Genetic Diseases 2/19/2023

Genetic Basis of Diseases Landmark References

Dr. Bhawna Kalra,

Ph.D.,(Genetics), Post-doctorate
Associate Professor, AIST, Dehradun
Ex-Women scientist, DBT-BioCare

Email: bk_geny@yahoo.com

Muscular disorders Cystic fibrosis

Duchenne Muscular dystrophy
Congenital Muscular dystrophy

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Cystic fibrosis-
A respiratory disorder
• Genetic disorders found in in humans of northern European descent.
It affects 1 in 2000 newborns of northern European heritage.
Symptoms:
• Benign effect of the mutant gene includes excessively salty sweat
in CF patient.
• Patients produce large quantities of thick, sticky mucus, which
plugs up the airways of the lungs and clogs the ducts leading from
the pancreas to the intestine, causing frequent respiratory infections
and digestive problems.
• It results in chronic infections and the eventual malfunction of
vital organs.

Genetic basis of disease

• Gene locus: Long arm of chromosome 7
• chromosome 7 at position 7q31.2–q31.3 • Cystic fibrosis: Chloride
• Autosomal recessive disease transporter inability to regulate
• Gene: It encodes a protein termed cystic fibrosis transmembrane ion balance across epithelial
conductance regulator (CFTR) The gene is huge, spanning 250 kb cells.
and containing 24 exons. The CF mRNA is about 6.5 kb in length • Leads to production of thick
and encodes a protein of 1480 amino acids. lung mucus and chronic lung
• Function of CFTR: It acts as a gate in the cell membrane and infections.
regulates the movement of chloride ions into and out of the cell. • The primary defect in the
chloride channels also disrupts
sodium channels. The result:
Salt trapped inside cells draws
moisture in and thickens
surrounding mucus.

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Multiple effects of CF Mutation

• Approximately 70 % of the CF cases result from a specific mutant allele of
the CF gene.
• CF gene encodes a protein called the cystic fibrosis transmembrane
conductance regulator (CFTR), which regulates ionic balance by allowing
the transport of chloride ions (Cl−) across epithelial cell membranes.
• The mutation that causes cystic fibrosis diminishes the function of this Cl−
transporter. The movement of Cl− affects water transport across
membranes, the most severe symptom of cystic fibrosis is thick mucus in
the lungs that occurs because of a water imbalance.
• In sweat glands, the normal Cl− transporter has the function of recycling
salt out of the glands and back into the skin before it can be lost to the
outside world. Persons with cystic fibrosis have excessively salty sweat due
to their inability to recycle salt back into their skin cells—a common test for
cystic fibrosis is measurement of salt on the skin.
• Another effect is seen in the reproductive system of males who are
homozygous for the cystic fibrosis allele. Males with cystic fibrosis
may be infertile because the vas deferens, the tubules that transport
sperm from the testes, may be absent or undeveloped. Taken together,
we can see that a defect in CFTR has multiple effects throughout the body

Cause of disease:
Patients with cystic fibrosis have a mutated,
dysfunctional form of CFTR that causes the channel to
stay closed, and so chloride ions build up in the cell.
This buildup causes the formation of thick mucus
and produces the symptoms of the disease.
Life expectancy: In 1940, the average life for a newborn
with CF was less than two years.
Today, the life expectancy for someone with CF is about
32-40 years, but the quality of life is poor.

