Analyzing Genetic Variation

Analyzing Genetic Variation

Overview of Genetic Variation

  • A staggering amount of variation exists in human genomes.

    • Genome length may differ by as much as 1% in healthy individuals.

    • Comparison of genomes from only three people has revealed over 5 million single nucleotide polymorphisms (SNPs) as compared to the reference genome.

    • Small insertions or deletions exist at over 100,000 genomic sites when comparing the genomes of Watson and Venter.

Genetic Polymorphisms

  • DNA Polymorphisms: Two or more alleles found at a single DNA locus.

    • Allele: In a narrow sense, a form (variant) of a gene; in a broader sense, a variant at a given position of the haploid genome.

    • More than 5.6 million SNPs exist when compared to the human reference genome.

Types of Genetic Variation

  1. Single nucleotide polymorphisms (SNPs)

  2. Deletion-insertion polymorphisms (INDELs)

  3. Simple sequence repeats (SSRs)

  4. Copy number variants (CNVs)

Single Nucleotide Polymorphisms (SNPs)

  • SNPs are the most common type of genetic variant.

    • Occur every 1 kb on average.

    • Over 300 million SNPs have been identified in the human genome, predominantly biallelic.

    • Common SNPs: These are SNPs with an allele frequency of at least 1% (approximately 15 million present in the human genome).

  • Mechanisms leading to SNPs:

    • Spontaneous mutations, including errors during DNA replication, having a per-base mutation rate of less than 1 in 30-100 million per generation.

    • Exposure to mutagenic chemicals or radiation.

    • SNPs serve as useful chromosome address labels for genetic mapping, disease-causing genes identification, and population genetics studies.

  • Ancestral vs. Derived Alleles:

    • Ancestral alleles: Present in the ancestral genome.

    • Derived alleles: Resulting from relatively recent mutations.

    • SNPs can provide insights into evolutionary history, with certain loci (1 and 2) being invariant in the chimp genome.

Deletion-Insertion Polymorphisms (INDELs)

  • INDELs occur approximately once in every 10 kb.

    • Range from one to hundreds of base pairs, with short INDELs (one or two nucleotides) being the most common type.

    • Mechanisms leading to INDELs: Issues during DNA replication, recombination, or repair processes.

    • INDELs in coding sequences can result in frameshift mutations unless the number of nucleotides affected is a multiple of 3.

Simple Sequence Repeats (SSRs)

  • SSRs arise spontaneously from rare events and are expanded through faulty DNA replication.

    • Most SSRs do not cause phenotypic changes, though longer tracts of trinucleotide repeats can lead to conditions like fragile X syndrome and Huntington's disease.

    • SSRs are highly polymorphic in repeat number at a single locus, serving as stable yet variable DNA markers.

    • Commonly referred to as microsatellites or short tandem repeats (STR); account for approximately 3% of total DNA in the human genome.

    • Occur every 30 kb on average.

Examples of Trinucleotide Repeat Diseases

Huntington's Disease

  • An autosomal dominant neurodegenerative disorder affecting about 1 in 10,000 people.

    • Caused by an excessive number of SSRs in the HTT gene.

      • Normal alleles: Less than 36 CAG repeats.

      • Disease-causing alleles: More than 36 CAG repeats; complete penetrance occurs at 42 or more repeats.

    • Alleles produce mutant proteins detrimental to nerve cells.

Fragile X Syndrome

  • Causing intellectual disability, with an incidence of about 1 in 4000 males and 1 in 8000 females.

    • The X-linked FMR-1 gene contains CGG repeats in its 5' UTR.

    • Disease alleles have expanded repeat numbers, which impacts transcription and/or translation of the gene due to methylation.

Gene Therapy and Huntington's Disease

  • A one-time gene therapy has shown potential to slow disease progression by 75% over three years in a trial of 29 participants.

    • The therapy employs a harmless virus to deliver a microRNA designed to silence the defective huntingtin gene.

    • It requires procedural surgery for administration.

    • No significant side effects detected, but complications arose from the surgical procedure.

    • Similar gene therapy programs are evolving, aiming for genetic corrections through CRISPR technology.

Copy Number Variants (CNVs)

  • CNVs are large blocks of DNA repeats (10 bp to 1 Mb).

    • Over 10,000 CNVs have been found in the human genome, occurring once every 3 Mb.

