Acrocentric Chromosomes
Chromosomes whose centromeres are located near the end of the chromosome; stalks and satellites on the short arms contain repetitive DNA sequences that code for rRNA
Chromsomes 13, 14, 15, 21, and 22
Imprinting
Non-coding DNA sequences (imprinting centers) recruit DNA methyltransferase complexes that methylate CpG dinucleotides near the IC on that chromosome; imprinting is established and maintained in one of the two germ lines
Chromosomal Microarray (CMA)
Flourescently labeled sample DNA and control DNA are mixed and hybridized to an array; visualization of the array gives information about the spots/color intensities for each probe, which correspond to variation in degrees of expression
Advantages: allows investigation of the whole genome simultaneously, reveals duplications and deletions
Limitations: detects gains & losses ONLY, not balanced rearrangements or SNPs
Heritability
The proportion of total variance in a trait that is due to variation in genes
Allelic Heterogeneity
Different alleles in the same gene resulting in the same trait; OR, different alleles in the same gene resulting in different traits
Ex: Many mutations in the CFTR gene (alleles) lead to CF; different “classes” of alleles lead to different presentation of disease
Locus Heterogeneity
Variants in different genes result in similar clinical presentation
Ex: Early onset Alzheimer’s results from mutations in 3 separate genes, all of which contribute to accumulation of AB42
Loss of Function Mutations - 5 Examples
Duchenne Muscular Dystrophy (DMD gene, dystrophin)
Alpha Thalassemia (alpha thalassemia gene, a-globin)
Turner Syndrome (loss of X chromosome)
HNPP (PMP-22 gene, peripheral myelin protein)
OI Type I (Col1A1 gene, COL1A1 type collagen)
Gain of Function Mutations - 4 Examples
Hb Kempsey (B-globin gene)
Achondroplasia (FGFR3 gene)
Alzheimer Disease in Trisomy 21 (APP gene)
Charcot-Marie Tooth (PMP 22 gene)
Novel Property Mutations - 2 Examples
Sickle Cell Disease (B-globin)
Huntington Disease (CAG repeats/polyglutamine tracts lead to novel toxicity of protein)
Compound Heterozygote
An individual who carries two different mutant alleles of the same gene
Ex: HbS/HbC
Replicative Segregation
During cell division, the multiple copies of mtDNA in each of the mitochondria in a cell replicate and sort randomly among the newly synthesized mitochondria, which are distributed randomly between the two daughter cells
Daughter cells may be homoplasmic (a daughter cell with a pure population of WT mtDNA) or heteroplasmic (a daughter cell with a mixture of WT and mutant mtDNA)
SINES & LINES
Short Interspersed Nuclear Elements (SINES) - Ex: Alu Family; ~300bp repeats, 10% of genome
Long Interspersed Nuclear Elements (LINES) - Ex: L1 family; ~6kb repeats, 20% of genome
Alpha satellite repeats
171 bp repeat unit found near centromeric region of all human chromosomes; likely important for chromosome segregation in mitosis and meiosis
Candidate gene association study
Markers within a candidate gene are identified and allele frequences are compared in cases vs. controls; association implies linkage disequilibrium with a causal mutation
Disadvantages: Cases & controls must be ethnically matched, population stratification may lead to false positives
Microsatellites & Short Tendem Repeat Polymorphisms (STRPs)
Repetitive DNA sequences 2-4 nucleotides long; short tandem repeat polymorphisms (STRPs) are different alleles that result from variation in the number of repeat units within a microsatellite region
Mini-satellites & Variable Number Tandem Repeats
Repetitive DNA sequences 10-100 nucleotides long; variable number tandem repeats (VNTRs) are different alleles that result from variation of the number of repeat units within a minisatellite region
Copy Number Polymorphisms (CNPs)
Recurring deletions or insertions of larger section of a chromosome, leading to gaps or duplications; may give rise to two discreet alleles (due to presence or absence of the chromosomal region) or to multiple alleles (due to variation in copy number of the chromosomal region)
Genome-Wide Association Studies (GWAS)
Same as candidate gene case-control association study but tests MANY markers across the genome searching for significantly different allele frequencies in cases vs. controls
Advantages: Can identify many genes, some new, that contribute to disease
Disadvantages: Requires a larger sample size (>1,000) to correct for multiple testing problem
Chromosomal Analysis
Detects gross anomalies in chromosome number as well as large structural rearrangements (deletions, insertions, etc.)
