1.

Changes to the DNA may have no consequences but sometimes lead to

observable differences in the individual.
a. Justify the above mentioned statement. (1.5)
b. In a particular family, children of a diseased mother are all affected, and their
maternal grandmother was also affected. But the cousins (children of maternal uncle)
are not affected. No disorders were recorded in the paternal side. Can you identify the
type of this disorder and provide justifications in support of your identification? (2.5)
c. Even without any changes in the genome, many disorders can occur and get inherited
into the next generation. How? (1)

- A. Any heritable changes to DNA sequence is usually known as “Mutation”, modern day
termed as variation. These changes or variation to DNA usually have no consequences due to
the fact that there is a high probability of these variations to be in a non-coding sequence as
only 3% of our gene is coding and even if the variation is on coding gene and damage gene
function, an individual may still remain healthy as for many genes only one functional copy is
required.
- However, sometime these variations may lead to observable differences in the individual
without any consequences as mentioned in the statement and this is because not all variations
are related to disease. This type of variation that cause phenotypic change in individual but not
necessarily give rise to any disease or cause any harm is called “benign variants”

B. This disease is caused by mitochondrial genome associated disease.

All the children of a diseased mother were affected while there was no record of disease from
the paternal side. This means the sole transmitter of the disease was the mother, which is
prevalent for mitochondrial disease. This is because Mitochondria remain in tail area of sperm
to facilitate motility, thus during fertilization, the tail is destroyed thus the mitochondria is not
passed to egg from father. It is also seen that the grandmother was affected, again affirming
that the mother line was the transmitter of the mitochondrial mutant genes. Also the uncle’s
children (i. e cousin) showing no disease proves that even if the uncle was affected from the
grandmother, he could not pass it on to his children as the disease was mitochondrial
associated. Therefore, the plausible explanation would be the grandmother had certain
homoplasmic mutant gene in her mitochondria in which she passed to her children, the mother
was thus affected and she passed the mutant genes to all her children but the uncle didn’t pass
the gene to any of his children.

This is an X-lined dominant disorder.

The question states that ALL children of a diseased mother are affected, thus it cannot be a
mitochondrial genome associated disorder because mitochondrial disorder is associated with
heteroplasmy thus it does not guaranty disease transmission to ALL children. The only plausible
case is if the diseased mother is “Homozygous dominant” for her X-linked disease. This means
she inherited pathogenic variants from each of her parents. The Father carried normal X variant
as there was no disorder in the paternal side.
 Mothers Genome: Xd Xd (Xn- nonpathogenic X variant, Xd- Pathogenic X variant)
Father Genome: Xn Y
Offspring genotype: XdXn XdXn XdY XdY
Offspring Phenotype: Affected Affected Affected Affected

In order for the mother to have Homozygous Dominant X variant, she had to receive The
pathogenic variant from both her parents, and it is established by the fact that the grandmother
was also affected. However, the Grandmother most probably was heterozygous for the
pathogenic variant and I reach to this assumption, keeping in mind that the maternal uncle was
not affected and thus none of his children. This can be possible only in the following scenario:

Grandmother Genotype: Xn Xd
Grandfather genotype: Xd Y
Offspring genotype: XdXd XdXn XdY XnY
Offspring Phenotype: affected(mother) affected affected not affected(uncle)
Thus the uncle’s genotype carried no variant of the pathogenic X variant, and thus His children
(i.e. the cousins) where non affected by the disease.

C. This can occur due to epigenetic changes where heritable changes in gene expression
take place without a change in DNA sequence i.e. changing the phenotype without changing the
genotype. Gene expression is controlled and it is turned on or turned off via two main epigenetic
methods like DNA methylation and Histone modification.

2.a. Gene “X” is responsible for breast cancer and can be inherited into the next
progeny. In one such case, one affected male parent who had only one affected parent
(can be either male or female) too came to a genetic counselor. Upon the testing
recommended by the counselor, it has been found that that male parent’s child has a
50% risk of developing the cancer. Can you discuss the inheritance pattern of this
disease for this male parent? (3)
b. In which Mandelian disorder, all the children of a mother (both of her alleles of a
gene on X chromosome are pathogenic) could also be affected? (2)

- A. According to the genetic counselor, the male parent’s child has 50% chances of
developing the cancer and this can be possible if the disease is an autosomal dominant
disease and the male parent genotype to be heterozygous dominant for the X gene.

