Lesson 2.

1: Genetic Testing and Screening

A brief review and introduction

In Unit 1, we were introduced to the Smith family for the first time. Sue, the college-age daughter of James and Judy
Smith, contracted bacterial meningitis and we worked towards treating her, handling the long-term effects (hearing loss) and
studied how future infections by pathogens can be prevented. In Unit 2, we find out some new and exciting information for the
Smith family – Judy Smith is pregnant again. Because Judy is a bit older than when her first two children were born, there are
some additional risks in the form of an increased chance of inherited diseases. In Unit 2, we looked at a new category of diseases
– diseases a person is born with (inherited diseases) rather than those they “catch.” We also look at genetic screening and
testing, the value of screening and examining DNA, the importance of prenatal care, and the future of genetic technology.

Current technology allows us to look past the surface of our cells and to understand their inner
workings, their most important part – DNA. DNA can now be isolated from cells and “picked apart” to
reveal disease. Genetic testing can be used to diagnose disease before a child is even born. We can test
ourselves for diseases and learn the likelihood of passing them on to children. Genetic testing is the use
of molecular methods (DNA sequencing with BLAST, karyotyping, etc.) to determine if someone has a
genetic disorder, will develop one, or is a carrier of a genetic illness. It involves sampling a person’s
DNA and examining the chromosomes or genes for abnormalities.

Genes, Chromosomes, and DNA

A bit of review: a chromosome is tightly coiled DNA. The

human body contains 23 pairs of chromosomes: 22 pairs of autosomes
and one pair of sex chromosomes. These chromosomes are inherited
from your parents, and from the moment of conception (fertilization)
they are your genetic code – your DNA. The chromosomes are typically
only visible during cell division – the rest of the time, DNA is a jumbled
mess that is invisible with a light microscope. This DNA, which forms
chromosomes, holds genes. Genes are the coding sections of DNA, and
their job is to provide the instructions for building proteins. Your body
is composed of proteins. They are the workers of your body and are
essentially responsible for every trait you have: hair color, eye color,
blood type, skin color, and diseases you have. Chromosomes
themselves can be the cause of disease, as can defective genes.

In short, too many chromosomes – bad. Not enough chromosomes – bad. Inheriting a copy of defective DNA (bad
genes) – also bad. So far so good? Great! Lets’s continue.

Genetic Testing Overview

By now, you probably realize that there are all kinds of things that can go wrong when a human being is created. It’s a
wonder more of us don’t have things wrong. Because of the possibilities that exist for pr oblems, many people have a strong
desire to know whether they have diseases, could pass them to children, or if their unborn children have a disease. That is w hat
genetic testing is all about – using DNA to help people find out what they want (and sometimes need) to know.

Genetic testing is often performed by a genetic counselor. A genetic counselor is a trained professional who helps
individuals and families understand and adjust to a genetic diagnosis or the possibility of having a hereditary disorder. Ge netic
counselors interpret family history information and educate patients and professionals about genetic diseases. As specialized
counselors, these professionals help patients and families understand genetic testing options and the implications of
undergoing genetic testing. In addition, genetic counselors address psychosocial and ethical issues associated with a genetic
disorder and/or a genetic test result. As members of a health care team, genetic counselors serve as educators to their patie nts,
to physicians, other health care providers, as well as to society. Genetic counseling can help a family
understand the risks of having a child with a genetic disorder, the medical facts about an already diagnosed
condition, and other information necessary for a person or couple to make decisions suitable to their
cultural, religious, and moral beliefs. To keep things simple, they help with the testing and provide
information people need to make informed choices.

Types of Genetic Disorders

Genetic testing reveals whether or not a DNA-based problem is present. These genetic disorders are caused by
abnormalities in an individual’s genetic material. We talked about four different types of genetic disorders: single-gene,
multifactorial, chromosomal, and mitochondrial. You may remember these – if so, feel free to skip ahead!

