The Genetic Testing

The Genetic Testing
By Dr rer. nat , Dr H.c. phil. Theo Giannaros T.U. Karlsruhe, (Kernforschungszentrum-Leopoldshafen) Department of Molecular & Nuclear Biology










Introduction

This article discusses genetic testing; that is, testing that looks at a person's genetic makeup for a variety of reasons. An increasing number of genetic tests are becoming available as a result of recent and rapid advances in biomedical research. It has been said that genetic testing may revolutionize the way many diseases are diagnosed. But genetic testing does not just help a physician diagnose disease. There are a number of different reasons genetic tests are performed. These include the following:
1. clinical genetic testing (diagnosing current or future disease),
2. pharmacogenomics (assessment of therapeutic drug treatment ),
3. identity testing for criminal investigations or forensics studies.
4. parentage testing tissue typing for transplantation,
5. cytogenetics (chromosome analysis),
6. infectious disease testing.
Clinical Genetic Testing
Clinical genetic testing refers to the laboratory analysis of DNA or RNA to aid in the diagnosis of disease. It is very important to understand that clinical genetic testing is quite different than other types of laboratory tests. Genetic testing is unique in that it can provide definitive diagnosis as well as help predict the likelihood of developing a particular disease before symptoms even appear; it can tell if a person is carrying a specific gene that could be passed on to his or her children; it can inform as to whether some treatments will work before a patient starts therapy. These are definite advantages. However, there are also some qualities of genetic testing that should be carefully thought out and perhaps discussed with a genetic counselor before undergoing any test. These aspects are reviewed in the section titled Pros and Cons of Genetic Testing. In an era of patient responsibility, it is important that you be educated in these matters to fully appreciate the value as well as the drawbacks of genetic testing.
Testing Genetic Material
Testing of genetic material is performed on a variety of specimens including blood, urine, saliva, stool, body tissues, bone, or hair. Cells in these samples are isolated and the DNA within them is extracted and examined for possible mutations or alterations. Looking at small portions of the DNA within a gene requires specialized and specific laboratory testing. This is done to pinpoint the exact location of genetic errors. This section will focus on the examination of a person’s genes to look for the one(s) responsible for a particular disease.
There are four basic reasons that genetic material is tested for clinical reasons. Presymptomatic testing identifies the presence of variant genes that cause disease even if the physical abnormalities associated with the disease are not yet present in an individual. Diagnostic genetic testing is performed on a symptomatic individual with symptoms sufficiently suggestive of a genetic disorder. This assists the individual’s physician in making a clear diagnosis.
Testing of genetic material can also be performed as a prenatal screening tool to assess whether two individuals who wish to become parents have an autosomal or X-linked recessive gene that, when combined in a child, will produce a serious disorder in that child. This type of genetic testing is referred to as carrier screening. Fetuses developing in the uterus can also have their genetic material tested to assess their health status if it is thought to be in jeopardy.
To test DNA for medical reasons, some type of cellular material is required. This material can come from blood, urine, saliva, body tissues, bone marrow, hair, etc. The material can be submitted in a tube, on a swab, in a container, or frozen. If the test requires RNA, the same materials can be used. Once received in the laboratory, the cells are removed from the substance they are in, broken apart, and the DNA in the nuclei is isolated and extracted.
The lab professionals who perform and interpret these tests are specially trained physicians and scientists. The extracted DNA is manipulated in different ways in order for the molecular pathologist or genetic technologist to see what might be missing or mutated in such a way as to cause disease. One type of manipulation is “cutting” the DNA into small pieces using special enzymes. These small pieces are much easier to test than the long strands of uncut DNA, and they contain the genes of interest. Another manipulation is to apply the extracted and cut DNA to an agarose gel, apply an electrical field to the gel, and see how the DNA moves on the gel. This can indicate differences in the size of the pieces of the cut DNA which might be caused by specific mutations.
Other manipulations to DNA include amplification, sequencing, or a special procedure called hybridization. When the results of these tests are examined and compared with results from a normal person, it is possible to see differences in the genes that might cause a disease.
Specific Genetic Diseases
There are many diseases that are now thought to be caused by alterations in DNA. These alterations can either be inherited or can occur spontaneously. Some diseases that have a genetic component to them include:
Alzheimer's Disease
Bone Marrow Disorders
Breast Cancer

Ovarian Cancer
Colon Cancer
Cystic Fibrosis

Down Syndrome
Hemochromotosis
Leukemia

Lupus
Lymphoma
Osteoarthritis

Pre-senilin Mutation Sickle Cell Anemia
Thalassemia

Several things can go wrong with the genes that make up the DNA, resulting in these and other diseases. The section below discusses what can happen to DNA, and specifically to genes, that might lead to a disease.
Genetic Variation and Mutation
All genetic variations or polymorphisms originate from the process of mutation. Genetic variations occur sometimes during the process of somatic cell division (mitosis). Other genetic variations can occur during meiosis, the cycle of division that a sperm cell or an ovum goes through. Some variations are passed along through the generations, adding more and more changes over the years. Sometimes these mutations lead to disease, other times there is no noticeable effect. Genetic variations can be classified into different categories: stable genetic variations, unstable genetic variations, silent genetic variations, and other types.
Stable genetic variations are caused by specific changes in single nucleotides. These changes are called single nucleotide polymorphisms or SNPs and can include
1. substitutions, in which one nucleotide is replaced by another,
2. deletions, in which a single nucleotide is lost, and
3. insertions, in which one or more nucleotides are inserted into a gene.
If the SNP causes a new amino acid to be made, it is called a “missense mutation.” An example of this is in sickle cell anemia, in which one nucleotide is substituted for another. The genetic variation in the gene causes a different amino acid to be added to a protein, resulting in a protein that doesn’t do its job properly and causes cells to form sickle shapes and not carry oxygen.
Unstable genetic variations occur when a nucleotide sequence repeats itself over and over. This is called a “repeat” and is usually normal; however, if the number of repeats increases too greatly, it is called an “expanded repeat” and has been found to be the cause of many genetic disorders. An example of a disease caused by an expanded repeat is Huntington disease, a severe disorder of a part of the brain that is marked by dementia, hydrocephalus, and unusual movements.
Silent genetic variations are those mutations or changes in a gene that do not change the protein product of the gene. These mutations rarely result in a disease.
Other types of variations occur when an entire gene is duplicated somewhere in a person’s genome. When this occurs, extra copies of the gene are present and makes extra protein product. This is seen in a disorder that effects peripheral nerves and is called Charcot-Marie-Tooth disease type 1. Some variations occur in a special part of the gene that controls when DNA is copied to RNA. When the timing of protein production is thrown off, it results in decreased protein production. Other variations include a defect in a gene that makes a protein that serves to repair broken DNA in our cells. This variation can result in many types of diseases, including colorectal cancer and a skin disease called xeroderma pigmentosum.
Testing for Products of Genetic Expression
Many inherited disorders are identified indirectly by examining abnormalities in the genetic end products (proteins or metabolites) that are present in abnormal forms or quantities. As a reminder, genes code for the production of thousands of proteins and, if there is an error in the code, changes can occur in the production of those proteins. So, rather than detecting the problem in the gene, some types of testing look for unusual findings related to the pertinent proteins, such as their absence.
An example of testing for genetic products includes those widely used to screen newborns for a variety of disorders. For example, newborns are tested for phenylketonuria (PKU), an inherited autosomal recessive metabolic disorder caused by a variation in a gene that makes a special enzyme that breaks down phenylalanine, an amino acid. When too much of this substance builds up in blood, it can lead to mental retardation if not treated early in life with a special, restricted diet. The test uses a blood sample from a baby’s heel to look for the presence of extra phenylalanine, rather than looking for the mutated gene itself. Other examples include blood tests for congenital hypothyroidism, diagnosed by low blood levels or absence of thyroid hormone, and congenital adrenal hyperplasia, a genetic disease that causes the hormone cortisol to be decreased in blood.

