modx - lec

MOLECULAR BIOLOGY AND DIAGNOSTICS | SECOND SEMESTER | MIDTERMS

MOLECULAR DETECTIONS OF INHERITED AND NON-INHERITED DISORDERS

● They produce secretions that are too thick and sticky
● We will be dealing with different disorders that are ○ sputum and other body fluids
diagnosed by molecular methods ● The problem here lies on the chromosome 7 gene
● usually because of genetic or cytogenetic problems that codes for the cystic fibrosis (CF) transmembrane
● direct observation of the source of some disorders conductance regulator protein (CFTR)
DISEASES WITH MENDELIAN INHERITANCE ● Gene on chromosome 7
● Codes for the cystic fibrosis (CF) transmembrane
● Inheritance pedigree that follows a dominant and conductance regulator protein (CFTR)
recessive pattern ● Over 1300 mutations have been identified.
○ dominant - one gene from the parent ● Most common mutation is delta-F508, which is a
○ recessive - need the two gene from each of three-base pair deletion.
your parent for you to express the phenotype ○ the first and most frequently observed
○ most of the phenotype would result from the mutation in the CFTR is a three-base pair
interactions of multiple genetic and deletion that removes a phenylalanine
environmental factors residue to position 508 of the protein.
○ some of the phenotypes are caused by ● Phenotypic expression will vary based on mutation.
alterations of a single gene. ● Sweat chloride test performed as part of diagnosis
● Patterns of inheritance or transmission pattern ○ Common test for the diagnosis of cystic
are generally determined by family history fibrosis
○ kadalasan gumagamit tayo ng pedigree
● Three patterns of Mendelian Inheritance ● HOW DO WE DETECT CYSTIC FIBROSIS?
○ autosomal dominant ○ PCR-RFLP (Polymerase Chain Reaction -
○ autosomal recessive Restriction Fragment Length Polymorphism
○ X-linked or sex-linked ○ Single-strand Conformation Polymorphism
● Punnett Square ○ Invader (Cleavage-based assay)
○ Recurrence risk ○ Mid-array (microarray) - important
● Genes carrying the mutation are on autosomes ○ Direct Sequencing Reaction
(chromosomes 1-22)
○ Typically equal frequency for being HEREDITARY HEMOCHROMATOSIS
affected
● Heterozygous: one normal gene (wild-type) and one
abnormal gene (mutation) ● Over Absorption of iron from the food.
● Homozygous: inherited two genes with the same ● Iron accumulation subsequently causes damage to
mutation pancreas, liver, and skin.
● Characterized by patients with heart disorders and
diabetes
AUTOSOMAL RECESSIVE DISEASES ● At a molecular level, Hemochromatosis is caused by
the dysfunction of hemochromatosis type 1 (HFE
CYSTIC FIBROSIS gene) or problem with the HLA-H gene
● Excess iron absorption
● Life-threatening autosomal recessive disorder ● Treatment is via therapeutic phlebotomy
● Causes severe lung damage and nutritional ○ treatment of choice
deficiency in patients. ○ measurement of blood iron levels
○ with earlier detection, people with cystic ■ Transferrin saturation and biopsy of
fibrosis could live comfortably surviving the liver.
beyond the 4th decade of their life ● Several common base substitutions in the HFE gene
● Respiratory failure is the most dangerous or have been found.
severe consequence of the disease

♡ Divina, Mikaela ♡ 1
 MOLECULAR DETECTIONS OF INHERITED AND NON-INHERITED DISORDERS

● G to A at amino acid 282 (C282Y) ○ found in the short arm of chromosome 4

○ most common observed mutation ● CAG would expand from 9 to 37 to 38 up to 86
○ detected by PCR-RFLP
● C to G in exon 63 (H63D) ● WHAT ARE THE CLINICAL MANIFESTATIONS OF
● A to T in codon 65 (S65C) HUNTINGTON'S DISEASE?
○ Impair judgment
● Individuals with mutations may be asymptomatic ○ Slurred speech
because of incomplete penetrance ○ Difficulty in swallowing food
○ Abnormal body movement
AUTOSOMAL DOMINANT DISEASES ○ Personality changes
○ Depression
○ Mood swings
FACTOR V LEIDEN ○ Unsteady WHAT
○ intoxicated appearance
● Hereditary hypercoagulability ● With onset in the 30s or 40s these symptoms do not
● Too much coagulation because factor V is also known become obvious until the 4th or 5hth decade of life
as labile factor is part of our normal coagulations (reduced penetrance)
● Factor V gene is on chromosome 1 (1q23) ● The child of a person with Huntington's disease has a
○ Leiden mutation is 1691 A → G (R506Q) 50% chance of inheriting the disease
■ changing of adenine to guanine
● Oral contraceptives increase risk for thrombosis
X-LINKED DISEASES
○ thrombosis = blood clotting
● Mutations detected by many platforms, including
○ Invader (Cleavage-based Assay) RECESSIVE X-LINKED DISORDERS
○ PCR followed by electrophoresis ● X-linked recessive females are carriers and usually
○ Real-time PCR using melting curve analysis not affected.
● Males will receive the mutated gene only from their
HUNTINGTON'S DISEASE mother and will be affected
○ Hemizygous
● Many cases are due to new mutations.
● Late-onset neurodegenerative disorder
○ affects the brain
HEMOPHILIA A
○ higher numbers of repeats in offsprings in
earlier onset
● Trinucleotide repeat ● Deficiency of coagulation factor VII (FVIII)
○ problem with too much repetition of CAG ● X-linked recessive
● Gene for Huntington's disease is huntingtin and is on ● Mostly male cases, but female cases have been
chromosome #4 reported
● Trinucleotide repeat ● In female cases, they are related to skewed
○ Expansion of CAG chromosomal error in chromosome 10
○ Normal repeat range from 10 to 27 copies ● Female cases might also be due to the mutated
○ Repeats of 28 to 35 “mutable” Factor VIII gene
○ Repeats of 36 to 39 “reduced penetrance” ● Factor VIII would be coded in the sec chromosome
■ di pa visible yung huntington's (chromosome 10)
disorder ● WHAT ARE THE METHODS USED TO DETECT
■ di pa totally nagmamanifest as FACTOR VIII PROBLEM
huntington's disease ○ PCR Assay - for inversion mutations
○ Repeats of 40 or greater are associated ○ Sequencing Technique
with disease
● Huntington's disease was first described by George DUCHENNE'S MUSCULAR DYSTROPHY
Huntington in 1872
● Nagkakaroon ng associated with expansion of CAG in
huntingtin’s structural gene

