A brief history of medical diagnosis and the birth of the clinical laboratoryPart 5a—The foundation of

molecular science and geneticsBy Carren Bersch, Editor

Parts 1-4 in this series — originally published from July through December 1999 — were written by Darlene Berger, MLO editor from 1998-2000.

A t the conclusion of “Fraud and abuse, managed care and lab consolidation” — Part 4 of this series of articles in December 1999

— the author wrote: “Only a few years ago, laboratory visionaries predicted that developments in molecular biology had the potential to change laboratory medicine in the same way that computed tomography and magnetic resonance imaging altered the practice of radiology. Speculation that routine hospital admissions testing done in the 21st century could include a panel of DNA probes in place of a chemistry pro-file or complete blood cell count now look more plausible than ever. … On the verge of the 21st century, the lab is providing more information about the human condi-tion faster and more accurately than ever. It is strategically positioned for success in the healthcare industry — in the business of supplying critical information in the information age.”1

Where do we stand?It is now 2006, and the future is here. The healthcare industry is burgeoning with new companies that are involved in the molecular “revolution,” many of which have come into being as recently as the completion of the sequencing of the human genome was announced on April 14, 2003. It was at this point that the so-called genetic blueprint of life was available to researchers and scientists

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C O N T I N U I N G E D U C A T I O N

To earn CEUs, see test on page 24.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Identify six scientists and explain how their discoveries have proven to be pivotal to the science of genetics.

2. Discuss three major discoveries in mo-lecular biology that have infl uenced the development of molecular diagnostics.

3. Describe four inheritable disorders for which genetic testing is useful in risk pre-diction and diagnosis.

4. List three goals achieved in the Human Genome Project.

5. State three challenges to widespread ap-plication of molecular diagnostics.

who began the continuing mad genomic scramble, this time to identify the approx-imately 30,000 human genes.2

Biomedical research has been driven by the prospect of discovering what genes are involved in diseases as complex as can-cer and diabetes. Already, new treatment methods and “designer drugs” — made to suit a particular genetic profile — are being enabled by the sequencing results, as is earlier diagnosis of certain diseases through genetic testing. In the past decade, with the availability of technology and with knowledge fueled by the investment and interest in the Human Genome project, molecular diagnostics has recently enabled laboratories to offer diagnostic and pre-dictive tests for inherited disorders.3 And while molecular testing seemingly arrived all in a flurry, there is a long history of unglamorous trial-and-error behind today’s movement toward personalized medicine, which involves pharmacogenomics and nutrigenomics.

Mendel to Morgan to modern DNAThe precursor to the current era of molecular genetic testing in humans reaches back 141 years to Gregor Johann Mendel’s 1865 publication of experimen-tal data. Mendel (1822-1884) joined the Augustinian Order of monks in 1843. His experiments with peapods in the mon-astery’s garden led him to formulate the basic principles of heredity. Between 1856 — three years before Charles

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Darwin’s Origin of Species was published — and 1863,4 Mendel cultivated and test-ed some 28,000 pea plants. His experi-ments brought forth two generalizations which later became known as Mendel’s Laws of Heredity or Mendelian inheri-tance.

His basic tenets related to the transmis-sion of hereditary characteristics from parent organisms to their children. When Mendel published his theory in 1865, biol-ogists who did not believe his results were especially important largely ignored them. Even Mendel himself believed that his results applied to only certain categories of species and did not thoroughly understand his theory’s applicability.

At the start of the 20th century, Mendel’s work was “rediscovered,” arousing much controversy. Linked in this confused redis-covery, European scientists Hugo de Vries, Carl Correns, and Erich von Tschermak brought Mendel’s early theory of hered-ity to light. Despite a number of detrac-tors and a few promoters, Mendel’s ideas were eventually merged with Thomas Hunt Morgan’s chromosome theory of inheri-tance in 1915 and, thus, became the core of classical genetics.5,6

Morgan was an American geneticist and embryologist (1866-1945). He received his bachelor’s degree from the State College of Kentucky (now the University of Kentucky), his PhD from Johns Hopkins University, and at Bryn Mawr worked on embryology during his tenure there. By 1910, following the rediscovery of the Mendelian model in which the chro-mosomes of cells were thought to hold the actual hereditary particles, Morgan’s research moved to the study of mutation in the fruit fly: Drosphila melanogaster. In Morgan’s famous Fly Room at Columbia University, he demonstrated that genes are carried on chromosomes and are the mechanical basis of heredity — forming the foundation of modern genetic science and guaranteeing Mendel’s place in scien-tific history.6

The move toward revealing DNAFrom this point on, many scientists not nearly so famous as Mendel and Morgan labored on with experiments that, one by one, continued to add to the body of knowledge concerning genetics. While Linus Pauling and his colleagues intro-duced the term "molecular disease" into the medical vocabulary in 1949 (based on

their discovery that a single amino acid change at the beta-globin chain leads to sickle-cell anemia),7 the next big public sensation in the field was the discovery of DNA by Watson and Crick. While the two men did not become household words, the three little letters D-N-A did.