A single-gene disorder is a change or mutation in one gene. Sickle cell anemia and cystic fibrosis are good examples of
these. Single-gene disorders may be classified as autosomal dominant, autosomal recessive, or sex-linked. A dominant trait is
one where one copy of a gene passed to a child causes an effect in the child – like dwarfism or Huntington’s disease. A recessive
trait (sickle cell and cystic fibrosis) is one where a child must inherit the defective gene from both parents in order to express
the trait. If the child only gets one copy, he or she is a carrier of the trait, but will not show it. A sex -linked trait is one that is
passed on the sex chromosomes (the X or the Y). Remember that if a child inherits two x chromosomes, they’re a girl. If a child
gets an X and a Y (only dad can give a Y) the child is a boy. Sometimes, these X’s and Y’s contain defects. If a child inheri ts the
defective chromosome, they are likely to express the trait. Sex-linked traits are a little confusing for some people because the
rules are different for boys or girls. An x-linked trait is passed on the x chromosome. Because girls have two x chromosomes,
they must inherit two defective x’s to show an x-linked trait. If they only get 1, it’s no big deal because they have a normal x to
perform all the functions of the x chromosome. If a boy gets a defective x chromosome, though, they automatically have
whatever bad trait was carried on that chromosome. This is because they only have one x chromosome, so there’s no backup to
perform x-related functions. This is why disorders like colorblindness, duchenne muscular dystrophy and hemophilia are much
more common in males than females.

Let’s look at another type of inherited disorder now: multifactorial disorders. These are caused by multiple bad genes
AND the environment in combination. Breast cancer is an example of this. People are more prone to breast cancer if they have
certain forms of certain genes, but they are not guaranteed to inherit that disease. Their chances go up a lot if they make certain
lifestyle choices like alcohol use or the use of deodorant. So, both the genes and the environment play a role in multifactorial
diseases. Current research is suggesting that MOST common chronic illnesses (diabetes, alzheimer’s, dementia, high blood
pressure, etc.) are multifactorial.

Mitochondrial disorders are fairly rare, and are caused by mutations in the DNA of mitochondria. If the m itochondria are
defective, the body have a difficult time making ATP, which is needed to fuel all cell processes. These are ONLY passed from
mother to child. Leber’s hereditary optic neuropathy is an example of this.

Chromosomal disorders involve inheriting either not enough chromosomes or extras. This happens when either a
sperm or egg are made with the wrong number of chromosomes. Diseases where you inherit extra chromosomes include
Down’s syndrome. Down’s Syndrome is also known as Trisomy 21. This is because a person with Down’s syndrome has inherited
an extra copy of chromosome 21. Tri- means three, and these people have three copies of a chromosome when they are only
supposed to have two. You have probably met a person with Down’s syndrome at some point. You know that the condition
causes them to have very distinctive traits. These traits are caused by that trisomy. The extra DNA makes extra proteins, and this
is what causes the unique physical features and internal problems seen in someone with Down’s syndrome. These disorders are
easily revealed with a karyotype, a picture of the chromosomes where they have been paired based on size, banding pattern,
and centromere position, then arranged from biggest to smallest.

Types of Genetic Screening

It should be clear by now that there are all kinds of genetic disorders. Because of this, there are people out there who
want or need to know if they carry these diseases, can pass them on to children, or have diseases themselves. There are several
types of genetic testing and screening used to provide people with that information: Carrier screening, preimplantation genetic
diagnosis, fetal screening/prenatal diagnosis, and newborn screening.

Carrier screening is a test that is typically done on adult couples who are considering
having children, and want to determine if those children could inherit any diseases. Most of the
time, there is a family history of something like cystic fibrosis or Tay Sachs disease in the family
that the couple wants to ensure they won’t pass to the child. Remember that a carrier is
someone who holds a bad gene, but doesn’t show it. This process is fairly simple: a blood sample
is drawn, the DNA is extracted and amplified using PCR, and the DNA undergoes testing for the
disease(s) they are concerned about. This may involve DNA sequencing or gel electrophoresis –
sometimes both.

