Ch 7 Genetic and Pediatric Diseases (p.

243-272, 291-296)

Nature of Genetic Abnormalities Contributing to Human Disease

1. Common Gene Mutations: Protein Coding Genes
Point missense (change AA) or nonsense (stop codon)
Frameshift
Trinucleotide Repeat Disorders: amplification (all repeat patterns with G and C)
2. Alterations in Protein-Coding Genes Other than Mutations
Involves structural variations coding genes (amplification, deletion, translocation)
Alt in noncoding RNAs important ncRNAs= microRNAs (miRNAs) and loVariang non-coding RNAs (lnRNA)
3. Major categories of genetic disorders
Mutation in single genes high penetrance
Multiple gene involvement and environmental factors
Chromosomal abnormalities
Non classical single gene disorders triplet repeat mutations, mitochondrial, epigenetics
Mendelian Disorders: Diseases Caused by Single Gene Defects

1. Possible patterns of inheritance: autosomal dominant/recessive, X-linked

2. Diseases that can be caused by multiple types of single defects=Marfan, retinitis pigmentosa
3. Modifier genes genetic loci shown to play role in diseases that are caused by multiple single gene defects

Transmission Patterns of Single Gene Disorders

1. Autosomal Dominant:
May be passed down or derived from new mutations; penetrance and expressivity can vary
Penetrance: have mutant gene but severity of disease varies
Variable expressivity: people w/ gene show different sets of traits
(Neurofibromatosis I can express w/ brown spots on skin to tumors, skeletal issues, etc)
Symptoms usually delayed (adult onset)
Clnical signs asso with 50% reduction in normal gene product
NOTE: 50% reduction in enzyme usually can be compensated so most aut dom disorder are not for enzyme encoding genes
Alternative=metabolic pathways, structural proteins, subunit of multimers (collagen for example)
Dominant Negative: mutant allele that affects function of normal allele (Ex: issue with one collagen subunit)
2. Autosomal Recessive: (largest group of mendelian disorders)
Tend to have more normal expression of defects compared to aut dominant w/ complete penetrance
Earlier onset
Enzymes are often affected by mutation heterozygote usually retain 50% enzyme so normal function
3. X-linked
Y-linked Rare Y-linked familial deafness
Most are X-linked recessive

Diseases Caused by Mutations in Genes encoding Structural Proteins

Marfan Syndrome: Aut Dominant of connective tissue
1. Defect in fibrillin-1 (FBN1) (extracellular glycoprotein) which is major component of myofibrils in the ECM
2. FBN-1 is important in sequestering TGF-B activation many mutations cause disease so diagnose via clinical findings
3. FBN-1 defect thought to be dominant neg b/c heterozygotes show symptoms
4. TGF-B connection: myofibrils need to be intact to prevent excessive TGF-B activation which can weaken smooth muscle development/integrity
(asso with predisposition to aortic aneurysm)
5. Clinical symptoms: tall+slender, ectopia lentis, CV issues, floppy valve syndrome (cardiac), mitral valve prolapse
Ehlers-Danlos =group of diseases with defect in collagen synthesis/structure
1. All are single gene disorders but w/ varying mode of inheritance
2. 6 recognized variants caused by a distinct mutation
 3. Common clinical feature: hyperflexible skin, hypermobile joints, ruptures in colon, cornea, arteries, poor wound healing
4. Common deficient components: Type 3 collagen (COL3A1 mut) aut dom, enzyme lysyl hydroxylase, Type 5 collagen (COL5A1 and 2) aut dominant
Diseases Caused by Mutations in Genes Encoding Receptor Proteins/Channels
Familial Hypercholestrolemia effect on homo/heterozygotes; autosomal domoinant
1. LOF of LDL receptor (transport and metabolism of cholesterol) can’t transport LDL and IDL into cell
2. Activating mutations of PCSK9 which degrades LDL can have similar symptoms
3. Increased cholesterol more risk of atherosclerosis, CV issues, cholesterol deposit in tendon sheath produce xanthomas
4. Class 2 most common impaired transport of receptor from ER to golgi causing protein fold defect
Other classes: Class 1= complete loss of synthesis, 3=receptor on surface but don’t bind, 4= bind but don’t internalize;
5=trapped in endosomes
5. Treatment: statins to lower plasma cholesterol (decrease HMG-CoA reductase to increase LDL receptor synthesis)
Cystic Fibrosisdisorder of epithelial ion transport of fluid secretion; aut recessive CFTR gene for CF transmembrane regulator
1. Most common cause of death=cardiopulm complication infection
prone (pseumonas, burkholderia), right side HF
2. Pancreatic insufficiency common (issue w/ pancreatic duct
secretions), liver disease (cirrhosis) risk
3. Effect of CFTR mutation is different between different tissues
because used for transport out or in depending on part of the body
4. Therapies: molecular approach to enhance transport/stability of
mutant CFTR; supportive measure with anti-microbial therapies,
pancreatic enzyme replacement, lung transplant
5. NOTE: CFTR also affects bicarbonate transport main mech of
pancreatic insufficiency
6. Most common severe mutation: F508
7. Diagnosis: elevated sweat electrolyte concentration, sequencing
gene is gold standard

Diseases Caused by Mutations in Genes Encoding Enzyme Proteins

Phenylketonuria (PKU): aut recessive; lack phenylalanine hydroxylase which normally metab Phe
1. Clinical features: mental retardation, seizure, decrease skin pigmentation
2. Tx=avoid food with Phe as early as possible after birth (esp patients that are pregnant to
prevent birth defects), enzyme replacement therapy
3. Benign hyperphenylalaninemia: deficient PAH activity but not completely lost

Galactosemia: aut recessive lack of GALT enzyme galactose-1-

phosphate and galactose accumulation
1. Clinical features: jaundice, liver damage, cataracts, neural
damage, vomiting, diarrhea, E.coli sepsis
2. Tx: dietary restriction of galactose (best if within first 2
years of life)
3. GALT is needed to convert galactose to glucose
LYSOSOMAL STORAGE DISEASES
1. Typically fatal; carriers may be at risk of other diseases
(Gaucher and Parkinson, Niemann Pick and Alzheimer)
2. Autosomal recessive
3. Commonly have hepatosplenomegaly and CNS
involvement neural damage
4. Cell dysfunction result of cascade of secondary events
5. Treatment approach: molecular chaperone therapy
help decrease misfolded proteins