Pharmacogenomics
There have been cases regarding individuals who are given a certain therapeutic drug to treat symptoms or to keep symptoms from occurring in which the individual has a very violent reaction to the drug or feels no affect whatsoever. Many times this happens because of the genetic makeup of the individual. The study of this phenomenon is called “pharmacogenomics” or “pharmacogenetics.”
As an example, a woman had a surgery to remove a tumor and was given codeine as a pain reliever. Although she was doing well after the surgery, as soon as she began treatment with codeine she developed a full-body rash, difficulty breathing, and an irregular heartbeat. When she was taken off the codeine, her reaction disappeared. Upon further study, it was found that she lacked the enzyme in her blood that metabolized (broke it down into different components) the codeine into morphine and other substances, so she was essentially being overdosed with codeine. The lack of the enzyme was directly related to a variation in the gene that produced it. This genetic variation is a polymorphism between normal individuals and those who carry it. Sometimes these polymorphisms can cause a very serious reaction in an individual that could lead to death.
In some cases, individuals “hypermetabolize” drugs. This occurs when there is too much of an enzyme present that breaks down the helpful drug too quickly, leading to a lack of response to the drug. This can happen when too many copies of the gene are made and too much enzyme is produced. In other cases, the special receptor that the drug binds to on cells or tissues is missing, again because of a variation in the gene that makes the receptor protein. When there is no receptor to bind the drug, the drug may not have any affect on the cells or tissues that it should.
Genetic testing to determine the polymorphisms that play a role in our response to a drug is typical of basic genetic analyses. DNA is removed from cells, manipulated to find a specific area on a specific chromosome, and compared to “normal” DNA. In this way, genetic variations can be seen that may play a role in the over- or under-responsiveness to a therapeutic drug. This testing can also determine an individual’s resistance or sensitivity to the effectiveness of certain drugs used in viral therapy (HIV or hepatitis C drugs, for example).
There are many, many enzymes in our blood that act to metabolize or break down specific drugs, allowing them to be excreted in urine or by other means. At present, there are no testing programs in place that can give us an overall picture of our specific genetic variations that may cause us to be unresponsive or over-responsive to a therapeutic drug.

Identity Testing
Identity testing is sometimes referred to as “DNA testing”, a term most frequently used in relation to criminal investigations. "DNA testing" is an unfortunate misnomer as all types of genetic analysis, whether for disease or identification or for tissue typing, involves assessment of DNA or RNA.
Identity testing focuses on the identification of an individual through the analysis of either nuclear or mitochondrial DNA extracted from some material: blood, tissue, hair, bone, etc. Any material that contains cells with nuclei can be used for nuclear DNA extraction and eventual identity testing. Mitochondrial DNA, which is “extra-nuclear,” is used when a sample is severely degraded or if only hair shafts with no attached cells are available.
Increasingly, identity testing is used to identify a suspect in a criminal investigation by comparing the DNA found at a crime scene to that of the suspected individual. If suspected individual is convicted of the crime, his or her DNA polymorphisms are put into a data bank system that is accessible by law enforcement officials. This system is referred to as CODIS, or “Combined DNA Index System.” This system has helped to solve many crimes and also to clear those wrongfully accused of a crime.
Other uses of identity testing are to identify individuals whose identity cannot be distinguished by other means, as with decomposed bodies. In this type of genetic testing, specific parts of DNA are examined for polymorphisms (differences) that are unique to the individual. These parts of the DNA strand are referred to as microsatellites or minisatellites and are composed of repeated subunits of the DNA strand. Sometimes these repeated units are called short tandem repeats (STRs) or variable number of tandem repeats (VNTRs). In forensics, these unique sequences are given the name “DNA fingerprint.”
Other types of identity testing include the determination of an individual’s parent or parents, often called “parentage testing”, and identifying organ donors by using genetic testing for tissue transplantation, called "tissue typing".

Parentage Testing
The primary goal of parentage testing is to identify the biological parent of a given child. It is done to determine an individual’s parent or parents in, for example, cases of adoption or alleged paternity. This determination must be looked at very carefully and must identify the alleged parent with at least 99% certainty.
Many different types of laboratory tests can be done to assess parentage, including examination of red blood cell antigens (blood typing), examination of polymorphic serum protein genes, and assessment of short tandem repeats (see above). The DNA testing techniques used are similar to those used in identity testing for a criminal investigation, that is, extracting DNA from cells and manipulating it in such a way as to be able to examine the individual uniqueness of it.
If, after testing multiple systems, the parent in dispute is not excluded as a possible parent, a mathematical estimate of the possibility that the tested person could be the biological parent must be calculated. This mathematical testing combines the results of the genetic tests with other “non-genetic events” (location of the alleged parent at the time of conception, phenotype of the parent and child, etc.) and results in a “parentage index.” This index is a percentage of the likelihood of parentage. Results of these tests are admissible as evidence in court.

Tissue Typing for Transplantation
In the past, it was difficult to tell exactly whether an organ or tissue, such as a kidney, lung or bone marrow, was an exact match for the transplant between a donor and recipient. If it was not, a serious rejection reaction could sometimes occur between the recipient patient and the transplanted organ.
Basic laboratory testing for tissue transplantation involves mixing the white blood cells (leukocytes) from the donor (or the donor tissue) and the recipient together and observing whether an immune response occurs. Proliferation of a specific population of leukocytes signals the onset of an immune response and the likely rejection of the tissue by the recipient’s body. Although this technique is still commonly used, analysis of the DNA in both the donor and the recipient (tissue typing) is used to diminish the likelihood of rejection in the case of tissue transplantation. In bone marrow transplants, DNA testing is done to determine whether the leukocytes and their precursors repopulating a recipient’s bone marrow are his own or those of the donor.
A very specific set of genes is examined when DNA testing is used for tissue typing. On chromosome 6, a large set of genes called the “Major Histocompability Complex,” or MHC, resides. These genes are very polymorphic (different) between individuals, and they code for the production of specific glycoprotein antigens located on the surface of many cells. It is these antigens that “recognize” our own organs and tissues from those of another individual. These antigens have the ability to begin an immune system response that results in organ or tissue rejection if the tissue looks foreign.
A distinct region within the MHC on chromosome 6 is used in the DNA analysis of tissue that could be used for transplantation. This region called the human leukocyte antigen, or HLA-D, region, and sets of genes located there are further subdivided into HLA-DR, HLA-DQ, and HLA-DP depending on the type of glycoprotein antigen they code for. Polymorphisms in these genes are carefully compared between donor and recipient to determine the appropriateness of the transplant.
The exact techniques used to test DNA for tissue typing are similar to those mentioned in the sections above. DNA is extracted from donor and recipient cells, then manipulated and fragmented in such a way as to isolate a specific region on a chromosome and within a gene. The fragments are subjected to further analysis that allows for comparison of the polymorphisms in the HLA-DP between the donor’s tissue and the recipient’s blood. This careful analysis of genetic material results in fewer rejection reactions and the chance for a successful transplant.