Preimplantation Genetic Diagnosis (PGD) is a bit different. This

procedure is often used by people with known autosomal dominant or sex-
linked conditions that they do not want to pass on to their children. Here,
eggs and sperm are harvested from prospective parents. The eggs are
fertilized by the sperm in vitro (in a petri dish) and the embryos are allowed to
develop to the 8-cell stage. After the embryos are that big, one single cell
from each embryo is removed. The DNA is extracted from that one cell,
amplified, and tested for the presence of the trait the parents do not want.
Healthy embryos are selected and implanted in the mother for development.
This technology has several ethical dilemmas surrounding it – remember
designer babies???

Fetal Screening/Prenatal diagnosis is performed on fetuses while they

are still in utero (inside mommy). Amniocentesis or chorionic villus sampling
are used to extract cells from the fetus for testing. Amniocentesis involves
inserting a large needle through the abdomen and into the uterus, where
amniotic fluid (the fluid surrounding and protecting the baby) is removed. This
fluid contains cells shed from the baby: skin cells, cells from the lining of the
small intestine, or cells from the bladder. The cells in this fluid provide the
DNA needed to perform genetic
testing. Typically, this procedure
requires the use of ultrasound to
locate the baby. It is normally
performed after the baby is 14
weeks old. Chorionic villus
sampling, on the other hand, can be done earlier. Here, chorionic villus cells are
removed from the placenta. This is done by inserting a needle vaginally and directing
that needle to the placenta. A small sample of those cells – which are identical to the
cells inside the baby – are removed and used for testing. Just like with amniocentesis,
ultrasound is used to locate the baby as well as the placenta so the procedure can be
done safely. Both procedures carry some risk of miscarriage.

Newborn screening is the testing of infants shortly after birth. A small sample of blood is taken from the baby, and DNA
is isolated from it for testing purposes. Newborn screening is often used to test for inherited diseases if the parents choose not
to implement measures that complete this testing while the baby is still in utero. Some don’t want to risk miscarriage, and test
their babies after birth instead. There are certain newborn screenings that are done automatically for most babies: for Afric an
Americans, sickle cell is commonly tested for; for Caucasians, cystic fibrosis may be tested for; for Ashkenazi Jews, Tay Sachs is
tested for. This testing allows parents to take measures to give their children the best lives possible if a disease is present, and
to start treating early.
Getting Enough DNA for Testing Purposes

Several times this section, we have brought up a key task that is part of genetic testing: amplifying DNA by PCR. Here,
we will take some time to review that procedure.

PCR stands for the polymerase chain reaction. This is a laboratory procedure that produces multiple copies of a specific
DNA sequence. This can be a copy of a single gene, a large segment of DNA, or the entire genome of an individual. PCR is a three
step process that usually takes place in a thermal cycler (a PCR machine – the purple thingy). Three “ingredients” are added to a
sample of DNA so that copies can be made: Taq polymerase, DNA primers, and DNA nucleotides. These are included in a little
pellet called a PCR bead. The three ingredients are discussed in the paragraphs below as PCR is reviewed.

The first step of PCR is known as denaturation. The temperature in the thermal cycler cranks up to 95 degrees C – nearly
boiling. The high temperatures break up the hydrogen bonds that hold the double-stranded DNA together. Think of a zipper
being completely unzipped, with the two halves falling away from each other. Denaturation is required so that new DNA can be
“grown”.

The second step of PCR is called annealing. The thermal cycler cools to 55
degrees C, and the DNA primers which were added to the DNA mixture early on in the
bead are ready to do their job. Think of annealing as gluing. In this stage, the DNA
primers (short sequences of DNA that target the beginning of the section of DNA
being copied) bind to the section of DNA that scientists wish to copy. The primer is
there so that the DNA is “primed” (readied) for copying. It tells Taq p olymerase,
described below, what section of DNA it should copy.

