Scholars in HeAlth Research Program

Molecular Genetic Testing Introduction, Evolution, and Future

Rami Mahfouz MD, MPH Why Study Genetics? Statistics

Spontaneous Miscarriages A chromosome abnormality is present in at least 50% of all first-trimester pregnancy loss

Newborn Infants 2% of all neonates have a single gene disorder or a chromosome abnormality

Childhood Genetic disorders account for 50% of all blindness, deafness, and mental retardation

Adult Life At least 10% of common cancers (such as breast and colon) have a genetic etiology

Genetics and Medicine

• Cancer

• Diabetes

• Cardiovascular Disease (Hypertension, Myocardial infarction, etc..)

• Psychiatric disorders (Schizophrenia, ..) Terms to familiarize with

GENE - The basic hereditary unit. It’s a DNA sequence required for production of a functional product.

ALLELE - One of the alternate versions of a gene present in a population.

LOCUS - Literally a “place” on a chromosome or DNA molecule. Usually refers to the location of the gene.

GENETICS – The study of genes.

GENOMICS – The study of genes interaction.

GENOTYPE - An individual’s genetic constitution.

PHENOTYPE - Observable expression of genotype.

Human Genome Project Estimated number of genes ~25,000

We humans are 99.9% identical at the DNA sequence level

Only 1 to 1.5% of the genome encodes proteins

DNA contains information equal to some 600,000 printed pages of 500 words each!!! (a library of about 1,000 books) DNA Structure

Source: cnx.org/content/m12382/latest Features of DNA structure

Storing and coding

Semi-conservative DNA replication

DNA repair

Re-annealing Human DNA

Nuclear DNA Chromosomes + proteins = Chromatin Proteins = Histones (basic) + non-Histones (acidic) 2m long!

Mitochondrial DNA 5-10 Circular chromosomes per mitochondrion 2-100 mitochondria per cell Maternally inherited 37 genes only DNA packing Mitochondrial DNA RNA Structure

Transcription Translation Genetic Code 1944 DNA is the genetic material 1949 Abnormal Hemoglobin in Sickle cell anemia 1953 Double Helix 1956 Glu….Val in sickle hemoglobin 1966 Completion of the genetic code 1970 First restriction enzyme 1972 Recombinant plasmids 1975 Southern blotting 1977 DNA sequencing 1981 Transgenic mice 1983 Huntington disease gene mapped 1985 PCR 1987 Knockout mice 1990 First NIH-approved gene therapy experiment 1995 First complete bacterial genome sequence 1996 Complete yeast genome sequence 2003 Human Genome Project

Restriction Fragment Length Polymorphism (RFLP)

Restriction Enzymes Restriction Enzymes

Bacterial enzymes that recognize and cut at or near a specific sequence (Restriction site)

Named after bacteria of origin (>430 different RE)

EcoRI recognizes 5’ GAATTC 3’

Restriction Enzymes (Cont’d)

The frequency with which a given restriction site occurs within a specific sequence depends in part on its length. Examples: – Specific 6-bp restriction site like GAATTC recognized by EcoRI would be expected to occur in a random stretch of DNA sequence ~ once every 46 (or 4096) nucleotides (since there are 4 possibilities at each of the 6 positions) – EcoRI : product average size is 4096 bp (?)….This is not the case actually

Some Restriction Enzymes

EcoRI 5’ G*AATTC 3’ BamHI 5’ G*GATCC 3’ HindIII 5’ A*AGCTT 3’ MstII 5’ CCTNAGG 3’ NotI 5’ GC*GGCCGC 3’ SmaI 5’ CCC*GGG 3’ AluI 5’ AG*CT 3’ HaeIII 5’ GG*CC 3’ MspI 5’ C*CGG 3’

Rsa I Rsa I

Normal 261 bp 59 bp

C282Y * 232 bp 29 bp 59 bp

Normal Heterozygous Homozygous mutant

261 bp 232 bp

59 bp 29 bp Sequencing Use of Molecular Techniques

Broadly divided into:

Indirect mutant gene tracking

Direct mutant gene analysis

Indirect mutant gene tracking

Utilizes DNA sequence variations close to a gene on a chromosome in order to follow the inheritance of mutant and normal alleles within a family This is done by establishing linkage between the mutant allele and a marker close to it. This is necessary in 3 cases: – Where the chromosomal location is known but the gene itself has not yet been isolated – Where the gene is known but mutations are very varied and hard to find – Where common mutations can be detected easily but rarer ones are very time consuming

Markers used in Linkage Analysis

Must vary in size in different individuals

The greater the variation the better, that is, the markers must be highly polymorphic in the population

Indirect mutant gene tracking (Cont’d)

The mutant gene needs to be “labeled” or “tagged” and its inheritance followed in order to identify affected individuals and carriers Such analysis can only be used in individual families, not in the population as a whole A marker (close to the disease locus) is analyzed in each family member Marker data is then put onto the family pedigree to establish linkage of the marker with the mutant alleles

Thus,

Linkage analysis uses highly polymorphic repeat markers in the region of a gene to determine the chromosomal inheritance of the gene region in family members

Two types of polymorphic repeat markers are used, requiring different methods of analysis. 1. Minisatellite locus or VNTR (Variable Number of Tandem Repeats) VNTRs have a unit of 8-80 bp repeated tens to thousands of times. Best analyzed by Southern blot rather than PCR.

2. Microsatellite loci or Short Tandem Repeats (STRs) The repeat unit of STRs is 2-7 bp long and is repeated up to ~100 times STRs are small enough to be amplified by PCR

Prenatal testing?

Linkage analysis can also be used for prenatal testing.

Prenatal diagnosis demonstrating that the fetus has inherited the same markers as the affected individual implies that the fetus is highly likely to be affected at birth or later in life. Indirect mutant gene tracking (Cont’d) Error of diagnosis

Recombination at meiosis (polymorphic site and gene) Error of the diagnosis: roughly dependent on the distance between the marker and the gene families with genetic disease have to be tested with each marker, and the number of recombinants and non-recombinants scored. The recombination fraction gives the error of diagnosis.

1 Mutation X

X 2 Normal gene

Meiosis with recombination Meiosis with no recombination Between marker and gene

1 Normal gene 1 Mutation X X X X 2 Mutation 2 Normal gene 1 2

1,2 3,4

1 2 3 4 5 6 7 8 9 10

* *

1,3 2,3 2,4 1,3 1,4 2,4 1,3 2,4 2,4 1,3

Number of Recombinants Recombination Fraction = Number of Recombinants + Non-recombinants

Here: RF=2/10=0.2 error is 20% Direct Mutant Gene Analysis In general

Fewer samples are needed Confirms the clinical diagnosis Is the procedure of choice for DNA analysis Examples: – Length mutations – Point mutations

TaqManR use

Real-time PCR Quantitative PCR

Applications: – Mutations (in terms of detection) – Microbiology (viral load) – Oncology (translocations) patient Molecular Pathology tests

They are used to: – Diagnose disease – Direct the choice of therapy – Detect Residual disease after therapy (MRD) – Provide prognostic information – Distinguish one person from another Positive & Negative Controls

A positive and a negative control demonstrate that the process from amplification to analysis has worked properly and demonstrate the expected positive and negative results. Sensitivity Control

For some tests, especially oncology tests for assessment of minimal residual disease, a sensitivity control also is included. A sensitivity control assesses that the specific test run can detect the presence of a minimal positive signal comparable to a few cancer cells in a large number of normal cells. The sensitivity of a PCR is established by testing different dilutions of a positive control (10-2, 10-3, 10-4,..). The lowest amount of positive template that generates a positive signal is referred to as the “threshold” of the assay. Interpretation

Absence of PCR products: Negative result Presence of PCR products: Positive result

Negative result: – Absence of target sequence – Degradation of template – Presence of PCR inhibitors

Each patient’s specimen is tested for the amplification of a second control sequence that should be present in all specimens:

“Housekeeping Genes” Qualitative

Quantitative Microarrays

Cancer Gene Mutation Panel by NGS

• Designed to target 739 mutations

• 46 key cancer genes:

ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL.

Ethical Issues

Who owns Genetic information? How should genetic information be used? Who decides who should be screened? Are there limits to prenatal testing? Who should be employed? Who should be insured?