Gene Mutations & Human Disease PDF

GENE MUTATIONS & HUMAN DISEASE Block: Foundations Block Director: James Proffitt, PhD Session Date: Wednesday, August 07, 2024 Time: 11:30 - 12:00 pm Instructor: Casey Romanoski, PhD Department: Cellular & Molecular Medicine Email: crom...

GENE MUTATIONS & HUMAN DISEASE Block: Foundations Block Director: James Proffitt, PhD Session Date: Wednesday, August 07, 2024 Time: 11:30 - 12:00 pm Instructor: Casey Romanoski, PhD Department: Cellular & Molecular Medicine Email: [email protected] INSTRUCTIONAL METHODS Primary Method: IM07: Discussion, Large Group (>12) ☐ Flipped Session ☐ Clinical Correlation Resource Types: RE18: Written or Visual Media (or Digital Equivalent) INSTRUCTIONS/READINGS Required: Review the LEARNING OBJECTIVES and NOTES prior to attending the session. LECTURE LEARNING OBJECTIVES 1. Identify the different classes of mutations in monogenic (Mendelian) genetic diseases. 2. Discuss the classification of gene mutations associated with cystic fibrosis. 3. Using cystic fibrosis as an example, explain how knowing the molecular effects of specific mutations in a genetic disease is used to select the most appropriate therapeutic options. CURRICULAR CONNECTIONS Below are the competencies, educational program objectives (EPOs), course objectives, session learning objectives, disciplines and threads that most accurately describe the connection of this session to the curriculum. Related Related Competency\EPO Disciplines Threads COs LOs CO-01 LO-01 MK-01: Core of basic Genetics H & I: Medical Genetics sciences CO-02 LO-02 MK-05: The altered Genetics H & I: Medical Genetics structure and function (pathology & pathophysiology) of the body/organs in disease CO-02 LO-03 MK-05: The altered Genetics H & I: Medical Genetics structure and function (pathology & pathophysiology) of the body/organs in disease Block: Foundations | ROMANOSKI [1 of 8] GENE MUTATIONS & HUMAN DISEASE USMLE 1 STUDY CONNECTIONS FIRST AID FOR THE USMLE STEP 1 – 2023 EDITION: Biochemistry – Genetics: pp. 57-58. CLINICAL CONTEXT There are multiple classes of diseases associated gene mutations. Knowing how each class leads to disease etiology provides insights into diagnosis, prognosis, and choices for therapeutic targets. LECTURE NOTES Contents I. Clinical Case Study II. Classes of Mutations Found Within the Genome III. Single Gene Mutations IV.Chromosomal Alterations V. Mutations and Human Disease VI.Clinical Case Discussion Clinical Case Study An Infant with Recurrent Respiratory Infections A six-month female infant is brought to the pediatric ED with a severe respiratory infection and is admitted to the children’s ICU. A chest X-ray followed by a bacterial culture confirms a diagnosis of staphylococcus aureus pneumonia. The child was born at home, did not receive newborn screening, and has had recurrent respiratory infections since birth. The mother says that the child has very thick mucus and smelly stools. The infection is treated with antibiotics, but the attending physician suspects that the child may have cystic fibrosis and orders a sweat chloride conductivity test. The test results come back positive, and the physician then orders a rapid targeted gene panel sequencing of the CFTR gene. CLASSES OF MUTATIONS FOUND WITHIN THE GENOME Single-Gene Mutations Single base changes (point mutations) silent, missense, nonsense – detected by genotyping and by short-read NEXGEN sequencing. Short insertions/deletions (indels) – detected by short-read NEXGEN sequencing. Gene copy number variants – detected by long-read sequencing. Short tandem repeat (STR) expansions – detected by long-read sequencing Chromosomal Alterations Translocations – detected by long-read sequencing. Inversions – detected by long-read sequencing. Duplications – detected by long-read sequencing. Block: Foundations | ROMANOSKI [2 of 8] GENE MUTATIONS & HUMAN DISEASE MUTATIONS & HUMAN DISEASE “Mendelian” monogenic genetic diseases are characterized by mutations in single genes or specific chromosomal aberrations, with a corresponding loss or gain of function of an essential protein or multiple proteins. In most cases, the disease-causing mutation is either dominant or recessive. Dominant mutations require that only one of the two homologous chromosomes carry the gene mutation for the disease to occur, while recessive mutations require that both homologous chromosomes carry a mutation in the same gene. If the mutations are on the 22 autosomes, the mutation is