Exam 2 Review

Allele

One of two or more versions of a gene at a given locus. A person inherits two alleles, one from each parent.

Wild type allele

The normal, most common version of a gene in a population.

Variant / mutant allele

An altered version of a gene that differs from wild type; may or may not cause disease.

Locus

The specific physical location of a gene on a chromosome (e.g., 7q31 for CFTR).

Haplotype

A set of DNA variations or genes on a single chromosome that tend to be inherited together.

Polymorphism

A variant present in at least 1% of the population; may be neutral or mildly affect function.

Genotype

The specific combination of alleles an individual carries at a given locus.

Phenotype

The observable physical or biochemical characteristics resulting from genotype and environment.

Pleiotropy

When a single gene mutation affects multiple seemingly unrelated traits (e.g., CFTR affects lungs, pancreas, sweat glands, and vas deferens).

Homozygous

Having two identical alleles at a locus (e.g., ΔF508/ΔF508 in CF).

Heterozygous

Having two different alleles at a locus (one normal, one mutant).

Compound heterozygote

Having two different mutant alleles at the same locus (e.g., ΔF508/G551D in CF). Different from homozygous.

Hemizygous

Having only one copy of a gene because the other copy is absent. Males are hemizygous for X-linked genes (e.g., G6PD, MECP2).

Haploinsufficiency

When one copy of a gene is lost and the remaining single copy cannot produce enough protein for normal function, causing disease in a heterozygote. Examples: ELN (Williams), RAI1 (SMS), TBX1 (DiGeorge), SHOX (Turner).

Dominant negative

A mutant protein that actively interferes with the normal protein from the other allele. Seen in Marfan - mutant fibrillin monomers impair normal fibrillin monomers.

Loss-of-function mutation

Reduces or eliminates protein activity. Usually recessive unless the gene is haploinsufficient. Examples: thalassemias, CF.

Gain-of-function mutation

Creates a new or enhanced protein activity not present in the normal protein. Usually dominant. Example: Hb S gains the novel property of polymerizing under low O2.

Reduced penetrance

Not all individuals with a disease-causing genotype show the associated phenotype.

Variable expressivity

Individuals with the same disease-causing genotype show different degrees of severity. Prominent in Marfan - same FBN1 mutation can produce different phenotypes within the same family.

Allelic heterogeneity

Different mutations within the same gene can all cause the same disease. Example: 1,900+ CFTR variants cause CF; 1,850+ FBN1 mutations cause Marfan.

Locus heterogeneity

Different genes at different loci can cause the same disease. Example: CF can also involve ENaC mutations; CMT involves 100+ genes.

Clinical / phenotypic heterogeneity

Different mutations at the same locus produce different phenotypes. Example: CFTR mutations range from classic CF to isolated CBAVD or pancreatitis only.

Missense mutation

A single nucleotide change resulting in a different amino acid. Example: βGlu6Val in sickle cell; ~60% of FBN1 mutations in Marfan.

Nonsense mutation

A point mutation that creates a premature stop codon, truncating the protein. Common in β-thalassemia and CFTR Class I mutations.

Frameshift mutation

Insertion or deletion of nucleotides not in a multiple of three, shifting the reading frame and usually causing a premature stop codon. Frameshift mutations in G6PD are lethal.

De novo mutation

A new mutation not inherited from either parent; arises in egg, sperm, or just after fertilization. Most cases of DiGeorge, Rett, and SMS are de novo.

Housekeeping protein

Present in every cell; maintains structure and function. ~90% of expressed genes. Example: G6PD (knockout is lethal).

Specialty protein

Tissue-specific; produced in limited cell types with unique functions. ~10% of expressed genes. Examples: fibrillin-1 (connective tissue), beta-globin (RBCs).

NAHR (Non-allelic homologous recombination)

Misalignment of low copy repeats (LCRs) during meiosis causing deletions or duplications. Mechanism behind SMS (17p11.2 deletion), CMT1A (PMP22 duplication), DiGeorge (22q11.2), and Williams (7q11.23).

Sensitivity

If a person has the disease, how often is the test positive? (True positive rate)

Specificity

If a person does not have the disease, how often is the test negative? (True negative rate)

Positive Predictive Value (PPV)

If the test is positive, what is the probability the patient actually has the disease? Increases with higher disease prevalence.

Negative Predictive Value (NPV)

If the test is negative, what is the probability the patient does not have the disease?

Thermal Inactivation Assay

Measures enzyme stability by assaying activity at increasing temperatures. Compares mutant vs. wild-type to reveal structural and functional differences. Used to characterize G6PD Class I variants.

