MCB Block IV Tarlton

DYNAMIC MUTATION DISORDERS − Classifications of dynamic repeat disorders o Diseases caused by altered RNA function:

" GOF at RNA level affecting transcripts of other genes " Phenotype is result of expansion itself " i.e. DM1, DM2, FXTAS

DYNAMIC MUTATION DISORDERS − Classifications of dynamic repeat disorders o Diseases caused by altered protein function

" GOF at coding region/protein level " Phenotype is result of expansion itself " i.e. HD, SCAs

What are inclusion for? Are there inclusions in vulnerable cell populations?

" Inclusions are the body's way of protecting its cells from mutant proteins - if the mutant protein cannot get to the nucleus, it cannot affect transcription - however, NO inclusions are seen in vulnerable cell populations!

DYNAMIC MUTATION DISORDERS − Classifications of dynamic repeat disorders o What is the diseases caused by loss of protein (null mutation)?

" Phenotype is result of absence of gene product " i.e. FRAX, FRDA

o Multiple pathogenic mechanisms for polyQ expansions:

" Proteolytic cleavage, aberrant interactions, aggregate formation, TRANSCRIPTION DISREGULATION

*TAKE HOME MESSAGE FROM OI TYPES:

*Very similar phenotypes arise by different means: failure to synthesize a protein, failure to secrete a protein, failure to modify a protein to functional form

DISORDERS OF PORPHYRIN METABOLISM: Dominant

-- EXCEPTION TO RULE: enzyme deficiency usually AR, but many porphyrin metabolism disorders are AD! − Deficiencies of haem biosynthesis: erythropoietic & hepatic types

Disease: Polydactyly Mode of Inheritance:

Autosomal Dominant

Disease: Campomelic dysplasia ("bent bones") Mode of Inheritance:

Autosomal dominant

Disease: Charcot-Marie-Tooth Disease Mode of Inheritance:

Autosomal dominant

Disease: Hereditary Neuropathy with liability to Pressure Palsies (HNPP) Mode of Inheritance:

Autosomal dominant

Disease: Lactose tolerance/persistence (LP) Mode of Inheritance:

Autosomal dominant

Disease: Smith Magenis Syndrome Mode of Inheritance:

Autosomal dominant Characteristics: Broad square face, prominent forehead, deep set eyes, speech delay, moderate MR

Disease: Williams-Beuren Syndrome (WBS) Mode of Inheritance:

Autosomal dominant [no pedigree will be seen]

Disease: Congenital Adrenal Hyperplasia (CAH) [Case 19] Mode of Inheritance:

Autosomal recessive

Disease: Williams-Beuren Syndrome (WBS) CHARACTERISTICS?

Characteristics: IQ <80, loquacious personality - strength in expressive language, very weak spatial skills − Comparing drawings between WBS & Downs: WBS child draws individual details but not in a recognizable shape - "sees a lot of trees but doesn't appreciate the forest"

Disease: Chromosome abnormality [Case 13]

Characteristics: Normal karyotype, but multiple dysmorphic features - severe MR, growth retardation, microcephaly, and dysmorphism of the face and hands (epicanthic folds, hypertelorism, arched eyebrows, low-set ears, short philtrum, open mouth appearance, clinodactyly of 5th finer and distal brachydactyly) Context: Normal karyotype, but multiple dysmorphic features ! suggestive of unbalanced chromosome abnormality - but no suspected diagnosis ! test entire genome for copy number changes (duplications or deletions) ! Use CGH

Disease: Charcot-Marie-Tooth Disease What is the one important characteristic?

Characteristics: Slowly progressive weakness & atrophy of distal leg muscles ! FOOT DROP; chronic motor & sensory polyneuropathy

Disease: Charcot-Marie-Tooth Disease What are the two context?

Context1: Contiguous gene deletion syndrome Context2: Deletion & duplication can result in contrasting phenotypes: deletion (LOS) in PMP22 causes HNPP

Disease: Williams-Beuren Syndrome (WBS) What are they three contexts?