Cytogenetics (Chromosome Analysis)
Everyone has 23 pairs of chromosomes, 22 pairs of autosomes and one pair of sex chromosomes. The science that relates to the study of these chromosomes is referred to as “cytogenetics.” Persons who look at chromosome preparations on slides are “cytogenetic technologists” or “cytogeneticists.” A trained cytogeneticist examines the number, shape and staining pattern of these structures using special technologies. In this way, extra chromosomes, missing chromosomes, or rearranged chromosomes can be detected.
Studies of chromosomes begin with the extraction of whole chromosomes from the nuclei of cells. These chromosomes are then placed on glass slides, stained with special stains, and examined under a microscope. Sometimes, pictures are taken of the chromosomes on the slides, and the picture is cut into pieces so the chromosome pairs can be matched. Each chromosome pair is assigned special number (from 1 to 22, then X and Y) that is based on their staining pattern and size.
There are many disorders that can be diagnosed by examining a person’s whole chromosomes. Down syndrome, in which an individual has an extra chromosome 21, can be determined by cytogenetic studies. When there are three chromosomes in one group instead of a pair, it is referred to as a “trisomy.” Missing chromosomes can also be detected, as in the case of Turner’s syndrome, in which a female has only a single X chromosome. When there is only one chromosome instead of a pair, it is referred to as a “monosomy.”
Abnormalities in chromosome structure are also observed with cytogenetic staining techniques. The Fragile X syndrome, the most common inherited cause of mental retardation, takes its name from the appearance of the stained X chromosome under a microscope. There is a site near the end of this chromosome that does not stain, indicating its fragility. The gene in the fragile region is important in making a special protein needed by developing brain cells.
Sometimes, pieces of a chromosome will break off and attach to another chromosome somewhere in a person’s genome. When this happens, it is referred to as a “translocation.” An example of a disease caused by a translocation would be chronic myelogenous leukemia (CML) in which a part of chromosome 9 breaks off and attaches itself to chromosome 22. Another example would be Burkitt’s lymphoma, in which a piece of chromosome 8 attaches to chromosome 14. These chromosomal translocations cause disease because the broken piece usually attaches to the new chromosome near a special gene that then becomes activated and produces tumor cells. Translocations can sometimes be seen under the microscope if a special stain is used.
A special technique called “fluorescent in situ hybridization” or FISH can be used to view changes in chromosomes that result from genetic variations. A mutated gene segment in a chromosome can be made to “light up” or fluoresce when it is bound by a special probe. Genetic changes in some cancers can be detected using this method. For instance, FISH is one of the methods used to determine increased expression of the gene HER2/neu. There are many other applications of FISH technology as well, such as chromosome microdeletions, in which a certain part of a chromosome is completely missing. In this case, the chromosome segment will not fluoresce compared to a normal set of chromosomes.

Infectious Disease Testing
When we hear the term “infectious disease”, we usually think of something that can infect us and cause a disease process to begin. That “something” can be a bacteria, virus, parasite, or fungus obtained from many different sources (other infected individuals, poor hygiene, transfusion with infected blood, shared needles between drug users, etc.). Disease-causing bacteria and viruses are known as infectious agents, and some of them can be quickly identified by using genetic testing techniques; however, common infectious agents, such as certain bacteria and viruses, are much less expensive to identify using standard laboratory methods that don’t involve genetic testing techniques.
Bacteria are one-celled organisms that contain their own DNA and in some cases can cause serious disease. Even those bacteria that harmlessly live inside our bodies and are involved in beneficial chemical processes can become mutated under unusual conditions and cause us to be very sick. By isolating the DNA from bacteria, breaking it into small pieces and amplifying them, the bacteria can be identified very quickly. Some of the bacteria that can be quickly identified using these genetic testing techniques include: Chlamydia trachomitis, which is an organism that causes a sexually-transmitted disease; Neisseria gonorrhea, which causes gonorrhea, Borrelia burgdorferi which causes Lyme Disease, Legionella pneumophilia which causes Legionnaire’s disease, Mycoplasma pneumoniae which leads to “walking pneumonia,” Mycopbacterium tuberculosis which can cause tuberculosis, and Bordetella pertussis which causes whooping cough. Specimens that might contain these bacteria include urine, blood, sputum, cerebrospinal fluid, and others.
Viruses are unusual organisms that sometimes insert their DNA into a host’s genome. The viral RNA or DNA utilizes the host’s cells to produce proteins and make more viruses. Viruses such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are examples of RNA viruses.
Other disease-causing viruses that contain DNA instead of RNA include Herpes simplex virus, cytomegalovirus, Epstein-Barr virus, parvovirus, and varicella-zoster viruses. All of these viruses can be identified by first removing the suspected viral DNA or RNA from a patient specimen and then using it to provide a “fingerprint” of the suspected virus. Specimens usually include blood, cerebrospinal fluid, sputum, other body fluids, amniotic fluid, tissue, or bone marrow. Much of the testing on donor blood that will be used in a blood transfusion utilizes genetic testing to inspect the blood for viral contamination.
Determining how many copies of a virus’ RNA are present in an individual’s blood is another use of infectious disease genetic testing techniques. The number of copies present is typically referred to as the “viral load” or “viral burden”. This testing is usually done after a drug therapy is initiated to assess whether it is working or not to remove or decrease the viral RNA load. The most common viral load tests are for HCV or HIV, and the tests require a sample of blood.
A parasite is a complex multi-cellular organism. Parasites usually infect an individual through the saliva of a biting insect, such as a mosquito, or through infected material. An example of a parasite that can be identified using genetic tests is Toxoplasma gondii which can cause encephalitis or congenital infections that lead to severe damage of a fetus (fetal toxoplasmosis).

The genes could help us to live longer

The genes could help us to live longer
By Dr Theo Giannaros Molecular & Nuclear Biologist University of Goettingen
/Germany

Doctor Giannaros says scientists are learning more about key genes that control longevity
The 'holy grail' of preventing ageing is coming ever closer with the discovery of key genes that control longevity, according to a leading Greek & German scientist.
It will help us understand the mechanisms behind illnesses linked to old age ranging from heart disease to Alzheimer's and lead to new drugs to tackle them, said Professor Wilfried Hanke.
She said gene mutations which extend the lives of animals such as worms, fruit flies and mice appear to play the same role in humans, holding out hope that we will soon be able to live longer while staying healthy.
Recent research has revealed that changes to single genes can give animals an extended lifespan, slowing down many diseases of ageing at the same time.
Dr Giannaros, scientific director of the Institute of Geno-Type biotechnology, said such research could lead to many diseases being treated or delayed simultaneously with medication.
The 'pathways' in the human body through which nutrients are processed also offer an opportunity for manipulating lifespan, he said.
Although simply eating less can extend life in a range of animals, this has proved difficult to achieve in practice.
However, drugs which inhibit the nutrient pathways could replicate the effects of a restricted diet, increasing healthy lifespan and affecting a broad range of conditions such as cardiovascular disease, cancers, diabetes and Alzheimer's.
Doctor Giannaros, who will present a public lecture at the Hilton Hotel of Athens next month, said tackling the causes of ageing rather than treating the symptoms 'piecemeal' offers the best prospect for dealing with the diseases that result from it.
He said: 'Research on the diseases associated with ageing is generally done by separate communities of research workers who read different journals, attend different conferences and generally do not communicate with each other. But by tackling the causes of ageing itself we could treat, or at least delay, a broad spectrum of conditions simultaneously.'

