Molecular Genetics and Otolaryngology: Molecular Genetics and Otolaryngology Michael E. Prater, MD Shawn D. Newlands, MD


Introduction: Introduction Chromosomal analysis Cytogenetics Molecular biology and genetics Biochemical genetics Clinical genetics Population genetics Genetic epidemiology Developmental genetics Immunogenetics Genetic counseling Fetal genetics


History: History Gregor Mendel, 1865 “Mendel’s Laws” of autosomal inheritence Work “lost” until early 1900’s Charles Darwin, 1859 “The Origin of Species” Jean Baptiste Lemarck


History, continued: History, continued Francis Galton (Charles Darwin’s cousin) The “father” of modern genetics rediscovered Mendel’s laws “nature versus nurture” “inborn errors of metabolism” responsible for biological abnormalities


History, Continued: History, Continued James Watson and Francis Crick DNA discovered in 1940’s Determined double helix in 1953 Nobel Prize in 1962 Human Genome Project Begun in 1990 Goal is to identify every human gene by 2005 9% completed as of 1999


Classification of Disorders: Classification of Disorders Single Gene Defects Usually single critical error in the genetic code Usually phenotypically obvious Examples: NF I and II, osteogenesis imperfecta, cystic fibrosis


Classification, continued: Classification, continued Chromosomal disorders not due to single defect usually due to deficiency in number of genes within chromosome classic example is Down Syndrome (Trisomy 21) other examples: Trimsomies 13, 18, Klinefelter’s Syndrome, Turner’s Syndrome phenotypically obvious usually incompatible with life


Classification, continued: Classification, continued Multifactorial inheritance multiple single code defects usually form a pattern classic examples: cleft lip/palate, neural tube defects possible example: head and neck cancer?


Chromosomal Structure: Chromosomal Structure 23 pairs of chromosomes approximately 7 million base pairs 100,000 genes DNA: five carbon sugar (deoxyribose; ribose in RNA) nitrogen base (purines, pyrimidines) 3’5’ phosphate linkage hydrogen bonded double strand


DNA Bases: DNA Bases


DNA Bases: DNA Bases


Transcription: Transcription The Central Dogma


Tools of Genetics: Tools of Genetics Revolutionary changes since late 1970’s restriction enzymes recombinant DNA vectors probes PCR DNA sequence analysis protein analysis


Tools of Genetics, cont.: Tools of Genetics, cont. Restriction Endonucleases enzymes which cleave DNA at specific sites almost always palindromic hundreds of known endonucleases Recombinant DNA an DNA fragment is combined with a known piece of DNA to form a plasmid plasmid inserted in vector (bacterium, virus, yeast) vector cultured and isolated


Tools, continued: Tools, continued Identification of recombinant fragments “Blotting” - southern, northern, western electrophoresis/chromotography of fragment hybridization with known radioactive fragment antibodies to known fragments may be used


Tools, continued: Tools, continued Polymerase Chain Reaction (PCR) simplest, most rapid, most effective enzymatic amplification of desired fragment DNA fragment formed by endonuclease known “primer” is annealed to fragment steps repeated approximately 30 times yields more than a billion copies of desired DNA fragment


Tools, continued: Tools, continued DNA Sequence Analysis Fred Sanger, Nobel Prize 1980 also won Nobel Prize in 1958 for protein analysis nucleotide analog with inhibits DNA synthesis endonuclease which cleaves at nucleotide site electrophoresis/chromotography radioactive tagging/antibodies


Genetic Mutations: Genetic Mutations Defn: Permanent chagne in nucleotide sequence occur in somatic cells or germline cells only germline cells inherited somatic mutations believed responsible for many medical problems many cancers, ?CAD


Gentic Mutations, cont.: Gentic Mutations, cont. Genome Mutations missegregation of chromosome results in aneuploidy Down Syndrome classic example 1:50 meiotic divisions usually incompatible with life


Genetic Mutations, cont.: Genetic Mutations, cont. Chromosome mutations usually involve translocations and rearrangements 1:1000 meiotic divisions almost uniformly incompatible with life Gene mutations (single gene defects) DNA replicates 20 bases/sec/polymerase Only one defect per ten million copies Repair enzymes repair 99.9% of defects Less than one defect per 10 billion bases!