Deoxyribonucleic acid (DNA) was actually isolated in 1869 by Swiss chemist Friedrich Miescher. He later demonstrated that DNA exists only in chromosomes, the site of hereditary material. By the 1930s, DNA was known to be a large molecule in the form of a long chain of nucleotides; but, other than that, its structures and func-tions were poorly understood. By then, geneticist George Beadle and biochemist Edward Tatum teamed up to investigate the relations between genes and enzymes. In 1943, Oswald Avery had identified the genetic role of DNA: that DNA carried genetic information and might well be the gene.8

Two teams, two approaches, one answerAlmost as fascinating as the scientific studies that unfolded from Mendel down to Avery is the tale of how James Dewey Watson and Francis Harry Compton Crick converged at the same time, in the same place and, in 1959, cracked the DNA code. Crick was a Briton who had studied physics, then chemistry and biology, and still had not earned a doctoral degree. At age 19, Watson — an American — had graduated from the University of Chicago and had received his doctorate at age 22. He had studied ornithology but changed career paths when he went to Europe for post-doctoral studies.

Another team — Maurice Hugh Frederick Wilkins and Rosalind Elsie Franklin — were working with DNA at King’s College in London. At a conference in Italy, Watson heard Wilkins speak and was introduced by him to a photograph of a DNA molecule which had been rendered by his colleague, Franklin, via X-ray crys-tallography. With this bit of knowledge, Watson was immediately keen to solve the riddle of DNA.9

Watson (now 23, became a research fel-low at Cambridge) and Crick (now 35, and a graduate student there) had admired the work of Linus Pauling who had discovered in 1948 "that many proteins take the shape of an alpha helix, spiraled like a spring coil."8 They were also aware of the stud-

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1858 Charles Darwin, Alfred Russel Wallace

Joint announcement of the theory of natural selection: that members of a population who are better adapted to the environment survive and pass on their traits.

1859 Charles Darwin Published The Origin of Species.

1866 Gregor Mendel Published the results of his

investigations of the inheritance of “factors” in pea plants.

1900 Carl Correns, Hugo de Vries, Erich von Tschermak

Mendel’s principles were independently discovered and verified, marking the beginning of modern genetics.

1902 Walter Sutton Pointed out the inter-relationships

between cytology and Mendelism, closing the gap between cell morphology and heredity.

1905 Nettie Stevens, Edmund Wilson Independently described the

behavior of sex chromosomes: XX determines female; XY determines male.

1908 Archibald Garrod Proposed that some human

diseases are due to “inborn errors of metabolism” that result from the lack of a specific enzyme.

1910 Thomas Hunt Morgan Proposed a theory of sex-linked

inheritance for the first mutation discovered in the fruit fly, Drosophila, white eye. This was followed by the gene theory, including the principle of linkage.

1927 Hermann J. Muller Used x-rays to cause artificial gene

mutations in Drosophila.

1928 Fred Griffith Proposed that some unknown

“principle” had transformed the harmless R strain of Diplococcus to the virulent S strain.

1931 Harriet B. Creighton, Barbara McClintock

Continued on page 18

Figure 1.

History of genetics timeline

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ies of Erwin Chargaff, a biochemist, who, in 1951, announced that "the arrangement of nitrogen bases in DNA varied widely, but the amount of certain bases always occurred in a one-to-one ratio. These dis-coveries were an important foundation for Watson and Crick’s later description of DNA."8 The two busied themselves by constructing physical models in order to create an accurate picture of the DNA mol-ecule.