New discoveries may allow people to live longer and remain in good health
Dr Giannaros said reducing the activity of a molecular signalling pathway in fat tissue could extend life by up to 50 per cent.
He said the insulin/insulin-like growth factor signalling pathway in mammals regulates blood sugar levels, growth and metabolic response to food intake.
Mutations in genes that encode the protein components of the pathway have proved to extend lifespan in a nematode worm, the fruit fly and mice.
Genetic variants for these genes in humans have proved to be associated with lifespan.
Doctor Giannaros said. 'Making the pathway less active helps worms, flies and mice to live longer. Given that this is a pathway that is present throughout the animal kingdom, these findings could offer important clues as to how humans could live longer.'
He said this research means a new approach to the treatment of age-related conditions. 'The major burden of ill health is in the older section of the population,' she added. 'The new discoveries about ageing have raised the prospect of increasing the number of years that people enjoy in good health, with broad-spectrum preventative medicines for the diseases of ageing.'
Doctor Giannaros has just been awarded a grant from Research into Ageing to advance knowledge on why bodies age.
'During the last decade, research into the biology underlying the ageing process has developed remarkably quickly,' he said.
'It is likely that during the next decade the nature of the major cellular and biochemical mechanisms that determine longevity and ageing will be identified.'
Doctor Giannaros was recently named a Scientist of Outstanding Achievement for 2009 by the UK Resource Centre for People in Science, Engineering and Technology.

Human Nutrition and Gene Interactions

Human Nutrition and Gene Interactions
A Symposium Introduction: Human Nutrition and Gene Interactions
Theo Giannaros, Willy Xylander, Hanna Assem and George Blackburn
Harvard Medical School, Division of Nutrition, Boston MA 02215



Nutritional genomics is the study of nutrient-gene interactions and the effect these interactions have on health. It includes how diet affects the way genes are expressed, the effect of genes on how the body uses nutrients, and the effects of nutrients on molecular level processes in the body. It is an area of science that seeks to help people improve their health by studying how genes influence responses of genetically similar
individuals or groups to foods. Knowledge of these interactions and variations can then be applied in the field of nutrigenetics to improve dietary guidelines for populations, or to tailor-make specific diets for individuals. Dietary patterns are known to be strongly linked to 7 of the
top 10 causes of sickness and death in North America, including heart disease, obesity, several cancers, and diabetes. A diet practiced by one individual may support his or her health and quality of life, yet the same diet practiced by another may lead to obesity and subsequent metabolic problems that compromise quality of life. The science of nutritional genomics should increase our understanding of diet-health-gene interactions, and provide a number of potential benefits for individuals,
groups, and societies. These include improved individual health, greater consumer choice and control, an increased role for prevention in health management, greater social equity, and health care savings through the prevention and slowing of diseases. However, translating the flood of information from the mapping of the human genome into useful knowledge is as low and painstaking process.

Dietary factors and related metabolic interactions have direct and indirect nutrient influence on specific gene regulation and expression. Research suggests that significant regulation appears to be at the level of transcription, with controlled modulation of messenger RNA levels. Data also indicate that nutritional factors, e.g., various vitamins regulated through dietary intake, can interact with other regulatory networks—
such as tissue-specific, developmental, and hormonal factors, as well as dietary fat or carbohydrate—to regulate gene expression. Other studies have demonstrated regulation of apoprotein gene expression by sucrose-rich diet, nutritional regulation of gene expression in lipogenesis, and suppression of fatty acid synthase transcription by PUFA.
Sanderson suggests that changes in the intestinal lumen can alter the expression of molecules in the intestinal epithelium that direct the mucosal immune system, with the intestinal epithelium acting as a relay for transducing the information of the intestinal environment to the mucosal immune system. Data indicate that this mechanism has advantages
over other forms of immune surveillance in the gut that require the breach of invading organisms. Such breaches can be manipulated by invading organisms such as the polio virus to enter the body. Sanderson et al. suggest that other mechanisms (e.g., enteral feeds used to treat Crohn’s disease in children in the UK) can also alter the luminal environment
radically enough to vary the signals from the intestinal epithelium
to the mucosal immune system. Girard reports that until recently, the regulation of gene expression in response to changes in nutritional environment was thought to be mediated primarily by hormones and/or the nervous system. However, the last decade has provided evidence
that major (glucose, fatty acids, amino acids) or minor (e.g., iron, vitamin) nutrients, or their respective metabolites, regulate gene expression in a hormone-independent manner. Recent experiments indicate that regulation of gene transcription is not a simple PPAR-mediated process.
Rather, data show that a great diversity of fatty acid-sensor proteins and potential transcription factors are involved in fatty acid mediated gene expression. Sensors are known to relay the transcriptional effects of fatty acids, mediating them either directly, through their specific binding to various nuclear receptors (PPAR, LXR, HFN-4alpha), or indirectly, via
modulations in the abundance of regulatory transcription factors (e.g., SREBP1-c, ChREBP). Each transcription factor makes a relative contribution to fatty acid-induced positive or negative gene expression. Fatty acids control the expression genes encoding regulatory protein involved in their own metabolism via molecular mechanisms. Nonesterified fatty acids or their CoA derivatives seem to be the main signals involved in the transcriptional effect of long-chain fatty acids. Growing knowledge of the
mechanisms by which fatty acids control specific gene expression may provide insight into the development of new therapeutic strategies for better management of whole body lipid metabolism and the control of blood levels of triglycerides and cholesterol, major risk factors for coronary heart disease. Salati notes that regulation of the activity of enzymes
involved in lipogenesis is key to understanding how a cell 1 Presented at the 6th Postgraduate Course on Nutrition entitled “Nutrition and Gene Regulation” Symposium at Harvard Medical School, Boston, MA, March 13–14, 2003. This symposium was supported by Conrad Taff Nutrition
Educational Fund, ConAgra Foods, GlaxoSmithKline Consumer Healthcare, Mc-Neil Nutritionals, Nestle Nutrition Institute, The Peanut Institute, Procter & Gamble Company Nutrition Science Institute, Ross Products Division–Abbott Laboratories, and Slim Fast Foods Company. The proceedings of this symposium are published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were: W. Allan Walker, Harvard Medical School, George Blackburn, Harvard Medical School, Edward Giovanucci, Harvard School of Public Health, Boston, MA, and Ian Sanderson, University of London, London, UK.

Downloaded from jn.nutrition.org by on September 25, 2009 adapts to dietary energy in the form of carbohydrate versus energy in the form of triacylglycerol. Changes in the activity of these enzymes are largely caused by changes in the rates at which their proteins are synthesized, with dietary nutrients signaling these changes via alteration of hormone concentrations or their own unique signal transduction pathways.