Tay-Sachs Disease (GM2 Gangliosidosis): Deficiency in

Hexosaminidase A)
1. Gangliosidoses: accumulation of gangliosides esp in the
brain deficiency in an enzyme for glycolipid catabolism
 2. Tay-Sachs is most common of the gangliosidoses; most common in Ashkenazi Jews
3. Ganglioside accumulation in neurons and central/autonomic nervous system and retina dominates clinical signs
4. Molecular basis of neuronal injury not fully understood mutant protein is misfolded and induces many responses
5. Treatments: molecular chaperone therapy

Niemann-Pick Diseases (Type A and B) primary deficiency of acid sphingomyelinase  Sphingomyelin accumulation
1. Common Ashkenazi Jews
2. Type A sphingomyelinase deficiency leading to sphingomyelin accumulation in phagocytic cells in neurons
Affects spleen, liver, bone marrow, lymph nodes, lungs b/c they have more phagocytic cells
Neurons become enlarged and vacuolated with lipids
3. Type B mutant sphingomyelinase with some function
Has organomegaly but no neurologic manifestations
Niemann Pick Disease Type C: mutation in genes NPC1 and ; lipid transport defect
1. More common than A and B
2. Cells accumulate with cholesterol and gangliosides (GM1 and 2)
3. Clinically heterogeneous gaze palsy, ataxia, psychomotor regression
Gaucher Disease: Glucocerebrosidase deficiency accumulation of glucocerebrocide in macrophages
1. Macrophages with glucocerebrocide accumulation Gaucher cells that look like “wrinkled tissue paper”
2. Leads to more macrophage activation and secretion of macrophage derived cytokines
3. Forms of Gaucher Disease
Type 1: Chronic nonneuronopathic: 99%--> has bone involvement usually, hepatosplenomegaly, no CNS involved; good prognosis
Type 2 and 3 have neurologic signs and symptoms (infancy type 2; later, more mile type 3)
4. Increased risk of Parkinson’s due to relationship with levels of alpha synuclein
5. Therapies enzyme replacement for Type 1 , substrate reduction therapy via inhibitor of glucocerebrosidase synthase
Mucopolysaccharidoses (MPS) defective degradation of mucopolysaccharides
1. Mucopolysaccharides are part of ECM
2. Examples of mucopolysaccharides Dermatan sulfate, heparan sulfate, keratan sulfate, chondroitin sulfate
3. Common features: hepatosplenomegaly, skeletal deformities, heart valve lesion, subendothelial arterial deposit, brain lesions
4. Myocardial ischemia most common link to deaths
5. Autosomal recessive (except Hunter which is X-linked recessive)
6. Hurler Syndrome=MPS Type 1 deficiency in a-L-iduronidase leads to accumulation in dermatin and heparan sulfates in fibroblasts vascular wall
endothelium and smooth muscle
7. Hunter Syndrome=MPS Type 2 x-linked recessive and milder than type 1; accumulation of heparan and dermatan sulfate from deficiency in L-
iduronate sulfatase

Glycogen Storage Diseases

General Aspects
1. Glycogen often accumulates in
cytoplasm or nucleus
2. Most are autosomal recessive (except
Pompe disease where accumulation is
in lysosome)
3. Categories=Hepatic, Myopathic,
Miscellaneous

Hepatic
1. Issue in hepatic enzymes for glycogen
metabolism
2. Signs: Liver enlargement, hypoglycemia
3. Von Gierke lack of G6P phosphatase

Myopathic
1. In striated muscle
2. Signs: muscle cramps after
exercise, myoglobinuria, don’t
make lactate because block in
glycolysis
3. McArdle Disease muscle
phosphorylase

Pompe lysosomal acid maltase

deficient
1. Causes deposition of
glycogen in almost every organ (heart most serious)
Complex Multigenic Disorders
1. Definition=multifactorial disorders caused by gene interactions and environmental factors
2. Common disease common variant hypothesis theory that these diseases arise when multiple polymorphisms that are mild by themselves are
co-inherited to become more serious/more physically obvious
3. Example= Type 1 Diabetes has 20-30 implicated genes, hair and eye color
4. Expression of complex traits is influenced heavily by environment Ex: Type 2 diabetes
5. These disorders don’t follow mendelian characteristics of disease transmission
Cytogenic Disorders Chromosomal abnormalities (numeric, structural)
Numeric Abnormalities
1. Polyploid: more than a pair for chromosome (normally diploid)
2. Aneuploid: abnormal number of chromosomes in cell most commonly caused by nondisjunction at first meiotic division or failure of sister
chromatids to separate during second meiotic division or mitosis in somatic cells
3. Monosomy involving autosome is incompatible with life, trisomies of some may be compatible
4. May result in mosaicism presence of multiple populations of cells with different complements of chromosomes in same individual (more
common in sex chromosomes)
Structural Abnormalities
1. Often leads to chromosomal breakage with loss/rearranged material
2. Designate chromosome arms using p (short) and q (long)
3. Translocation: transfer part of one chromosome to another (often reciprocal)
Example: 46,XX,t(2;5)(q31;p14)= long arm (q) of chromosome 2 at reg 3,
band 1, and the short arm of chromosome 5, reg 1, band 4
No harm if balanced (don’t lose fragments in process)
4. Robertsonian/Centric Fusion type Translocation: break at/near centromere
leading to one large chromosome and one very small leading to loss of the
short fragments
5. Isochromosomes: when centromere divides horizontally instead of vertically
causing loss of one of the arms
6. Deletion
7. Inversion
8. Ring Chromosome arms form ring

General Features of Chromosomal Disorders

1. Loss usually more serious than gain of chromosome
2. Often from de novo changes (exception is translocation form of Down syndrome)

Cytogenic Disorders Involving Autosomes: Trisomy 21, 18, 13, and Cri du chat
Trisomy 21 (Down syndrome)
1. Most common chromosomal disorder
2. Most common cause=meiotic nondisjunction (usually maternal origin)
3. Other types= Translocation (Robertsonian) which has familial link
and mosaic from mitotic nondisjunction which has milder clinical
manifestation
3. Clinical features: risk of congenital heart disease, atresias of
esophagus, leukemia risk, Alzheimer link, abnormal immune response