Randall-Selitto Paw Pressure Test

Measures mechanical pain sensitivity in lab animals by applying increasing pressure to the feet until vocalization. Used in CMT1A gene therapy studies to assess sensory function rescue.

MLPA (Multiplex Ligation-Dependent Probe Amplification)

Detects copy number variants. Probes hybridize to target sequences; ligation only with perfect complementarity; products PCR-amplified and sized simultaneously ("multiplexing"). Used to detect 17p11.2 deletions in SMS diagnosis.

PARIS2

Psoralen Analysis of RNA Interactions and Structures. Chemically freezes RNA-RNA interactions inside living cells, then sequences them to identify exactly where RNAs are interacting.

Marfan Syndrome - Gene and Inheritance

Gene: FBN1 (15q21.1), encodes fibrillin-1. Autosomal dominant. 75% inherited, ~25% de novo. Over 1,850 mutations (usually family-specific). Shows allelic, locus, and phenotypic heterogeneity.

Marfan - Fibrillin-1 Function

Fibrillin-1 is a large glycoprotein that forms microfibrils, providing structural support for elastic fibers (especially in the aorta and ligaments) and regulating TGF-β signaling.

Marfan - Molecular Mechanism (two types)

FBN1 mutations cause fibrillin deficiency via: (1) haploinsufficiency - not enough fibrillin for normal microfibrils; (2) dominant negative - mutant monomers impair normal monomers. Both lead to weakened elastic fibers AND overactivation of TGF-β → cardiovascular, skeletal, and ocular phenotypes.

Marfan - Key Phenotypes

Skeletal: tall stature, arachnodactyly, arm span:height >1.05, pectus deformity, scoliosis. Ocular: ectopia lentis (50%). Cardiovascular: aortic root dilation, risk of aortic dissection (leading cause of death). Lungs: spontaneous pneumothorax. Skin: stretch marks.

Marfan - Diagnosis

Revised Ghent Criteria: clinical manifestations in at least two of ocular, musculoskeletal, and cardiovascular systems plus FBN1 mutation analysis. Clinical signs: positive wrist sign (Walker) and thumb sign (Steinberg).

Marfan - Treatment

Activity restriction. Beta-blockers (standard of care - delay aortic aneurysm). Doxycycline (inhibits matrix metalloproteinases). Eyeglasses or surgery for lens dislocation. Life expectancy improved from 37 to ~78 years with proper management.

What is the difference between a hemoglobinopathy and a thalassemia?

• Hemoglobinopathies: abnormal globin chain structure (gain of function).

• Thalassemias: reduced quantity/synthesis of globin chain (loss of function).

Hemoglobin Structure

O2 carrier in RBCs. Four subunits: 2α + 2β chains, each with a heme (iron) group. HbA (α2β2) = 97%; HbA2 (α2δ2) = 2%; HbF (α2γ2) = 1%. β-like genes on chr 11; α-like genes on chr 16. Gene dosage: 4 α-globin genes, 2 β-globin genes - β mutations are more clinically significant.

Three Mutation Classes in Hemoglobinopathies

(1) Structural variants / gain of function - alter globin structure without changing synthesis rate (e.g., Hb S, Hb C). (2) Thalassemias / loss of function - decreased synthesis or extreme instability. (3) Heterochronic expression - hereditary persistence of fetal hemoglobin (HPFH).

Sickle Cell Disease - Gene and Mutation

Gene: HBB (chr 11). Mutation: βGlu6Val (GAG→GTG) - structural variant/missense, gain of abnormal function. Autosomal recessive. Hb SS = disease; Hb SA = trait (asymptomatic). Incidence in African Americans: 1/625.

Sickle Cell - Molecular Mechanism (how they become sickled)

Under low O2 tension, deoxygenated Hb S is 1/5 as soluble as Hb A → aggregates into stiff fibrous polymers → sickle-shaped RBCs. Sickled cells are fragile, have shortened lifespan, cannot squeeze through capillaries → hemolytic anemia and vaso-occlusion.

Sickle Cell - Key Phenotypes

Anemia, jaundice, dactylitis, splenomegaly, recurrent infections (splenic dysfunction), vaso-occlusive crises (stroke, necrosis, ulcers), renal and pulmonary failure. 11% mortality in first 6 months.

Sickle Cell - Treatment / Gene Therapy

Supportive: immunization, folic acid, fluids, O2, analgesics. Hydroxyurea or butyrate increase HbF. Allogenic bone marrow transplant. Casgevy (CRISPR/Cas9 - edits BCL11A to upregulate HbF; FDA approved Dec 2023). Lyfgenia (lentiviral vector - produces HbAT87Q; BLACK BOX WARNING for hematologic malignancy).