Context1: Contiguous gene deletion syndrome Context2: Deletion & duplication can result in contrasting phenotypes: duplication associated with speech delay Context3: Heterozygous loss of function in ELN (elastin coding gene) - detectable by FISH using BAC probe

Disease: Hereditary Neuropathy with liability to Pressure Palsies (HNPP) What are the two contexts?

Context1: Contiguous gene deletion syndrome Context2: Deletion & duplication can result in contrasting phenotypes: duplication causes Charcot-Marie-Tooth

Disease: Smith Magenis Syndrome What are the three context?

Context1: Contiguous gene deletion syndrome Context2: Deletion & duplication can result in contrasting phenotypes: duplication causes Potocki-Lupski Context3: Mechanism is the same as for WBS

Disease: Campomelic dysplasia ("bent bones") What are the two contexts?

Context1: Distant chromosomal rearrangements can disrupt gene expression − Translocation separating coding regions & proximal promoter from enhancers responsible for expression in developing bone but not for expression in developing genital ridge ! able to express SOX9 normally for sex differentiation but not in developing bone [SKELETAL PHENOTYPE WITHOUT SEX REVERSAL] Context2: Different enhancers can direct expression of the same gene in different tissues. − Developing bone & genital ridge

Disease: Congenital Adrenal Hyperplasia (CAH) [Case 19] What is the one context?

Context1: Gene conversion: sequence from 1 gene copied into another − Unequal recombination results in a 30kb deletion (detectable by SOUTHERN BLOT) − No gain/loss of material but sequence is copied from ψ-gene into functional gene ! CAH allele/ nonfunctional CYP21 gene − It is equally likely that sequence is copied from functional gene into ψ-gene ! no change in CYP21 activity

Disease: Polydactyly What are the three contexts?

Context1: Locus heterogeneity Context2: Mutation in distal elements & enhancers Context3: Different enhancers can direct expression of the same gene in different tissues (lobar HPE)

Disease: Lobar Holoprosencephaly (HPE) Context1:

Different enhancers can direct expression of the same gene in different tissues (polydactyly)

Disease: Polydactyly Genetic mutation:

Enhancer (intronic) mutation in gene upstream of SHH (controlling lower limb development) Characteristics: Common birth defect, part of many different syndromes

Disease: 22q11 Deletion Syndrome (DiGeorge Syndrome, Velo-Cardio-Facial Syndrome) [Case7]

Genetic mutation: 22q11 deletion Characteristics: Variable - pts have same deletion but variable symptoms - multiple congenital abnormalities, normal karyotypes; short stature, cleft palate, prominent nose, hypotonia, MR, VSD, absent or malformed kidneys Context: Multiple congenital abnormalities with a normal karyotype ! use FISH analysis with BAC probe to find deletion on Chr 22 (Due to variability of syndrome, always look at parents because they may be mildly defected.)

Disease: Congenital Adrenal Hyperplasia (CAH) [Case 19] GM?

Genetic mutation: Deficiency of steroid 21-hydroxylase on CYP21A ! impairs feedback control of ACTH secretion ! SALT WASTING; accumulated precursors shunted through uninhibited pathways ! EXCESSING ADRENAL ANDROGEN BIOSYNTHESIS; impaired feedback ! chronic stimulation of adrenal cortex ! ADRENAL HYPERPLASIA Characteristics: Virilized external genitalia in female; a male baby with hypospadias who clearly has testes in scrotal sac; adrenal hyperplasia

Disease: Hereditary Persistence of Fetal Hemoglobin (HPFH)

Genetic mutation: Deletion of δ & β genes removes promoter elements that LCR would interact with so LCR continues to interact with γ promoting synthesis of γ hemoglobin postnatally; can also result from mutations in silencer elements around γ genes ! γ continues to be expressed even though β is present Characteristics: Normal phenotype unless exposed to low O2 Context: No competition for γ-gene promoter so production continues postnatally; functionally equivalent to nonsense mutation or deletion of β-globin − Female with HPFH becomes pregnant: fetus will not get oxygen because cannot compete o Treat mother with adult hemoglobin − Treatments for β-thal: create HPFP in β-thal patients tricking body into producing γHb throughout adulthood

Disease: Williams-Beuren Syndrome (WBS) GM?