22q11.2 Deletion Syndrome (spectrum of disorders)

1. Clinical features: congenital heart disease, palate abnormalities, facial
dysmorphism, dev delay, thymic hypoplasia T cell decrease,
Parathyroid hypoplasia hypocalcemia
2. Increased Schizophrenia risk
3. Suspected to involved impaired expression of TBX1 trans factor gene
4. Disorders: DiGeorge Syndrome and Velocardiofacial Syndrome
5. Digeorge Syndrome
-Have T cell immunodeficiency and hypocalcemia as dominant
features
4. Velocardiofacial Syndrome
-mild immunodeficiency but pronounced dysmorphology and
cardiac effect
Cytogenic Disorders Involving Sex Chromosomes
Reasons these have better outcomes than autosomal issues (especially for female)
1. Lyonization of X chromosomes (X-inactivation) inactivates all but one X chromosome
2. Small amount of genetic info in Y so extra Y can be well tolerated
Klinefelter Syndrome: XXY
1. From nondisjunction meiotic error in either parent
2. Mosaic pattern possible and associated with milder condition
3. Range of clinical manifestations: hypogonadism, elongated body appearance, reduced facial/body
hair, low testosterone, sterile
Turner Syndrome: X
1. Features: primary hypogonadism, short stature, swelled neck, hypothyroidism
2. Partial or complete monosomy of short arm of X-chromosome (complete leads to more serious
symptoms)
3. Most common cause of early death=cardiovascular abnormalities
4. Fail to develop normal secondary sex characteristics
5. Normal mental status
6. SHOX (short stature homeobox) involved
7. 43% are mosaics or have structural abnormalities in X making partial monosomy
Most common deletion leads to formation of isochromosome of long arm

Single Gene Disorders with Atypical Patterns of Inheritance

General types: triplet repeat mutation, mitochondrial, imprinting

Triplet Repeat Mutations=repeating sequences of 3 nucleotides

Fragile X Syndrome (CGG repeat): FMR1 gene silence FMRP (familial mental retardation protein)
1. Group of many diseases
2. Second most common cause of mental retardation
3. CCG repeat over 230 leads to abnormal methylation
4. Shared features: neurodegenerative (severity depends on length of repeated region), large ear and mandible, long face, macroorchidism
5. Number of repeats can amplify from generation to generation leading to worse clinical outcome
6. Unique aspects of pedigree patterns: related to dynamic nature of mutation (oogenesis can amplify repeat)
Approx. 20% males that are carriers don’t have neuro symptoms or phys characteristics
Affected females 30-50% carriers show some mild cog impairment
Anticipation symptoms worsen w/ each generation
Fragile X Tremor/Ataxia
1. From CGG permutation on FMR1 gene that leads to toxic gain of function
2. Intention tremors and cerebellar ataxia that can progress to parkinsonism
3. Permutation that causes more transcription instead of silencing of the gene and FMR1 accumulation in nucleus and form inclusions
Additional notes on trinucleotide repeat disorders
1. Not all have expansion during oogenesis Huntington expansion of repeats is during spermatogenesis
2. Expansion can involve any part of gene Fragile X is in a untranslated region while Huntington is in a translated region
Untranslated region usually leads to loss of function
Translated region usually leads to misfolded protein
Mitochondrial Gene Mutations
1. Maternal inheritance
2. Generally encodes enzymes associated with oxidative phosphorylation mainly effect on skeletal muscle, CNS, cardiac, liver, kidney that depend
on oxidative phosphorylation
Diseases Cuased by Alterations of Imprinted Regtions: Prader-Willi and Angelman
1. Functional differences between maternal and paternal copies of same genes via genomic imprinting where certain genes are “inactivated”
differently during paternal and maternal gametogenesis; generally via methylation of promotor, histone binding, etc
2. Maternal imprinting transcriptional silencing on maternal allele
3. Paternal imprinting inactivation of paternal allele
Prader Willi: deletion of band q12 in long arm of chromosome 15; PATERNAL
1. Mental retardation, short stature, hypotonia, obesity, hypogonadism
Angelman Syndrome “happy puppet”: q12 on chromosome 15 too: MATERNAL
1. Mental retardation, ataxic gait, seizure, inappropriate laughter
2. UBE3A is imprinted in paternal  make Ub ligase (rely on maternal usually) this is associated with mental retardation in Angelman
NOTE: Prader willi and angelmen have different features showing there is difference between maternal and paternal imprinting effects
Mechanism: when one is missing (Example, deletion in maternal band), there will be expression of the Angelman gene in the paternal chromosome
Molecular diagnosis of Mendelian and Complex Disorders (p 291-296) Ch. 7

New Tools to Aid in Molecular diagnostics

1. Sequencing of human genome
2. “off the shelf” PCR kits
3. Gene chips high resolution microarrays that can analyze DNA and RNA in genomewide scale
4. NextGen: sequence technology

Indications for Genetic Analysis

1. Inherited diseases can test pre or post natally cytogenetics, FISH, molecular diagnostics
2. Newer technique maternal blood using NextGen sequencing

Molecular Diagnosis of Copy Number Abnormalities

1. Karyotype analysis of chromosomes by G banding=classic approach to analysis at chromosomal level (low resolution though)
2. FISH=focused analysis of chromosomal regions
Uses fluorescent dyed DNA probes to recognize seq specific to chromosomal regions
Can use on metaphase or interphase nuclei more rapid diagnosis
Can see microdeletions, numeric abnormalities that karyotyping and gene amplification can’t
3. CCH=comparative genomic hybridization; global genomic approach
4. Array Based Genomic Hybridization
aCGH=array comparative genomic hybridization=test DNA with normal reference DNA labeled with dif dyes coloring will be different
in hybrid if there is a genetic abnormality
SNP chips use a similar technique can use to provide zygosity (if someone has uniparental diosomy)
5. PCR Analysis
Ways to analyze PCR products: Sanger is common gold standard
Sanger sequencing: mix amplified DNA with DNA poly, primer, and di-deoxynucleotides and use fragments to determine seq
Use different fluorescently labeled nucleotides to add to PCR mixture more sensitive than Sanger for identifying mutation
Droplet Digital PCR: ultrasensitive variant of the fluorescent method
6. Next Generation Sequencing
Low cost way of genetic analysis with a high volume of sequencing data
Can sequence multiple fragments of human genome in parallel
Fluorescently labeled nucleotides are incorporated that are complementary to DNA template to map