Genetics and Cancer: Genetics and Cancer Tumor cells are clone of abnormally dividing cell usually from single/multiple point mutations rarely from translocations Protooncogenes normal growth genes Oncogenes a protooncogene which has undergone somatic mutation and is oncogenic


Genetics/Cancer, cont.: Genetics/Cancer, cont. Tumor Suppressor Genes genes that regulate cell growth/genomic expression p53, Bcl-2 are classic examples p53: arrests growth in G1 (growth 1) phase allows repair of DNA defects induces apoptosis (programmed cell death) found in 40% of HNSCCa have NOT shown correlation with prognosis


Genetics/Cancer, cont.: Genetics/Cancer, cont. Bcl-2 tumor suppressor gene normal Bcell lymphoma/leukemia gene (Bcl-2) prevents apoptosis (programmed cell death) somatic mutations present HNSCC, usually resulting in overexpression Friedman’s study: retrospective study of Stage I/II HNSCCa overexpression of Bcl-2 lead to 50% cure versus 90% in normal expression others unable to reproduce (see Gallo)


Treatment : Treatment Most disease treated at phenotypic level medicines surgery genetic counseling Molecular level gene therapy


Treatment, continued: Treatment, continued Gene Therapy attempted modification of abnormal cell function involves transfer of functioning genes gene therapy via addition more practical insertion into cell (not necessarily into genome) of functioning gene gene therapy via replacement theoretical goal is to replace abnormal gene with inserted gene


Treatment, continued: Treatment, continued Gene therapy, continued Transfer strategies recombinant DNA in vector viral versus bacterium retroviral vectors with reverse transcriptase not inserted into host genome problems: inability to maintain expression under/overexpression adenine deaminase deficiency (ADA)


Genetic Disease in ENT: Genetic Disease in ENT Cystic Fibrosis chromosome 7q, spans 250,000 bases 70% have deletion of phenylalanine at position 508 (point mutation) frameshift versus point mutation most common fatal autosomal disease in whites phenotypic expression results from failure of membrane transport (Cl, Na) and from exocrine function (pancreas) Tx at phenotypic level


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Cleft Lip and Palate one of the most common malformations CL and P genetically distinct from isolated CL failure of fusion of frontal process with maxillary process at 35 days gestation classically described as multifactorial, although single gene froms, chromosomal forms (Trisomy 13) teratogenic forms (rubella, thalidomide) are known


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Human papilloma virus strains 16, 18 and 31 carcinogenic in GU tract exact role in HNSCCa not fully known, although 46% of post mortem specimens contained HPV strains E6 HPV protein binds to p53 forming mutation which suppresses gene function in vivo


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Thyroid carcinoma Medullary thyroid carcinoma (MTC) neoplasm of parafollicular C cells (ultimobranchial body) produce calcitonin sporadic and familiar forms familial MTC associated with MEN 2A and 2B MEN 2A: pheo, hyperparthyroid, MTC MEN 2B: pheo, MTC, Marfan’s, NFI RET protooncogene associated with familial forms 10p Aggressive papillary CA associated with aneuploidy noninvasive dz uniformly diploid


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Salivary Gland Neoplasms Aggressive adenoid cystic Ca associated with aneuploidy all patients with aneuploidy recurred after resection versus only 2/14 with diploid genome (Sugano) Salivary gland adenocarcinoma with overexpression of Bcl-2 were more difficult to resect, recurred more frequently and metastasized more frequently (Sugano)


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Acoustic Neuroma 5% are familial and associated with NF II often bilateral NF II defect on 22p therapy at phenotypic level


Genetic Dz inENT, cont. : Genetic Dz inENT, cont. Congenital Hearing Loss 60% of congenital hearing loss is genetic most associated with phenotypic anomaly Waardenburg Syndrome autosomal dominant - variable penetrance dystopia canthorum, hyperchromatic iris, white forelock and SNHL PAX3 locus of chromosome 2 treatment at phenotypic level


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Congenital hearing loss, continued Usher’s Syndrome autosomal recessive five different classifications (Usher’s Types I through V) all subtypes on different chromosomes associated with retinitis pigmentosum therapy at phenotypic level


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Congenital Hearing Loss, continued Pendred’s Syndrome autosomal recessive with variable penetrance located on chromosome 7q associated with thyroid goiter and carcinoma tx at phenotypic level


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Congenital hearing loss, cont. Alport’s Syndrome two forms: X linked, autosomal recessive X linked on 5p, produces mutant alpha 5 protein recessive form on 2p, produces mutant Type IV collagen treatment at phenotypic level


Genetic Dz in ENT, cont.: Genetic Dz in ENT, cont. Head and Neck Cancer heavily associated with p53 underexpression, Bcl-2 overexpression, HPV types 16, 18 and 31 None of these proven prognostic Ultimate goal: gene therapy to correct somatic mutation


Future Directions and Conclusion: Future Directions and Conclusion Rapidly expanding field Ultimate goal: correction of somatic defect which would correct phenotypic abnormality. Would eliminate surgical intervention.