Bioactive food components and molecular targets
Milner reports mounting evidence pointing to dietary habits as an important determinant of cancer risk and tumor behavior. At the same time, he notes inconsistencies in the literature that are probably due to variable abilities of bioactive constituents to reach or affect critical molecular targets.
Data indicate that fluctuations in the foods consumed not only influence the intake of particular bioactive components, but may also alter metabolism, and potentially influence the sites of action of both essential and nonessential nutrients. Genetic polymorphisms are increasingly recognized as another factor that can alter the response to dietary components (nutrigenetic effect) by influencing the absorption, metabolism, or sites of action. In addition, variation in the ability of
food components to increase or depress gene expression (nutrigenomic effect) may account for some observed inconsistencies
in response to dietary change. A host of food components known to influence phosphorylation and other posttranslational events make it is likely that these, and other proteomic modifications, account for at least part of the response and variation reported in the literature.
The term nutritional genomics has been used to describe work at the interface of plant biochemistry, genomics, and human nutrition. Genetic changes arising as a result of single point mutations, rearrangements, or copy number involving either deletions or additions likely influence the response to various dietary components. The basis for nutrigenomics arises
from rather compelling evidence that a variety of nutrients interact with specific molecular targets. Invariably, studies reveal that a host of messages are modified by the presence or absence of a food component.
Proteomic techniques are being developed and refined to assist in the identification of the proteome, which is defined as all of the proteins present in a particular cell at a particular moment. The importance of these types of investigations stems from the fact that gene expression does not always correlate with protein expression, and the influence of food
components may be either translational or posttranslational rather than at the transcriptional level. Proteomic-based studies, although technically challenging, complement genomic studies and are essential in any comprehensive research strategy aimed at examining the molecular
processes and factors involved in modulating disease risk. The use of current proteomic tools will not only advance the field of nutritional sciences, but will make the discipline more valued for its involvement in disease prevention including cancer. Metabolomic science derives from the fact that responses to a bioactive food component cannot be considered to occur in isolation, but must be evaluated in the context of an entire diet. Metabolomics involves the systematic estimation of metabolomes, i.e., the characterization of all metabolites and small molecular weight compounds occurring in an organism. Several methods, including principal component analysis and clustering, are being examined to analyze metabolomic data.
Metabolomic subsets, such as the lipid and amino acid metabolome, already suggest that some very useful information can be obtained from metabolomic analyses.

Fatty acid binding proteins
Hotamisligil’s research indicates that fatty acid binding proteins (FABPs) are members of a family of proteins highly
conserved with the task of protecting the delicate lipid balance of a cell. However, when faced with metabolic or inflammatory stress, they fail, turning the cytosol into an inhospitable environment with less than ideal outcomes. Recent studies focus on how FABPs direct lipid traffic and simultaneously control metabolic and inflammatory pathways under the pressures of the metabolic syndrome.
Under normal physiologic conditions, mice do not have a compromised phenotype when FABPs are deleted, but they benefit enormously when faced with systemic stresses, particularly of metabolic and inflammatory origin. Evidence suggests an evolutionary role for the existence of this protein. For survival of the most successful organism, efficiency in metabolic and immune responses is crucial to resist starvation as well as infection. Evolutionary selection has clearly preserved the FABP from yeast to humans, indicating that the close link between the inflammatory and metabolic responses underlies the conservation of FABP function. Data suggest the concept of a biological role for FABPs as a potential central regulator of common pathways controlling metabolic and inflammatory signaling under physiological and pathological conditions. For many signaling systems acutely activated, such as during inflammation, regulatory mechanisms have evolved to amplify and/or attenuate the response. For example, inflammatory stress increases lipolysis in the adipocyte and fatty acid synthesis by the liver, while decreasing oxidation of fatty acids by the liver, heart, and muscle, thereby flooding the system with excess fatty acids. While FABPs appear necessary to evoke a strong inflammatory response, too strong a response can be overwhelming and damaging. Data suggest that FABPs may be master regulators, necessary to fine-tune the balance between the availability of metabolic resources, a robust inflammatory response, and its resolution. Further understanding of the mechanism of action, and eventual modulation of FABP activity, might lead to opportunities to regulate lipid-sensitive pathways. Based on the limited expression pattern of FABPs, interference with the protein’s ability to orchestrate lipid signals may provide clinicians with pharmaceutical specificity in a highly cell-typerestricted manner. In time, the true nature of FABPs will yield to scientific inquiry, paving the way for the development of
therapeutic options for a broad range of pathologies, including obesity, insulin resistance, type 2 diabetes, atherosclerosis, and perhaps other inflammatory conditions such as arthritis, asthma, or Alzheimer’s disease.

Cancer prevention
Genetically engineered mouse strains with over expressed or inactivated cancer-related genes have recently been developed. These provide investigators with powerful tools for studying carcinogenesis and for testing preventive strategies that can offset increased genetic susceptibility to cancer in humans due to specific genetic lesions. Hursting reports work
on the development of relevant animal models for cancer prevention research that aims to: 1) characterize the molecular mechanisms that underlie effective modulators of cancer risk; 2) capitalize on mechanistic information to develop effective combination regimens; and 3) develop surrogate endpoint biomarkers that can be translated to human studies. Research to date has focused on preventing cancer by dietary interventions, particularly obesity prevention/energy

INTRODUCTION 2435S
Downloaded from jn.nutrition.org by on September 25, 2009 balance modulation, in mice deficient in the p53 tumor suppressor gene, the most frequently altered gene in human cancer. Given the impact of obesity on cancer development, and the paucity of mechanistic data on this association, studies of energy balance and cancer are essential. Hursting also proposes to capitalize on the availability of new tools (e.g., geneticallyengineered mice, gene expression microarrays, and proteomics)
to identify additional targets that can be modulated. Hursting is currently comparing and combining caloric restriction and exercise in p53-deficient mouse models, as well as in other tumor models. They are also investigating the role of IGF-1, other hormones, and body composition in the energy balance and cancer relationship. Together, findings clearly demonstrate that the increased susceptibility to cancer as a result of a genetic lesion, such as loss of p53 tumor suppressor function, can be offset, at least in part, by preventive approaches. Halperin reports that the composition of diets critically influences the expression of many genes. Long chain _-3 PUFA are of particular interest since they represent one dietary component that appears to have a significant impact on the expression of specific genes. The molecular mechanism of the anti-cancer activity of _-3 PUFA includes the partial depletion of ER Ca__ stores, which inhibits translation initiation and preferentially downregulates the synthesis and expression of oncogenes and growth-promoting proteins that block the progression of the cell cycle in G1. Long chain _-3 PUFA also induce the expression of pro-apoptotic proteins through gene-specific regulation mediated by transcription factors.
Halperin notes that perhaps the most important aspect of his research is the generation of tools that allow assessment of whether the anti-cancer effect demonstrated in vitro and in animal models also operates in human subjects. He expects accreditation of the translation initiation machinery as the effector of the anti-cancer properties of _-3 PUFA in humans to foster
clinical trials to test their therapeutic and preventive effect in human cancers. Furthermore, since the expression of other genes involved in the pathogenesis of several diseases may also be highly regulated at the level of translation, it is conceivable that_-3 PUFA may help treat and perhaps reduce the risk of some chronic pathological conditions that burden the human population.

Leptin
In his keynote address, Friedman discussed the role of leptin as a central mediator in a negative feedback loop regulating energy homeostasis. He that noted that although elucidation of leptin’s role enables a more detailed view of the biology underlying energy homeostasis, most obese individuals are leptin resistant. According to Friedman, the development of effective treatment for obesity and the metabolic syndrome will require more complete understanding of the molecular components of the leptin pathway.
Friedman and Cohen’s recent review of studies on the identification of one such component, stearoyl-CoA desaturase-1 (SCD-1), suggests that leptin’s metabolic effects are, to a large extent, mediated by repression of this gene. The specific mechanism by which leptin represses SCD-1 is currently unknown, but it could involve both transcriptional and post-translational modulation. Moreover, SCD-1 appears to be a critical metabolic control point partitioning fats towards storage when activity is high, and towards oxidation when activity is low. Greater insight into the role of SCD-1 in other aspects of the metabolic syndrome, such as diabetes and atherosclerosis, will require further study. Research is also necessary to determine whether the absence of SCD-1 confers any adverse health risks. Data indicate that increased fatty acid oxidation may raise the levels of toxic free radicals, which could predispose to cancer or reduced longevity. While a mouse model algorithm has found SCD-1 to be the most potently leptinregulated gene, other genes identified as being either specifically
repressed or induced by leptin may also have critical physiological roles. According to Friedman, future work will determine whether inhibition of SCD-1 could be a therapeutic target in the treatment of obesity, hepatic steatosis, and other components of the metabolic syndrome.
Dr.Mantzoros reported on the role of leptin in the regulation of several neuroendocrine axes, such as the hypothalamic-pituitary- gonadal and the hypothalamic-pituitary-thyroid axes in human, and its potential pathophysiologic role in eating disorders. Recent discoveries indicate that leptin levels above a certain threshold are required to activate the hypothalamicpituitary- gonadal and hypothalamic-pituitary-thyroid axes in
men, whereas the hypothalamic-pituitary-adrenal, renin-aldosterone,
and growth hormone-IGF-1 axes may be largely independent of circulating leptin levels in humans. Leptin is a hormone that communicates information on the body’s fat stores/energy reserves to the brain, thus maintaining normal function of several neuroendocrine axes. Several conditions, including eating disorders such as anorexia nervosa and
bulimia nervosa, are associated with altered serum levels of leptin
as well as abnormalities in neuroendocrine functions. According to Mantzoros, recently completed interventional studies propose that leptin acts as a “master hormone” in regulating neuroendocrine function in normal healthy volunteers. These studies have already provided the basis for a better understanding of the mechanisms underlying the hormonal abnormalities of subjects with eating disorders, and may lead to the development of new therapeutic strategies for these conditions.