Genetic mutation: Recombination after misalignment in WBSCR on Chr7 ! DELETION of ELN at 7q11.23

Disease: Hereditary Neuropathy with liability to Pressure Palsies (HNPP) GM?

Genetic mutation: Recombination due to misalignment ! deletion (LOF) of PMP22 on Chr 17p11.2 Characteristics: Repeated focal pressure neuropathies like carpal tunnel syndrome; demyelination

Disease: Charcot-Marie-Tooth Disease GM?

Genetic mutation: Recombination due to misalignment ! duplication (GOF) of PMP22 on Chr 17p11.2

Disease: Lactose tolerance/persistence (LP) What is context two and three?

Genetic mutation: Single nucleotide change (C!T) upstream of lactase gene ! increases binding of TF Characteristics: Maintains intestinal expression of lactase throughout life in Europeans Context1: Lactose tolerance is a mutant phenotype. Context2: Variations in sequence controlling gene expression are important causes of human variation & disease Context3: Gain of function

Disease: Variegate Porphyria (VP)

Mode of Inheritance: Autosomal dominant Genetic mutation: Deficiency of protoporphyrinogen oxidase Characteristics: Photosensitivity, variable neurological & visceral findings triggered by drugs Context1: Founder Effect amongst Afrikaners (~1/300) Context2: Haploinsufficiency Context3: Enzyme deficiency with AD inheritance

Disease: Apert Syndrome

Mode of Inheritance: Autosomal dominant Genetic mutation: FGFR2 (expressed predominantly in skull) Characteristics: Skull defects - premature fusion of skull bones Context1: Gain of function Context2: Similar mutations in similar genes can result in different phenotypes - genes expressed in different cell types or at different times of development [FGFR3 in limbs vs FGFR2 in skull]

Disease: Nail Patella Syndrome (NPS)

Mode of Inheritance: Autosomal dominant Genetic mutation: G!A at R200Q (loss of restriction site); C!T at R198X of the TF LMX1B gene ! prevents synthesis of full length protein *all affecteds in a family have the same lmx1b mutation but can have different symptoms Characteristics: Nail dysplasia, absent patella, elbow winging/ptyerigia, iliac horns*, contractures & hyperextensibility of interphalandeal joints, kidney problems due to podocyte underdevelopment, anterior chamber defects in eyes - increased risk for glaucoma Context1: Use DNA sequencing - Affecteds are heterozygotes & have 2 bands at same position on the gel; OR Southern blot - heterozygotes will have a fainter band due to weaker signal (less target DNA) Context2: Haploinsufficiency - loss of function Context3: Pleiotropy Context4: Complete penetrance: everyone with mutation has same phenotype Context5: Variable expressivity: all have same mutation, but different signs & symptoms due to variation at other genes that influence a person's genetic predisposition & stochastic events in development

Disease: Acute Intermittent Porphyria (AIP)

Mode of Inheritance: Autosomal dominant Genetic mutation: Haploinsufficiency of porphobilinogen deaminase Characteristics: Latent prior to puberty; females>males; neurologic symptoms, not photosensitivity - abdominal pain, weakness, hallucinations; exacerbated by hormones, drugs, & diet - via activation of ALA synthase; acute because there must be some change to cause build up of prophobilinogen Context1: Haploinsufficiency, penetrance ~10% Context2: Enzyme deficiency with AD inheritance

Disease: Hypochondroplasia

Mode of Inheritance: Autosomal dominant Genetic mutation: Missense mutation in FGFR3 Characteristics: Lesser degree of shortening as compared to achondroplasia & thanatophoric dysplasia; approaches normal height Context1: Gain of function

Disease: Osteogenesis imperfecta (OI) type III [severe, but not lethal]