Male Parent Genotype: B b (B- Gene X for Breast cancer, b- normal non-pathogenic gene)
Female Parent Genotype: b b
 Offspring genotype: Bb Bb bb bb
Offspring Phenotype: Affected Affected Not-Affected Not-Affected
Percentage- 50% affected, 50% not-affected

As mentioned, one of his parent was affected, thus in order for the male parent to end
up with heterozygous dominant condition, the affected parent had to be heterozygous
dominant, while the other parent was homozygous recessive to these pathogenic gene
“X”. And just like the above punnet square, the male parent too, inherited the Gene X
from one of his Parent via the 50% chance.

- B. X- linked dominant disorder.

- The mother who is homozygous dominant in her X- linked disease, i.e. she has inherited
pathogenic variants from each of her parents, all her children will also be affected. This is
because, all her children will contain one of her X chromosome which contain the pathogenic
allele, irrespective of son or daughter. It is shown below:
Mothers Genome: Xd Xd (Xn- nonpathogenic X variant, Xd- Pathogenic X variant)
Father Genome: Xn Y
Offspring genotype: XdXn XdXn XdY XdY
Offspring Phenotype: Affected(daughter) Affected(daughter) Affected(son)
Affected(son)
-

3.a. Diseases which are caused by recessive variants in loci located on the X

chromosome affect females and males differently. How? (2)
b. Lifestyle choices and environmental exposures can also shape up a disease, how?
(Discuss from the aspects of molecular biology) (2)
c. In a designed Microarray experiment, in which the probes for the control cell’s
transcripts are labelled with green dye and probes for cancer cell’s transcripts are
labelled with red dye. What color intensities will you expect in the spots of oncogenes
and tumor suppressor genes? (1)
-

A. X-linked recessive disease are riskier for male compared to female, this is because, in order for the
recessive allele to be not expressed, there has to be a presence of the normal dominant allele, which the
male cannot have.

In other words, as the male has only 1 X chromosome, if he inherits the recessive pathogenic allele,
there is no other X chromosome present to compensate its effect, thus all his Daughter will inherit the X
chromosome and be a carrier. Although the sons will remain unaffected.
On the other hand, when it comes to female, they always have two X chromosome, thus the recessive X
chromosome will only express in homozygous condition (inheriting 2 copies of the pathogenic variant)
and if there is her other X chromosome is normal, she will not be affected but just a carrier. The female’s
daughter will also never be affected unless their father is affected.

B. Lifestyle choices and environmental exposures can also shape up a disease because these factors
lead to epigenetic changes, where heritable changes in gene expression take place without a change in
DNA sequence. Epigenetics show gene control by either turning on or off certain gene expression and
this is done by two modification methods: DNA methylation and Histone modification. Especially when it
comes to multifactorial diseases, lifestyle and environment can cause several gene expressions that can
lead to diseases.

For instance, Tobacco smoking lead to decreased methylation in several genes associated with T2D,
including KCNQ1. As we know, Methylation tighten the histone and DNA interaction and thus decreasing
methylation lead to the expression of the gene associated with the T2D, leading to the development of
the disease. Also, exercise has been shown to promote these methylation changes in T2D-associated
genes (reducing the gene expression) and also altering histone deacetylase expression- reducing
acetylation and thus reducing the gene expression.

C. Oncogenes are genes whose activation contributes to the development of cancer therefore,
“Red” color intensity is expected in the spot of oncogene as they would be expressed in cancer cell.
Whereas, in the spot of tumor suppressor gene, “Green” color intensity is expected as they are
expressed in in normal cell only while in cancer cell, their expression is inhibited. It might show yellow
color too if there is slight expression of the Tumor suppressor.

4. Using PCR technique, molecular diagnostic approaches have entered into a new
era.
a. In conventional PCR applications, a heat-resistant polymerase is used. Why? (1.5)
b. You want to identify and compare the gene expression profile of several genes in a
disease. Will you be able achieve this using Q-PCR technique? (2)
c.  Can you identify a potential mutation (the nucleotide change) within a gene
sequence using conventional PCR? If not, then what approach you should take? (1.5)

