Molecular Biology - Lecture - 13 - Applications of Molecular Techniques in Genetic Testing and Forensics - Diseases - Part 2 - Complete

Cystic fibrosis

Autosomal recessive disorder caused by mutations in the CFTR gene in chromosome 7

Autosomal recessive

Cystic fibrosis inheritance pattern

CFTR gene

Gene affected in Cystic fibrosis

Chromosome 7

CFTR gene location

Cystic fibrosis transmembrane conductance regulator

CFTR gene code for the:

Cystic fibrosis transmembrane conductance regulator

It is an ion channel essential for proper chloride and bicarbonate transport across the epithelia

Defective or absent

CFTR gene mutation in Cystic fibrosis

3-nucleotide deletion of phenylalanine codon 508

Most common mutation in Cystic fibrosis in the CFTR gene

Delta F508

Designation for 3-nucleotide deletion of phenylalanine codon 508

3-nucleotide deletion of phenylalanine codon 508

CFTR gene mutation in Cystic fibrosis that leads to a misfolded CFTR protein that is degraded or non-functional, resulting in defective chloride and water transport across cell membrane, which causes decreased chloride secretion and increased sodium and water reabsorption.

Decreased, Increased

Cystic Fibrosis CFTR Delta F508 mutation

Chloride secretion: Increased/Decreased

Sodium and water reabsorption: Increased/Decreased

Dehydrated, thick, sticky mucus

Cystic Fibrosis CFTR Delta F508 mutation

Produces this mucus appearance in the epithelial-lined organs

Cystic Fibrosis CFTR Delta F508 mutation

The dehydrated, thick, sticky mucus in the epithelial-lined organs leads to different symptoms such as:

Recurrent respiratory tract infection, excessively salty skin, Pancreatic insufficiency

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Diagnosis for cystic fibrosis includes a wide variety of molecular methods which incorporate at least the core panel of the 25 most prevalent mutations

SO probe hybridization, Microbead arrays, Allele-specific PCR, Sanger sequencing, Next-generation sequencing

Molecular methods for diagnosis of Cystic fibrosis

Cystic fibrosis

One of the first genetic diseases for which specific mutation-targeted therapies have come to market

CGTR modulators

Collective term for the drugs which are designed to circumvent the biochemical or post-translational process defect caused by the mutations in Cystic fibrosis

Duchenne muscular dystrophy

X-linked progressive myopathy caused by the mutations in dystrophin gene

X-linked

Duchenne muscular dystrophy inheritance pattern

Dystrophin gene

Duchenne muscular dystrophy affected gene

Deletion

Most common mutation in the Dystrophin gene in Duchenne muscular dystrophy which more often produce frameshift mutations and more truncated protein product

Point mutations, Microdeletions, Insertion

The other 1/3rd of Duchenne muscular dystrophy have these mutations in the Dystrophin gene

Multiplex PCR, Next-generation sequencing, Western blot or immunohistochemistry in muscle biopsy tissue

Diagnostic techniques for Duchenne muscular dystrophy

Multiplex PCR

Diagnostic technique for Duchenne muscular dystrophy which allows rapid and inexpensive identification of more than 98% of dystrophin deletions and their localizations to specific exons of this gene

Next-generation sequencing

Diagnostic technique for Duchenne muscular dystrophy which directly detect mutations

Western blot or immunohistochemistry in muscle biopsy tissue

Diagnostic technique for Duchenne muscular dystrophy which will reveal decreased or absent dystrophin.

Duchenne muscular dystrophy

Progressive muscle weakness that will lead to loss of ambulation, and later on there would be respiratory decline that will lead to respiratory and or cardiac complications that eventually results to mortality.

Spliceosome

Primary therapeutic target in Duchenne muscular dystrophy which is designed to fix the dystrophin reading frame and enable the production of a shortened but functional protein

Antisense oligonucleotide-mediated splice modulation

Spliceosome mechanism of action in Duchenne muscular dystrophy

Spinal muscular atrophy

Neuromuscular disorder that is considered the second most common lethal autosomal recessive disorder after cystic fibrosis.

