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Molecular basis of
hematology Chromosome nomenclature 2
The normal somatic cell has 46 chromosomes and is called diploid
ova or sperm have 23 chromosomes and are called haploid. The chromosomes occur in pairs and are numbered 1–22 in decreasing size order; there are two sex chromosomes, XX in females, XY in males. Karyotype is the term used to describe the chromosomes derived from a mitotic cell which have been set out in numerical order. A somatic cell with more or less than 46 chromosomes is termed neuploid; more than 46 is hyperdiploid less than 46 hypodiploid; 46 but with chromosome rearrangements, pseudodiploid. Each chromosome has two arms: the shorter called ‘p’, The longer called ‘q’. These meet at the centromere and the distal ends of the chromosomes are called telomeres. On staining each arm divides into regions numbered outwards from the centromere and each region divides into bands When a whole chromosome is lost or gained, a − or + is put in front of the chromosome number. If part of the chromosome is lost it is prefixed with del (for deletion). If there is extra material replacing part of a chromosome the prefix add (for additional material) is used. Chromosome translocations are denoted by t, the chromosomes involved placed in brackets with the lower numbered chromosome first. The prefix inv describes an inversion where part of the chromosome has been inverted to run in the opposite direction. An isochromosome, denoted by i, describes a chromosome with identical chromosome arms at each end; for example, i(17q) would consist of two copies of 17q joined at the centromere. Telomeres 2
Telomeres are repetitive sequences at the ends of chromosomes.
They decrease by approximately 200 base pairs of DNA with every round of replication. When they decrease to a critical length, the cell exits from cell cycle. Germ cells and stem cells, which need to self‐renew and maintain a high proliferative potential, contain the enzyme telomerase, which can add extensions to the telomeric repeats and compensate for loss at replication, and so enable the cells to continue proliferation. Telomerase is also often expressed in malignant cells but this is probably a consequence of the malignant transformation rather than an initiating factor. Anatomy of the gene1
Structure of DNA DNA is a complex, double-stranded molecule composed of nucleotides. Each nucleotide consists of a purine (adenine or guanine) or pyrimidine (thymine or cytosine) base attached to a deoxyribose sugar residue. Each strand of DNA is a succession of nucleotides linked through phosphodiester bonds between the 5` position of the deoxyribose of one nucleotide and the 3` position of the sugar moiety of the adjacent nucleotide. The two strands are connected through hydrogen bonds between strict pairs of purines and pyrimidines; that is, adenine must be paired with thymine (A-T) and guanine must be paired with cytosine (G-C). This is known as Watson–Crick base pairing. Consequently, the two strands of DNA are said to be complementary, in that the sequence of one strand determines the sequence of the other through the demands of strict base pairing. The two strands are joined in an antiparallel manner so that the 5` end of one strand is joined with the 3` end of the complementary strand. The strand containing the codons for amino acid sequences is designated as the sense strand, whereas the opposite strand that is transcribed into messenger RNA (mRNA) is referred to as the antisense strand. Structure of the gene
DNA dictates the biologic functions of the organism by the
flow of genetic information from DNA to RNA to protein. The functional genetic unit responsible for the production of a given protein, including the elements that control the timing and the level of its expression, is termed a gene. The gene contains several critical components that determine both the amino acid structure of the protein it encodes and the mechanisms by which the production of that protein may be controlled. The coding sequence, which dictates protein sequence, is contained within exons; these stretches of DNA may be interrupted by intervening noncoding sequences, or introns. In addition, there are flanking sequences in the 5` and 3` ends of the coding sequences that often contain important regulatory elements that control the expression of the gene. Genes are arrayed in a linear fashion along chromosomes, which are long DNA structures complexed with protein. Within chromosomes, DNA is bound in chromatin, a complex of DNA with histone and nonhistone proteins that “shield” the DNA from the proteins that activate gene expression. Flow of genetic information 1 Transcription
RNAs are mostly single-stranded molecules that differ from