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• Normal: Two copies of the normal allele

Deletion of phenylalanine
for CFTR and produce only functional
CFTR protein. • The wild-type CF gene and gene
• Diseased: Those with cystic fibrosis product have the nucleotide and amino
possess two copies of the mutated acid sequences in the region altered
by the ΔF508 mutation
CFTR allele and produce only the
• The mutant allele, CFΔF508, contains
defective CFTR protein. a three-base deletion that eliminates a
• Heterozygotes/Carriers: having one phenylalanine residue at position 508
normal and one defective CFTR allele, in the polypeptide product.
produce both functional and defective
CFTR protein. One functional allele
produces functional CFTR protein to
allow normal chloride ion transport, the
heterozygote exhibits no adverse
effects, as mutated CFTR allele is
recessive

Detection of CF allele Basic Procedure

• Lap-Chee Tsui and colleagues synthesized oligonucleotides spanning
this region of the mutant and wild-type alleles of the CF gene and
tested their specificity. In the late 1980s, the gene for cystic fibrosis
was identified.
• They demonstrated that at 37 °C under a standard set of conditions:
• oligonucleotide probe (oligo-N: 3´-CTTTTATAG TAGAAACCAC-5´)
hybridized only with the wild-type allele.
• whereas another
• (oligo- F: 3´-TTCTTTTATAGTA . . . ACCACAA-5´) hybridized only
with the ΔF508 allele.
• Their results showed that the oligo- F probe could be used to detect
the Δ F508 allele in either the homozygous or heterozygous state.
• PCR was used to amplify the CF loci in genomic DNAs
isolated from individual family members.
• Separation by gel electrophoresis,
• Transferred to nylon membranes,
• Denatured, and hybridized to the radioactive
oligonucleotide probes.
• Duplicate Southern blots were prepared; one blot was
hybridized to the probe specific for the wild-type CF
allele (top lane), and the other was hybridized to the
probe specific for the F508 allele (bottom lane).

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In the pedigrees at the top, filled symbols represent individuals who carry two
mutant CF alleles, and half-filled symbols represent individuals who carry
mutant and wild type CF alleles.
The mutant genes that cause cystic fibrosis were identified by positional cloning.
Isolation of CF genes
• Cloning the Cystic Fibrosis Gene
• Identifying RFLP markers linked to the CF gene
• The CF gene was first mapped to the long arm of chromosome 7 by its cosegregation
with RFLPs
• The two RFLP markers closest to the CF gene were then used to initiate
chromosome walks and jumps and to begin construction of a detailed physical map of
the region.
• Identifying the chromosome on which the CF gene is located
• Identifying the chromosome region where the CF Gene is located
• Cloning the CF gene between the Flanking markers
• Identifying the CF gene in the Cloned DNA
• The Gene defects in Cystic Fibrosis
Bhawna Kalra
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Three kinds of information were used to
narrow the search for the CF gene
1. Human genes are often preceded by clusters of cytosines and guanines
called CpG islands. Three such clusters are present just upstream from
the CF gene.
2. Important coding sequences usually are conserved in related species.
When exon sequences from the CF gene were used to probe Southern
blots containing restriction fragments from human, mouse, hamster, and
bovine genomic DNAs (often called zoo blots), the exons were found to be
highly conserved.
3. A cDNA library was prepared from mRNA isolated from sweat gland
cells growing in culture and screened by colony hybridization using exon
probes from the CF gene (candidate CF gene at the time).
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The mutations in the CF gene are pleiotropic; they cause a number of distinct phenotypic effects.
Malfunctions of the pancreas, liver, bones, and intestinal tract are common in individuals with CF.

• The presence of mucus on the lining of the respiratory tract leads to chronic,
progressive infections by Pseudomonas aeruginosa, Staphylococcus
aureus, and related bacteria.
• CFTR interacts with a number of other proteins and undergoes
phosphorylation/dephosphorylation by kinases and phosphatases. Thus,
CFTR should be considered multifunctional.
• Indeed, some of the symptoms of CF may result from the loss of CFTR
functions other than the chloride channels. Although 70 percent of the cases
of CF are due to the F508 trinucleotide deletion, over 900 different CF
mutations have been identified .
• These tests can be performed on fetal cells obtained by amniocentesis or
chorionic biopsy. They have also been done successfully on eight-cell pre-
implantation embryos produced by in vitro fertilization. The diversity of the
mutations that cause CF makes it very difficult to devise DNA tests for all of
the mutant CF alleles.