said to be autosomal, while mutations on the X or Y sex chromosomes are said to be sex-linked. Common X-linked monogenic diseases include Duchenne muscular dystrophy and hemophilia A, and Rett syndrome. Generally, these monogenic diseases display a comprehendible genotype-phenotype relationship. For example, in Duchenne muscular dystrophy, the disease- causing mutation occurs in the gene located on the X chromosome within the muscle cell structural gene dystrophin, which attaches the muscle cell membrane to the sarcomeres of the muscle cell cytoskeleton. However, even in these relatively “simple” cases, genetic variants in separate genes can modify the expression of a phenotype and cause a wide spectrum of disease severity in patients harboring the same monogenic mutation. Identification and characterization of single gene mutations that cause inherited human diseases was first carried out by a method referred to as haplotype mapping. A haplotype refers to a set of DNA sequence variants that tend to be inherited together because they are in close proximity together on the chromosome; thus, recombination between these variants is rare. Haplotype mapping is based on determining the physical proximity of one gene to another by observing how closely the two genes segregate together or remained linked as a unit during meiotic homologous recombination: thus, the higher the co- recombinational frequencies, the closer the proximity of the two genes. Two genes that always segregate together during recombination are said to be in linkage disequilibrium. Genes that are not in close proximity will segregate independently and are said to be in linkage equilibrium. Genes belonging to the same linkage group are said to be part of the same “haplotype.” By using reference genes scattered throughout the genome, along with family disease pedigrees, haplotype maps provided the first glimpse into the locations of disease-associated genes and which other genes were in general proximity. Complex multifactorial genetic disorders are the most common class of genetic diseases. Most diseases with an inherited component do not involve monogenic mutations inherited in a Mendelian manner. Diseases such as cancer, cardiovascular, metabolic, neurological, and immunological disorders that have a mixture of genetic, environmental, and age-related components. In these so-called complex multifactorial genetic disorders, there may be familial risk factors caused by multiple genetic variants, any one of which does not cause the disease by itself. Such variants are said to be of low penetrance. Penetrance is a term that is defined as the likelihood that a patient with a specific genotype will display the disease phenotype and is often given as a percentage or ratio. In a disease with 100% penetrance, all patients carrying the genotype will show the disease phenotype. Incomplete penetrance will cause only a percentage of individuals that have the genotype to display the phenotype. (Note: The term penetrance is not to be confused with the term variable Block: Foundations | ROMANOSKI [3 of 8] GENE MUTATIONS & HUMAN DISEASE expressivity, which refers to the level of expression of a disease phenotype, which can be due to a variety of factors including modifying genes, environment, and age. A disease with 100 percent penetrance may have significantly different severities of symptoms and age of presentation, or variable expressivity, in different individuals. SINGLE GENE MUTATIONS Point Mutations Point mutations (SNPs or SNVs) are single base substitutions that arise predominantly due to base modifying chemicals and ionizing radiation and DNA replication errors. If point mutations occur in the open-reading frame (ORF) of a protein-encoding gene, one of three results may occur. (1) If the mutation changes a codon, but due to the degeneracy of the genetic code, the amino acid corresponding to the codon is not changed, there is usually no effect. This is referred to as a synonymous mutation. (2) If the mutation results in a codon that codes for a different amino acid, it is referred to as a missense mutation. Missense mutations may or may not produce a phenotypic change in the protein. If the new amino acid is closely related in chemical properties to the amino acid normally encountered at that position in the protein (e.g., a glycine instead of an alanine, an arginine instead of a lysine, or glutamic acid instead of an aspartic acid), we call this a conservative substitution