-A. Conventional PCR application is performed at various temperature and especially

involve raising the temperature to 90-95°C, in order to denature and separate the two strands
of DNA. In this case, a heat-resistant polymerase is necessary in order to withstand this
temperature and not be damaged and perform polymeration at 75°C after this changing
temperature. This heat stable DNA polymerase is obtained from a thermophilic bacteria Thermus
aquaticus, the inhabitant of hot springs.
B. Yes, Q-PCR technique can be used to widely to identify and compare the gene-
expression profile of several genes in a disease, in fact it is known as the most effective
method to analyze modulations in gene expression because of its efficiency to detect
and precisely quantify the target genes, even at low expression levels. Q-PCR uses cDNA
as template thus cDNA of the gene transcripts can be used. During the qPCR reactions,
a dye or probe binds to and is incorporated into amplified double-stranded DNA
(dsDNA), acting as a fluorescent reporter during amplification. The computerized Q-PCR
instrument measures after each cycle the amount of fluorescence emitted. The amount
of fluorescence is directly proportional to the copy number and the cycle in which this
critical copy number reaches depends on the original number of transcript produces by
the gene expression. The reactions of qPCR enable us to measure the mRNA expression
levels in numerous kinds of samples and as it produces computerized graphs, the
different gene expression profile can be identified and compared in a rapid quantitative
way.

C. No, conventional PCR alone cannot really identify potential mutation within a gene
sequence. Although it plays crucial role in amplifying the number of gene sequence. In
order to identify mutation, many methods can be used, among them microarray is a
very well-known technique. Fluorescence tagged mRNA of the genes are produced for
both normal cell and mutated cell with different color tag, ie. Green for normal cell and
red for mutate cell and they are let to hybridize with oligonucleotide microarray. The
hybridized result is scan to compare color to observe expression of gene, thus
identifying whether mutation occurred or not.

5. a. Suppose; you want to detect the CAG repeat expansion within a particular gene (30
repeats in normal changes to 250 repeats in disease) in a certain disease. How will you
diagnose this disease condition? (2)
Allele FISH
b. Can you identify Y chromosome microdeletion (which involves the deletion of AZF
locus) using conventional karyotyping? If not, then why? (1)
c. How will you diagnose a chromosomal translocation event? (Discuss any one of the
processes) (2)

A. Special fluorescent tagged oligonucleotide primer is designed for the CAG repeat
expansion that will bind to both end of the repeat sequence. Then PCR is used to
amplify the targeted gene. For polymerization, fluorescent tagged nucleotide can be
used. This same procedure is done for both normal and diseased cell. The PCR product
is then analyzed via capillary electrophoresis instrument, which tracks the fluorescence-tagged
sequence. Computers are used to detect the fluorescence to generate peak at the location of
migration of the PCR product. Longer sequence will travel slower thus the length of the
sequence can be compared with the normal control cell or against standardized electrophoresis
ladder to identify particular length.

B. Conventional karyotyping cannot identify this microdeletion because this deleted

portion in the Y chromosome would be smaller than the threshold resolution of
karyotype which is approximately 3–4Mb.

-C. Chromosomal translocation can be observed via spectral karyotyping.

Spectral karyotyping is a karyotype in which the homologous pairs of chromosomes are
manipulated to have distinctive colors. Thus translocation can be easily detected by
observing the transposition of colored segment of chromosome. As each chromosome
had its distinct color, the change in color can easily identify the chromosomal structural
translocation.

6. a. For differential gene expression analysis for a disease state, how will you utilize the
microarray technique? Discuss briefly. (3)
-
b. Define karyotyping using one sentence. Why karyotyping is performed on metaphase
chromosomes? (1)
c. Why FISH is more advantageous compared to the conventional karyotyping? (1)

A. For a disease state, if we want to analyze the different gene expression to

understand which of the genes are being expressed in a certain disease cell,
microarray is a very efficient tool. Microarray can be used to

B.

Karyotyping is a diagnostic study of the structure and properties of chromosomes and

cell division.
Karyotyping involves observing chromosome, thus, performing it during metaphase is
the based option as during metaphase of mitosis or meiosis the chromosomes condense and
become easy distinguishable as they align in the center of the dividing cell. Condensed
chromosomes can be stained and can be clearly observed under a light microscope

C. FISH is more advantageous than conventional karyotyping because FISH allows

detection of much smaller chromosomal abnormalities than it can be detected with karyotyping,
for instance FISH can help in study of chromosomal deletions and translocations.
Also, Conventional karyotyping requires a time-consuming cell culture step and can only be
performed with fresh tissue samples which is rarely available whereas, FISH can be performed
older tissue sections.

Also, karyotyping is needed to be done in metaphase while FISH can be done in both
Metaphase and interphase.