DNA in two ways: by a sugar backbone composed of ribose rather than deoxyribose and by containing the pyrimidine uracil rather than thymine. The first step in the expression of protein from a gene is the synthesis of a premessenger RNA (pre-mRNA). The transcription of pre-mRNA is directed by RNA polymerase II, which in conjunction with other proteins generates an RNA copy of the DNA sense strand. This transcribed mRNA is complementary to the DNA antisense strand. The pre-mRNA contains the sequences of allof the genes exons and introns. The introns are then removed by a complex process called mRNA splicing. This process involves the recognition of specific sequences on either side of the intron that allow its excision in a precise manner that maintains the exon sequence. Splicing is controlled by the spliceosome, a large complex of proteins and five small nuclear ribonuclear proteins (snRNPs). mRNA splicing is an important mechanism for generating diversity of the proteins produced by a single gene. Some genes exhibit alternative splicing, a process by which certain exons are included in or excluded from the mature mRNA, depending on which splice sequences are used in the excision process. Mutations in the sequences of either introns or exons can derange the splicing process by either creating or destroying a splice site so that the intron sequence is not removed or the exon sequence eliminated. If abnormal splicing results in a premature stop codon (nonsense mutation), then a surveillance pathway known as nonsense mediated decay may result in degradation of the abnormal mRNA. This mechanism generally applies to stop codon mutations in the first one-third to one- half of the mRNA and works to prevent synthesis of mutant peptides. When mutations occur in the last one-third of the mRNA molecule, abnormal peptides may be produced. Translation
The mature mRNA is transported from the nucleus to the cytoplasm, where it undergoes translation into protein. The mRNA is “read” in a linear fashion by ribosomes, which are structures composed of ribonucleoprotein that move along the mRNA and insert the appropriate amino acids, carried by transfer RNAs (tRNAs), into the nascent protein. The amino acids are encoded by three base triplets called codons, the genetic code. The four bases can encode 64 possible codons; because there are only 20 amino acids used in protein sequences, more than one codon may encode the same amino acid. For this reason, the genetic code has been termed degenerate. An amino acid may be encoded by more than one codon; however, any single codon encodes only one amino acid. The beginning of the coding sequence in mRNA is encoded by AUG codon that has variable translation initiation activity determined by the neighboring nucleotide sequences (Kozak sequence). In addition, there are three termination codons (UAA, UAG, and UGA) that signal the end of the protein sequence. Single base-pair alterations in the coding sequence of genes may have a range of effects on the resultant protein. Because the genetic code is degenerate, some single base-pair changes may not alter the amino acid sequence, or they may change the amino acid sequence in a manner that has no effect on the overall function of the protein; these are predicted to be phenotypically silent mutations. Other mutations may change a codon to a termination codon, resulting in premature termination of the protein (nonsense mutation). Finally, single or multiple base-pair insertions or deletions can disrupt the reading frame of genes. These frame shift mutations render the gene incapable of encoding normal protein. These latter two abnormalities account for some β-thalassemias and for polycythemia due to a gain of function in the erythropoietin receptor. Clinically important mutations also may occur in the noncoding region of genes, such as in the regulatory elements upstream of the initiation codon or within intronic splicing sites. Control of gene expression
biologic processes are critically dependent on gene regulation, the control of
gene expression such that proteins are produced only at the appropriate time within the appropriate cells. Gene regulation is the result of a complex interplay of specific sequences within a gene locus, chromatin, and regulatory proteins (transcription factors) that interact with those sequences to increase or decrease the transcription from that gene. DNA sequences that lie in proximity to and regulate the expression of genes, which encode protein, are termed cisacting regulatory elements. Nearly all genes have a site for binding RNA polymerase II that is within the first 50 bases 59 to the structural gene and is called the promoter region. Other sequences that regulate the level of transcription of the gene are located at less predictable distances from the structural gene. Such sequences may increase (enhancers) or decrease (silencers) expression. A special type of enhancer is locus control region (LCR), which was first and best defined in the β-globin cluster of genes on chromosome 11. It is located approximately 50 kilobases (kb) upstream from the β-globin gene, controls all genes in the β-globin locus, and also has a strong tissue- specific activity (erythroid specific). Control of gene expression is exerted through the interaction of the cis-acting elements described previously with proteins that bind to those sequences. These nuclear DNA binding proteins are termed transcription factors. Most of these proteins have a DNA binding domain that can bind directly to regulatory sequences within the gene locus; many of them contain common motifs, such as zinc-fingers or leucine zippers, which are shared by many transcription factors. In addition, they frequently have unique domains that allow them to interact with other transcription factors. Thus, a complex pattern is emerging whereby the expression of different transcription factors, which may interact both with one another and with specific regions of DNA to increase or decrease transcription, determines the unique tissue, and stage-specific expression of the genes within a given cell. Epigenetics