Finally, extension occurs. The temperature here is 72 degrees C, and requires

both Taq polymerase and DNA nucleotides. You may remember talking about Taq
polymerase in class. This is an enzyme that originated in the bacteria of hot springs, so
they are able to survive the hot temperature used in PCR. In bacteria, Taq polymerase
is used to copy bacterial DNA before bacterial cells divide. Scientists use this
polymerase to copy DNA during PCR. Taq polymerase attaches to the DNA at the site
of the primer. After attaching, it flows down the DNA strand, adding complementary
nucleotides to the DNA so that it becomes double-stranded. When Taq polymerase is
done doing its job, there are two double-stranded pieces of DNA made from the
original one.

This three-step process repeats over and over. Each time it occurs, the amount of DNA doubles. This exponential
growth of DNA allows lots of DNA to be made really really quickly. Within an hour and a half, 1 copy of DNA can be turned int o
more than 2 billion. A graph was made of this process in activity 2.1.2 – it may be useful to review it.

Testing for Disease

Genetic testing is not complete when DNA copies have been made. PCR makes DNA, but another process is required to
use it to diagnose disease. Diagnosis of disease requires healthcare professionals to look inside cells and decode the message
buried in the sequence of nucleotides. The genotype, what is written in our DNA, predicts phenotype, what we see as a result of
that code. Genotype is the genetic code for the traits we have – eye color, dimples, or diseases. Testing for these traits can be
done with the process of gel electrophoresis. When starting from scratch, this can be a fairly complicated process. It is
described in the text that follows.

The process begins with the isolation of DNA. Cells are taken from somewhere (blood, saliva, cheek swabbing) and the
cells are lysed (blown up). The blown up cells and their contents are all mixed together, so a new procedure is used to separ ate
the DNA from the cell waste. This is centrifugation. Centrifugation (fast spinning) separates the heavy cell components from the
from other waste products (plasma, spit, etc.). At the end of the process, a small pellet of cell parts – including DNA – can be
found at the bottom of the spun tube, while the supernatant (fluid on top of the pellet) is merely waste that can be discarded.
To that tiny tube, a small amount of Chelex is added. Chelex forces the DNA to precipitate out and separate itself from the
remainder of the cell waste in the tube. After this happens, the supernatant (which in this case contains the desired DNA) is
moved to a new tube, while the pellet full of cell garbage is discarded. So far… get cells  blow up cells  spin cells  dump
waste  add Chelex  move DNA-holding supernatant to new tube

Now that we have a small sample of DNA, it must be amplified with PCR. As this process is described in the previous
section, I’m not going to repeat it again here. I think you guys would shoot me! Just know the section of DNA carrying the
gene(s) of interest will be copied with PCR.

After the section of DNA we want has been copied, we next need a way to find out if people have the “bad” version of
it or not. It is important to do something so that the different versions of the gene can be distinguished. This is don e with
restriction enzymes. Restriction enzymes are molecular scissors that recognize specific DNA sequences and cut the nucleotide
strands. This allows identification of the single nucleotide polymorphisms, or SNPs. Single nucleotide polymorphisms are tiny
differences in the DNA of individuals that make them unique. They are 1-nucleotide differences in the DNA. SNPs can be the
reason for disease, so being able to identify them is incredibly useful for scientists. This is one method used to detect the “bad”
genes that cause some diseases. Let’s try to make this
simpler. Restriction enzymes are used to cut DNA. They
are “picky” enzymes whose only job is to seek out the
specific DNA they recognize and cut it. If scientists know
the sequence of DNA they are testing, they can use
restriction enzymes to cut it into fragments to reveal
SNPs. Restriction enzymes may cut one version of the
gene, but not another. They may also cause cuts in
different places, creating different fragments of DNA
that can be used to determine what version of the gene
an individual has.