SMN1 gene

Gene affected in Spinal muscular atrophy

Homozygous deletion of exon 7

Most common mutation in Spinal muscular atrophy in the SMN1 gene

SMN2 gene

The most common mutation causing spinal muscular atrophy is a homozygous deletion of exon 7 of the SMN1 gene, which is difficult to determine by DNA sequencing because of the presence of one or more copies of a highly homologous pseudogene called:

SMN2 gene

In Spinal muscular atrophy, number of copies of this gene can modify the severity of the symptoms

Quantitative and allele-specific PCR, Multiplex ligation-dependent probe amplification

Detection of the SMN1 deletion and the SMN2 copy number usually requires some form these tests

Next-generation sequencing

Method that enables the detection of the small percentage of Spinal muscular atrophy patients who carry a small intragenic mutation in one allele.

Thalassemia

Disease that involve both qualitative and quantitative alterations in one or more globin chains.

Alpha-thalassemia

Thalassemia caused by the deletion of either or both HBA genes on one or both of chromosome 16

Southern blot, MLPA, Quantitative PCR

Tests to detect Alpha-thalassemia allowing differentiation of different types

Silent carrier

Type of Alpha-thalassemia wherein only one alpha gene is missing

Alpha-thalassemia minor

Type of Alpha-thalassemia with two mutated alleles

Hemoglobin H disease

Type of Alpha-thalassemia with three mutated alleles

Alpha-thalassemia major or Hydrops fetalis

Type of Alpha-thalassemia wherein all four genes are missing or mutated.

Beta-thalassemia

Thalassemia caused by mutations in the HBB gene on the chromosome 11.

Beta-thalassemia

Molecular diagnosis for this type of Thalassemia is more complicated because of the wide variety of identified mutations.

HBA gene

Gene affected in Alpha-thalassemia

Chromosome 16

HBA gene location

HBB gene

Gene affected in Beta-thalassemia

Chromosome 11

HBB gene location

Autosomal recessive

Sickle cell anemia inheritance pattern

HBB gene

Gene affected in Sickle cell anemia

DNA-based techniques

Sickle cell anemia

Techniques can be used for diagnosis, carrier screening, or prenatal diagnosis on amniocytes

Trinucleotide repeats

Three nucleotides that repeat consecutively

Trinucleotide repeat disorders

Caused by 3 nucleotides that repeat consecutively and are unstable and tend to increase in size in subsequent generations

Earlier

Trinucleotide repeat disorders

As the repeat size increases, the (older/earlier) age of symptomatic onset and the worse the symptoms

Gene silencing, Inhibition of RNA processing, Toxic gain of function by mutant protein

Mechanisms by which these expanded repeat sequences (Trinucleotide repeat disorders) produce disease

Fragile X Syndrome, Myotonic Dystrophy, Friedreich Ataxia, Neurodegenerative Disorders

Enumerate Trinucleotide repeat disorders

Fragile X Syndrome

FRAXA meaning

X-linked

Fragile X Syndrome inheritance pattern

Fragile X Syndrome

Most common single gene defect causing moderate to severe mental retardation

Expanded triple repeat (CGG)n in the 5'-non-coding region of the FMR1 gene at chromosome Xq27.3

Cytogenetic hallmark of FRAXA

CGG

Fragile X Syndrome

Trinucleotide unit that gets repeated

Number of times the trinucleotide gets repeated

Fragile X Syndrome

(CGG)n

Meaning of n

54

Fragile X Syndrome - (CGG)n

In normal individuals, n ranges up to about:

55-199

Fragile X Syndrome - (CGG)n

Number of n when it is referred to as premutation

X chromosome

Individuals who carry an this chromosome with premutation do not show signs of classic fragile X syndrome but are at risk of passing an allele of even larger size to their children

Full mutation

Fragile X Syndrome - (CGG)n

N = >200

Male

Fragile X Syndrome primarily affects what sex?