because the chemical properties (small aliphatic hydrocarbon, acid, base) are conserved. However, if the mutation results in a new amino acid with different chemical properties (non- conservative substitution), the function of the resultant protein may be significantly affected. (3) If the mutation changes a codon into a stop codon (also referred to as a nonsense codon), the mutation is referred to as a nonsense mutation. There are three nonsense codons (UGA, UAA, and UAG), which can be easily remembered by a device: UGA (U Go Away), UAA (U Are Away), and UAG (U Are Gone). It is difficult to predict the effects of missense, frameshift, and nonsense mutations: The effect of an amino acid substitution depends on many factors including the chemical nature of the amino acid and its location within the protein sequence. For nonsense mutations, there is an additional complexity. A common misconception is that a nonsense mutation within the coding region will lead to a truncated protein. However, such proteins would most likely be extremely toxic to the cell, often competing with the wild-type protein generated from the heterozygous allele on a homologous chromosome. To minimize the generation of toxic truncated proteins due to nonsense mutations and out-of-reading frame changes that result in stop codons, the cell has a mechanism for the recognition of nonsense codons in the exons of mRNAs that results in degradation of the mRNA before a toxic protein is translated; the process is called nonsense mediated decay (NMD). Thus, in most mutations that result in the generation of a nonsense codon, a truncated protein is not produced, although exceptions can occur. Insertion/Deletion (INDEL) Mutations Various processes including replication and recombination errors, and transposons can result in insertions and deletions (INDELs). INDELs can range from one or two nucleotides up to several hundred nucleotides. Unless by chance the INDEL is a multiple of three nucleotides, INDELS in the protein encoding exonic regions of genes will always result in a frame shift. Evolution has preserved the integrity of open reading Block: Foundations | ROMANOSKI [4 of 8] GENE MUTATIONS & HUMAN DISEASE frames of protein encoding genes by not placing nonsense codons in alternative exons. However, if a change in reading frame occurs due to an INDEL, there is no mechanism to prevent a stop codon from occurring in the new reading frame. Because there are three stop codons out of 64, without any evolutionary constraints, there is a probability that once every three out of 64 codons (approximately once every 20 base pairs) there will be a stop codon in the new reading frame; thus, generation of a premature stop codon is virtually guaranteed when an INDEL occurs within a protein-encoding portion of an exon. However, as in the case of stop codons generated by nonsense mutations, INDEL-generated premature stop codons rarely give rise to truncated proteins. Truncated proteins are very toxic to the cell because they can compete with the normal full-length proteins, causing dysfunctional protein assemblies. The cell guards against these toxic truncated protein by destroying mRNAs with premature stop codons using the nonsense-mediated decay (NMD) surveillance machinery before translation occurs. Short Duplications, Inversions and Translocations There are several different mechanisms that give rise to duplication of part or all of a gene or a noncoding region. As discussed earlier in the description of repetitive DNA within the human genome, gene duplications occur through transposon and recombination mechanisms throughout evolution, resulting in gene copy number variations (CNVs). DNA replication errors occur within short repetitive sequences that give rise to larger microsatellite DNA sequences, which can undergo further expansion and deletions. Short Tandem Repeat Expansions There are multiple loci in the human genome with multiple repeats of 2-10 base pairs with repeat sizes of less than 20 up to hundreds. These sequences are found in the 5’ and 3’ untranslated regions, protein coding regions, and in introns. The expansions are due to DNA replication errors and unequal crossing over during meiosis. Over 40 human diseases have been identified that are due to nucleotide repeat expansions. Figure 1. Nucleotide repeat expansion disorders (NREDs) are caused by expansions of short tandem repeats that may occur in any region of the gene’s coding or noncoding regions. There are over 40 known NREDs. Chromosomal Alterations Block: Foundations | ROMANOSKI [5 of 8] GENE MUTATIONS & HUMAN DISEASE Figure 2. Chromosomal Alterations. Source: Liu et al.