So, we have DNA. We’ve made copies. We’ve

cut the copies. What’s next? We have to be able to SEE
the results. The best way to do this is with gel
electrophoresis. Here, an agarose gel is prepared and
placed into a buffer solution. The gel contains wells at one end to which DNA is added. Markers (standard fragments of known
lengths) are added first. Following this are the samples from the patient or patients being tested. After the DNA is placed into
the wells in the agarose gel, it is charged with an electrical current that separates the fragments. This occurs because DNA has a
slightly negative charge. Due to the placement of the agarose gel in the buffer, DNA is pulled based on attractive forces to the
positive end of the electrophoresis chamber. The pulling separates the DNA fragments, which are stained after the process is
completed, and can then be read. Part of this involves determining the size of the DNA fragments. To do this, the “known”
bands of the markers are used as a comparison for the sizes of the other fragments. The sizes are used to figure out which ge ne
versions (Alleles) a person has inherited. The gel results can be used to determine which version of a gene a patient has,
revealing their SNPs and answering many questions about the presence of disease or the ability to pass disease on to children.

The end result of this entire process is a gel that reveals the genotype of the individual being te sted. It may reveal that a person
is the carrier of a disease, is affected by a disease, or that he/she has nothing to worry about. Remember that genotype
determines phenotype, the physical characteristics of an individual. If genotype says a person carrie s a disease, it means they
hold the gene, but aren’t affected themselves. If genotype says a person has two copies of a disease gene, they have a disease.
 Healthy Pregnancy and a Healthy Baby

Recall that at the beginning of this, we revealed that Judy Smith was
pregnant with an oopsie baby. Genetic testing was used to determine
whether or not that baby had some sort of disease. Judy Smith underwent an
amniocentesis, then chromosomal analysis with a karyotype. She and her
husband did not undergo carrier screening, although that option was likely
made available to them. The karyotype revealed that her baby was fine. Still,
the fact that the baby was genetically normal did not mean that nothing
could go wrong. If a woman is not careful during pregnancy, a baby that is
perfectly healthy genetically can be born with life-long complications.

Throughout the pregnancy, Judy’s health and that of her son will be
monitored. Maternal health will affect the health of the baby. The first
trimester of pregnancy is especially critical, as all major body systems are
formed during this time, and outside agents like alcohol, cigarette smoke,
and drugs can have a drastic effect. Thankfully, Judy is smart enough to avoid
these substances, but she still needs to monitor her eating habits, consume
enough folate, and take her prenatal vitamins. Throughout pregnancy,
chemical substances can have a nasty effect on a baby and should be avoided. Exercise and a healthy diet ensure that both mom
and baby have what they need to stay healthy.

Lesson 2.2: Our Genetic Future

A Brief Introduction

Given how much we are able to do NOW when it comes to understanding DNA and its effects, it is not difficult to see
that the future may reveal some amazing changes. Understanding genetics will alter the way doctors and scientists treat
disease, as well as the way humans reproduce. Think about it: what if we could correct genetic diseases in children before they
were born? What if we could ensure that future children would never have a major health problem like sickle cell or cystic
fibrosis? What if we could make people healthier, smarter, and longer-living? These are all distinct possibilities, and will likely be
topics of high interest in your lifetime. We will discuss them here.

Gene Therapy

Gene therapy is a type of disease treatment in which faulty genes are replaced by functional copies. It involves replacing
“bad” genes with “good” ones, or flipping genetic switches that are making things go wrong. Gene therapy has the potential to
eradicate certain inherited diseases by providing people affected with diseases with normal genes that will produce what the
body needs.