Large ears, Long face, Prominent jaw, Macroorchidism

Dysmorphic features in males with Fragile X Syndrome

Myotonic dystrophy

Caused by an expansion of a CTGn repeat in the 3 prime untranslated region of the myotonin protein kinase, DMPK gene on chromosome 19q13.3

Expansion of a CTGn repeat in the 3' untranslated region of the myotonin protein kinase, DMPK gene on chromosome 19q13.3

Myotonic dystrophy hallmark

>100

Amount of repeats that would likely cause a typical Myotonic dystrophy phenotype

Progressive myotonia, Weakness, Distal extremities and face muscle atrophy

Myotonic dystrophy manifestation in late childhood or early adulthood

Friedreich Ataxia

Most common of the hereditary ataxias

Autosomal recessive

Friedreich Ataxia inheritance pattern

None

Friedreich Ataxia evidence of anticipation

Expanded (GAA)n repeat in the 1st intron of the FRDA gene

Friedreich Ataxia hallmark

Frataxin

Its expression is reduced in Friedreich Ataxia

Frataxin

Affected in Friedreich Ataxia, it is involved in mitochondrial respiration, iron balance, and response to oxidative stress.

Selective neuronal degeneration in the central nervous system

Characteristics of Neurodegenerative Disorders that are Trinucleotide repeat disorders

Huntington disease, Spinocerebellar ataxias, Dentatorubral-pallidoluysian atrophy

Examples of Neurodegenerative Disorders that are Trinucleotide repeat disorders

Autosomal dominant

Inheritance pattern of Neurodegenerative Disorders that are Trinucleotide repeat disorders

Spinal and bulbar muscular atrophy

Neurodegenerative Trinucleotide repeat disorder that is X-linked

X-linked

Spinal and bulbar muscular atrophy inheritance pattern

(CAG)n trinucleotide repeat in the coding region of the respective gene that produces abnormal elongation of a polyglutamine tract

Trinucleotide repeat of Neurodegenerative Trinucleotide repeat disorders

Polyglutamine tract

In Neurodegenerative Trinucleotide repeat disorders, there is abnormal elongation of a:

Intranuclear neuronal inclusions

In Neurodegenerative Trinucleotide repeat disorders, the resultant abnormal protein produces:

Apoptosis

In Neurodegenerative Trinucleotide, it is postulated that expanded polyglutamine sequence in each of these proteins leads to alterations in their folding and binding characteristics and a gain of function that is toxic to neurons in a selective manner causing ___.

Diagnostic, Predictive

Purpose of the molecular diagnosis of trinucleotide repeat neurodegenerative disorders

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T/F

Trinucleotide repeat neurodegenerative disorders have no documented treatment or preventive intervention

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T/F

Pre-symptomatic testing for these Trinucleotide repeat neurodegenerative disorders must be accompanied by pre- and post-test genetic counseling along with mental health support if indicated

Prader-Willi syndrome, Angelman syndrome

Both commonly caused by deletion of the same region in the proximal long arm of chromosome 15.

Deletion at the 15q12 region in the paternally inherited chromosome

Prader-Willi syndrome gene mutation

Paternal

Prader-Willi syndrome

Which genes are normal? Paternal or Maternal?

Maternal

Prader-Willi syndrome

Which genes are imprinted or silenced? Paternal or Maternal?

Maternal uniparental disomy

Other cause for Prader-Willi syndrome

Paternal

Prader-Willi syndrome

Which genes are deleted if there is Maternal uniparental disomy?

Deletion of the UBE3A gene exclusively on the maternally inherited chromosome

Angelman syndrome mutation

Paternal

In Angelman syndrome, which UBE3A gene is imprinted or silenced? Paternal or maternal?