Linkage Analysis and Genomewide Association Studies

1. Analyzing disease associated genes for genetic disorders that can’t be identified via direct genetic diagnosis often b/c multifactorial
2. Approaches use surrogate markers in genome (marker loci) are used to localize chromosomal regions of interest
3. Marker loci are naturally occurring polymorphisms (SNPs usually)
4. Use markers for “gene hunting”
5. Divide human genome into haplotypes: contain varying numbers of contiguous SNPs on same chromosome that are inherited as cluster

Linkage Analysis
1. Assess shared marker loci in family members with disease/trait of interest assuming SNP/linkage disequilibrium is transmitted via pedigree
2. Help with cloning and localization of disease allele
3. Useful for mendelian disorders related to one gene w/ profound effect/high penetrance

Genomewide Association Studies (GWASs)

1. For polygenic diseases which can’t be analyzed well with Linkage analysis
2. Use cohorts of patients with and without disease (not families) to examine across entire genome for variant SNPs overrepresented in people with
the disease
3. Variants are identified as “candidate genes” and assessed based on how tightly they’re associated with disease
Genetic Basis of Disease Susceptibility Ch 5 (p. 124-128, 145-148, 150-154) Ch 15 (621-623) Ch 20 (774-776)
Ch. 5: Cells and Tissues of the Immune System
Lymphocytes
1. Each T or B lymphocyte expresses a single antigen receptor; diversity via lymphocyte maturation
2. Antibodies=antigen receptors expressed on B-cells
3. T-cell receptors are the counterpart to the antibodies
4. Clonal selection: when foreign antigens bind to and activate lymphocytes
5. Naïve lymphocytes: express antigen receptors but haven’t reacted to an antigen yet
6. Effector Lymphocytes: induced via lymphocyte activation and function to eliminate microbes
7. Memory lymphocytes: also induced via activation but are functionally silent to respond rapidly in the future
T-Lymphocytes
1. Help B-cells make antibodies
2. Location: splenic periarteriolar sheath and lymph node interfollicular zone, circulation
3. Receptor complex: TCR, CD3 complex (y, d, e proteins) and 2 ζ Chains TCR binds antigens, CD3 help with signal activation
4. Other molecules expressed on T-cells
CD4: bind invariant portion of MHC 2 molecules Helper T cells which secrete cytokines to help B cells produce Abs and macrophages
CD8: binding to invariant portion of MHC 1 molecules cytotoxic T lymphocyte (CTL) which function more for directly killing virus
infected cell
Co-stimulators: other invariant proteins on T-cells
CD28: receptor for costimulatory molecules
Costimulators are induced on APCs by microbes and adhesion molecules to strengthen bond between T-cell and APCs
Treg: suppresses the immune response
5. Most T cells have TCRs composed of a and b chains, and a minority have yd chains
The yd TCR T-cells are mainly in mucosal surfaces and don’t express CD4 or 8 and recognize non-protein molecules (don’t need MHC)
6. NKT Cells: small population of T cells that express marker for T and NK cells recognize microbial glycolipids (Don’t need MHC)

MHC Molecules: Major Histocompatibility Complex Molecules

1. MHC restriction: in each person, a person’s T-cells only recognize peptides displayed by their own MHC molecules
2. HLA= human leukocyte antigen (human MHC) on a cluster of genes on chromosome 6
3. Class 1 MHC: express on loci HLA-A, B, C
Composition: polymorphic a chain noncovalently associated with invariant B2 microglobulin peptide
Antigen binding on extracellular portion of alpha chain with polymorphic residues and a conserved region to bind CD8+ T cells
4. Class 2 MHC: express on loci HLA-DP, DQ, DR
Composition: heterodimer of non-covalently linked a and b subunits
Expressed on APCs (dendritic, macrophage, B cells)
Antigen binding: extracellular portion has cleft for antigen and CD4 binding
5. Other proteins encoded on MHC locus: C2, C3 and Factor B for complement system and TNF and lymphotoxin cytokines
6. Each person expresses only one set of HLA genes polymorphism for great diversity
7. HLA haplotype: refers to each set of maternal and paternal HLA genes (inheritance “en bloc” that act like single locus)
Important to match when doing transplantation
Only identical twins can accept grafts without fear of transplantation rejection
Minor histocompatibility loci lead to risk of matched sibling donors still causing transplant rejection
B-Lymphocytes (Bone-marrow derived lymphocytes)
1. B-cell= mediator of humoral immunity and cells that produce antibodies
2. Recognize antigen via membrane-bound antibody of IgM class expressed on surface with signal molecules to form BCR complex
3. Can respond to soluble and cell associated proteins, lipids, polysaccharides, etc
4. Associated Molecules
CD21: complement receptor (AKA CR2) to recognize complement breakdown products which promotes B-cell activation
EBV: utilizes CD21 for infecting B cells
5. B cells can differentiate to plasma cells to release antibodies IgG, M, A (more than 95% circulating antibodies), IgE, D

NK Cells and Innate Lymphoid Cells

1. NK cells: arise from same lymphoid progenitor that gives rise to T and B cells but work for innate immunity don’t need activation
2. NK cells only have 2 receptor types: inhibitory and activating (basal inhibitory state)
Inhibitory recognize self MHC 1
Activating recognize molecules expresses/upregulated during stress (infection related to downreg of MHC 1)
Secretes: IFN-y to activate macrophages
3. Innate Lymphoid Cells lymphocytes that lack TCR but make cytokines similar to T-cells
Th1 IFN-y, TH2 IL-5, TH17 IL-17
Antigen Presenting Cells
1. Dendritic Cells capture antigens; called Langerhans cells in the epidermis, express many receptor to capture microbes (TLR, C lectin)
 Express high levels of MHC/other molecules needed for antigen presentation/activation of T cells
2. Plasmacytoid DCs subset of DCs that resemble plasma cells major sources of anti-viral cytokine Type 1 interferon
3. Follicular dendritic cells: in germinal center contain Fc receptors for IgG and receptors for C3b to trap antigen/complement and promote
antibody responses (don’t display antigen to T-cells)
4. Other APCs: macrophages, B-cells present to helper T