Mode of Inheritance: Autosomal dominant Genetic mutation: Missense mutation in Type I collagen ! gly substitution *mutation in N-terminal part of triple helix: minor disturbance of packing Characteristics: Severe, but not lethal; gross deformities Context1: Dominant- negative - mutant collagen interferes with any normal collagen Context2: Missense mutation = DN ! null/LOF mutation = milder phenotype (OI type I) Context2: Varying degrees of severity - different types of mutation result in phenotypes of different severity - correlates with size of mutant amino acid & position of substitution (N or C term)

Disease: Osteogenesis imperfecta (OI) type II [severe & lethal]

Mode of Inheritance: Autosomal dominant Genetic mutation: Missense mutation of COL1A1 or COL1A2 of Type I collagen ! Gly substitution *mutation in C-terminal part of triple helix: major disturbance of packing Characteristics: Neonatal lethal, multiple in utero fractures Context1: Dominant-negative - mutant collagen interferes with any normal collagen Context2: Missense mutation = DN ! null/LOF mutation = milder phenotype (OI type I) Context3: Varying degrees of severity - different types of mutation result in phenotypes of different severity - correlates with size of mutant amino acid & position of substitution (N or C term)

Disease: Craniosynostosis

Mode of Inheritance: Autosomal dominant Genetic mutation: Missense mutation of MSX2 ! excessive DNA binding by TF Characteristics: Premature closure of sutures Context1: Gain of function Context2: Missense mutation=GOF ! null/LOF mutation=opposite phenotype (parietal foramina)

Disease: Osteogenesis imperfecta (OI) type I [mild]

Mode of Inheritance: Autosomal dominant Genetic mutation: Null mutation of COLA1 or COL1A2 of Type I collagen Characteristics: Multiple fractures but no gross deformity - XR would show multiple healed fractures; blue sclera; bisphosphonates can be used for treatment Context1: Haploinsufficiency - 50% of normal collagen [no expression of mutant protein] Context2: Varying degrees of severity - different mutations result in phenotypes of different severity Context3: Rare time a more severe mutation (null) has a milder phenotype as compared to type II & III (missense) − This is because sometimes having no protein is better than having mutant protein

Disease: Stickler Syndrome [Case 15]

Mode of Inheritance: Autosomal dominant Genetic mutation: Null mutation of Type II Collagen [COL2A1 gene] Characteristics: Short, severe myopia, cleft palate, retinal detachment Context1: Frameshift/null mutation ! premature termination codon (PTC) ! termination of transcript ! NO production of shortened polypeptides! [haploinsufficiency] − More severe chondrodysplasias when mutant type II collagen is made [dominant negative]

Disease: Thanatophoric Dysplasia

Mode of Inheritance: Autosomal dominant (100% sporadic!) Genetic mutation: FGFR3 mutation: dimerization of TK domain ! signaling in absence of ligand ! excessive inhibition of bone growth = stunted bone growth Characteristics: "death loving" = usually stillborn; micromelic limb shortening, severe frontal bossing, very small chest; TD-1: curved femurs +/- cloverleaf skull; TD-2: straight femurs & cloverleaf skull Context1: Gain of function

Disease: Achondroplasia

Mode of Inheritance: Autosomal dominant (80% are sporadic) Genetic mutation: Missense mutation in FGFR3 [G380R]: dimerization of TK domain ! signaling in absence of ligand ! excessive inhibition of bone growth = stunted bone growth Characteristics: Short stature, depressed nasal bridge, pronounced lumbar lordosis; compression of foramen magnum Context1: Gain of function Context2: Homozygous achondroplasia - 1:4 children of achondroplast couple will have this; severe phenotype comparable to TD-1 (lethal) Context3: Missense mutation=GOF ! null/LOF mutation=opposite phenotype (Spider Leg Syndrome)

Disease: Parietal foramina

Mode of Inheritance: Autosomal dominant (heterozygous LOF) Genetic mutation: Null mutation of MSX2 ! reduction of DNA binding ! inhibits bone growth Characteristics: Open skull Context1: Heterozygous loss of function - haploinsufficiency

Disease: Fibrodysplasia ossificans progressive (FOP)