Summary
This symposium provided an overview of research in the field of nutritional genomics, including developments in nutrigenics, proteomics, and metabolomics. Speakers reviewed nutrient-gene-health processes that have the potential to help prevent many diseases, and greatly affect our optimal nutrition and health. Humans differ in their metabolic regulation, and the optimal diet for one person is not necessarily the optimal one for another. Determining which diet is best for each individual will require personalized assessment.
Progress towards the goal of individualized diets and health care will make diagnostic use of single biomarkers of diseases inadequate for accurate surveillance and intervention in problems of metabolic regulation in healthy individuals. At a 2003 symposium on next-generation nutritional assessment, Bruce German (1) noted that measuring entire metabolic pathways is the ultimate scientific goal, and that modern analytic techniques are in a position to deliver such capabilities. His challenge to us at that time was to build the metabolic knowledge to understand metabolism as a whole, and provide guidance to individuals to change their diets and lifestyles to affect metabolism in a net positive direction (2). This symposium shows impressive advances in that direction, with the promise of accelerating progress as our knowledge base in nutritional genomics continues to grow.

LITERATURE CITED
1. German, J. B., Roberts, M. A. & Watkins, S. A. (2003) Personal metabolomics
as a next generation nutritional assessment. J. Nutr. 133: 4260–4266.
2. Ward, R. E. & German, J. B. (2004) Understanding milk’s bioactive
components: a goal for the genomics toolbox. J. Nutr. 134: 962S–967S.

A Genetic Profile of Prognosis using PLS

A Genetic Profile of Prognosis using PLS
Dr rer. nat. Theo Giannaros, Medifit S.A., K. Paleologou 12 N. Smyrni, Athens /Greece Reference Laboratory (Geno-Type biotechnology) Kifisias.Ave 48-50, Athens/Greece

ABSTRACT
In a typical clinical study based on a microarray gene expression experiment, there are often more genes than subject samples, and many genes are correlated. Partial least square regression, though not originally designed for classification purposes, can be used to build a classifier to predict outcomes based on the high dimensional correlated gene expression data. In this article, using a publicly available breast cancer study data, we show the process of using PROC PLS in SAS to construct a metastasis risk classifier from a training dataset. The classifier is further used to assign patients from an independent validation set into high- and low- risk groups based on their gene expressions. The result confirms that patients in the two predicted risk groups show significant difference in their survival outcome.


INTRODUCTION
With the maturing of DNA microarray technology, many gene expression based applications in the cancer diagnostics field are becoming more acceptable to physicians and the regulatory agencies. On February 14 2007, the FDA approved the first gene expression based in vitro prognostics tool, MammaPrint, in US allowing it to proceed to the market to help doctors assess young breast cancer patients’ risk of distant metastasis. This approval will certainly encourage more clinical studies involving microarray technology, and more gene data analysis methods and tools will be developed as a result. In a DNA microarray experiment dataset, the number of subjects (observations) is usually much smaller than the number of genes (variables), and many genes are correlated, i.e., they are not independent. Theoretically, statistical methods having an independency assumption cannot be used directly in the above scenario. Many machine learning methods such as the Support Vector Machine, C5.0 Decision Tree and Neural Networks have been used to find gene expression patterns related to disease [1], Partial Least Square is also a good method for exploring information hidden in the huge amount of gene data. While Ordinary least squares regression has the goal of minimizing sample response prediction error and seeking linear functions of the predictors that explain as much variation in each response as possible, Partial Least Square has the additional goal of accounting for variation in the predictors under the assumption that directions in the predictor space that are well sampled should provide better prediction for new observations when the predictors are highly correlated [2]. Also, the Partial Least Square method has been implemented in SAS and is easy to use. It is a simple approach to first use PROC PLS to analyze the data and see if a gene expression difference exist among patient groups of interest in a study. Then other methods may be brought in to refine models and build more powerful tools. In this paper we show the detail of using PROC PLS for classification applications in SAS.

EXAMPLE: GENE EXPRESSION OF YOUNG BREAST CANCER PATIENTS
Breast cancer is the most common cancer among women, except for non-melanoma skin cancers, and it is the second leading cause of cancer death in women, exceeded only by lung cancer. According to the American Cancer Society, an estimated 178,480 new cases of invasive breast cancer will be diagnosed among women in the United States this year and over 40,000 women are expected to die from the disease. Death rates from breast cancer continue to decline, with larger decreases in women younger than 50. It is believed that early detection through screening based on various technologies, refined prognosis, increased awareness, as well as improved treatment have contributed to the death rate decrease. Microarray technology is widely used to provide a better screening tool and refine prognosis to provide physicians with accurate information and guide tailored treatment for patient. Using DNA microarray analysis on primary breast tumors of 117 young patients, Van’t Veer et al reported a 70-gene prognosis profile associated with the risk of early development of distant metastasis in patients with lymph-node negative breast cancer [3].

Van de Vijver et al further evaluated the 70-gene classifier on a series of 295 consecutive patients with stage I or stage II breast cancer and made those 295 patients gene expression data available to the public [4]. The log ratio column in the dataset represents the normalized expression level of a gene. The dataset consists of log ratios of over 20000 genes for 295 patients who were diagnosed with breast cancer between 1984 and 1995 and treated by modified radical mastectomy or breast-conserving surgery, followed by radiotherapy at the hospital of the Netherlands Cancer Institute. The median follow up time was 7.2 years and the median survival time was 3.8 years for those 295 patients. We first used the following macro to randomly split the 295 patients into two independent set, 70% training set and 30% validation set. The all295 is the input data set, and 1224 is a seed for the random process. Since a random split is relatively easy to code in SAS, the detail of the macro is omitted here.

