Molecular Basis of Human Disease

What are the uses of novel DNA variants?

Use in silico tools to predict the pathogenicity of the mutation and if it causes disease  

What are the uses of known mutations?

Understand how the mutation causes the disease  

What is the importance of understanding the disease mechanisms?

• Understand the progression of the disease 

• Predict disease progression and outcomes  

• Guide management of patients  

• Identify drug targets for therapies  

How do mutations in DNA, mRNA, proteins, subcellular organelles and cells impact?

What are the types of DNA variants?

• Loss of function mutation  

• Gain of function mutation  

• Dominant negative function  

How do mutations cause clinical pheotypes?

• Functional consequences of DNA variants  

• Specific type of DNA variants  

• Modification by genetic background  

What are loss of function mutations?

Mutations that result in reduced or abolished function of the mutant protein  

What are the features of thalassemia?

• Autosomal recessive condition  

• Alpha version has HBA mutation and beta version has HBB mutation  

How does thalassemia cause disease?

• Point mutations or deletions reduce the rate of synthesis or no synthesis of one of the globin chains that makes the haemoglobin  

• Too few globin synthesised leading to anaemia  

What are the features of sickle cell?

• Autosomal recessive

• Distort the red blood cells into sickle shapes, rigid and less flexible  

• Microvascular thrombosis  

• Abnormal function of B-globin leading to anaemia  

• A qualitative problem  

• Still has the same amount of protein  

How is sickle cell caused?

• Single nucleotide change in the B-globin gene HBB at codon 6  

• GAG glutamic acid to GTG valine  

• The mutant B-globin tend to stick together  

• Valine is hydrophobic and causes the molecules to get stuck together  

What are the features of haemophilia B?

• X-linked recessive  

• Mutation at promoter region of F9 gene encoding factor IX  

What are the features of the binding sites in F9?

• Regulate the expression of factor IX  

• Mutations in this region cause Haemophilia B Leyden  What

What are the features of the ARE region?

• Androgen regulated  

• Increase the factor IX expression at puberty  

• Mutations at ARE region cause Haemophilia B Brandenburg  

What are DMD and BMD?

• DMD and BMD are caused by mutations in then DMD gene  

• This encodes the protein dystrophin  

• Mostly large deletions  

What are the features of DMD?

• X-linked recessive condition  

• Affects 1 in 3,000 boys  

• Mainly affects 3-5 year olds, progressive muscle weakness, wheelchair in teens and die in 20s due to respiratory and cardiac issues  

• No effective treatment  

What are the features of BMD?

• X-linked recessive condition  

• Affects 1 in 20,000 men  

• Symptoms are much milder and occurs much later 

• Can have a normal lifespan  

How do mutations cause DMD?

Deletion of exons that create a frameshift completely abolish the synthesis of dystrophin  

How do mutations cause BMD?

Deletions that leave the reading frame intact result in synthesis of a smaller put partially functional protein  

What is the structure of the dystrophin protein?

Has N terminal binding sites, actin cytoskeleton and C terminal that binds to other proteins and anchors the cell membrane to the extracellular matrix  

What happens to the dystrophin protein in DMD?

• Long middle structure produces strong and flexible structure  

• Without the protein the contractile machinery falls apart

What happens to the dystrophin protein in BMD?

• Some regions are deleted but the machinery is still held together  

• Produces a milder disease phenotype  

What is a gain of function mutation?

A mutation that results in an abnormally increased activity of the mutant protein

What are the features of familial hypercholesterolemia?

• Can be caused by mutations in PCSK9 gene  

• Autosomal dominant  

• High blood level of LDL cholesterol  

• Early atherosclerosis, xanthomas  

How does PCKS9 cause FHC?

• PCKS9 reduces LDL-Rs on liver cells  

• Mutation repress the recycling of LDL-R to the membrane and causes them to be degraded in the lysosome  

• Leads to high cholesterol  

• Increases the protein produced and then overactivated the repression of the cycle  

What are the treatments for FHC?

• PCSD9 inhibitors

• mAbs

• siRNA

What are dominant negative mutations?

• Mutant products not only lose its own function but adversely affect the function of the normal product in heterozygote  

• Specific type of loss of function mutation  

What are the features of colagen?

• 19 human collagens encoded by 30 genes  

• Each collagen is a triple helix  

How does collagen cause disorders?

• Repetitive Gly-X-Y motif is important for triple helix  

• Majority of mutations happen in this region  

• Mutations disrupt the collagen processing causing a dominant negative effect on the triple helix  

How do different mutations impact collagen production?

• Abnormal collages and incorporated into 2 normal chains and produces dysfunctional protein chain  

• Severe mutations abolish the protein so won’t interfere with the other protein 

What are the features of osteogenesis?

• Mutations in COL1A1 or COL1A2 genes  

• Severe bone fragility, bone malformations, short stature, macrencephaly, hearing loss and blue sclarea 

What are the features of stickler syndrome?