α-Thalassemia - How it works

Four α-globin genes (two per chr 16). Mutations are usually deletions via NAHR. 1 deleted = silent carrier. 2 deleted = mild anemia (α-thal trait). 3 deleted = Hb H disease (β4 tetramers, cannot release O2). 4 deleted = hydrops fetalis (lethal - Hb B parts γ4 cannot release O2).

β-Thalassemia - Gene and Mechanism

Gene: HBB (chr 11). Usually point mutations, not deletions. Excess α chains precipitate → destroy RBCs. β0 (two mutant alleles) = thalassemia major (severe). β+ (one mutant allele) = thalassemia minor/intermedia (mild).

Hemoglobin E (Hb E)

Coding sequence mutation activating a cryptic splice site → reduced β-chain synthesis → mild anemia. Most common structurally abnormal hemoglobin worldwide. As compound heterozygote with β-thalassemia → more severe phenotype.

Hereditary Persistence of Fetal Hemoglobin (HPFH)

Mutations in β-globin LCR or enhancers leave γ genes active after birth → continued HbF production (18–100%).

Smith-Magenis Syndrome (SMS) - Gene and Inheritance

Autosomal dominant. ~90% have interstitial 17p11.2 deletion (1.5–9 Mb, ~80 genes). 10% have pathogenic RAI1 point mutations only. De novo in most cases via NAHR between seven LCR elements (Alu family + AT-rich repeats).

SMS - Causative Gene: RAI1

RAI1 (Retinoic Acid Induced 1) haploinsufficiency is responsible for the SMS phenotype. Transcriptional regulator that regulates synaptic plasticity (neural homeostasis) and PER2 → circadian rhythm.

SMS - Molecular Consequence

RAI1 haploinsufficiency → neural network cannot respond normally to changes in neuronal activity → neurobehavioral dysregulation. PER2 disruption → inverted melatonin secretion → fragmented sleep cycles and circadian disruption.

SMS - Key Phenotypes

Craniofacial: broad forehead, mid-face retrusion, short upturned nose. Neurobehavioral: self-injurious behaviors (95%), repetitive actions (95%), intellectual disability (6–8 yr level), inverted melatonin → severe sleep disturbances. 17p11.2 deletion adds: hearing loss (79%), congenital heart defects (25–45%), short stature. RAI1-only: mainly neurobehavioral + obesity.

Rett Syndrome - Gene and Inheritance

X-linked; primarily affects females (>95%). 99% de novo, most often from the paternal X. Gene: MECP2 (Xq28). Over 300 loss-of-function variants; 8 recurrent hotspots = >60% of cases.

Rett - MeCP2 Protein Function

Key epigenetic regulator. Primary role: transcriptional repressor - binds methylated CpG sites, recruits NCoR/SMRT co-repressor complex → closed chromatin, gene silencing. Secondary role: transcriptional activator - drives BDNF and IGF1 expression. Loss → widespread gene dysregulation and synaptic dysfunction.

Rett - Key Phenotypes (4 Stages)

Stage 1 (6–18 mo): appears normal ("too good"). Stage 2 (18 mo–5 yr): regression - loss of purposeful hand skills, loss of language, stereotypic hand movements, seizures, microcephaly, gait abnormalities. Stage 3 (5–12 yr): GI dysmotility, scoliosis, feeding difficulties, anxiety. Stage 4 (12+ yr): stabilization, social withdrawal. ~70% survive into their 50s.

Rett - Why Males Are More Severely Affected

Males have only one X → all cells carry the mutation → severe brain dysfunction, usually fatal before adulthood. Females have random X inactivation → ~50% of cells express normal MECP2 → partial compensation. Severity depends on degree of X inactivation skewing.

Rett - Why Symptoms Appear Later (Not at Birth)

MeCP2 expression is low during early development → mutant cells can compensate. Expression increases as neurons mature after birth. Regression begins at 6–18 months when MeCP2 demand peaks.

Rett - MECP2 Dosage Sensitivity

Too little MeCP2 → Rett syndrome. Too much MeCP2 → MECP2 Duplication Syndrome (severe intellectual disability, seizures). Must be "just right" - the main challenge in gene therapy development.

Rett - Treatment

Supportive care: speech and motor therapy, mobility aids, etc. FDA-approved: Trofinetide (Daybue, 2023) - synthetic IGF-1 fragment supporting neuronal signaling. Gene therapies in trials: TSHA-102 (AAV9, mini-MECP2, miRARE dosage control) and NGN-401 (AAV9, full-length MECP2, EXACT system).