Ch. 5 Autoimmune Diseases (p. 145-154)

Signs of Autoimmune Disease
1. High affinity autoantibodies detected
2. Activation of pathogenic self-reactive T-cells
3. Exact etiologies are difficult to determine
4. Variations: organ-specific, systemic (lesions primarily affect connect tissue, RBCs, blood vessels collagen vascular diseases even though immune
reactions are not directed against connective tissue/blood vessels
5. Autoimmunity= failure of self tolerance

Immunologic Tolerance
1. Immunologic Tolerance: state of unresponsiveness to antigen via exposure of specific lymphocytes to the antigen
2. Self-tolerance: lack of immune response to one’s own tissue antigens
3. Mechanisms for selecting against self-reactivity=central tolerance and peripheral tolerance

Central Tolerance
1. Principle Mechanism: antigen-induced death of self-reactive T-lymphocytes and b-lymphocytes during maturation
2. Thymus present self protein antigens via thymic APCs
AIRE Protein: autoimmune regulatorstimulates expression of some peripheral tissue-restricted self antigens in thymus
Autoimmune polyendocrinopathy: associated with mutations in AIRE gene
3. Bone marrowB cells with high affinity either are killed via apoptosis or go through receptor editing

Peripheral Tolerance
1. Mechanisms
Anergy: functional inactivation (not death) of lymphocytes (can be caused by lack or co-stim signal or exposure to an inhibitory co-
signal in addition to APC presentation
Suppression by regulatory T-cells
Treg function to prevent immune reactions against self antigens
Treg cells can be can be induced in peripheral lymphoid tissues
Treg CD4+ cells with high levels of CD25, a chain of IL-2 receptor and FOXP3 (NEED FOXP3)
IPEX: Immune dysregulation , polyendocrinopathy, enteropathy, X-linked
Deletion by Apoptosis
Evidence that T-cells that recognize self antigens upregulate Bim (in Bcl-2 fam of pro-apoptotic family)
Fas and FasL binding  mutations are associated with Autoimmune lymphoproliferative syndrome (ALPS)
Some self antigens are sequestered from immune system (don’t communicate with blood/lymph)  immune privileged sites
Believed to be the case for antigens in testis, eye, brain

Mechanisms of Autoimmunity: General Principles

Common theory autoimmunity occurs via breakdown of self-tolerance in combo with susceptibility genes affecting lymphocyte tolerance and
environmental factors (infections, tissue injury, etc); overall alteration in how self antigens are displayed

Genetic factors in autoimmunity

1. Most are multigenic and tend to run in families
2. Links to HLA locus, especially for class II alleles HLA-DR, HLA-DQ (mechanism of link still unclear)  leads to increased relative risk
3. Associated diseases: Rheumatoid Arthritis, Type I Diabetes, MS, SLE, Ankylosing spondylitis, Celiac
4. GWAS: Genome wide association studies: identifying polymorphisms associated with dif diseases

Environmental Factors
1. Microbes can induce autoimmune reactions via different mechanisms
a. Microbial infections causing tissue necrosis/inflammation can stim expression of co-stim molecules on APCs to favor T-cell
tolerance (instead of anergy) leading to T-cell activation
b. Viruses and other microbes may share cross-reacting epitopes with self antigens to create responses to self tissues example of
molecular mimicry
c. Example: Rheumatic Heart Disease: antibody response streptococci cross reacting with cardiac antigens to cause rxn to cardiac
tissues
d. Some evidence that gut and skin microbiome may contribute to autoimmunity risk by affecting relative proportion of effector and
regulatory T-cells (still unclear)
2. NOTE: Microbe effect might not all be to increase risk of autoimmunity
a. Crohn, MS, Type 1 diabetes: there is evidence that infections paradoxically protect individuals from these autoimmune diseases
3. Ultraviolet Radiation causes cell death and may lead to exposure of nuclear antigens that elicit pathologic responses seen in lupus may be a
reason why lupus flare increases in risk with sunlight exposure
4. Smoking risk factor for rheumatoid arthritis possibly via chemical modification of self-antigens
 5. Gender bias: more common in women; may have hormonal link
6. Tissue injuries: may lead to exposure of concealed self antigen epitopes epitope spreading

Autoimmunity application to disease: Systemic Lupus Erythematosus (SLE)

1. Main antibodies: antinuclear antibodies (ANAs0
2. Injury mechanism=deposition of immune complex and antibody binding to various cells and tissues effect on basically every organ
3. Heterogeneous disease and can present with variety of symptom combinations; often presents during 20s or 30s and more in women

Spectrum of Autoantibodies in SLE HALLMARK of SLE is autoantibody production

1. Different autoantibodies are directed against different cell components nuclear, cytoplasmic, cell surface, etc
2. Immune complex mediated glomerulonephritis commonly seen in SLE, highly associated with autoantibody production
3. Anti-Nuclear Antibodies (ANAs)
a. Generally group via what they target: DNA, histones, non-histone proteins bound to RNA, nucleolar antigens
b. Detection indirect immunofluorescence for detecting “generic ANAs”; may use fluorescence pattern to determine type
c. Fluorescence patterns
Homogeneous/diffuse: antibodies to chromatin, histones, dsDNA
Rim/peripheral stain: usually dsDNA, sometimes nuclear envelope
Speckled: commonly seen and pretty non-specific; abs to non-DNA
nuclear constituents
Nucleolar pattern: abs for RNA commonly seen in systemic
sclerosis
4. Other detection: now have quantitative assays for abs against dsDNA and Smith (Sm)
antigen  used to diagnose SLE

Other Autoantibodies
1. Other autoantibodies seen in SLE Ab against blood cells, anti-phospholipid, against different plasma proteins, abs against phospholipid B-
glycoprotein (may lead to accidental syphilis diagnosis)
2. Phospholipid binding may increase the partial thromboplastin time b/c the test require phospholipids  “lupus anti-coagulant”
3. Clotting application: anti-coag results in VITRO, but in patients actually have a hypercoagulable state

