The Molecular, Biochemical, and Cellular Basis of Genomic Disease PDF

The Molecular, Biochemical, and Cellular Basis of Genomic Disease 1  Objectives of This Lecture to illustrate the fundamental mechanism behind genetic variation to explain different type of mutations to describe the effect of mutation on protein functio...

The Molecular, Biochemical, and Cellular Basis of Genomic Disease 1  Objectives of This Lecture to illustrate the fundamental mechanism behind genetic variation to explain different type of mutations to describe the effect of mutation on protein function to describe the protein classification based on their expression patterns and tissue specificity to understand genetic heterogeneity and phenotypic heterogeneity to describe the relationships between the site of protein expression and disease to describe the diseases discussed in the lecture 2 Recommended Reading 3  Genetic Variation Among Humans Substantial variation exist among humans, for example, in skin color, height, hair color, eye color, blood pressure, susceptibility to certain diseases… How? Mutation: technically, ‘any change in DNA sequence’ In human genetics, mutations often refer to DNA changes that cause genetic disease; often affect only single genes; not microscopically observable (for example, through karyotyping or hybridization techniques); can take place in coding DNA or in regulatory sequences Germline cells (gamete producing): transferred to next generation Somatic cells (adult cells): are not transferred to next generation Allele: the alternative forms or versions of a gene; e.g. HbA or HbS Locus: location of a gene on a chromosome Polymorphism: existence of multiple alleles of a gene in the population 4  Molecular Basis of A Disease Molecular disease is a disease where pathology can be traced back to a single molecular factor (i.e. protein or a polypeptide) Cause? mutation, either inherited or acquired, is the primary cause of molecular disease Knowledge of molecular pathology is fundamental for therapy, management of genetic diseases, and understanding of normal functions Biochemical genetics studies the relationships between genes and the observable traits or phenotypes at the levels of protein production, protein function, biochemistry (e.g. modifications), and metabolism (e.g. turnover). 5  Types of Mutations Base-pair substitutions/point mutations Fig. 3.3 | Base-pair substitution. Missense mutations A, produce a single amino acid change, whereas nonsense mutations B, produce a stop codon in the mRNA. Stop codons terminate translation of the polypeptide. 6  Types of Mutations Base-pair(s) deletions or insertions Fig. 3.4 | Frameshift mutations 7  Types of Mutations Splice-site mutations 5' 3' Splice Sites: Consensus sequences recognized by the spliceosome, a complex of RNA and protein molecules responsible for splicing… Major sites are: 5' Splice Site (Donor Site): splice site at the beginning of the intron: GT (or GU for mRNA).. 3' Splice Site (Acceptor Site): splice site at the end of the intron: AG Fig. 3.5 | A, Normal splicing. B, C, Splice-site mutations. 8  Types of Mutations Base-pair substitutions/point mutations Base-pair(s) deletions or insertions Splice-site mutations Gene duplications/deletions Promoter mutations Mobile element insertion Expanded repeats 9  Dominant alleles and recessive alleles Say height allele is designated: H (tall) or h (short) Dominant: HH, Hh: tall Recessive: hh: short Fig. 3.1 | Punnett square illustrating a cross Fig. 3.2 | Punnett square illustrating a cross between HH and hh homozygote parents. between two Hh heterozygotes. 10  Dominant alleles and recessive alleles Parent: AaBb Let us assume, there are two genes, and each has two different alleles Based on Mendel's law of independent assortment: Parent: AaBb aa: 1/4 bb: 1/4 aabb : 1/16 Applicable if two genes are located on different chromosomes 11  Linked Loci Combinations during meiosis: A1C1 or A1C2: 50% A2C1 or A2C2: 50% A1B1 or A1B2: ? A2B1 or A2B2: ? > depends on physical distance between A and B loci Fig. 8.1 | Loci A and B are linked on the same chromosome, so alleles A 1 and B 1 are usually inherited together. Locus C is on a different chromosome, so it is not linked to A and B, and its alleles are transmitted independently of the alleles of A and B. 12  Linked Loci The genetic distance between two loci is measured in centimorgans (cM), in honor of T. H. Morgan, who discovered the process of crossing over in 1910. 1 cM = 1% recombination frequency. 13  Red Blood Cells; Structure and Function RBCs (Erythrocytes): a biconcave disc without a nucleus RBCs contain hemoglobin Each RBC contains ~270,000,000 hemoglobin units Each hemoglobin contains 4 heme units 2 α units 2 β units Active site of a heme unit has an Iron (Fe) ion; this is where O2 will attach. 