G6PD Deficiency - Gene and Inheritance

X-linked (Xq28). Most common enzyme deficiency worldwide (~400 million). 1 in 10 African American males. Mostly missense mutations (200+ variants); frameshift mutations are lethal.

G6PD - Molecular Mechanism

G6PD = first enzyme of the pentose phosphate pathway (PPP) → produces NADPH. NADPH protects RBCs from reactive oxygen species (ROS). G6PD is NOT synthesized in mature RBCs (fixed reservoir). In deficiency: low G6PD → low NADPH → oxidative stress from fava beans, primaquine, or infections → hemoglobin denatures into Heinz bodies → macrophages remove them creating "bite cells" → acute hemolytic anemia.

G6PD - Key Phenotypes

Most asymptomatic unless triggered. Neonatal: jaundice (NNJ), kernicterus, AHA. Adults: episodic AHA triggered by fava beans, drugs, or infection. Class I = most severe (chronic AHA); Class II/III = episodic; Class IV/V = clinically insignificant. Complete G6PD inactivation is lethal.

G6PD - Diagnosis

Fluorescent Spot Test (FST) for newborns - less accurate in females due to X inactivation mosaicism (some cells have normal G6PD). G6PD enzymatic assay for children and adults, typically after an AHA episode or before prescribing trigger drugs.

G6PD - Treatment and Research Techniques

No direct cure; avoid triggers. Gene therapies in development.

Charcot-Marie-Tooth (CMT) - Overview

Most common inherited neuromuscular disorder. Multigenic (100+ genes) - classic locus heterogeneity. CMT1/2 = autosomal dominant; CMT3 = autosomal recessive; CMT4 = X-linked. Progressive distal peripheral neuropathy affecting motor/sensory axons and Schwann cells.

CMT - Gene and Mechanism (CMT1A)

Most common cause: PMP22 duplication at 17p11.2 (via NAHR). PMP22 encodes peripheral myelin protein 22, produced by Schwann cells. Duplication → protein overload → disrupts myelin assembly → demyelination. Molecular consequences: misfolded proteins, altered Schwann cell signaling, impaired axonal transport.

CMT - Key Phenotypes

Adult: high arched feet (pes cavus), hammertoes, foot drop, distal muscle atrophy ("champagne bottle" calves), weakness, decreased sensation, "clumsy" gait, scoliosis. Childhood: failing milestones, frequent falls. Progressive but not fatal.

CMT - Treatment

No cure. Management: physical therapy, orthotics, pain management, surgical foot correction. Gene therapy research.

Cystic Fibrosis (CF) - Gene and Inheritance

Autosomal recessive. Gene: CFTR (7q31), encodes a 170 kD chloride channel on the apical membrane of epithelial cells.

CF - Molecular Mechanism (Lungs)

Loss of CFTR → hyperabsorption of Na+ and reduced Cl- secretion → depleted airway surface liquid → thick mucus adheres to airways → impairs mucociliary clearance → recurrent infection → inflammation → bronchiectasis → respiratory failure. Leading cause of CF morbidity and mortality.

CF - CFTR Mutation Classes

Class I: defective protein production (premature stop codon). Class II: defective protein processing/misfolding - ΔF508, retained in ER. Class III: defective channel regulation. Class IV: defective Cl- conduction. Class V: reduced CFTR transcripts (abnormal splicing). Class VI: unstable protein at cell surface.

CF - ΔF508 Mutation

Class II: protein misfolds → retained in ER, degraded → no functional CFTR at cell surface. Also has secondary defects in stability and channel activation.

CF - Key Phenotypes

Lungs: chronic cough, recurrent infections, bronchiectasis, respiratory failure. Pancreas: enzyme retention → fibrosis → malabsorption (95% pancreatic insufficient). Sweat: Cl- >60 mmol/L (CFTR loss prevents Cl- reabsorption in sweat duct). Reproductive: 95% of males have CBAVD → infertility.

CF - Allelic, Locus, and Clinical Heterogeneity

Allelic: ~1,900 disease-associated CFTR variants; most common is ΔF508. Locus: ENaC mutations (different gene) cause CF-like pulmonary disease. CFTR genotype predicts pancreatic phenotype well but poorly predicts lung severity (modified by TGF-β1).

CF - Treatment

Airway clearance + antibiotics. Pancreatic enzyme replacement + nutrition. CFTR modulators: Ivacaftor (potentiator - increases channel opening, Class III); Lumacaftor (corrector - chaperone for ΔF508, Class II); Trikafta combines corrector + potentiator. Lung transplant for end-stage disease.