Pathogenesis of SLE
1. Fundamental defect= failure of mechanisms that maintain self-tolerance
2. Genetic Factors: association with HLA-DR2 and HLA-DR3, deficiency in classical path (C1q, C2, C4), polymorphism of inhibitory Fc receptor
3. Environmental Factors: UV light exposure, association with X chromosome (gender bias), certain drugs (hydralazine, procainamide, D-
penicillamine)
4. Immunologic Factors:
a. Defective elimination of self-reactive B-cells from bone marrow
b. Escape of CD4+ cells specific for nucleosomal antigens patients have increased helper T-cells in follicles
c. Abnormally large amounts of IFN-a
d. TLR9 (recognize DNA) and TLR7 (recognize RNA) produce signals to activate B cells specific for self nuclear antigens
e. Other cytokines leading to B-cell activation: BAFF (promote B-cell survival), etc
f. Generally a cycle of antigen release and immune activation leading to production of high affinity autoantibodies

Mechanisms of Tissue Injury

1. Most of the systemic lesions are caused by immune complex (involve the ANAs) Type III hypersensitivity; mainly glomeruli + small blood
vessels
2. Type II Hypersensitivity autoantibodies specific for RBCs, WBCs, platelets; ANAs can’t penetrate cell so only act if nuclear material exposed
when nuclei of damaged cells react with ANA and lose chromatin pattern, become “LE bodies” aka hematoxylin bodies
3. LE cells: phagocytes (neutrophil or macrophage) that has engulfed denatured nucleus of injured cell; presentation of LE cells was used as
diagnostic test for SLE (older testing method)
4. Anti-phospholipid antibody syndrome can lead to venous and arterial thrombosis, ischemia; clinical features associated with lupus may be
referred to as secondary anti-phospholipid antibody syndrome
5. Neuropsych manifestations: attributed to antibodies that cross the blood brain barrier
Most common morphological signs for SLE
1. Blood vessels necrotizing vasculitis, fibrosis
 2. Kidney highly involved (50% patients), many complications asso with deposition of immune complex in glomeruli 6 associated classes of
glomerular disease is associated with SLE (most common in SLE is class 4 Diffuse lupus nephritis)

Inflammatory Bowel Disease (IBD) p. 621-623

IBD= chronic condition due to complex interaction between intestinal microbiota and host immunity (genetic predisposition) leading inappropriate
mucosal immune activation comes in form of Crohn disease and ulcerative colitis

Crohn Disease: aka regional enteritis can involve any area of the GI tract and is often transmural (across entire wall of organ/vessel) ; often has ileal
involvement

Ulcerative Colitis: limited to the colon and rectum and only extends to the mucosa and submucosa

Epidemiology
1. Usually present in adolescence/young adults
2. Common in whites, esp Ashkenazi Jews  more common in more developed countries (hygiene hypothesis)

Pathogenesis
1. Combined effect/alteration of host interaction with intestinal microbiota, intestinal epithelial dysfunction, aberrant mucosal immune response,
altered composition of gut microbio
2. Genetics: NOD2=susceptibility gene encodes protein that binds to intracellular bacterial peptidoglycans and activates NFKB leading to more
bacterial entering intestinal
3. Other associated polymorphisms: ATG16L1 (autophagy related), IRGM in the autophagosome pathway
4. Mucosal immune response connection one therapies involve immunosuppressive and immunomodulating agents
Polarization of helper T cells to Th1
Th17 involvement hypothesized and IL-23 (involved in Th17 differentiation)
Agents that block Th17 and Th1 have been shown to have some benefit
Defects in Treg (esp the Il-10 producing subset)
5. Epithelial Defects, association with Paneth cell granules
6. Microbiota: probiotics may help people with IBD

Type 1 Diabetes: p.774-776

Type 1 Diabetes: autoimmune disease causing mainly islet destruction by immune effector cells reacting against beta-cell antigens (T-cell failure mainly)
1. Can occur at any age (mainly childhood) and patients require exogenous insulin to survive
2. Complications Ketoacidosis, coma
3. Disease manifests quickly, but is the result of a gradual chronic destruction of B-islet cells via autoimmunity over years  clinical manifestation
often when at least 90% of cells have been destroyed
4. Genetics: association with HLA-DR3 and 4, polymorphisms within CTLA4 and PTPN22 (normally inhibit T-cell responses) and genes that encode
insulin itself (may reduce expression in thymus leading to decreased elimination of T-cells self reactive to insulin gene
5. Environmental link: exposure to certain viruses (potentially via mimicry initiating triggers), microbiome
Main mechanism failure of T-cell self tolerance
1. May be due to combo of defective clonal deletion in thymus during education
2. Leads to autoantibody production against Beta cell antigens such as insulin and glutamic acid decarboxylase
3. Can have islets that show necrosis of beta cells and lymphatic infiltration

Type 2 Diabetes
1. Not an autoimmune issue
2. Characterized by decreased ability of peripheral tissues to response to insulin (Insulin resistance), Beta cell dysfunction causing inadequate insulin
secretion in the face of insulin resistance and hyperglycemia
3. Often presents with beta cell hyperfunction and hyperinsulinemia early on but later lose compensatory mechanism
4. GENETICS many susceptibility loci Diabetogenic genes (none of these are related to immune tolerance/regulation)
Consequences of Insulin Resistance
1. Fail to inhibit endogenous glucose production in liver leads to high glucose levels
2. Low glucose uptake and glycogen synthesis in skeletal muscle leading to high blood glucose
3. Fil to inhibit hormone sensitive lipase in adipose leading to excess circulating free fatty acids
Obesity Link
1. State of fat excess is associated with insulin resistance
2. Metabolic syndrome: associated with insulin resistance, glucose intolerance, hypertension, abnormal lipid profile high type 2 diabetes risk
3. Excess FFAs intracellular triglycerides are potent inhibitors of insulin signaling leads to acquired insulin resistance
4. Adipokines: adipose has endocrine function and releases hormones known collectively as adipokines; some promote hyperglycemia by increasing
insulin sensitivity some of these hormone levels decrease in obesity
 5. Inflammation: associated with insulin resistance and beta cell dysfunction; excess FFA in macrophages can form inflammasome that leads to
secretion of Il-1B that promotes inflammatory cytokines
B-cell Dysfunction
1. Essential component in development of overt diabetes
2. Function increases early in the disease as a compensatory measure but then can’t adapt to the long term demands and there is loss of the
compensatory hyperinsulinemic state to a state of insulin deficiency
3. Many implicated mechanism FFA excess, chronic hyperglycemia, amyloid, polymorphisms