%ran_split(all295, eventmeta,train, valid, 0.7,1224,idvar=sampleID, stratify=yes);


BUILDING A CLASSIFER FROM THE TRAINING SET
There are many ways to select genes that might be associated with clinical outcomes. It is out of the scope of this article to discuss and compare the difference methods of feature selection. As a demonstration about using of PROC PLS, we used the 70 genes identified in the original paper of Van’t Veer et al as our predictor variables. The number of factors to extract depends on the training data. However, the extraction of excess factors must be avoided in order to prevent overfitting. The PLS procedure enabled us to choose the number of extracted factors by cross validation. Various methods of cross validation are available, including one-at-a-time validation, and splitting the data into blocks.

proc pls data= train cv=split ;
model eventmeta = &pls_mvars ;
run;
cv=split option sets every 7th ( default ) observation aside as validation set. This number can be optimized according to the size of the sample set. The above pls_mvars macro variable includes all the 70 genes. The output is as follows:




The PLS Procedure

Split-sample Validation for the Number of Extracted Factors

Number of Root
Extracted Mean
Factors PRESS

0 1.079415
1 1.025679
2 1.017247
3 1.050493
4 1.073688
5 1.081337
6 1.090805
7 1.103451
8 1.113767
9 1.12401
10 1.128974
11 1.139266
12 1.143572
13 1.149865
14 1.154781
15 1.15846

Minimum root mean PRESS 1.0172
Minimizing number of factors 2


PREDICT RISK OUTCOME IN THE VALIDATION SET
It is recommended to extract 2 factors for the model based on the training data. The following code brings in the independent validation, specifies 2 factors to extract and output the predicted value to an output data set pred.
data all;
set train valid(rename=(eventmeta=meta));
run;
proc pls data=all nfac=2 ;
model eventmeta= &pls_mvars;
output out=pred p=p_meta;
run;

It is necessary to mention, when combining the training set with the validation set, the above “rename” option is important, because we want the model to predict the outcome of the observations from the “valid” set. If observations from the validation set have the eventmeta values, they will affect the PROC PLS model building process and a new model is rebuilt and we want to avoid this.
In the output dataset, the predicted values of response p_meta range from 0 to 1, we could chose difference cutoff value to dichotomize them, thus tuning the sensitivity and specificity. The following data set and two PROC FREQs were then used to see how well the model works on the training set and the validation set.

data pred;
set pred(keep=SampleID EventMeta TimeSurvival Meta P_meta) ;
if P_meta>0.37 then pls=1;
else pls=0;
run;
proc freq data=pred(where=(eventmeta>.));
le eventmeta*pls/nopercent nocol;
tabrun;
proc freq data=pred(where=(eventmeta=.));
table meta*pls/nopercent nocol;
run;

The output is shown in the following Table 1. and Table 2. As seen in Table 1, when we chose the above 0.37 cutoff value, on the training set, the sensitivity is 73.24% and specificity is 76.12. They are quite balanced. On the validation set, the sensitivity is 63.33%, while the specificity is 75.44%. It is reasonable that sensitivity, specificity or both are not as high as those on the training set. Increasing the sensitivity by lowering the cutoff value results in a decrease in specificity.


SURVIVAL DIFFERENCE OF THE PREDICTED RISK GROUPS
When the full clinical data is not available but survival data is provided, the survival data can be utilized. The classifier will divide patients based on gene data into high and low risk groups. The survival information can then be used to confirm the risk classification. The following is an example:

proc lifetest data=pred(where=(eventmeta=.))
method=KM plots=(s) ;
time timesurvival*eventdeath(0);
strata pls;
run;

The Kaplan-Meier curve plot is shown in Figure 1. The predicted high risk group ( pls=1, red) has significantly lower survival rate than the predicted low risk group(pls=0, black), as shown by a log rank test p-value <0.0001. The survival result verifies that the gene expression difference we found from the training set is truly related the biological difference.






Table 1. Training set result.

Table of EVENTmeta by pls
EventMeta pls

Frequency Row Pct 0 1 Total
0 102 76.12 3223.88 134
1 19 26.76 5273.24 71
Total 121 84 205


Table 2. Validation set result

Table of meta by pls
Meta pls
Frequency Row Pct 0 1 Total

0 43 75.44 14 24.56 57
1 11 36.67 19 63.33 30
Total 54 33 87


CONCLUSION
The SAS procedure PLS allow for building a classifier using many (greater than number of observations) variables as input predictors. The number of factors to be extracted can be determined by cross validation. The sensitivity and specificity of the model can be tuned by choose cutoff values when dichotomizing the predicted response value.

REFERENCES
[1] Brown et al. Knowledge-based analysis of microarray gene expression data by using support vector machines. Proc Natl Acad Sci U S A. 2000 January 4; 97(1): 262–267.
[2] SAS/STAT manual, http://support.sas.com/onlinedoc/912/docMainpage.jsp.
[3] Van’t Veer LJ, Dai H, Van de Vijver MJ, He YD, Hart AA, Mao M, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002; 415:530-6.
[4] Van de Vijver, M.J., He, Y.D., et al. (2002). A Gene-expression signature as a predictor of survival in breast cancer. New England Journal of Medicine 347, 1999-2009.
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
This Research was supported [in part] by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

CONTACT INFORMATION
Your comments and questions are valued and encouraged. Contact the author at
Theo Giannaros, theogiannaros@in.gr

Over 20 Years of Hair Transplant Struggle: Chronicle of a Hair Transplant Victim's Inspirational Transformation

The use of the Transstem Method
This is a true story…

This story of a former hair transplant victim who has spent over 20 years living with the consequences of some very bad hair transplant decisions. After living with a pluggy and unnatural hairline for a good part of his life, this hair transplant victim eventually found salvation from the Transstem Method, created by the known Molecular and nuclear biologist Dr Theo Giannaros in Athens Greece and is now the proud author of this incredible story.

Few corrective surgeries can rival what was actually involved in giving this hair transplant victim the ultimate transformation. In this story, you will read about the finest in micro surgical techniques, skin grafting, excision free transplant as well as revolutionary and ground breaking stem cell injection.

The following is an account of my experiences over the last 22 years. I am not a doctor and I am not promoting hair transplantation as all my efforts were concerned with correcting the unsightly condition of seven previous and hair transplant surgeries. I am not affiliated with any physician or represent any company or organization with regard to hair related matters and receive no compensation from anyone. I have gone too great effort and personal expense to seek an acceptable corrective surgical result. It is my opinion that corrective hair transplant surgery should be considered a specialty within the hair transplant process. It is also my opinion that hair transplanting a virgin head is a much easier process when compared to correcting a botched hair transplant and this is not an easy task for any doctor!

I had my first three hair transplant surgeries in 1988 via the "Black & Decker" punch graft method over a five-month period. Needless to say, the end result was something less than the image of the pictures in the color brochure with glowing testimonials and a picture of the medical director in a three-piece suit sitting behind his hardwood desk. I won't get into a rant about the many registered letters of complaint and phone calls that were never responded to. It would be 15 years before I put my faith in another physician in the hopes of correcting a wall of plugs protruding from my frontal hairline.

In 1994, I approached another clinic that advertised they performed hair transplant surgery for an evaluation of my condition. I was informed that I had sufficient donor area and that a series of surgeries would correct the condition. Throughout the 15 years, I would visit other doctors, not related to hair transplantation surgery, in the hope of getting some insight to my condition and had no luck in doing so. There was one surgery performed from the doctor at this clinic and I never returned. I would later find out that this particular doctor was responsible for many disfiguring hair transplant surgeries.