For a gene to be expressed, chromatin must be unwound and the DNA made more accessible to regulatory proteins. This is controlled by epigenetic processes, or modifications to the genome that regulate gene expression without altering the underlying nucleotide sequence. These changes may be modulated by nutrition or drugs and may be heritable. Two common forms of epigenetic changes are DNA methylation and histone modifications. DNA methylation
In addition to being complexed with protein, the DNA of inactive genes is
modified by the addition of methyl groups to cytosine residues. Methylation normally occurs throughout the genome. It is generally a marker of an inactive gene, and changes in gene expression often can be correlated with characteristic changes in the degree of methylation of the 5 ̀ regulatory sequences of the gene. This type of epigenetic modification is performed by enzymes called DNA methyltransferases and is associated with alterations in gene expression and processes, such as X chromosome inactivation, imprinting, and carcinogenesis. Some human genes are transcriptionally active on only one copy of a chromosome (such as the copy inherited from the father), whereas the other copy of the chromosome inherited from the mother is transcriptionally inactive. This mechanism of gene silencing is known as imprinting, and these transcriptionally silenced genes are said to be “imprinted.” When genes are imprinted, they are usually heavily methylated in contrast to the nonimprinted copy of the allele, which typically is not methylated. As DNA methylation modulates gene activity, aberrant methylation may contribute to cancer. For example, in one form of hereditary colorectal cancer, methylation of the promoter region of the MLH1 gene, whose protein product repairs damaged DNA, results in colon cancer. Likewise, methylation- associated silencing of the DNA repair gene BRCA1 is associated with breast and ovarian cancers Small molecule inhibitors of DNA methyltransferases (eg, 5-azacitidine, decitabine) are used in the treatment of hematologic disorders that are characterized by aberrant DNA methylation (eg, myelodysplastic syndrome [MDS], acute myeloid leukemia [AML]). Histone modification
Histones are DNA packaging proteins that organize DNA into structural units called nucleosomes. Histones are subject to multiple modifications, including methylation, acetylation, ubiquitination, phosphorylation, and others Like methylation, histone modifications regulate gene activity and therefore disruptions of the normal pattern of these modifications can contribute to cancer and other diseases. For example, hypoacetylation of histones H3 and H4 are associated with silencing of the cell cycle regulator p21WAF1, a gene whose expression is reduced in multiple tumor types. Small molecule inhibitors of the enzyme that removes acetyl groups from histone tails histone deacetylases) are being tested in a variety of hematologic malignancies, and the histone deacetylase inhibitor Vorinostat is used in the treatment of cutaneous T-cell lymphoma. Molecular basis of hematological malignancies Introduction3
Cellular growth and differentiation are carefully controlled processes that are regulated by several interconnected pathways in order to facilitate diverse biological phenomena ranging from development, responses to normal and abnormal stimuli and the replacement of dying cells. Oncogenesis represents the progressive corruption of this order and the stepwise escape of an individual cell and its progeny from checks on their growth. This corruption occurs in the form of the serial acquisition of genetic mutations, which disrupt the genome of the fateful cell and morph it into a cancer genome. Oncogenes
Oncogenes are genes that have the potential to cause cancer, and they arise from mutations in their normal counterparts termed proto-oncogenes. Proto-oncogenes generally code for proteins or ncRNAs that regulate such processes as proliferation and differentiation, and activating mutations or epigenetic modifications that increase the expression or enhance the function of these genes confer a growth or survival advantage to a cell. A classic example of an oncogene is the BCR-ABL1 fusion gene found in chronic myelogenous leukemia (CML). This fusion results from a translocation between the BCR gene on chromosome 9 and the ABL1 proto-oncogene on chromosome 22 and confers constitutive activation of ABL1 and enhanced cell proliferation. Tumor suppressors
In contrast to oncogenes, tumor suppressors are genes that encode for proteins or ncRNAs whose normal function is to inhibit tumor development through the promotion of such processes as apoptosis, DNA repair, cell cycle inhibition, cell adhesion, and others. Loss of the expression or function of these genes is associated with cancer, and generally both copies of the tumor suppressor gene must be altered to promote neoplasia. Tyrosine kinases 2
These are enzymes which phosphorylate proteins on tyrosine residues and they are important mediators of intracellular signalling. Mutations of tyrosine kinases underlie a large number of haematological malignancies and they are the targets of many extremely effective new drugs. Common examples : include ABL1 in chronic myeloid leukaemia (CML) JAK2 in myeloproliferative neoplasms FLT3 in AML KIT in both systemic mastocytosis and AML Bruton kinase in chronic lymphocytic leukaemia and other lymphoproliferative disorders. Classes of DNA mutations3