• Mutations in COL2A1 or COL11A1  

• Hereditary progressive artho-opthalamopathy  

• Distinctive facial abnormalities, ocular problems, hearing loss and joint problems  

• High myopia, retinal detachment and cataracts 

What are the features of COL4A1/A2?

• Mutations mainly in COL4A1 or COL4A2  

• Multiple haemorrhagic strokes, leukoencephalopathy, seizures, and migraine with auras  

What are the features of repeats in the genome?

• Over 50% of the genome is repetitive sequences  

• Microsatellite can be trinucleotide repeats  

• If they expand they can meet the threshold and cause Mendelian disease 

What are the features of dynamic mutations?

• Caused by expansion of existing polymorphic DNA repeat sequences beyond a copy number threshold  

• Usually trinucleotide  

• Disorders caused by dynamic mutations are commonly called TREDs  

What are the features of poly-glutamine diseases?

• Progressive neurodegenerative or neuromuscular diseases  

• Caused by modest expansion of CAG repeat (35-100)  

• In the coding sequence encodes a poly-Q tract  

• Dominant diseases  

• Likely pathogenic mechanism in protein aggregation  

• Repeat expansion leads to gain-of-function  

What causes Huntington’s?

• Autosomal dominant  

• Expansion of CAG repeats in HD gene  

• Late onset normally 40s  

What are the features of Huntington’s?

• Involuntary movements (40s)  

• Memory loss 

• Apathy  

• Changes in personality and mood  

• Depression, anxiety and seizures  

What are the features of neuronal intranuclear inclusions?

• HTT encodes the Huntingtin protein  

• Typical pathological change causes Huntingtin protein aggregation  

How do Poly-Q mechanisms cause disease?

• Sequesters important proteins  

• Affects mitochondrial function  

• Toxic titration of chaperones  

• Cell apoptosis  

• Inhibiting proteosome function  

• Blocking vesicle trafficking  

What are the features of fragile X syndrome?

• Intellectual disability  

• 20% autistic  

• Long face, log jaw, large ears  

• Affects 1 in 4,000 males  

What is the mechanism of fragile X?

• FMR1 encodes protein FMRP  

• Full expansions of CGG triggers DNA methylation, switching off the gene expression  

• CGG expansion in FMR1 impairs synapse function  

• Loss of FMRP is the underlying mechanism of fragile X syndrome  

What is the role of FMPR?

• FMPR is an RNA binding and associates with polyribosomes  

• Repressing translation of a subset of dendritically localised mRNAs  

• Play a critical role in dendritic local protein synthesis and synaptic plasticity  

What are the features of fragile X-associated tremor?

• Neurodegenerative disorder, adult onset >50 years  

• Tremors, ataxia, parkinsonism, memory loss and cognitive decline  Wha

What causes FXAT?

• CGG 55-200 expansion in 5’ UTR region of the FMR1 gene  

• Significant elevation of FMR1 mRNA resulting in intranuclear inclusions that sequences 20 proteins involved in 

• RNA binding  

• Protein folding  

• Nuclear membrane integrity  

What are the features of anticipation?

• The symptoms of the genetic disorder become apparent at an earlier age, are severer as it is passed onto the next generation  

• The size and expansion correlates with disease severity and age of onset  

What causes anticipation?

• Repeats are unstable and tend to expand  

• Polymerase slippage and don’t know where in the sequence it is  

What are the features of genotype-phenotype relationships?

• Genotype is the genetic make up  

• Phenotype is observable characteristics  

• Determined by nature of DNA variants and genetic background  

How does globin genes have DNA variants?

• Glutamic acid to valine at 6^th aa position of HBB causes sickle cell anaemia  

• Other aa substitutions cause 

• Reduced haemoglobin synthesis like Thalassemia  

• Instability of haemoglobin molecule  

• Interfere with a and b subunit interactions  

• Some can have high oxygen affinity – polycythaemia  

What is the DNA variants?

Same gene but different mutations can have different phenotypes  

What are the features of FHC genetic background?

• FHC have two mutations in the cis MYH7 gene  

• One is a low penetrance missense variant, the other is a pathogenic mutation  

• Results in a particularly severe phenotype  

What is genetic background in CFTR?

Two milder mutations in CFTR gene can act in concert leading to a more severe phenotype  

What are the features of sick sinus syndrome?

• SSS has SCN5A mutations and polymorphism modulate disease phenotype  

• If a variant is combined with another mutation can it rescue the gene  

What are the features of Angelman syndrome?

Slow development, unusual movement, sever learning difficulties, epilepsy, happy face, poor communication skills and little or absent speech  

What are the features of Prader-Willi syndrome?

Low muscle tone, short stature excessive appetite, obesity, immature physical development, learning disabilities and emotional instability, almond shaped eyes, left-handed  

How is Angelman and Prader-Willi related?

• Both are caused by 15q11 deletions  

• Prader-Willi is the paternal deletion and Angelman is the maternal deletion  

• Due to genetic imprinting