Meanwhile, Wilkins and Franklin continued experimenting with X-ray dif-fractions images of DNA. By using the technique, "the locations of atoms in any crystal can be precisely mapped by look-ing at the image of the crystal under an X-ray beam."10 Franklin was the first to state that the sugar-phosphate backbone of DNA lies on the outside of the molecule and described the helical structure of the molecule.10

Watson attended one of Franklin’s lec-tures in 1951 during which she outlined her work to date. With a rather fuzzy memory of what Franklin had described, Watson returned to the Cavendish Laboratory at Cambridge and, with Crick, constructed yet another failed model. At this point, the head of the laboratory directed them to end their DNA research.8 That order was ignored, and the two labored on in what had become their self-generated competi-tion with Pauling as well as Wilkins and Franklin.

Two years later, in 1951, a frustrated

Wilkins — having already made Watson’s acquaintance in Naples, Italy9 — showed Franklin’s results to Watson, reportedly without her knowledge or consent. The results of Franklin’s work with X-ray diffractions were that DNA had all the characterisitcs of a helix.6 With the evi-dence that Wilkins delivered, Watson and Crick resumed their construction of physical models, combining Franklin’s and Pauling’s information on helical struc-ture with that of Chargaff’s latest evidence about base pairs. By 1952, much was known about DNA, including the fact that it was the sole substance capable of storing all the information needed to create a living being. What was not yet known was what the elusive DNA molecule looked like, or how it performed this amazing hereditary function. This would change in the course of a single year.10 Their model ended up having matching base pairs interlocked in the middle of a double helix, so that the distance between the chains was consistent throughout.8

DNA and the ongoing search for answersThe two eager scientists, Watson and Crick, demonstrated that each strand of the DNA molecule was a template for the other. “During cell division, the two strands separate and on each strand a new ‘other half’ is built, just like the one before. This way, DNA can repro-duce itself without changing its structure

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Demonstrated the cytological proof for crossing-over in maize.

1941 George Beadle, Edward Tatum Irradiated the red bread mold,

Neurospora, and proved that the gene produces its effect by regulating particular enzymes.

1944 Oswald Avery, Colin MacLeod,

Maclyn McCarty Reported that they had purified the

transforming principle in Griffith’s experiment and that it was DNA.

1945 Max Delbruck Organized a phage course at Cold

Spring Harbor Laboratory which was taught for 26 consecutive years. This course was the training ground of the first two generations of molecular biologists.

late 1940s Barbara McClintock Developed the hypothesis of

transposable elements to explain color variations in corn.

1950 Erwin Chargaff Discovered a one-to-one ratio of

adenine to thymine and guanine to cytosine in DNA samples from a variety of organisms.

1951 Rosalind Franklin Obtained sharp X-ray diffraction

photographs of DNA. 1952 Martha Chase, Alfred Hershey Used phages in which the protein

was labeled with 35S and the DNA with 32P for the final proof that DNA is the molecule of heredity.

1953 Francis Crick James Watson Solved the three-dimensional

structure of the DNA molecule. 1958 Matthew Meselson, Frank Stahl Used isotopes of nitrogen to prove

the semiconservative replication of DNA.

1958 Arthur Kornberg Purified DNA polymerase I from

E coli, the first enzyme that made DNA in a test tube.

1966 Marshall Nirenberg, H. Gobind

Khorana Led teams that cracked the genetic

code: that triplet mRNA codons specify each of the twenty amino acids.

1949 Characterization of sickle-cell anemia

as a molecular disease

1953 Discovery of the DNA double helix

1958 Isolation of DNA polymerases

1960 First hybridization techniques

1969 In situ hybridization

1970 Discovery of restriction enzymes and

reverse transcriptase

1975 Southern blotting

1977 DNA sequencing

1983 First synthesis of oligonucleotides

1985 Restriction fragment length polymor-

phism analysis

Molecular diagnostics

1985 Invention of PCR

1986 Development of flourescent in situ hybridization (FISH)

1988 Discovery of the thermostable DNA

polymerase: Optimization of PCR

1992 Conception of real time PCR

1993 Discovery of structure-specific endo-

nucleases for cleavage assays

1996 First application of DNA microarrays

2001 First draft versions of the human

genome sequence

2001 Application of protein profiling in

human diseases

Continued on page 21

The timeline of the principal discoveries in the fi eld of molecular biology, which infl uenced the development of mo-lecular diagnostics. This adapted chart originally appeared in Molecular Diagnostics: Past, Present, and Future by George P. Patrinos and Wilhelm Ansorge. Permission to reprint has been requested from Corporate Relations, Else-vier.

Figure 2.