    • Caused by unequal crossing over in meiosis, most CNVs are inherited rather than newly mutated.

Genetic Variation Categories

  • Single Nucleotide Polymorphisms (SNPs): 1 base pair changes.

  • Deletion-Insertion Polymorphisms (INDELs): insertions/deletions from a single base pair to several hundred base pairs.

  • Simple Sequence Repeats (SSRs): 1-10 base sequences repeated multiple times in tandem.

  • Copy Number Variants (CNVs): Variable copies of large genetic blocks up to 1 Mb.

Techniques to Detect Genetic Variation

  • Targeted Genotyping: Detects polymorphisms in specific regions.

  • High Throughput Genotyping: Detects polymorphisms genome-wide through various methods:

    • PCR Genotyping

    • Taqman SNP Assays

    • SNP Microarrays

    • Next Generation Sequencing (NGS)

Detection of SNPs and INDELs

  • Detection using PCR and DNA Sequencing: Amplify specific PCR regions and sequence them.

    • Example: Mutations in the Hbβ gene can lead to sickle cell anemia.

  • Detection of SSRs: Utilize PCR followed by gel electrophoresis to assess product size differences.

DNA Fingerprinting with SSRs

  • Utilize multiple SSR loci to create unique fingerprints, with a probability of 1 in 10^13 for a match among 13 unlinked loci.

    • Used in forensic science, paternity testing, and identification of human remains.

    • Parents share 50% of SSR alleles with their children.

    • The Combined DNA Index System (CODIS) is a database that collects data from felons and missing persons based on 13 SSR loci.

Prenatal Genetic Diagnosis

  • Uses PCR to analyze fetal DNA for genetic abnormalities via methods like:

    • Genotyping fetal cells in amniotic fluid (amniocentesis).

    • Genotyping fetal DNA from maternal blood (cell-free fetal DNA analysis).

    • Preimplantation embryo testing (IVF embryos).

Allele-Specific Probes

  • Allele-Specific Oligonucleotides (ASOs): Short hybridization probes that can identify single-base mismatches.

    • Stable hybrids under stringent conditions indicate no mismatches,

    • Unstable hybrids indicate a mismatch, highlighting the presence of SNP variability.

Taqman SNP Assays

  • Utilize allele-specific probes for SNP detection to monitor the presence of specific alleles through fluorescent signaling during PCR reactions.

High-Throughput SNP Genotyping with SNP Arrays

  • Arrays containing allele-specific oligonucleotides (ASOs) can detect SNPs by fluorescent output proportional to allele copies.

    • Example: Affymetrix Genome-Wide Human SNP Array features 1.8 million markers.

Databases for Genetic Information

  • dbSNP: Repository for human single nucleotide variations and small-scale insertions/deletions.

    • Presents publication references, population frequencies, molecular consequences, and genomic mapping data.

  • Additional databases: Ensembl, Santa Cruz, PharmGKB, etc.

Positional Cloning in Research

  • Uses DNA polymorphism markers closely linked to disease-causing genes to narrow the search for disease-associated genetic loci.

    • Classic Linkage Analysis: Two loci are linked if they are close together on the same chromosome.

    • Recombination frequency as a measure of distance (1 cM = 1% recombination frequency).

    • Statistical tools such as the Lod score help ascertain whether a marker is linked to a disease gene.

Identifying Genotype-Phenotype Associations

  • Investigators use pedigree data to assess SNP linkage to disease—restricting the search for disease-causing mutations based on inheritance patterns.

  • Genetic conditions like neurofibromatosis rely heavily on SNP genotyping and analysis of family pedigrees to establish connections between genotypes and phenotypes.

Implications of Genetic Research

  • Most genetic polymorphisms do not directly affect phenotypes and are often neutral variations that do not alter protein function.

  • Allelic Heterogeneity: A phenomenon where the same phenotype may arise from different mutations within the same gene, necessitating careful genetic mapping and analysis to pinpoint exact mutations responsible for conditions like cystic fibrosis.

Search for Disease-Causing Mutations: A Case Study

  • Nicholas Volker Case: A severe inflammatory bowel disease case leading to the discovery of a missense mutation in the XIAP gene that altered an evolutionarily conserved amino acid, which ultimately resulted in successful treatment through bone marrow transplant.

  • This case underscores the potential and power of genomic sequencing in diagnosing and treating genetic disorders.