DNA polymorphism and inherited disease

What is the mutation nomenclature at the DNA level

DNA level = Sequence changes are expressed by base substitutions (e.g., 1691 A→G in F5 for Factor V Leiden).

What is the mutation nomenclature at the RNA level

RNA level = Described by the corresponding codon change using cDNA numbering (e.g., c.1521_1523delCTT for CFTR F508del).

What is the protein nomenclature at the protein level

Protein level = Expressed as the resulting amino acid change (e.g., R506Q means arginine replaced by glutamine at position 506; F508del means phenylalanine deleted at position 508)

What is a SNP and what are the types?

Single-nucleotide polymorphisms = Variations at a single base pair site in DNA that occur commonly in a population (may be benign or pathogenic).

Types:

-          Missense: Substitution that changes one amino acid (e.g., HFE C282Y in hemochromatosis).

-          Nonsense: Substitution introducing a stop codon, truncating the protein.

-          Frameshift: Insertion or deletion (not a multiple of 3 bases) that shifts the reading frame, altering downstream amino acids

What are the phenotypic consequences of GOF mutations

Gain-of-function mutations: Usually dominant; mutant allele produces abnormal protein causing disease (e.g., oncogene activation, Factor V Leiden).

-          Less common than LOF mutations

-          Include gene-expression/stability defects that generate gene products at inappropriate sites or times

What are the phenotypic conseqeunces of LOF mutations

Loss-of-function mutations: Usually recessive; functional protein is lost (e.g., cystic fibrosis, hemochromatosis).

-          Complex metabolic pathways are susceptible to LOF because of extensive interactions between and among proteins

What are the phenotypic consequenecs of dominant negative mutations

Dominant-negative mutations: Mutant protein interferes with the function of the normal protein (e.g., p53 tumor-suppressor defects)

-          Seen in cases of multimeric proteins 

-          Even though one allele is mutated, the mutated protein can interfere with the function of the tetramer, producing an abnormal phenotype

What are Common molecular detection methods

Common molecular detection methods include:

-          PCR-RFLP (e.g., used for Factor V Leiden and HFE C282Y mutations).

-          Sequencing (direct or cDNA sequencing).

-          SSP-PCR (Sequence-Specific Primer PCR) – detects allele-specific bases.

-          High-resolution melt-curve qPCR, microarray, bead array, and Invader technology for SNP screening

Describe Single nucleotide polymorphisms (SNPs) and their relevance to molecular diagnostics.

SNPs represent common, stable genetic markers used for disease association, pharmacogenomics (e.g., CYP450 polymorphisms), and personalized medicine.

-          In diagnostics, they can predict disease risk, drug response, and inheritance of pathogenic variants

Described X’linked inheritance patterns

X-linked = Carried on the X chromosome; expressed more in males who are hemizygous. Example: Duchenne muscular dystrophy

-          Primarily affects males; transmitted by carrier females

-          Relatively few genes are carried on the Y chromosome (reason most diseases are X linked)

-          Usually recessive, but there are some that are dominant

Describe autosomal dominant inheritance patterns

Autosomal dominant = One copy of mutant allele produces phenotype (50% chance of inheritance). Example: Prothrombin mutation

-          Appears in every generation.

-          Codominant offspring = simultaneously demonstrate the phenotype of both parents 

o   Ex = ABO blood types

Describe autosomal recessive inheritance patterns

Autosomal recessive = Two copies of mutant allele needed; carriers asymptomatic (25% recurrence risk). Example: Cystic fibrosis, Hemochromatosis, MTHFR deficiency

-          Skips generations; often appears with consanguinity

-          Recurrence risk of 25% if a sibling is affected

-          New mutations rarely detected in transmission patterns (usually a result of two individuals heterozygous for the same mutation producing offspring)

-          Inborn errors of metabolism are usually autosomal recessive

Define Congenital

Congenital = Condition present at birth (not necessarily inherited) “born with”

-          Result when some factor (drugs, chemicals, infection, or injury upsets the development process)

-          Spina bifida

Define polyploidy

Polyploidy = >2 complete sets of chromosomes (more than 2 of any autosome)

- Usually results in infertility and anormal appearance

- Triploidy (3n)

Define mosaicism

Mosaicism = Two or more genetically distinct cell populations from one zygote in an individual, results from mutation events affecting somatic or germ cells

-          Early segregation errors during fertilized egg divisions occasionally gives rise to mosaicism

-          45,X/47,XXX female

Define triploid

Triploid = 3 copies of each chromosome (3n).