14  Red Blood Cells; Structure and Function RBCs (Erythrocytes): a biconcave disc without a nucleus RBCs contain hemoglobin Each RBC contains ~270,000,000 hemoglobin units Each hemoglobin contains 2 α units 2 β units 4 heme units Active site of a heme unit has an Iron (Fe) ion; this is where O2 will attach. 15  Sickle Cell Disease α subunits are encoded by HBA on chromosome 16 β subunits encoded by the HBB gene on chromosome 11 Sickle cell disease: autosomal recessive disorder of hemoglobin where β subunit genes have a missense mutation that substitutes Valine for Glutamic acid at amino acid 6. The Glu6Val mutation in β-globin decreases the solubility of deoxygenated hemoglobin and causes it to form a sickle shape (see Fig. 11-5 , below). 16  Sickle Cell Disease Initially, oxygenation causes the hemoglobin polymer to dissolve/erythrocyte to regain its normal shape; repeated sickling and unsickling, however, produce irreversibly sickled cells that are removed from the circulation by the spleen: hemolytic anemia. Sickled erythrocytes occlude capillaries and cause infarctions (i.e. tissue death). gamma (γ) Replace defective beta unit (adult) with normal gamma unit (fetal) of the hemoglobin gamma (γ) 17  Importance of understanding disease at molecular level Autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood Transfusion independence Elimination of vaso-occlusive episodes (in the patient with SCD) Transfusion-dependent β-thalassemia (TDT), sickle cell disease (SCD) Frangoul et al., CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. The New England Journal of Medicine (2021) 18  Genotype vs Phenotype February 2nd, 2024 7,480 Assignment: 4,878 Check for 2025? Over 7,000 phenotypes Over 4,000 genes 19 OMIM = Online Mendelian Inheritance in Man https://www.omim.org/statistics/geneMap  Genotype vs Phenotype February 2nd, 2024 7,480 Assignment: 4,878 Check for 2025? Over 7,000 phenotypes Over 4,000 genes 3,419 Over 3,000 genes 888 319 associate with a 252 single phenotype 20 OMIM = Online Mendelian Inheritance in Man https://www.omim.org/statistics/geneMap  Effect of Mutation on Protein Function Mutations can cause disease through 4 different effects on protein function: loss-of-function of a protein gain-of-function of a protein acquisition of a novel property Misexpression: heterochronic expression of a protein (i.e. expression of a gene at the wrong time), ectopic expression of a protein (i.e. expression of a gene in the wrong place) Figure 11-1A HPFH, Hereditary persistence of fetal hemoglobin 21  Loss-of-Function Mutations alternations of a gene’s sequence by either substitution, insertion, deletion, or rearrangement can lead to loss-of-function of the gene mutations that cause loss of function: introduction of a premature stop codon in the coding sequence (nonsense) missense mutations in the coding sequence mutations that cause protein instability, reducing the protein abundance the severity of a disease can be correlated with the degree/amount of function lost 22  Loss of Protein Function Mutations Thalassemia The term thalassemia is derived from the Greek word thalassa (“sea”).. Because first described in populations living near the Mediterranean Sea.. also common in portions of Africa, the Mideast, India, and Southeast Asia. α-thalassemia: α-globin chains are deficient, so β chains (or γ chains in fetus) are in excess, forming β- or γ-homotetramers, with greatly reduced oxygen-binding capacity (hypoxemia). β-thalassemia: β-globin chains are deficient, forming α-homotetramers that precipitate (i.e. form solid) and damage the cell membranes of RBC precursors.. premature erythrocyte destruction and anemia. So, in thalassemia, mutations reduce α globin or β globin quantity!  Loss-of-Function Mutations The loss of function of a gene may result from alteration of its coding, regulatory, or other critical sequences due to nucleotide substitutions, deletions, insertions, or rearrangements. Examples of mutations that leads to some forms of hemoglobinopathies:. C->T (Gln39Stop) creates a nonsense mutation in β-globin that leads to cause β-thalassemia. Promoter mutations that decrease expression of the β-globin mRNA to cause β-thalassemia.. Deletions of the α-Globin Genes that lead to α-thalassemia 24  Loss of Protein Function Mutations Other examples: Monosomies: 45X (X is partially or completely missing); called Turner syndrome.. Phenotypes are variable; can result in a variety of medical and developmental problems, including short stature, failure to form ovaries, heart defects, and certain learning disabilities,… Tumor Suppressor Mutations: e.g. p53 (encoded by TP53 gene in human).. found in all forms of human tumors… incidence: >50% of all human tumors Retinoblastoma is caused by deletions in a tumor-suppressor gene RB1 (RB Transcriptional Corepressor 1, also called Retinoblastoma-Associated Protein) 26 what is name for this type of mutation? 