FISH
Flourescence In-situ hybridization; flourescently labeled, locus-specific probe can identify micro-deletions or multiple copies of loci of interest in patient DNA
Detects chromosomal microdeletion, microduplication, chromosomal rearrangements, and gene copy numbers of a known loci of interest
Single Nucleotide Polymorphism (SNPs)
A difference in a single DNA nucleotide base, within a particular gene, that gives rise to 2 discreet alleles; SNPs occur at a rate of ~ 1 per 1,000 nucleotides between any two individuals
Genetic Linkage Studies
Used to study genome segments that are disproportionately co-inherited along with disease to determine if the loci are linked; “multiplex” families with multiple cases of a disease are studied to determine the frequency of recombination events between two loci
Viral approaches to gene therapy
- Retroviral; can accomodate up to 8kb of DNA but requires target cell division for integration of recombinant DNA into host genome (i.e. limited use in non-dividing cells)
- Adenoviral: can accomodate up to 35kb and infect dividing or non-dividing cell but may lead to strong immune response
Non-viral approahces to gene therapy
- Naked DNA (i.e. cDNA with regulatory elements introduced in a plasmic)
- DNA packaged into liposomes
- Protein-DNA conjugates
- Artificial chromosomes
Reciprocal Translocations
An exchange of segments between non-homologous chromosomes; meiosis procedes through formation of a quadrivalent figure by one of three mechanisms: alternate (normal/balanced gametes), adjacent 1 and adjacent 2 (partial trisomy/monosomy gametes)
Meiosis Nondisjunction I and II
Nondisjunction during Meiosis I produces a gamete with 24 chromosomes including both the paternal and maternal homologues
Nondisjunction during Meiosis II produces a gamete with 24 chromosomes including both copies of either the maternal or paternal chromatids
Mosaicism
Two or more chromosome complements are present within an individual; most often numerical, resulting from non-disjunction in an early postzygotic mitotic (somatic) division producing two different surviving cell lines
Robertsonian Translocation
Fusion of two acrocentric chromosomes near the centromere with loss of the short arms; may segregate via a trivalent structure along 3 pathways to produce normal/balanced gametes or partially trisomic/monosomic gametes
*Considered a balanced translocation because only repetitive DNA coding for rRNA is lost
Maternal Age Effect (Two Hit Model and Cohesin Model)
Two Hit Model: Diminished meiotic recombination caused by a lack of chiasmata or mislocated chiasmata, followed by faulty segregation
Cohesin Model: Degradation of cohesin complexes throughout the course of extended Meiosis I arrest in oocytes, allowing “terminalization” of chiasmata and premature separation of homologs and/or sister chromatids
Paracentric vs. Pericentric Inversions
Paracentric: Inversions of a region of a chromosome excluding the centromere; crossover may generate both dicentric and acentric chromosomes in gametes
Pericentric Inversions: Inversions of a region of a chromosome including the centromere; crossover may result in chromosomes with duplications & deletions in gametes
*Balanced Rearrangements
Paracentric vs. Pericentric Interstitial Deletion
Paracentric: Produces one chromosome with lost genetic material in the region of the deletion and one acentric fragment that is not transmissible
Pericentric: Produces two acentric fragments that are not transmissible and one stably transmissible centric ring (marker) chromosome
*Unbalanced rearrangements
Isochromosome
An acrocentric chromosome in which the short arm is missing and the long arm is duplicated; may result from exchange between one arm of a chromosome and it’s homolog or by misdivision through the centromere in Meiosis II
Uniparental disomy
Most often occurs as a result of trisomy followed by loss of the extra chromosome leaving either 2 maternal or 2 paternal homologs, where normal development is dependent on the expression of genes at the missing loci
Gametes of a reciprocal translocation carrier - Adjacent-1 pathway
Adjacent-1 pathway leads to the formation of two unbalanced gametes with segregated homologs; following fertilization, each gamete will be partially trisomic & partially monosomic for one of the chromosomes involved in the translocation