-          Triploid fetus (45, XY/47,XY,+21)

Define aneuploid

Aneuploid = Gain or loss of one or more chromosomes (autosome)

-          Caused by erroneous separation of chromosomes during egg or sperm production

-          Sex chromosome aneuploidy is more frequently tolerated, but is associated with phenotypic abnormalities

-          Trisomy 21

Define monosomy

Monosomy = One copy of a chromosome instead of two

-          Results from fertilization of gametes missing a chromosome

-          Generally, but not always, incompatible with life

-          Turner syndrome (45,X)

Define trisomy

Trisomy = Three copies of a chromosome instead of two

-          Results from fertilization of gametes containing an extra chromosome

-          Down syndrome

Define hemizygous

Hemizygous = Only one copy of a gene is present (X-linked in males).

-          X-linked disorders

-          Males are hemizygous because they only have one copy of the X chromosome (more likely to manifest the disease phenotype)

What is factor V Leiden? and the mutation that causes it?

Factor V Leiden = hypercoagulation phenotype resulting in mutations in the factor V gene

-          A>G, R506Q in coagulation factor V gene F5 causes a hypercoagulable phenotype

-          Tested with... PCR, PCR-RFLP, or PCR with sequence specific primers

What is Prothrombin? and the mutation that causes it?

Prothrombin = the precursor to thrombin in the coagulation cascade and is required for the conversion of fibrinogen to fibrin

-          Mutation in the 3’ untranslated region of the gene that codes for prothrombin or coagulation factor II, F2 (results in autosomal dominant increased risk of thrombosis)

-          Labs test for F2 and F5 mutations

-          Tested with... multiplex PCR-RFLP

What is Methylenetetrahydrofolate reductase (MTHFR)? and the mutation that causes it?

Methylenetetrahydrofolate reductase (MTHFR) = results in increased homocysteine levels causing a predisposition to venous and arterial thrombosis

-          Caused by deficiency of the 5, 10-methylenetetrahydrofolate reductase (MTHFR) gene

o   Enzyme co-substrates the conversion of homocysteine to methionine

-          Mutations = 667C>T (p. A222V) & 1298 A>C (p.E429A)

-          Tested with... standard or multiplex PCR with RFLP & multiplex qPCR

What is Hemochromatosis? and the mutation that causes it?

Hemochromatosis = causes overabsorption of iron from food, causing pancreas, liver, heart disease, diabetes, and skin damage

-          Caused by dysfunction of the hemochromatosis type I HFE or HLA-H gene product

o   HFE codes for membrane bound protein that binds with B2-microglobulin and transferring on the membrane of cells in the small intestine/placenta

o   Absence of HFE function, intestinal cells do not sense iron stores, and iron absorption continues into overload

-          Mutation = C282Y & H63D/S65C

-          Tested with PCR-RFLP

What is cystic fibrosis? and the mutation that causes it?

Cystic Fibrosis = disease causes severe lung damage and nutritional deficiencies. Affects cells that produce mucus, sweat, saliva, and digestive juices

-          Mutation causes secretions to become thick and sticky

-          Caused by a loss of function of the CF transmembrane conductance regulator (CFTR gene (7q31.2))

o   Gene codes for chloride channel membrane protein

o   Most frequent mutation = 3-bp deletion that removes a phenylalanine residue from position 508 of the CFTR protein (F508del)

-          Testing with... RFLP, PCR-RFLP, heteroduplex analysis, temporal temperature gradient gel electrophoresis, single-strand conformation polymorphism (SSCP), SSP-PCR, cleavase, bead array technology, and direct sequencing

Know these diseases and their mutations

Disease	Gene / Mutation	Molecular Alteration	Notes
Factor V Leiden	F5 (1q23), 1691A→G (R506Q)	Missense point mutation causing hypercoagulable state	Detected by PCR-RFLP or SSP-PCR
Prothrombin	F2 (11p11-q12)	Mutation in 3′ untranslated region increases prothrombin mRNA → thrombosis risk	Often tested with Factor V Leiden
MTHFR Deficiency	MTHFR (1p36.3), 677C>T (A222V) and 1298A>C (E429A)	Reduces enzyme activity, elevates homocysteine	PCR-RFLP or sequencing detection
Hemochromatosis	HFE (6p21.3), C282Y, H63D, S65C	Missense mutations impair iron sensing, causing overload	PCR-RFLP
Cystic Fibrosis	CFTR (7q31.2), ΔF508 (F508del)	3-bp deletion removes phenylalanine, disrupting chloride channel	Detected by sequencing, RFLP, or allele-specific assays