27  Gain-of-Function Mutations alternations of a gene’s sequence can enhance the normal functions of a protein gain-of-function mutations can: enhance one normal function of a protein e.g. gain-of-function mutation in FGFR3 leads to short stature disorder called achondroplasia increase production of a normal protein e.g. trisomy 21 (Down syndrome) 28  Gain-of-Function Mutations Charcot-Marie-Tooth disease type 1A (CMT1A): > duplication of PMP22 (Peripheral Myelin Protein 22) gene on chromosome 17; mutation leads abnormal protein production.. damages myelin sheath (protective covering around nerve fibers). > Inherited neurological disorder characterized by a progressive loss of muscle tissue and touch sensation across various parts of the body. > difficulties with balance, gait, and fine motor skills. Other symptoms may include foot deformities, decreased ability to run, difficulty lifting the foot at the ankle (foot drop), awkward or higher than normal step (gait), and frequent tripping or falling. Hb (Hemoglobin) Kempsey: > single amino acid substitution, beta-globin: Asp(Aspartic acid)99Asn(Asparagine). > disturb heme–heme interactions at α1/β1 subunit interaction point, > high Oxygen affinity  Novel Property Mutations a change in the amino acid residue can cause disease by conferring a novel property on the protein without altering its normal functions e.g. sickle cell disease (β-globin Glu6Val mutation): ▪ mutation does not affect the ability of sickle hemoglobin to transport oxygen ▪ mutated globin chains aggregate to form polymeric fibers that deform red blood cells 30  Mutations Associated with Heterochronic or Ectopic Gene Expression mutations alter the regulatory regions of a gene to cause inappropriate expression at an abnormal time (heterochronic) or place (ectopic) e.g. > 1) Oncogenes (KrasG12D; A glycine [G] to aspartate [D] mutation) result in malignancy > 2) Mutations that disrupt binding site of transcriptional silencer enabling expression of fetal γ-globin in adults in hereditary persistence of fetal hemoglobin (HPFH ) NuRD: Nucleosome Remodeling and Deacetylase Complex LCR: Locus Control Region, NF-Y: Nuclear transcription factor Y Benign, asymptomatic anomaly 31  Ways for Mutations to Disrupt the Synthesis or Function of A Normal Protein 1> 2> 3> 4> 5> 6> 7> body can't process the amino acid methionine. 8> 32 how to classify mutations that disrupt cell and organ functions? 33 Protein Abnormalities and Genetic Diseases examples of mutations in functional class of proteins that lead to genetic disorders 34  Classification of Proteins 2 classes of proteins based on their expression patterns: housekeeping proteins: present in virtually every cell fundamental for the maintenance of cell structure and function account for 90% of the mRNAs expressed in human tissue-specific specialty proteins: expressed in only one or a limited number of cells types have unique functions account for 10% of mRNAs expressed in human 35 Relationship Between Site of Proteins Expression and Disease mutation in a tissue-specific protein may cause: disease restricted to that tissues (e.g. sickle cell disease) disease in a secondary site, where the protein is NOT expressed e.g. phenylketonuria (PKU): caused by the absence of phenylalanine hydroxylase in the liver the brain (which does not express the enzyme), but not liver, is damaged 36  Relationship Between Site of Proteins Expression and Disease (Cont’d) mutations in housekeeping proteins rarely cause panoramic pathological changes: mutations in some housekeeping proteins are lethal e.g. mutations in actin and DNA polymerase are not compatible with live birth in most cases, pathologies caused by mutations in housekeeping proteins are limited to one or few tissues potentially due to: genetic redundancy: other genes with overlapping activities reduce the impact of the loss-of-function of the mutant gene the abundance of protein expressed: e.g. Tay-Sachs disease: the enzyme, hexosaminidase A (helps break down fatty substances), is expressed ubiquitously (i.e. in all cells); plays critical role in CNS; most disorders retain residual enzymatic activity absence of the enzyme leads to a fetal neurodegeneration leaving non-neuronal cells intact 37  Genetic Heterogeneity vs. Pleiotropy different mutations can result in the same or similar phenotype genetic heterogeneity is a phenomenon in which a single phenotype or a genetic disorder is caused by mutations in one of a multiple number of alleles or loci pleiotropy is a phenomenon in which mutation of a single gene leads to multiple phenotypes or genetic disorders 38