Molecular Diagnosis of Mendelian and Complex Disorders p. 291-296

1. Factors that enable rapid extension of molecular diagnostics
a. Sequencing of human genome and available
database
b. “Off the shelf” PCR kits
c. Microarrays (gene chips) that can interrogate both
DNA and RNA on a genome wide scale
d. NextGen automated high throughput sequencing
technology
2. Rising areas of focus for molecular diagnostics cancer
screening/risk screening, identification of infectious agents

Indications for Genetic Analysis: Prenatal: performed via

amniocentesis, chorionic villus biopsy, liquid biopsies that compare
maternal blood with NextGen sequencing
1. Advanced maternal age (34+)
2. Confirmed carrier status for a balanced reciprocal
translocation, Robertsonian translocation, or inversion
3. Fetal abnormalities observed on ultrasound or abnormal
result on routine maternal blood screening
4. Chromosomal abnormality or mendelian disorder affecting a
previous child
5. Determination of fetal sex when parent or partner is
confirmed carrier of an X-linked genetic disorder

Indications for Genetic Analysis: Postnatal: often via peripheral blood

lymphocytes
1. Multiple congenital anomalies
2. Suspicion of a metabolic syndrome
3. Unexplained mental retardation and/or developmental delay
4. Suspected aneuploidy or other syndrome of chromosomal
abnormality
5. Suspected monogenic disease

Molecular Diagnosis of Copy Number Abnormalities

1. Karyotype analysis of chromosomes via G banding= classic
approach to identify changes at chromosomal level
2. FISH- Fluorescence in Situ Hybridization
a. Uses DNA probes that recognize sequences specific to chromosomal
regions of over 100 kilobases
b. Probes are labeled with fluorescent dyes and hybridize to its
complementary sequence on the chromosome to label specific regions of
the chromosome that can be visualized on fluorescence microscope
c. Making the probe requires knowledge of one or a few specific
chromosomal regions suspected of being altered in the sample
d. Analysis can be done on prenatal samples, peripheral blood lymphocytes,
and archival tissue sections
3. Array based Genomic Hybridization
a. Addresses the setback in FISH due to the requirement of knowing the
specific chromosomal regions thought to be abnormal
b. Performs a global genomic survery
c. CGH= comparative genomic hybridization labels DNA with 2 different
fluorescent dyes (red and green) which are cohybridized to array with DNA
probes to compare to the sample
d. Result interpretation: yellow= sample is identical to DNA probes; red or
green= there is either a deletion or expansion in the sample
 e. SNP Chips: Single nucleotide polymorphism probes are designed to identify SNP sites genomewide; can be used to follow genetic
markers being passed from parent to child; can provide zygosity data preferred method over CGH

Direct Detection of DNA Mutations by PCR

1. PCR  involves exponential amplificiation of DNA
2. Sanger sequencing amplified DNA is mixed with a DNA polymerase, a DNA primer, nucleotides, and 4 dead end nucleotides (A, T, C, G)
a. Size separation detected on electrophoresis used to determine the DNA sequence of sample
3. Adding fluorescently labeled nucleotides to a PCR mixture can be used to identify mutations at a specific nucleotide position  more sensitive
than Sanger method

Next Generation Sequencing (NextGen)

1. Cost efficient
2. Sequence multiple fragments of human
genome in parallel
3. Incorporate fluorescently labeled nucleotides
complementary to DNA template strands to
map findings
4. Can detect variant alleles that occur at low
frequency in samples containing heterogenous
mix of cells  higher detection than other
methods like Sanger
5. Can measure DNA, transcriptome, and
genomewide binding sites for transcription
factors or histones

Linkage Analysis and Genomewide Association Studies

1. Use surrogate markers in genome (marker loci)
to localize chromosomal regions of interest
2. Marker loci naturally occurring variations in
DNA sequences (polymorphisms)
3. Used to “Gene hunt” in cases where the genetic
disorder’s causal gene has not yet been identified or if there is a multifactorial cause involving multiple genes
4. Divides genome into haplotypes to analyze SNPs in groups instead of looking for every single SNP
5. Linkage analysis assesses shared marker loci in family members exhibiting disease to define the “disease haplotype”
6. Genomewide Association Studies addresses fact that linkage analysis can’t assess disease (like HTN, diabetes, asthma, etc) that involve multiple
genetic loci; GWAS uses large cohorts of patients with and without disease and examines entire genome and identify SNPs overrepresented in
those that have disease that may be used to determine genetic susceptibility to a disease
Lecture Notes: Prenatal Diagnosis and Genetic Counseling

Indications for Prenatal Diagnosis

1. 35+ maternal age at delivery
2. Previous affected pregnancy (prior aneuploidy)
3. Family history/if either parent is a known carrier, abnormal screenings/ultrasounds
Terminology
1. Screening determine if there is increased risk; not determining diagnosis
Screening Tools= ultrasound, nuchal translucency, serum markers, cell free DNA screening
2. Diagnostic testing goal to diagnose as accurately as possible
Tool: Ultrasounds
1. General uses= non-invasive method of determining gestational age,
number/location of fetus
2. Screening: indicate anatomic abnormalities
3. 2 Ultrasounds Generally performed
First: between 11-13 weeks to determine gestational age
Second: between 18-22 weeks to look at anatomical structures
4. Application: Trisomy 21 Features
Increased nuchal thickness, echogenic bowel and intracardiac focus, short
humerus/femur, cardiac defect, cystic hygroma, duodenal atresia
5. Can also use to visualize polydactyly, omphalocele
SCREENING TOOLS
Tool: Nuchal Translucency
1. Measure fluid beneath skin behind neck; measure between 11-13 weeks gestation
2. Use in combo with other prenatal blood tests to test for chromosomal
abnormalities Trisomy 13,18, 21
Tool: Maternal Serum Screening
1. AFP (alpha fetoprotein)
2. hCG (human chrorionic gonadotropin)
3. PaPP-A (pappalysin-1 or pregnancy associated plasma protein A
4. Unconjugated Estriol (E3)-Inhibin A-Fetal nuchal translucency