Mutations that change the nucleotide sequence of DNA are broadly classified as substitutions and indels (insertions or deletions). When substitutions affect coding exons, they can be synonymous (no change in the coded amino acid), missense (change of the coded amino acid to another amino acid) or nonsense (change of the coded amino acid to a stop codon). if the size of an in/del is not a multiple of three nucleotides the resulting change is a frameshift mutation. Frameshift mutations lead to the translation of a novel aberrant sequence of amino acids terminating at an endogenous stop codon sequence. When both substitutions and indels do occur outside coding exons.When they affect splice junctions of exons, they can lead to reduced usage or complete exclusion of an exon from the final mRNA. When they occur elsewhere in the genome they may not have a significant functional effect, but in some cases they can affect gene regulatory sequences. Mutations altering the copy number of regions of the genome are referred to as amplifications and deletions. They can affect part of a gene, several genes, large chromosome segments or whole chromosomes. Chromosomal translocations and inversions are a recurrent type of structural genomic rearrangement in haematological and other cancers. With these mutations, two regions of the genome situated far away from each other are juxtaposed as a result of breakages in their host chromosomes, followed by illegitimate fusions between them. These events can result in the formation of a fusion oncogene, such as BCR-ABL1 in the t(9;22) in CML or lead to marked over-expression of a gene such as CCND1 or MYC in the t(11;14) and t(8;14) respectively . In each of the latter two cases, the target gene is over-expressed as a result of coming under the control of the strong IGH promoter in mantle and Burkitt lymphomas, respectively. From genotype to phenotype
Regardless of how mutations are generated, most of them will not
impart a growth advantage and are deemed to be ‘passengers’ In most cancers only a minority of mutations are ‘drivers’, a term used to signify mutations that impart oncogenic properties to the host cell(s) and drive cancer growth; either by activating oncogenes or inactivating tumour suppressor genes. NOTCH-signalling and lymphoid malignancies
NOTCH1 is a cell surface receptor with a central role in T-lymphopoiesis.
It regulates interactions between adjacent cells and is activated by ligand binding which triggers heterodimerization of the receptor and proteolytic cleavage of an intracellular peptide (ICN1=intracellular domain of NOTCH1) that migrates to the nucleus where it affects DNA transcription. In the bone marrow, NOTCH1 signalling is required to promote the generation of T-lymphoid precursors from early haemopoietic progenitors, at the expense of B-lymphopoiesis. NOTCH1 pathway mutations
Point mutations affecting the heterodimerization domain of
NOTCH1 result in ligand independent activation while truncating mutations of the C-terminal regulatory PEST domain result in increased half-life of ICN1. NOTCH1 is involved in the chromosomal translocation t(7;9), which results in the expression of a truncated and constitutively active form of the protein. loss-of function mutations of FBXW7, protein ligase that targets ICN1 for degradation, similarly result in its constitutive activation. The Core - Binding Factor Complex And AML
These principles are exemplified by the core - binding factor (CBF) complex, comprising RUNX1 (also known as AML1 or CBF α ) and CBF β . The normal CBF dimer recognizes and binds specific DNA sequences through the RUNX1 subunit and regulates the expression of many genes important for the differentiation of haemopoietic cells The genes for the two CBF subunits represent the most commonly involved genes in acute leukaemia translocations, with : t(12;21) ( ETV6 – RUNX1 ) found in 25% of childhood ALL, t(8;21) ( RUNX1 – ETO ) in 15% of AML, and inv(16) ( MYH11 –CBFB ) in 10% of AML. It appears that the leukaemogenic effects of these fusion genes are mediated in large part through dominant - negative inhibition of CBF function For example, in vitro experiments confirm that binding of wild - type RUNX1 to gene enhancers is inhibited in the presence of RUNX1 – ETO . Moreover, mice carrying the RUNX1 – ETO fusion gene and a wild - type RUNX1 allele have exactly the same embryonic lethal phenotype as mice completely lacking RUNX1 . The fusion partners of the CBF genes appear to recruit nuclear co-repressor complexes, leading to transcriptional inhibition of target genes. In fact, in the case of the RUNX1 – ETO fusion, the ETO moiety recruits a complex comprising three proteins, NcoR, SIN3 and a histone deacetylase, resulting in histone deacetylation, a change in chromatin structure and target gene repression. Similarly, the CBF β – MYH11 fusion protein of inv(16) forms a complex with the normal RUNX1 subunit through the CBF β moiety, and recruits several transcriptional repressors via the MYH11 component The end result is similar for both fusion genes, namely transcriptional silencing of genes required for normal differentiation and a consequent maturation arrest. Reference