— except for occasional errors, or muta-tions.”8 They published these findings in Nature in 1953; Rosalind Franklin’s find-ings were published in the same issue as a “supporting article.”8 Because their model had fit experimental data so well, Watson and Crick’s structure was widely accepted. In 1962, Watson, Crick, and Wilkins won the Nobel Prize for physiology/medicine. Rosalind Franklin had, by this time, died at age 37 of ovarian cancer, and by the

www.mlo-online.com MLO ■ September 2006 21

conditions required by the Nobel Prize Committee that only living persons be recognized, she could not be one of the recipients.8

“The discovery of DNA, the explanation of its construct, has been acknowledged as the most important biological work of the last 100 years, and the field it opened may be the scientific ‘frontier’ for the next 100.”8 In the 50 years since Watson and Crick revealed their DNA findings, the field of molecular diagnostics has flour-ished (see Figure 2). The 1985 invention of polymerase chain reaction (PCR) provided the boost that scientists needed to improve their capabilities to diagnose inherited dis-eases on the DNA level.9

“PCR opened the door to eliminating complexities, costs, and time requirements of available technologies like cloning and sequencing. Its ability to generate expo-nential copies of a target sequence means that a known mutation can be identified within a day rather than months. PCR also eliminated the necessity for radioactivity for routine molecular diagnosis, so that the clinical laboratory is now able to provide genetic services for carrier or population screening for known mutations, as well as prenatal diagnosis of inherited diseases. From PCR, mutation-detection techniques developed can be categorized under enzy-matic-based methods, electrophoretic-based methods, and solid phase-based tech-niques.”9

Some of the disease-related gene muta-tions are recessive (they must be present in both gene copies, one from each parent) before they cause dysfunction, while others are dominant (a single altered gene copy can cause disease). Some are X- or sex-linked, associated with the X or Y chro-mosome that determines our gender, and are found only in males or females. Some mutations have arisen and been passed down in specific families and some are more prevalent in individuals of certain ethnic descent.10

For example, carriers of certain muta-tions of the BRCA1 or the BRCA2 gene (especially Ashkenazi Jewish women) are at a higher risk of both breast cancer and ovarian cancer, often at an earlier age than the general population.11 Individuals of Ashkenazi Jewish descent are also at increased risk for inheriting Tay-Sachs, Gaucher, Canavan disease, and Familial Dysautonomia — genetic diseases that can occur when both parents have an abnor-

1970 Hamilton Smith, Kent Wilcox Isolated the first restriction

enzyme, HindII, that could cut DNA molecules within specific recognition sites.

1972 Paul Berg, Herb Boyer Produced the first recombinant

DNA molecules. 1973 Joseph Sambrook Led the team at Cold Spring Harbor

Laboratory that refined DNA electrophoresis by using agarose gel and staining with ethidium bromide.

1973 Annie Chang, Stanley Cohen Showed that a recombinant DNA

molecule can be maintained and replicated in E coli.

1975 International meeting at Asilomar,

California urged the adoption of guidelines regulating recombinant DNA experimentation.

1977 Fred Sanger Developed the chain termination

(dideoxy) method for sequencing DNA.

1977 The first genetic engineering company (Genentech) is founded, using recombinant DNA methods to make medically important drugs.

1978 Somatostatin became the first

human hormone produced using recombinant DNA technology.

1981 Three independent research

teams announced the discovery of human oncogenes (cancer genes).

1983 James Gusella Used blood samples collected

by Nancy Wexler and her co-workers to demonstrate that the Huntington’s disease gene is on chromosome 4.

1985 Kary B. Mullis Published a paper describing the

polymerase chain reaction (PCR), the most sensitive assay for DNA yet devised.

1988 The Human Genome Project

began with the goal of determining the entire sequence of DNA composing human chromosomes.

Challenges for widespread application of molecular

diagnostics

• Defining the appropriate circumstances for ordering

• Developing therapies that correct or address specific genetic defects or genetic risk factors

• Developing consensus on standards of care

• Educating physicians and patients concerning the potential information from testing

• Providing access to affordable testing and services

• Providing adequate controls to prevent discrimination at work, within communities and for insurance coverage

• Performing tests on platform technologies that are easy to control

• Developing adequate proficiency testing and consistency among laboratories

• Training a sufficient number of medical technologists to perform highly complex testing

• Interpreting the results in the context of the clinical history and other results

• Integrating results with other family members and ethnicity, while complying with HIPAA

• Providing adequate genetic counseling support for physicians and patients

• Obtaining adequate reimbursement for performing the tests and the potential liability

• Obtaining necessary regulatory approval and coding for reimbursement

• Convincing carriers and payers of the merits of providing payment coverage

Challenges for widespread application of mo-lecular diagnostics. Originally printed in MLO July 2003,”From peapods to laboratory medicine: mo-lecular diagnostics of inheritable diseases, p. 30.