Gametes of a reciprocal translocation carrier - Adjacent-2 pathway
Adjacent-2 leads to the formation of two unbalanced gametes in which homologous centromeres are unsegregated; following fertilization, each gamete will be partially trisomic & partially monosomic for one of the chromosomes involved in the translocation
X inactivation
XIST gene is located within the X inactivation center on Xq and expressed only on the inactive X; XIST produces a non-coding RNA which associates in cis to signal DNA methylation and histone modification leading to expanded CpG methylation of the promoters of many (85-90%) genes on the inactive X chromosome
X inactivation is usually random, except for in the case of a damaged X chromosome (preferentially inactivated) or an X:autosomal translocation (preferentially remains active)
Mesonephric (Wolffian) ducts
Thickenings of the genital ridges that give rise to the epididymal duct and ductus deferens under the influence of androgens secreted by Leydig cells in the testes
Sertoli cells
In the embryonic testes, produce Mullerian Inhibitory Substance (MIS) hormone that suppresses formation of the paramesonephric ducts
SRY
Sex determining region of Y; lies near the pseudo-autosomal boundary of the Y chromosome and encodes a TF that is crucial for proper testes development
Paramesonephric (Mullerian) ducts
Thickenings of the genital ridges that give rise to the female duct system (fallopian tubes, uterus, and upper vagina) in the absence of male hormonal signals (MIS and testosterone)
DAX1
Located on Xp, encodes a TF that is dosage-sensitive in affecting gonadal sex; duplication of DAX1 suppresses the normal male-determining function of SRY and ovarian development results
Duplication of DAX1 can lead to XY sex reversal
SOX9
Located on 17, required for normal testes formation; in it’s absence, testes fail to form; duplicated copies lead to XX sex reversal (phenotypic males in the absence of SRY)
Deletions lead to camtomelic dysplasia, a disease of skeletal malformation also characterized by XY sex reversal (phenotypic females)
Androgen insensitivity syndrome
X-linked mutation of the AR gene which codes for the androgen receptor; individuals are XY but will present with phenotypically female genitalia; infants present with undescended testicles in the inguinal canal
Congenital Adrenal Hyperplasia
Caused by mutation in the 21-hydroxylase enzyme which normally functions in the synthesis of glucocorticoids; precursors accumulate and are shunted toward the testosterone synthesis pathway leading to in-utero testosterone exposure
46 XX individuals present with ambiguous/masculinized genitalia
Drug Metabolism - Phases I & II
Phase I - Attaches a polar group (usually a hydroxyl group) onto the compound to make it more soluble
Phase II - Attaches a sugar/acetyl group to detoxify the drug and make it easier to excrete
Cytochrome P450
Liver & intestinal enzymes that participate in the Phase I metabolism (inactivation) of 90% of all commonly used medications; CYP3A4 is particularly active
Ex: CYP3A (Cyclosporin), CYP2D6 (Tricyclics, codeine), CYP2C9 (Warfarin)
N-acetyltransferase (NAT) gene
NAT enzyme is important for Phase II metabolism; rate of acetylation is determined by polymorphism in the NAT genes
Thiopurine methyltransferase (TPMT) gene
TPMT enzyme is important in the metabolism of 6-mercaptopurine and 6-thioguanine; giving children with deficient TPMT enzyme activity standard doses of ALL drugs will kill them by hyperimmunosuppression
Vitamin K Epoxide Reductase Complex (VKORC1 gene)
VKORC1 protein reduces & recycles Vitamin K so that it can contribute to the carboxylation (activation) of clotting factors; deficient individuals are at greater risk for severe bleeds with anti-coagulants like Warfarin
Patau Syndrome
Trisomy 13; characterized by severe intellectual disabilities, failure of the embryonic forebrain to divide properly (holoprosencephaly), facial clefts, polydactyly, renal anomalies, congenital heart & urogenital defects
Edward’s Syndrome
Trisomy 18; characterized by receding jaw, low-set ears, intrauterine growth retardation, clenched fists, valvular heart disease, diaphragmatic hernia, renal anomalies