(2021) J. Human Genet. 66: 879–885. Clinical Case Discussion Cystic fibrosis (CF) is one of the most common inherited single gene disorders. CF is inherited in an autosomal recessive manner. The mutations associated with CF are located in the CFTR gene, which is located on chromosome 7 at q31.2. The CF carrier frequency for individuals of European descent in the U.S. population is approximately 1/25. The prevalence of CF is 1/2500 to 1/3500 newborns of European descent in the U.S. The disease is much less common in other U.S. ethnic populations with 1/17,000 in African Americans and 1/31,000 in Asian Americans. CF among the most common diseases that is part of newborn screening across the world. For neonates, a blood sample is drawn from an infant’s heel by collecting a spot of blood onto filter paper; for older children and adults, a blood sample is obtained by standard venous puncture. For prenatal testing, a sample of amniotic fluid may be collected using amniocentesis or a chorionic villus sample. Recommendations by the American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG) have led to the adoption of a standard CF gene mutation panel. While over 2000 mutations in the CFTR gene, 23 of the most common mutations account for 99.9% of mutations encountered in the U.S. population. Many affected patients are compound heterozygotes, i.e., the patient carries two different pathological variants in the CFTR gene. In normal lung epithelial cells, the chloride channel encoded by the CFTR gene functions to allow chloride ions into the cells, prompting the absorption of water and production of normal hydrated mucus. Mutations in the CFTR gene can lead to the absence or inhibition loss of function of the CFTR channel leading water efflux through the epithelial cell lining and thickening of mucus, causing airway obstruction, and providing a rich environment for bacterial growth and leading to repeated respiratory infections. Block: Foundations | ROMANOSKI [6 of 8] GENE MUTATIONS & HUMAN DISEASE Figure 3. Mechanism of mucus dehydration in cystic fibrosis. Source: BioRender Genetic results from the gene panel sequencing revealed that the patient is heterozygous for the two common CFTR mutations. F508del and G551D; F508del is a deletion of the phenylalanine codon for amino acid 508 in exon 10, and G551D is a missense mutation where a glycine codon for amino acid 551 is mutated to an aspartic acid codon. Figure 4. The six classes of mutations in the CFTR gene. Source: ResearchGate The F508del mutation is the most common CF mutation in patients of European descent and the most common mutation in the U.S. population. F508del causes misfolding of the CFTR protein in the endoplasmic reticulum disrupting intracellular trafficking to the Golgi and leading to CFTR degradation. There are six classes of CFTR mutations, each of which requires a different therapeutic strategy. After resolution of the respiratory infection, the patient is placed on a regimen of DNase 1 nasal spray plus an anti-inflammatory drugs. Based on the gene mutation analysis, the triple combination drug, Elexacaftor-Tezacaftor-Ivacaftor is prescribed. A lung transplant may be an option later in the patient’s life. Practice Exam Questions 1. The most common mutation associated with cystic fibrosis is one that affects which one of the following? A. CFTR gene transcription B. CFTR protein stability C. CFTR intracellular trafficking Block: Foundations | ROMANOSKI [7 of 8] GENE MUTATIONS & HUMAN DISEASE D. CFTR mRNA stability E. CFTR protein synthesis 2. A two-base insertion mutation associated with familial breast cancer occurs in the first exon of the BRCA1 gene. Which one of the following is most likely? A. A BRCA1 protein with a length increased by one amino acid can be detected B. A BRCA1 protein with an entirely different sequence from the normal BRCA1 can be detected C. A BRCA1 mRNA with a length increased by two bases can be detected D. No BRCA1 mutant protein can be detected E. A BRCA1 protein with a shorter length can be detected 3. Using the table below (Figure 5), what is the classification for a point mutation where the codon AAA is changed to TAA? A. Missense mutation B. Nonsense C. Deletion D. Insertion E. Synonymous Figure 5. The Genetic Code. Source: BioRender Block: Foundations | ROMANOSKI [8 of 8]