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Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy
• Duchenne muscular dystrophy (DMD), which was first described by the French
neurologist Guillaume Duchenne in the 1860s. It affects about one in 3300
newborn males
• X-linked recessive (Single Gene inheritance pattern)
• Inheritance of single genes is also called Mendelian, or monofactorial
inheritance.
Symptoms:
• Affected individuals show signs of muscle weakness as early as age 3.
• The disease gradually weakens the skeletal muscles and eventually affects the
heart and breathing muscles.
• Symptoms of (DMD; OMIM 310200) develop in children between 2 and 5 years
of age. Individuals with DMD undergo progressive muscle degeneration starting
early in life.
• They are usually confined to wheel chairs by their teens and commonly die in
their late teens or early twenties.
Life expectancy: Survival is rare beyond the early 30s.
Gene: The gene for DMD, found on the X chromosome, encodes a protein called
dystrophin that is required inside muscle cells for structural support.

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Dystrophin
• Dystrophin was identified in 1987. The majority of mutations of the
dystrophin gene are deletions of one or more parts of it.
• DMD occurs because the mutated DMD gene fails to produce
virtually any functional dystrophin.

• Dystrophin is thought to strengthen muscle cells by anchoring

elements of the internal cytoskeleton to the plasma membrane.
Without it, the plasma membrane becomes permeable and may
rupture.

• The DMD gene is composed of many exons and introns. The DMD
gene spans 2.5 million nucleotide pairs and contains 79 exons,78
introns.

• DMD gene also identified through chromosome walking.

• The dystrophin protein transfers the force of muscle contraction from the
inside of the muscle cell outward to the cell membrane.
• Because it connects the center of the muscle cell to the edge of the cell, the
dystrophin protein is extremely long. One end is specialized for linking to
the muscle cell interior and the other end is specialized for linking to a
variety of proteins at the cell membrane. The long middle section, called
the rod domain, is taken up by a series of repeating units called spectrin
repeats.
• The repeated spectrin units in the middle of the protein play an important
role in linking the two ends.
Harmful effect of absence of dystrophin are:
• Fibrous tissue begins to form in the muscle
• Body’s immune system increases inflammation
• muscle damage and progressive weakness, beginning in early childhood.

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Genetic basis regarding gene

expression

Inheritance pattern
• X-linked recessive
• Several males are affected by this disorder, as indicated by filled squares. The
mothers of these males are presumed heterozygotes for this X-linked recessive
allele.
• This recessive disorder is very rare among females because daughters would have to
inherit a copy of the mutant allele from their mother and a copy from an affected
father

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• Disease caused by mutations in the gene that encodes dystrophin
• Many of the mutations that cause DMD produce premature stop
codons, which bring protein synthesis to a halt, resulting in a
greatly shortened and nonfunctional form of dystrophin.
• Genetic Treatment:
Introduction of small RNA molecules that cause the spliceosome to
skip the exon containing the stop codon. The introduction of the
small RNAs will produce a protein that is somewhat shortened
(because an exon is skipped and some amino acids are missing) but
may still result in a protein that has some function.
The small RNAs used for exon skipping are complementary to
bases in the pre-mRNA.
Bhawna Kalra
2/19/2023
Why don’t girls usually get
DMD?
• When a girl inherits a flawed dystrophin gene
from one parent, she usually also gets a healthy
dystrophin gene from her other parent, giving her
enough of the protein to protect her from the
disease. Males who inherit the mutation get the
disease because they have no second dystrophin
gene to make up for the faulty one.
• Early in the embryonic development of a
female, either the X chromosome from the
mother (maternal X) or the one from the father
(paternal X) is inactivated in each cell.
Chromosomes become inactivated at random.
In each cell, there is a 50 percent chance that
either the maternal or paternal X chromosome
will be inactivated, with the other left active.
• Usually, girls do not experience the full effects of
DMD the way boys do, although they still have
symptoms of muscle weakness.
How to find information about
Dystrophin gene?
• At the NCBI home page (click on each of the following)
• → Human genome resources
• → Gene Database
• → Search with query DMD AND human[orgn]
• → 1. DMD → Primary Source: HGNC:2928
• → under Gene Symbol Links,
• GENATLAS → DMD → See the exons.
• Also, search the OMIM (the Online Medical Inheritance
in Man) database for more information about Duchenne
and Becker muscular dystrophies.
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An unaffected woman (i.e., without disease symptoms) who is

heterozygous for the X-linked allele causing Duchenne muscular dystrophy
has children with a man with a normal allele. What are the probabilities of
the following combinations of offspring?