Unstable repeat expansion

Expansion of a short DNA repeat (CAG, CGG, GAA, CTG) that grows as the gene is passed between generations. Mechanism: slipped mispairing during replication or unequal crossing over. Can expand in meiosis, mitosis, or non-dividing neurons (DNA repair). Causes anticipation.

Repeat expansion - Class 1

Noncoding repeat expansion causing loss of protein function. Examples: Fragile X (CGG in 5' UTR of FMR1) and Friedreich Ataxia (GAA in intron 1 of FRDA - impairs transcription elongation).

Repeat expansion - Class 2

Noncoding repeat expansion conferring toxic properties on the RNA itself. Example: Myotonic Dystrophy (CTG in 3' UTR of DMPK - CUG RNA sequesters RNA-binding proteins).

Repeat expansion - Class 3

Coding region repeat expansion producing a novel toxic protein. Example: Huntington disease (CAG in exon 1 of HTT - polyglutamine tract causes toxic gain of function).

SCA - Overview and Phenotypes

Autosomal dominant neurodegenerative disorder. Progressive cerebellar degeneration (Purkinje, molecular, granule cell layers) causing uncoordinated gait, limb/eye movement problems, slurred speech, dysphagia. Shows anticipation. Death often from pneumonia.

SCA5 - Gene and Inheritance

Autosomal dominant. Gene: SPTBN2 (beta-III spectrin) on chromosome 11q13. Pure cerebellar ataxia - Purkinje cell loss and thinning of molecular layer. Normal life expectancy.

SCA5 - Beta-III Spectrin Function

Expressed specifically in Purkinje cells. Stabilizes membrane proteins - especially EAAT4 (Purkinje cell-specific glutamate transporter) and mGluR1-alpha (perisynaptic metabotropic glutamate receptor critical for cerebellar synaptic plasticity).

• Mutations cause loss of membrane stabilization → Purkinje cell degeneration.

• Purkinje cell degeneration → uncoordinated gait, limb/eye movement problems, slurred speech, dysphagia.

SCA5 - Molecular Mechanism

Mutant beta-III spectrin causes: (1) loss of EAAT4 membrane stabilization - reduces glutamate clearance at synapses; (2) mGluR1-alpha mislocalization from Purkinje cell dendritic spines - disrupts synaptic plasticity. Result: progressive Purkinje cell death and cerebellar degeneration.

Rotarod Test

Behavioral test of motor coordination in mice. Animal must balance on an accelerating rotating rod; latency to fall is measured. Used to assess cerebellar function and disease progression in SCA5 transgenic mouse models.

Huntington Disease - Gene and Inheritance

Autosomal dominant. Gene: HTT (4p16.3). CAG repeat expansion in exon 1 of huntingtin protein (3300 aa). Average age of onset: 40-45 years. Death 15-20 years after onset. Anticipation more pronounced with paternal transmission.

HD - CAG Repeat Thresholds

Normal: 9-35 repeats. Borderline: 36-39 (usually affected, may not show disease). Full mutation: 40+ repeats = disease. 40-55 = adult onset (90% of carriers). 60-70 = juvenile onset (Westphal variant). Larger expansion = earlier onset.

HD - Paternal Transmission

HD expansions preferentially expand during paternal transmission - sperm undergo more rounds of replication. 73% of sperm show expansion, 23% contraction. Explains why anticipation is more pronounced through the paternal line.

Huntingtin - Normal Function

Ubiquitous cytoplasmic protein involved in neuronal survival, transport, transcription, and synaptic function; essential for development.

HD - Molecular Mechanism

Toxic gain of function. Expanded polyglutamine (poly-Q) tract causes huntingtin misfolding, intranuclear and cytoplasmic insoluble aggregates, and defects in axonal transport. Disrupts transcriptional regulation, synaptic function, and anti-apoptotic activity. Aggregates appear AFTER disease onset - relationship not fully understood.

HD - Key Phenotypes

Three domains: (1) Motor: chorea (brief involuntary movements from random muscle contractions), progressing to dystonia and rigidity. (2) Psychiatric: depression (25% attempt suicide), anxiety, obsessive/compulsive, psychosis. (3) Cognitive: gradual decline to dementia. End stage: rigid, akinetic, mute. Death from aspiration pneumonia.

HD - Age of Onset Variation

CAG repeat length accounts for ~72% of age of onset variation. Remaining variance influenced by modifier loci and environment. Individuals with the same repeat number can still have different ages of onset.