16) (p13q22)MYH-11 CBF fusionCML t (9,22) (p34q11)ABLBcr-abl fusion 11/27/2012 23 24. Philadelphia chromosomePartial karyogram of chromosomes 9 and 22 from apatient with CML, showing t(9;22). The normal chromosome ofeach pair is on the left and the abnormal (translocated)chromosomes are on the right (arrowed)11/27/201224 25. The most consistent chromosomal abnormalityassociated with a haematological malignancy is thePhiladelphia chromosome (Ph).1. CML, this translocation is found in 92% of patients.2. BCRABL fusion gene.3. Breakpoints within BCR occur within a 5.8-kb region, termed the major breakpoint cluster region (M- BCR),4. This transcribes an aberrant 8.5-kb mRNA, encoding a chimeric p210 protein with enhanced tyrosine kinase activity.11/27/201225 26. AML t(8;21)1. Good-risk cytogenetic groups2. This translocation occurs predominantly in FAB type M2 (acute myeloid leukaemia with granulocytic maturation at or beyond promyelocyte stage) and M4 (myelomonocytic leukaemia). 3. It fuses the core binding factor alpha gene (CBF, AML1 or RUN1) onchromosome 21 with ETO on chromosome 8 to produce a novelchimeric gene.11/27/201226 27. AML inv (16)1. These abnormalities of chromosome 16 are found in AML M4 and are notable for their association with abnormal eosinophilia (M4Eo) and a good prognosis.2. Fusion of the smooth muscle myosin heavy-chain gene, MYH11, normally on 16p13 and CBF, normally on 16q22.3. FISH and RT- PCR have become routine detection methods. 11/27/2012 27 28. PML-RARA fusion gene11/27/2012 28 29. AML t(15,17)1. The translocation t(15;17)(q22;q21) (is specic for AML M3 and M3v (acute promyelocytic leukaemia).2. APML patients has a tendency to develop bleeding tendencies because of the presence of promyelocytes.3. patients < 35 years old.4. Rearrangement of the retinoic acid receptor alpha (RAR) gene, located at 17q21, which is fused to the PML gene on chromosome 15, a gene which is transcribed in normal haemopoietic cells.11/27/201229 30. 5. PML-RARA fusion transcript blocks the differentiation.6. The prognosis in these patients is good.7. Response to treatment with all-trans-retinoic acid (ATRA). This acts by converting the PMLRAR- fusion protein from a transcriptional repressor to a transcriptional activator, thus inducing terminal differentiation of the leukaemic clone. (i.e. Maturation of promyelocytes)8. ATRA has now been adopted as a component of rst-line therapy for this disease, highlighting the importance of accurate identication of this chromosomal abnormality.9. FISH and RT-PCR 11/27/201230 31. Other techniques: FISHFISH (uorescence in situhybridization).Four interphase nuclei showingindividual signals specic for theABL (red) and BCR (green) geneson the normal chromosomes 9 and22 respectively and the BCR/ABLfusion (red/green collocalizedsignals appearing yellow) indicatingthepresence of the Philadelphiachromosome. 11/27/2012 31 32. Other techniques: multiplex FISHKaryogram of anormalmale painted inseven colours insucha way as toproduce colouredbandsalong thechromosome arms(Rx-FISH)allowingidentication of theindividualchromosome pairs.11/27/2012 32 33. Beta ThalassaemiaDefinition: Thalassemia is inherited disorderscharacterized reduced or absent amounts of hemoglobin,the oxygen-carrying protein inside the red blood cells. 34. Two Basic Groups of ThalassemiaDisorder Alpha Thalassemia Beta Thalassemia: A person with thisdisorder has two mutated genes 35. There are 3 types of BetaThalassemia Thalassemia Minor Thalassemia Intermediate. Thalassemia Major or CooleysAnemia 36. Thalassemias In the case of beta thalassemias, in contrast to alpha -thalassemias, the most frequently encountered molecular abnormalities are point mutations and short insertions or deletions limited to a few nucleotides chain is still synthesized. The quantity ofTwo situations have clearly to beglobin chain, which is made, varies largelydistinguished: from one molecular defect to another, this In beta + thalassemias, the mutated chain may be structurally normal orgene encodes for a small amount of abnormalIn beta 0 thalassemias , the gene isnormal mRNA and, thus, a low amount unable to encode for any functional mRNAof and therefore there is no beta chain synthesize 37. Beta Thalasemia It is caused by a change in the gene for the betaglobin component of hemoglobin It can cause variable anemia that can range frommoderate to severe.Beta thalassaemia trait is seen most commonly inpeople with the following ancestry: Mediterranean(including North African, and particularly Italian andGreek), Middle Eastern, Indian, African, Chinese,and Southeast Asian (including Vietnamese,Laotian, Thai, Singaporean, Filipino, Cambodian,Malaysian, Burmese, and Indonesian 38. Symptoms of Beta Thalassemia It is characterize by severe anemia that canbegin months after birth Paleness Delays in growth and development Bone marrow expansion. Untreated Beta Thalassemia major can leadto child death due to heart failure. 39. Alpha and Beta Thalassemias The thalassaemia are, therefore,considered quantitative hemoglobindiseases. Because all types of thalassaemiaare caused by changes in either thealpha- or beta-globin gene. Thesechanges cause little or no globin to beproduced. 40. 11/27/2012 40 41. Types of mutation that can occur in globingenes and adjoining sequences. Point mutations -Within coding sequence, i.e. within an exon Within non-coding sequence, i.e. In an intron Mutation 5 or 3 to the gene (i.e. outside the gene) Deletion or duplication of one or more genes Deletion of genes with downstream enhancer being juxtaposed to remaininggene Duplication of a gene Triplication of entire a globin gene cluster Abnormal cross-over during meiosis leading to gene fusion Deletion of DNAsequences but without a frame shift in coding sequence Deletion plus inversion Deletion plus insertion Frame shift mutation Types of mutation that can occur in globin genes and adjoining sequences.11/27/201241 42. A famous pedigree 43. A nicer pedigree 44. A modest pedigree 45. X linked recessive, normal father,carrier mother1 carrier daughter1 normal daughter1 affected son1 normal son 46. Clotting cascade 47. Hemophilia A Clinical syndrome Easily prone to hemorrhage from minor trauma Hemarthroses common - result in degenerative joint disease Ecchymoses, but not petechiae Laboratory Prolonged PTT, normal PT & bleeding times Normal platelet function A treatable genetic disease: Plasma (90 % of thosetreated with donor blood products developed AIDS inthe 1980s), recombinant factor 8 (10 -15 % developantibodies) Over 620 different mutations known to affect the factorVIII clotting factor gene (allelic heterogeneity) Gene lies at Xq28 48. One common Factor VIIImutation 49. Knowing genetic syndromescan help prevent treatablecomplications of untreatablediseases 50. Ethnic background andgeographic origins areimportant risk factors for somegenetic conditions 51. Glucose-6-phosphate dehydrogenasedeficiency Common among Africans, Asians and around the Mediterranean Discovered that about 10 % of African American servicemen during WWII developed hemolytic anemia when given certain drugs, such as sulfonamides, antimalarials or when they ate fava beans Caused by deficiency of the enzyme, which is needed to generate NADPH 52. G6PD deficiency Affects the G6PD gene at Xq28 Many mutations and polymorphismshave been discovered Heterozygosity (technicallyhemizygosity) in women appears toconfer resistance to malaria 53. Prevalence of G6PD 54. Old World Malaria prevalence 55. X linked recessive, affectedfather2 carrierdaughters2 normal sons:Never any Male-to-Maletransmission!