•Deletion in the gene: 60% to 70% of affected individuals

•Duplication within the gene: 10% to 15%.
•Point mutation: 15% to 30% patients carry a which causes premature chain
termination during translation.

Congenital Muscular dystrophy Genetics

• Autosomal recessive inheritance
• Autosomal recessively inherited, in some
• Group of heterogeneous disorders
cases of de novo gene mutation and
Symptoms Ullrich congenital muscular dystrophy.
• Muscle weakness which is present at birth • Both parents are carriers of a CMD gene
and the different changes on muscle
• For CMDs more than 35 genes have been
biopsy. discovered resulting from mutations.
• Muscular dystrophy
• Increase muscular breakdown with age

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Different forms of CMDs based

upon changes in protein level
1. Merosin-deficient congenital muscular dystrophy (MDC1A):
It accounts for 1/3rd of all CMD cases
• Gene Mutation : LAMA2 is caused by mutations in the LAMA 2 gene on the 6q2
chromosome, encoding for the laminin-α2 chain.
• Function: Laminin-α2 is an essential part of proteins like laminin-2 and Laminin-4
that have important functions in muscle movement, and most patients with a
mutated LAMA2 gene have no expression of Laminin-α2 in muscle tissue.
2. Ullrich congenital muscular dystrophy
• Gene mutation: COL6A1, COL6A2 and COL6A3 genes that encode for three of the
alpha chains making up Collagen VI
• Function: Collagen VI is important in muscle, tendon, and skin tissue, and functions
to attach cells to the extracellular matrix.
• Ullrich CMD can be caused by both autosomal recessive or autosomal dominant
mutations.
• Recessive mutations often lead to a complete absence of Collagen VI in the
extracellular matrix, while there are different types of dominant mutations that can
cause partial function of Collagen V1.
3. Rigid Spine Congenital Muscular Dystrophy (RSMD1), or Rigid Spine
Syndrome
• Gene mutations: SEPN1 gene encoding for selenoprotein N.
• Function: It is expressed in the rough ER of Skelton muscles, heart, brain,
lung, and placenta tissues, as well as at high levels in the diaphragm.
• RSMD1 is characterized by axial and respiratory weakness, spinal rigidity
and muscular atrophy
4. CMDs are dystroglycanopathies caused by glycosylation defects of α-
dystroglycan (α-DG), which helps link the extracellular matrix and
the cytoskelton . Dystroglycanopathies are caused by mutations in genes
encoding for proteins involved in modifying α-DG after translation of the
protein, not mutations in the protein itself.
• 19 genes have been discovered that cause α-DG-related dystrophies

• 5. Walker-Wurburg syndrome : It is the most severe

dystroglycanopathy phenotype, with the POMT1 gene. There have
been 11 additional genes implicated in WWS. These genes
include POMT2, FKRP, FKTN, ISPD, CTDC2,

Thanks
TMEM5, POMGnT1, B3GALnT2, GMPPB, B3GnT1, and SGK196,
many of which have been identified as involved in other
dystroglycanopathies
• Symptoms: Patients display muscle weakness and cerebellar and
ocular malformations, with a life expectancy of less than 1 year.

6. Fukuyama congenital muscular dystrophy (FCMD): It is

caused by a mutation in the Fukutin (FKTN) gene,. It leads to
muscle weakness, abnormal eye function, seizures, and intellectual
disability

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