Klinefelter Syndrome
47 XXY as a result of errors in either maternal meiosis I or II, or paternal meiosis I; characterized by tall stature, hypogonadism, gynecomastia, sterility, and learning disabilities
Turner Syndrome
X0, most often resulting from loss of the paternal X chromosome; characterized by short stature, webbed neck, broad chest, ammenorrhea, renal & cardiovascular anomalies
Hemophilia A
X-linked disorder of coagulation caused by mutation in the F8 gene which codes for clotting Factor VIII; characterized by spontaneous bleeding into joints, muscles, intracranial bleeding, and excessive bruising
Hemophilia B
X-linked disorder of coagulation caused by mutation in the F9 gene which codes for clotting Factor IX
Hemophilia B-Leyden results from point mutations in the F9 promoter; affected males produce only 1% of the normal amount of Factor IX until puberty, when production increases to 60% normal
Chromosomal abnormalities underlying Down Syndrome
Trisomy 21, usually as a result of maternal nondisjunction (95%)
Robertsonian translocation between 21q and the long arm of one of the other acrocentric chromosomes; 46 chromosomes but effectively trisomic for genes on 21q (4%)
Isochromosome 21 resulting from a 21q21q translocation; all gametes are either monosomic (lethal) or trisomic (Down)
Mosaic - cell populations have both normal and Trisomy 21 karyotype (1-2%)
Neurofibromatosis Type I
Autosomal dominant condition resulting from loss of function mutation in the NF1 gene, which codes for neurofibromin tumor suppressor protein
Example of pleiotropy: characterized by growth of benign fleshy tumors (neurofibromas), cafe au lait spots, & freckling of the axillary & inguinal regions
XYY Syndrome
Results from paternal nondisjunction at meiosis II, producing YY sperm; generally indistinguishable from XY males except for slight learning disability
Trisomy X
XXX resulting from errors in maternal meiosis I; two of the X chromosomes are inactivated and so individual are phenotypically normal; may exhibit slight learning disability
Charcot Marie Tooth
Autosomal dominant disorder caused by a duplication of the PMP-22 gene on chromosome 17; characterized by muscle atrophy of the foot, lower leg, and hand muscles, hammertoe; causes peripheral nerve demyelinating neuropathy
Hereditary Neuropathy with Predisposition to Pressure Palsy (HNPP)
Autosomal dominant condition resulting from deletion of PMP-22 gene on 17; presents as focal pressure neuropathy
Prader Willi Syndrome
Caused by deletion of 15q11-13 on the paternal homolog (or by uniparental disomy of the maternal homolog); characterized by almond-shaped eyes, excessive & indiscriminate eating, obesity, short stature, strabismus & nystagmus, hypotonicity, and mild to moderate cognitive disability
Angelman Syndrome
Caused by deletion of 15q11-13 on the maternal homolog; characterized by short stature, spasticity, seizures, autism, and intellectual disability
Acute Promyelocytic Leukemia (PML)
Characterized by translocation involving the retionic acid receptor-alpha (RARA) gene on 17 and the promyelocytic leukemia (PML) gene on 15; PML-RARa fusion protein binds to the RAR element in the promoters of certain genes necessary for proper myeloid differentiation
Treatment: Differentiation therapy with retinoic acid (vitamin A)
Chronic Myeloid Leukemia (CML)
Characterized by translocation between the breakpoint cluster region (BCR) gene on 22 with the ABL gene on 9 - the “Philadelphia chromosome”; this translocation produces the BCR-ABL fusion protein, a constitutively active tyrosine kinase, leading to myeloproliferative disease
Treatment: Gleevac (a tyrosine kinase inhibitor)
Down Syndrome Phenotypic Features
Flattened facial features Upslanting palpebral fissures Epicanthal folds Small Ears Low muscle tone/ increased joint mobility Transverse Palmar Crease
Medical issues associated with Down Syndrome
Cardiac anomalies (30-50%), especially atrioventricular septum defects Esophageal/duodenal atresia Hirschprung Disease GERD Nystagmus Cataracts Chronic ear infections & nasal congestion Obstructive apnea Thyroid disease (hypothyroidism) Increased risk of leukemia Early onset Alzheimer's
Fragile X Syndrome