How does this

technology work? Gene therapy
involves using vectors to deliver
healthy genes to affected cells.
This method is still highly
experimental in most cases,
although it has been used to
successfully treat certain diseases
in humans. There are several
types of vectors: retroviruses,
adenoviruses, adeno-associated
viruses, herpes simplex viruses,
liposomes, and naked DNA.
 You may have noticed that many vectors are viruses. This is because the entire existence of viruses involves finding a
specific cell types and delivering genetic material to it. Given that this is the goal of gene therapy, using viruses makes sense!
Regardless of the type of vector used, they all have the same purpose: getting unaffected genes to people carrying a specific
disease. This involves trying to deliver DNA to the right type of cell, not to all cells. Consider someone with an inherited form of
blindness. This could result from a defect in the protein meant to form the retina. If a person doesn’t have a retina, they don’t
see. If, in theory, a vector could be used to deliver normal “retina DNA” to the eye, it might produce a retina and give a pe rson
vision. It would not be necessary to deliver retina DNA to the brain – our brains don’t need retinas. So, that is one problem we
are working to solve: finding a way to get vectors to deliver DNA to target cells. Another problem is complete integration. It
might be possible to get DNA to the right type of cell, and to get that DNA into the cell, but what if the DNA is not incorporated
into the genome? If the retina DNA doesn’t become part of the rest of DNA, a retina will never be produced. Additionally, that
DNA has to be part of that cell – and every other future cell it divides into – for the life of the person. Can you imagine being
given vision for the first time in your life, the having the cells that made it possible die and going back to being blind? A method
of fixing that new DNA permanently into the DNA of the patient is important. Finally, we need to make sure that the DNA is
fixed in a safe spot. If, in the process of incorporating Retina DNA, the DNA responsible for producing the retina disr upts
another eyeball process? Suddenly, there’s a retina, but no eyeball to put it in! Though this example may be a bit silly, it should
illustrate the idea that, right now, gene therapy is far from perfect. Still, it has a lot of potential for curing illnesses and
conditions that are seen as incurable right now.

Designer Babies

There is a dark side to this technology. If we can fix blindness, what else can we do? Could we create the perfect
athlete? The perfect student? The perfect soldier? Gene therapy involves tweaking genes, and there is the potential for this to be
abused in the process of genetic enhancement. Remember talking about designer babies? How far is too far when it comes to
gene therapy?

Current Applications of Reproductive Technologies

Current technologies allow parents to make certain selections for their babies. It is possible to choose babies of a
certain gender, and to do testing to choose babies who do NOT carry diseases of concern to parents. It is easy to see that
choosing gender and healthy babies could be taken too far. If someone has enough money, what else might they choose in a
child? If a parent wants to choose the gender of their child, it’s possible right now. There is a procedure called sperm sort ing that
involves taking a sperm sample and centrifuging it. Sperm carrying an X chromosome are heavier, so sink during centrifugation,
while sperm carrying a Y chromosome end up on top. By “skimming” from the top or the bottom and completing artificial
insemination or in vitro fertilization, a boy or girl baby can be selected. There is also the possibility of preimplantation genetic
diagnosis, which can reveal baby gender as well as tested defects. Since preimplantation genetic diagnosis was discussed
earlier, we won’t repeat the discussion here.

Future Applications of Reproductive Technology

Do you remember discussing Dolly the sheep, the first mammal to be cloned? It’s likely she won’t be the last. How long
do you think it will be before we hear about the first cloned human? Reproductive cloning is cloning to make a copy of an
individual. It is cloning in the most traditional sense of the word. This is very different from therapeutic cloning, which involves
making a clone of a certain body part, like the kidney or the arm. One day, technology may allow a person with failing kidneys to
have a sample of their DNA taken, and a brand new, healthy kidney to be grown. One day, rather than a prosthetic, we might be
able to just grown an amputee a new arm. Therapeutic cloning is cloning for healing purposes rather than reproductive ones.
Again, the question we need to ask ourselves is “How far is too far? Where does healing end and toying with nature begin?”

A final review

The focus of Unit 2 has been diagnosis and future prevention of inherited diseases. Parts of the puzzle are already in
place: we know the human genome, we know what many genes do and their role in disease. We are beginning to learn the
methods needed to change the behavior of damaged genes. In the future, this could lead to a world very different than the one
we live in today.