The next year, in 1995, I had an evaluation from another doctor that said he could help me and that he performs corrective surgery on an ongoing basis and has been performing hair transplant surgery for many years. Once again I put my faith in another physician and submitted to a series of three corrective surgeries with this doctor. The results of the corrective surgery are evident in my before pictures.
In 1998, at 50 years old I retired from my business and decided I would try a different approach to acquiring a corrective surgery other than putting my faith in another physician involved in standard hair transplant surgery. I now had the time and financial resources to acquire a capable physician and a true corrective surgery, if available. Anger is a great motivator! I listened to radio advertising from two of the previous physicians responsible for my hair transplant surgery proclaiming they were expert/specialist and the "new technology" in hair transplantation is superior. The clinic responsible for my first three plughair transplants advertised "you have seen people with bad hair transplants and we would not do that to you!" After 20 years I walked into this clinic and had a chat with the head honcho. Nothing is worse for a hair transplant doctors business when there is someone like me in the waiting room verbalizing and frightening the prospects waiting for an evaluation. I told the doctor and manager I would do some advertising of my own. And I did!
I took out advertising in all Greek newspapers for "Bad Hair Transplant" recipients in the hope of making contact with someone that had the bad hair transplant and was able to locate a physician capable of repair. At the same time with the same advertising, I offered free information to anyone looking into the hair transplant process. The responses I received from prior hair transplant recipients were nothing but horror stories. Not one person could give me any insight to a physician concerning a corrective method. In fact, it seems that many people, including myself, have been playing musical chairs, a better term is Russian roulette, with the same doctors responsible for other people's bad hair transplants.
Had the internet been available even seven years ago, to the extent it is today, things might have been different. The various forums on the internet have provided the good, the bad and the ugly with regard to information. The internet is a tool and I have learned to read between the lines to source out viable information. I would visit many doctors over the next two years and I was interviewing them as much as they were evaluation me. There are doctors of note that have satisfied clients concerning corrective surgery. The prognosis with regard to my situation came down to a ravaged donor area and not enough elasticity for suturing the donor site. Every evaluation ended with "something could be done to improve the condition." I received proposals that ranged from a single surgery to a series of four surgeries. More confused than ever, I still surfed the net and participated in forums and voiced my opinions concerning hair transplants. There are proponents of hair transplants and there are people satisfied with the surgery and I wish them well. However, the people that responded to my advertising and the people emailing me were recipients of a botched hair transplant and have been living with the adverse effects from a surgery that was supposed to improve your life.
I first heard of Dr Theo Giannaros and his team in Athens Greece the summer of last year and that his approach was different. I kept his name and a printout of his website among the many doctors in my file. I emailed my "before" pictures to Dr. Giannaros. I was contacted and informed that the removal of the plugs could be achieved with a series of 4 or 5 surgeries over a ten day period. The elasticity concern in the donor area was not an issue as there is no suturing and the hair follicles are removed within the entire donor area with a minimum of surrounding tissue removed. I am intrigued but I'm not ready to hop on a plane just yet! I kept surfing and getting feedback from people, especially from Greece and Southern Europe, and once again decided to put my faith in another doctor. He wasn’ t actually a Medical Doctor, But A Molecular Biologist specialized in the Biology of Beauty

Monday February 5th, 2008

Athens, here I come! My plane leaves Boston Logan airport February 5th and I have a two-hour layover in Los Angeles before the 14-hour flight to Athens - Greece. It's a long flight and I have pretty good case of jet lag when the plane lands at 6:00am Athens time February 6th.

Wednesday February 7th, 2008

I clear customs and crash in a hotel for a day. I am picked up the next day and meet Dr. Aris Tzikos and get transported to my accommodations at the surgery center where I met Dr. Theo Giannaros. I was examined by both Dr. Tzikos, & Dr Giannaros then settle in and prepared for surgery the next day.

Friday February 9th, 2008

As prepared as I was and with the information I had, it really comes down to putting yourself in the hands of the doctors. Surgery starts at noontime! Extensive videotaping and then discussion between the two doctors. The left frontal side was damaged from prior surgeries as the thick wall of plugs and scaring was severe in this area. The anesthetic is applied to the frontal area by Dr. Kamarinos in very small amounts. The "ouch factor" is minimal and I tolerate it well. The doctors proceed with the labor intensive task of plug removal and skin grafting the area where the plugs were removed. The follicles from the plugs are disbursed into other areas. The two doctors work together and independently. Breaks are taken for lunch and resting. Surgery finishes 13-1/2 hours later at 1:30am. I take it easy over the weekend and I feel OK considering the marathon surgery I just went through and prepare myself for the next surgery.

Monday February 12th, 2008

The right frontal side is addressed in the same manor as the left side. This area was very pluggy but was not as damaged as the left side. Surgery time with lunch and breaks is 10 hours.
The next day is a rest day. There is some minor swelling and this is normal with hairline frontal surgery due to the anesthetic.

Wednesday February 14th 2008

This was a short day with the top area approached away from the frontal hairline and hair follicles are extracted from the donor area and implanted into this region. Surgery time is 8 hours.

Thursday February 15th, 2008

Another marathon surgery! Follicles were removed from the donor area and implanted in the frontal area establishing a new hairline. The hair follicle has to be inserted at the proper angle in the recipient site. A very long tedious process! Surgery time is another 11 hours.


Saturday February 17th, 2008

A touch up of plug removal in the back area and disbursement of the follicles. The injection of the Stem cells taken from my own fat were injected in to the skin of my head. This was the final step or my …restoration Surgery time is 6 hours.
I want to point out that this method of surgery is exhausting for the physicians as they are bent over you for hour after hour wearing binocular visual aids.

NOW LET AS DISCRIBE THE RESULTS...
I was a corrective patient and a worst case scenario and should not be confused with a new virgin head having hair transplant surgery. My goal was to diminish the unsightly effects of the prior seven hair transplant surgeries. I am told by Dr.Tzikos that I will look worse before I start to look better. However, the thick plugs that were staring at me for 22 years are gone and considering what I looked like there is already a definite improvement and I am encouraged with the results.

Considering the surgery was performed 5 weeks ago, I am greatly encouraged with the results. I don't expect to have an Elvis head of hair when I am fully healed and the new hair grafts have grown in as that was not my intention for having the surgery. My greatest hope was to have the thick wall of plugs completely removed and re-establish the frontal hairline
I wet my hair on the top of my head and combed my hair directly back and took the pictures at a closer distance than the other photo's. I do not comb my hair in this manner but I felt as though this was necessary to expose the area fully where the frontal corrective surgery was performed. The area is still healing and it will take several months to completely heal.
It has been 11 weeks since my corrective surgery with Dr. Giannaros in Greece. The skin grafting in the area where the former pencil eraser sized plugs, that had formed my hairline, has healed and blended in nicely. The area you see in the picture on the upper hairline with my hair wet, was severely damaged with a raised wall of scar tissue and thick plugs. The plugs had been placed at the wrong angle on the hairline and grew outwardly in an unnatural direction and amplified the unsightly condition.
Another benefit of the corrective surgery, besides the cosmetic improvement, was the cobblestoning and raised plugs would constantly get irritated and even more so in the hot summer months. I can report that I feel better as there is no longer any irritation via the removal of the raised plugs and scar tissue. The majority of the follicular units that were transplanted are just starting to break through the surface and I am looking forward to the future when they have grown to length and blend in.
I will return to Dr. Giannaros and the rest of his team next year for corrective donor scar revision from prior hair transplant surgeries. Dr. Giannaros and his team is able to utilize your chest hair and transplant the follicles into the prior donor scars to diminish the appearance and save any valuable donor hair for the top of your head. I also have enough donor hair for another 500 grafts and will utilize this for a touch up. But, actually, the best part of my reconstruction was the injection of my own Stem cells, which are …still working in my scalp producing hair follicles
Once again, I am greatly encouraged with the results. It also takes getting use to looking at yourself, especially when I look at a "before" picture of myself. I am now a raving fan of the TRANSSTEM METHOD, of Dr. Giannaros team and of Greece… Thank you very much guys!