Caused by an expansion in the number (>200) of CGG repeats in the 5’ UTR of the FMR1 gene, causing excessive methylation of the promoter and failure to express the FMR1 protein; characterized by intellectual disability, dysmorphic features, and autistic behavior
Genetic anticipation through the maternal germ line
Fragile X associated tremor/ataxia syndrome (FXTAS): X linked pre-mutation triplet repeat expansion (59-200 CGG) in FMR1 gene; characterized by adult onset ataxia, tremor, memory loss, peripheral neuropathy
Acute Lymphoblastic Leukemia (ALL)
Caused by translocation between the TCF3 gene on 1 and the PBX1 gene on 19; Hyperdiploidy (>51 chromosomes) is associated with a good prognosis
Cystic Fibrosis
Autosomal recessive disease caused by mutation in the CFTR gene causing build up of thick mucus secretions in the lung and pancreas
Ex: of allelic heterogeneity: mild/mild or mild/severe alleles lead to 15% pancreatic insufficiency whereas severe/severe alleles lead to 85% insufficiency
Phenylketonuria (PKU)
Autosomal recessive condition resulting from defect in the phenylalanine hydroxylase (PAH) gene, a liver enzyme that catalyzes the breakdown of Phe; 1-3% have normal PAH but are defective in genes needed for the synthesis of BH4, a cofactor for PAH
PKU presents as microcephaly, seizure, tremor, gait disorders, and cognitive disability, as well as high plasma [Phe] and low plasma [Tyr]
Maternal PKU & PKU testing
Pregnant women with PKU have increased risk of miscarriage/congenital malformation, cognitive disability, and growth impairment (regardless of fetal genotype) due to elevated Phe levels in maternal circulation; these women should be maintained on a low-Phe diet
Bacterial growth in Guthrie test is diagnostic for PKU
alpha 1 antitrypsin deficiency (ATD)
Autosomal recessive disaese caused by mutation in the SERPINA1 gene, which codes for a serine protease inhibitor that targets elastase; if left uninhibited, elastase can destroy the connective tissue proteins in the lung, causing emphysema; misfolded protein also builds up in the liver, causing liver cirrhosis/carcinoma
Ex of allelic heterogeneity, with Z/Z genotype being the most severe and S/S genotype being the most mild
Tay Sach’s Disease
Autosomal recessive disorder caused by HEXA gene mutation, which codes for the alpha subunit of hexosaminidase A (HexA), a heterodimer of alpha and beta subunits; HexA is needed to metabolize GM2 ganglioside; GM2 accumulates in the lysosomes of CNS neurons causing neurodegeneration
Symptoms present around 3-6 months: muscle weakness, seizures, vision/hearing loss, diminishing mental function, and death by 3-4 years
Fetuses can be screened by amniocentesis in utero
A-B Variant of Tay Sachs
Rare form of T-S in which both HexA and HexB are notmal but GM2 accumulates due to a defect in the GM2 activator protein (GM2-AP) which facilitates interaction between the lipid substrate GM2 and the HexA enzyme
Sickle Cell Anemia (HbSS)
Autosomal recessive condition caused by a missense mutation in the B-globin gene resulting in a Glu –> Val substitution; HbS is 80% less soluble than HbA and polymerizes into long fibers that distort the RBC
HbS can be diagnosed by digestion with restriction enzyme MstII yielding only one 1.35kb fragment or by hemoglobin electrophoresis
Heterozygotes are phenotypically normal except under conditions of low pO2
Hemoglobin C Disease (HbC)
Autosomal recessive condition caused by a missense mtuation in the B-globin gene resulting in a Glu –> Lys mutation; HbC is less soluble than HbA and forms crystals that reduce the deformability of the RBC
a-Thalassemias
Anemic disorders caused by deletion of one or both copies of the a-globin gene on the a-cluster of chromsome 16
a-Thalassemia alleles
a-thal-1-allele is caused by deletion of both copies of a-globin genes; homozygotes (–/–) are stillborn whereas heterozygotes (aa/–) have a-thalassemia trait (mild anemia); allele common in SE Asia
a-thal-2 allele is caused by deletion of only one copy of a-globin gene in the cluster; homozygotes (a-/a-) have a-thalassemia trait and heterozygotes (aa/a-) are phenotypically normal; allele is common in Africa, Meditteranean, and Asia
HbH Disease
Caused by compound heterozygosity for a-thal-1 and a-thal-2 alleles (a-/–); individuals produce only 25% of the normal a-globin levels, resulting in severe anema; marrow compensates by producing higher levels of HbH (B4) which precipitates and may lead to hydrops fetalis (edema)