First Trimester Combined Screening (10-13 weeks)

1. Tests= nuchal translucency, blood samples between 10-13 weeks
2. Risk of chromosomal defect increased hCG, decreased PAPP-A, increased nuchal translucency
3. “Positive test” is considered risk of being over 1/300
4. Positive test predictability 79-90% detection of Trisomy 13,18,21 and 5% normal (False positive)

Second Trimester Maternal Serum Screen (15-20 weeks if there is moderate/high risk, don’t do if low risk via first screening)
1. Triple or quad screen of markers AFT, hCG, unconjugated estriol,
sometimes inhibin A
2. Determine risk in combo with maternal age, family history, weight,
race, diabetic status

AFP and Neural Tube Defects

1. Neural tube defects lead to opening in spine that allows for AFP to pass into the mother’s blood
Prenatal Cell-Free DNA screening
1. Examines the fetal DNA in maternal blood stream to determine risk of abnormalities Does not asses neural tube or abdominal wall defect risk
2. This is a screening test so if positive, would need to confirm with diagnostic testing
3. Has recently become a pretty standard procedure for screening
Diagnostic Tools
Overview:
1. Chorionic villus sampling (CVS)
2. Amniocentesis
3. PUBS (percutaneous umbilical blood sampling) take blood from umbilical cord
4. PGD (Pre-implantation genetic diagnosis) no longer widely used
 5. Possible sources of error: lab error, maternal cell contamination of CVS and amniotic fluid, mosaicism, poor cell sampling/cell growth
Tool: CVS and Amniocentesis
1. Directly samples fetal cells and uses direct DNA testing to analyze risk of disease
2. Limitation= can only be done after implantation of the embryo
3. CVS performed 10-12 weeks gestation via transcervical or transabdominal approaches; results in 2-4 weeks
Sample: chorionic villi from placenta contains same genetic material as fetus
Uses of Sample: chromosome analysis, DNA analysis
Limitations: complications, mosaicism (may not reflect condition of fetus could be result of sample containing multiple genetic lines),
can’t detect neural tube defects
NOTE: give Rhesus or Rh negative women anti-D immunoglobulin after procedure to prevent formation of Abs to Rh positive fetus
4. Amniocentesis performed 16-18 weeks using fine needle through abdominal wall into amniotic fluid
Fluid collected: cells that directly shed from fetal skin, lungs, bladder
Uses of Sample: chromosome, DNA, biochemical analysis
LIMITATIONS: complications, leakage of amniotic fluid from vagina, performed later in pregnancy so may be less time for management
NOTE: Rh neg women receive Anti-D Ig post procedure too to prevent production of Abs to Rh positive fetus this procedure will
cause blood cell exposure between mom and baby that could trigger immune response
Test: Pre-Implantation Genetic Diagnosis
1. Combined with in-vitro fertilization to offer patient possibility to implant an unaffected embryo
2. Used more by couples who have been found to be carriers of disease
3. Technique: uses single cell from 6-8 cell blastomere from IVF to analyze for genetic defects via cytogenic techniques/chromosome analysis or
direct DNA mutation analysis or chromosome microarray
4. LIMITATIONS: PCR may fail to amplify enough DNA for testing, sperm contamination, cost (not insurance covered), limited availability
Genetic Counseling
1. General indications: background, history, maternal age, patient
request
2. Process: initial intake, pedigree analysis (Back 3 generations),
research conditions, interpret results and communicate results,
recommendation for further studies if indicated, summary letter for
patient/physician and other involved specialists
3. Obstacles to counseling incomplete history, not enough tissue to
test, poor medical history records, paternity issues, adoption
4. Carrier screening test blood or saliva to determine if mutation
present more done for cystic fibrosis, fragile X, sickle cell, Tay-
Sachs

Newer Technologies:
1. Development of preconceptual DNA panels do preconceptually
and not prenatally
2. May do Targeted mutation analysis to focus on diseases associated
with ethnicity vs full DNA sequencing

GINA: Genetic Information Nondiscrimination Act

1. Prohibits genetic discrimination in health insurance
2. Prohibit discrimination by employers (doesn’t covers employers with less than 15 employees, military, people receiving VA health benefits or
Indian health services)
3. Does not cover life, disability, long term care insurance companies
Lecture Notes: An Evidence Based Approach to the Diagnosis of Child Abuse

General Categories of Analysis

1. Skin: pattern/location of injury, mechanism reported, age/development level, dating of bruises is not possible (depth/color/location), potential
diseases that could cause skin lesions
2. Skeletal: Fractures look at age/development level, mechanism reported, type and location, workup with skeletal survey; potential causes of
fracture susceptibility are systemic disease, nutritional issues, birth trauma, endocrine/genetic disorders; determine if it is due to abuse,
accidental trauma; rib fractures are generally not seen until healing process is happening; injuries near bony prominences are more likely to be
from accidental injuries

3. Head: think about mechanism reported, differential, additional indications of abuse in workup, timing of injury/past injury
4. Abdomen/Thorax
5. Genital/Anal
6. Behavior
7. Statements/Disclosures

Indicators of Sexual Abuse

1. Disclosure
2. Physical signs: genital bruising, anal bruising/bleeding, genital bleeding, STD/Pregnancy
3. Behavioral signs: behavior change, school performance change, feer/anxiety, somatization
4. Other causes of vaginal bleeding: tumor, urethral origin, infection, vascular abnormalities, bleeding disorders, estrogen, labial adhesion
5. Reason most exams may be normal despite trauma
Vulvar coitus, delayed disclosure so time for healing,
hymen and anus flexible, lubricants, hormonal factors
Reporting
1. Physically abused child
2. Sexually abused child includes adults who commit/allow
offense to occur
3. Neglected child
4. Mandated reporting required to report suspected child abuse
or maltreatment when, in professional capacity are presented
with reasonable cause to suspect abuse/maltreatment
Safe Infant Sleep:
1. A sleeping alone (no other people , blankets/toys/etc in crib)
2. B place on back (tummy not recommended)
3. C in a crib
Children Advocacy Center:
1. Children will be interviewed and examined for abuse to prevent making the child go to multiple places in these cases

SUID: Sudden Unexplained Infant Death