♡ Divina, Mikaela ♡ 2
 MOLECULAR DETECTIONS OF INHERITED AND NON-INHERITED DISORDERS

● Most common neuromuscular disorder characterized ■

mutations on the ribosomal RNA
by progressive myopathy weakness, elevated serum (rRNA) gene
creatinine kinase (CK) ■ those that affect mitochondria
● Diagnosis can be made by immunohistochemical protein synthesis.
studies ● HOW DO WE DETECT MITOCHONDRIAL
● Molecular Method: MULTIPLEX PCR DISEASES?
○ DNA sequencing of the mitochondrial DNA
● X-linked recessive ○ PCRSouthern Blotting techniques
● Mostly male cases, but female cases have been
reported IMPRINTING DISEASE
● Largest gene in the human genome with length of
2.2 megabases. ● Histone or DNA modification
● Dystrophin is the protein product ○ Results in transcriptional silencing
● ○ Deletion of allele during egg and sperm
production (gametogenesis)
FRAGILE X SYNDROME ● Different phenotypic presentation depending on
maternal or paternal deletion inheritance
● Problem with FMR-1 gene
● X-linked dominant disorder with reduced ● THE TWO PHENOTYPIC PRESENTATION OF
penetrance IMPRINTING:
● Penetrance increases in subsequent generations ○ Paternal Inheritance - Prader-Willi
● Common inherited form of learning disability syndrome
● Name derived from cytogenetic abnormality of ■ learning disability
breakpoint or fragile spot within the telomere of a ■ short stature and behavioral issues
metaphase X chromosome. ○ Maternal Inheritance - Angelman
● Trinucleotide repeat expansion of CGG Syndrome
○ Normal range contains about 5-45 repeats ■ learning disability
○ 50-200 - premutated ■ attacks of laughter (?)
○ >200 - mutated ■ absence of speech
● Common sa patients have learning disability and
penetrance increases from generation ● Imprinting on chromosome 15,del(q11q13)
○ Deletion on the long arm of chromosome 15
● HOW DO WE DETECT FRAGILE X SYNDROME? ● Mutations detected by many platforms, including
○ Southern blotting for the genotype analysis ○ Cytogenetic detection
○ PCR with capillary electrophoresis for the ■ for deletion detection
detection of number of CGG ■ Karyotyping - to detect
chromosomal rearrangements
■ FISH (Fluorescence In Situ
DISEASES WITH NON-MENDELIAN INHERITANCE Hybridization) - incorporates probe
to detect deletions, amplifications,
deletions, inversions in a
MITOCHONDRIAL DNA DISEASES chromosome
● Mitochondria generate energy by producing ATP ○ Methylation-specific PCR (mPCR)
through oxidative phosphorylation. ○ PCR for STRs for uniparental disomy
● Mitochondria contain their own circular DNA ■ unipaternal ang sabi ni sir sa
molecule, mtDNA recording
○ 16,569 base pairs containing 37 genes ○ Sequencing
● Heteroplasmy: normal and mutated mtDNA copies
can coexist in a cell INHERITED BREAST CANCER
○ Two types of mtDNA mutations:
■ mutations on the transfer RNA ● Familial breast cancer accounts for only 5% to 10% of
(tRNA) gene all breast carcinomas

♡ Divina, Mikaela ♡ 3
 MOLECULAR DETECTIONS OF INHERITED AND NON-INHERITED DISORDERS

● Mutations in two major breast cancer genes

predispose individuals to breast and ovarian cancer.

● Inheriting the gene does not mean that an individual

will develop cancer
● We cannot tell what type of cancer will develop
● We cannot at what age or onset the cancer will occur
● Men who carry the gene have greater risk at
developing the cancer

TUMOR-SUPPRESSOR GENEs
● BRCA1 gene also associated with increased risk for
prostate and colon cancer.
● BRCA2 gene also associated with increased risk for
pancreatic cancer
● Mutations with these will decrease the chance of
suppressing the oncogenes
● BRCA1 and BRCA2 regulates the oncogenes,
oncogenes yung gene na pwede magdevelop or
nagdedevelop ng cancer

● Mutations in BRCA1 and BRCA2 are inherited in an

autosomal dominant manner

● HOW DO WE DETECT INHERITED BREAST

CANCER?
○ DNA mutations can be detected through
DNA sequencing techniques, sequencing
yung BRCA1 and BRCA2 and kadalasan po
we need genetic counseling for individuals
before testing

♡ Divina, Mikaela ♡ 4