Categorizations of B-thalassemia
Thalassemia Major (Cooley’s Anemia): genotype (-/-) leading to severe anemia; patients are treated with transfusion & iron chelation
Thalassemia Intermedia: mild-moderate anemia caused by inheritance of 2 abnormal b-globin genes
Thalassemia minor: clinically insignificant anemia caused by genotype (b/-)
Simple B-thalassemia: only b-globin genes are affected
Complex B-thalassemia: b-globin genes AND other genes on the B-cluster are affected
B+ thalassemia: some B-globin is made and some HbA is present
B- thalassemia: No b-globin is made and no HbA is present; Hb present at <5% normal levels and is mostly a2y2 and a2d2 variants; mostly lethal
Hereditary Persistent Fetal Hemoglobin (HPFH)
Caused by deletions in the promoter region of the b-globin gene OR by large deletions in the B-cluster including d- and B-globin genes; 100% of Hb is HbF (a2y2) which is usually phenotypically normal
*Heterochronic mutation mechanism
Duchenne Muscular Dystrophy (DMD)
X-linked recessive disorder caused by mutation of DMD gene coding for dystrophin protein; boys present with calf hypertrophy, abnormal gait at 3-5 years with progressive involvement of respiratory muscles and death around 18 years
DMD-associated cardiomyopathy: Weakening & enlargement of the heart as a result of a lack of dystrophin in the myocardium; usually presents between 20-40 years of age without skeletal muscle involvement
Osteogenesis Imperfecta Types I & II
OI Type I: Autosomal dominant condition caused by mutation in the COL1A1 gene; normal collagen trimers are made in reduced amounts; patients present with brittle bones, increased fractures, blue sclerae
OI Type II: Caused by mutation in COL1A2 gene leading to production of protein with an abnormal (novel) structure; half of collagen triple helices will incorporate this abnormal protein and produce an abnormal collagen trimer, leading to severe phenotype, often fatal
Hb Kempsey & Hb Kansas
Hb Kempsey: Missense mutation in the b-globin gene that prevents T/R switching; Hb remains “locked” in the R state which has higher O2 affinity and cannot unload O2 in the tissues, leading to production of more RBCs (polycythemia)
Hb Kansas: Hb remains locked in the low O2 affinity “T state” leading to low O2 levels in the tissues presenting as cyanosis
Huntington Disease
Autosomal dominant disorder characterized by > 40 CAG coding repeats and expanded polyglutamine tracts in the HTT gene on 4 coding for Huntingtin protein; the number of repeats corresponds to absence, presence, and severity of the disease and genetic anticipation occurs through the paternal line
Myotonic Dystrophy
Autosomal dominant condition caused by expanded CTG repeats in the 3’ region of the DMPK gene, leading to production of abnormal RNA; characterized by facial weakness, ptosis (eyelid drooping); genetic anticipation occurs through the maternal line
Achondroplasia
Autosomal dominant disorder caused by gain of function mutation in FGFR3; constitituve activation of this tyrosine kinase receptor inhibits chondrocyte proliferation within the growth plate
Characterized by rhizomelic shortening of the limbs, midface hypoplasia, 10% risk of brainstem compression
Spontaneous mutations occur in the father’s germline and increase in frequency with advanced paternal age
Marfan syndrome
Autosomal dominant condition caused by mutation in the FBN1 gene coding for fibrillin, an ECM protein component of connective tissue; characterized by tall stature, mitral valve prolapse, and aortic dilation/rupture
Polycistic Kidney Disease (PKD)
Autosomal dominant condition caused by mutation in PDK1 or PDK2 encoding polycistin protein; characterized by bilateral renal & hepatic cysts & progressive renal failure
Ex: of locus heterogeneity
Familial Hypercholesterolemia
Autosomal dominant condition resulting from mutation in the LDLR gene; LDL deposits in the coronary arteries (atheromas) and in the skin and tendons (xanthomas)
Fabry Disease
X-linked disorder caused by mutation in the alpha-galactosidase A enzyme leading to lipid accumulation; presents with microvascular disease, neuropathy, nephropathy, cardiomyopathy