Figure 3.

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Continued on page 22

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Visit www.rsleads.com/609ml-020

1989 Alec Jeffreys Coined the term DNA fingerprinting

and was the first to use DNA polymorphisms in paternity, immigration, and murder cases.

1989 Francis Collins, Lap-Chee Tsui Identified the gene coding for the

cystic fibrosis transmembrane conductance regulator protein (CFTR) on chromosome 7 that, when mutant, causes cystic fibrosis.

1990 First gene replacement therapy:T cells of a four-year old girl were exposed outside of her body to retroviruses containing an RNA copy of a normal ADA gene. This allowed her immune system to begin functioning.

1993 FlavrSavr tomatoes, genetically

engineered for longer shelf life, were marketed.

mal gene that can cause the disease in the child.10 Testing for diseases such as cystic fibrosis, Fragile X syndrome, Marfan’s syndrome, and a host of other diseases are also available today.

What is on the molecular diagnostics horizon?The 21st century is barely underway, and molecular diagnostics is the hottest topic in the clinical laboratory field. Making molecular diagnostics widely available means overcoming obstacles and resolv-ing issues that have surfaced in its devel-opment. In the next segment of “A brief history of medical diagnosis and the birth of the clinical laboratory,” Part 5b will detail the most up-to-date testing methods and pertinent automation technologies, and examine the impact these will have on everyday healthcare decisions.

References

1. Berger D. A brief history of medical diagnosis and the birth of the clinical laboratory; Part 4—Fraud and abuse, managed care and lab consolidation. Medical Laboratory Observer. December 1999;

2. Human genome sequence completed. http://cnn.health.printthis.clickability.com/pt/cpt?action+cpt&title=CNN.com+-+Human+g... Accessed August 24, 2006.

3. Kaufman HW, Strom CM. From peapods to labora-tory medicine: molecular diagnostics of inheritable diseases. Medical Laboratory Observer. July 2003;30. on peapods

4. Auyang SY. Scientifi c convergence in the birth of molecular biology. 7. http://www.creatingtechnology.org/biomed/dna.pdf. Accessed August 8, 2006.

5. Wikipedia contributors. Gregor Mendel. Wikipedia, The Free Encyclopedia. August 28, 2006, 05:09 UTC. Available at: http://en.wikipedia.org/w/index.php?title=Gregor_Mendel&oldid=72331071. Accessed August 28, 2006.

6. Wikipedia contributors. Thomas Hunt Morgan. Wiki-pedia, The Free Encyclopedia. August 1, 2006, 03:28 UTC. Available at: http://en.wikipedia.org/w/index.php?title=Thomas_Hunt_Morgan&oldid=66975748. Accessed August 28, 2006.

7. Patrinos GP, Ansorg W. Molecular Diagnostics: Past, Present, and Future. Elsevier Inc. 2005;1, 2-4.

8. A Science Odyssey: People and Discoveries: Watson and Crick describe structure of DNA 1953. http://www.pbs.org/wgbh/aso/databank/entries/do53dn.html. Ac-cessed August 8, 2006.

9. Ardell D. Rosalind Franklin. http://www.accessexcel-lence.org/RC/Ab/BC/Rosalind_Franklin.html. Ac-cessed August 25, 2006

10. Wright R. TIME 100: James Watson and Francis Crick. http://www.time.com/tim/time100/scientist/profile/watsoncrick.html. Accessed August 8, 2006.

11. Genetic Testing for Inherited Diseases. http://www.labtestsonline.org/understanding/conditions/preg-nancy-12/html.

12. Fergus K, Simonsen J. The Testing Process in the Ash-kenzai Jewish Population. http://www.genetichealth.com/BROV_Genetic_Testing_in_People_of_Ashke-nazi_Jewish_Descent.shtml.

This adapted chart was developed by Jo Ann Lane, a 1994 Woodrow Wilson National Fellowship Foun-dation’s National Leadership Program for Teachers participant, and is used through the courtesy of WWNFF Leadership Program for Teachers (www.woodrow.org/lpt/LPTnational.php) and Access Excel-lence @ the National Health Museum (www.acces-sexcellence.org/AE/AEPC/WWC/1994/geneticstln.html ).