Jumping Into Your Gene Pool: Understanding Genetic Test Results
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1 Jumping Into Your Gene Pool: Understanding Genetic Test Results Susan A. Berry, MD Professor and Director Division of Genetics and Metabolism Department of Pediatrics University of Minnesota
2 Acknowledgment American Society of Clinical Oncologists Curriculum - educational materials used for this session (plus a few of my own!) Look for Understanding Gene Testing - National Cancer Institute, 1996
3 Chromosomes, DNA, and Genes Gene Cell Nucleus Chromosomes Protein Adapted from Understanding Gene Testing, NIH, 1995
4 The DNA Double Helix Sugar phosphate backbone Bases Base pair Adenine (A) Thymine (T) Cytosine (C) Guanine (G)
5 Genes are on chromosomes p Centromere q Chromosome 5
6 The Human Genome 23 pairs of chromosomes made of 700 million base pairs Extragenic DNA l Repetitive sequences l Control regions l Spacer DNA between genes l Function mostly unknown 70% 30% 20,000-25,000 genes = the exome
7 The Cell Cycle M (mitosis) G1 (cell growth) G2 S (synthesis) G0 (resting)
8 Mitosis and Meiosis Mitosis 2N 2N Meiosis (germ cells only) N N N N
9 Genetic Code A codon is made of 3 base pairs 64 codons total 1 codon (AUG) encodes methionine and starts translation of all proteins 61 codons encode 20 amino acids (redundant code) 3 codons stop protein translation A U G Met G C A Ala U A A
10 DNA Transcription and Translation Growing chain of amino acids mrna Ribosome Protein DNA Nuclear membrane Cell membrane Adapted from Understanding Gene Testing, NIH, 1995
11 Gene Structure RNA transcription start site Splice sites Stop site Promoter Exon 1 Intron Exon 2 Intron Exon 3 5' end 3' end Exon 1 Exon 2 Exon 3 mrna
12 RNA Processing DNA Exon Intron Exon Intron Exon Transcription Primary mrna Mature mrna Processing Translation Protein
13 Alleles: What are they!? A a a a A a A a a a a a Alleles: variant forms of the same gene (A a)
14 Autosomal Recessive Inheritance Noncarrier individual Non-affected carrier Affected individual Two germline mutations (one from each parent) to express the condition Equally transmitted by men and women
15 Your Dr. wants to phase your family. Why? Child s result? Is it this? or This?
16 Carrier Frequency Prevalence of an altered disease gene in a given population Carrier frequency = 20%
17 Founder Effect A high frequency of a specific gene mutation in a population founded by a small ancestral group Original population Marked population decrease, migration, or isolation Generations later
18 Environmental Factors Affecting Genes Modifier genes Carcinogens Response to DNA damage Hormonal/ reproductive factors Not every altered gene has the same effect on each person that inherits it
19 Genotype/Phenotype Correlation Different mutations in the same gene cause different effects Where is the genetic change in the sequence? What kind of change is it?
20 Variations in DNA sequence: What kinds are there? A variant is a change in the normal base pair sequence Can cause a change that does NOT alter protein function OR We might not be able to tell if the change is harmful OR Can be a change that makes a protein not work
21 Disease-Associated (Pathogenic) Variants Alter Protein Function Functional protein Nonfunctional or missing protein
22 Point Mutations Normal Missense Nonsense Frameshift (deletion) Frameshift (insertion) THE BIG RED DOG RAN OUT. THE BIG RAD DOG RAN OUT. THE BIG RED. THE BRE DDO GRA. THE BIG RED ZDO GRA. Point mutation: a change in a single base pair
23 Silent Sequence Variants Normal mrna Protein A U G Met A A G Lys U U U Phe G G C Gly G C A Ala U U G Leu C A A Gln Sequence variant mrna Protein A U G Met A A G Lys U U U Phe G G U Gly G C A Ala U U G Leu C A A Gln Sequence variant: a base pair change that does not change the amino acid sequence Adapted from Campbell NA (ed). Biology, 2nd ed, 1990
24 Missense Mutations Normal mrna Protein A U G Met A A G Lys U U U Phe G G C Gly G C A Ala U U G Leu C A A Gln Missense mrna Protein A U G Met A A G Lys U U U Phe A G C Ser G C A Ala U U G Leu C A A Gln Missense: changes to a codon for another amino acid (can be harmful or neutral) Adapted from Campbell NA (ed). Biology, 2nd ed, 1990
25 Nonsense Mutations Normal mrna Protein A U G Met A A G Lys U U U Phe G G C Gly G C A Ala U U G Leu C A A Gln Nonsense mrna Protein A U G Met U A G U U U G G C G C A U U G C A A Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein Adapted from Campbell NA (ed). Biology, 2nd ed, 1990
26 Frameshift Mutations Normal mrna Protein A U G Met A A G Lys U U U Phe G G C Gly G C A Ala U U G Leu C A A Gln mrna Frameshift A U G A A G U U G G C G C A U U G C A A Protein Met Lys Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein Leu Ala Adapted from Campbell NA (ed). Biology, 2nd ed, 1990
27 Splice-Site Mutations Exon 1 Intron Exon 2 Intron Exon 3 Exon 2 Altered mrna Exon 1 Exon 3 Splice-site mutation: a change that results in altered RNA sequence
28 Other Types of Mutations Mutations in regulatory regions of the gene Large deletions or insertions Chromosome translocations or inversions
29 Deciphering Clinical Gene Tests MMMMM Good! D G G E SSCP P C R
30
31 Preparing DNA for Analysis Blood sample Centrifuge and extract DNA from white blood cells DNA for analysis
32 Polymerase Chain Reaction (PCR) Isolate and denature DNA Anneal and extend primers Repeat as necessary Amplified segments Sequence to be amplified
33 Principle of Microarray (Chip) Assay Prehybridization Posthybridization Synthetic DNA probes Probes with hybridized DNA
34 By Suspencewl - Own work, CC0,
35 Pathogenic: associated with known disease state Likely pathogenic: strong evidence in favor of pathogenicity Uncertain ( VUS ): limited and/or conflicting evidence of association with disease Likely benign: strong evidence against pathogenicity Benign: very strong evidence against pathogenicity
36 What kinds of evidence? Published variant associated with condition Segregation in a family or in others with the condition Variant is very rare in an appropriate control population Loss of gene function is proven Prediction based on data tools Variant highly likely to not yield protein
37 Summary FAOD conditions follow an autosomal recessive pattern of inheritance Parental testing is needed to phase the child s result Results can be ambiguous (VUS) but often can be clarified by assessment of phenotype (how the child presented biochemically)
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