Mode of Inheritance: Autosomal dominant (most sporadic) Genetic mutation: R206H mutation in ACVR1 gene - All FOP patients have the same mutation Characteristics: Congenital malformation of big toes, progressive heterotopic ossification of skeletal muscles, tendons, ligaments, & fascia Context1: Gain of function of the BMP receptor - being activated when it shouldn't be ! mutant protein made

Disease: Sickle Cell Disease aka HbS [Case 11]

Mode of Inheritance: Autosomal recessive Genetic Mutation: Missense mutation [A!T; Glu!Val] on β-globin gene - everyone with sickle cell disease is homozygous for the same mutation Characteristics: Splenomegaly, autosplenectomy, bony infarctions ! unequal finger length, limb pain, abdominal pain, bone marrow expansion, hemolytic anemia, painful crises; common in West Africa (1/4) Context1: Pleiotropy - one gene influencing multiple, seemingly unrelated traits Context2: Single base pair substitution so use PCR & restriction digest to identify HbS allele (because loss of restriction site) ! then use ASO because we know mutation & it's single base change; could use Southern blot but PCR needs less DNA and is cheaper Context3: HbS heterozygotes are generally healthy but may be symptomatic under stressful conditions

Disease: Maple Syrup Urine Disease

Mode of Inheritance: Autosomal recessive Genetic mutation: Deficiency of branched chain ketoacid decarboxylase ! excretion of Val, Leu, Ile Characteristics: Urine smells sweet due to excretion of Val, Leu, Ile; diffuse hypoattentuation of cerebral & cerebellar white matter Context1: Enzyme deficiency = loss of function = recessive Context2: Overall incidence of 1/180,000 BUT > 1/200 in PA Mennoites [Founder's Effect]

Disease: Homocystinuria

Mode of Inheritance: Autosomal recessive Genetic mutation: Deficiency of cystathionine β-synthetase (CBS); defected Met ! Cys Characteristics: Mental retardation, fits, osteoporosis, low-Met diet helps Context: Enzyme deficiency = loss of function = recessive

Disease: Autosomal Recessive OI

Mode of Inheritance: Autosomal recessive Genetic mutation: Deficient for enzymes essential for posttranslational modification of collagen (no mutations in Collagen Type I) Characteristics: Severity depends on the enzyme - varies from neonatal lethal to mild

Disease: α-thalassemia

Mode of Inheritance: Autosomal recessive Genetic mutation: Deletion of α-globin genes due to homologous pairing & unequal crossover during meiosis Characteristics: SE Asia populations; hemolytic anemia, hyperplasia of bone marrow Context1: Deletions of α-globin genes due to unequal crossover during homologous pairing ! excess β-globin Context2: Southern blot ! deletions will change distance between restriction sites

Disease: Hyperphenylalanemia

Mode of Inheritance: Autosomal recessive Genetic mutation: Few are due to deficiency of co-factor tetrahydrobiopterin (BH4) - by different genes on different chromosomes Characteristics: High levels of Phe, but low-phe diet will not prevent MR because BH-4 is needed for neurotransmitter synthesis Context1: Absence of cofactor = absence/deficiency of enzyme = loss of function = recessive Context2: Locus heterogeneity - mutations in different genes lead to same phenotype

Disease: β-thalassemia (Cooley's anemia) [Case 14]

Mode of Inheritance: Autosomal recessive Genetic mutation: Null mutation in β-globin gene Characteristics: Mediterranean & African populations because protection against malaria; usually presents as severe anemia before 12mos; enlarged cheek bones & maxilla; hepatosplenomegaly Context1: Majority due to null mutations of β-globin gene ! reduction in β-globin synthesis (stop codons, frameshifts, mutation at TATA box, splicing mutation, etc.) - specific mutation identified by DNA sequencing Context2: Can also use ASO panels to test if parents are carriers for common mutations in the population Context2: Allelic heterogeneity - different mutations in the same gene cause the same disease − β+-thalassemia o Genetic mutation: trace (10%) β-globin synthesis (leaky mutations) o Context: posttranslational defects − β0-thalassemia o Genetic mutation: no β-globin synthesis (major mutations) o Characteristics: "hair-on-end" appearance of the skull o Context: deletion of β-globin gene, RNA splicing defects, nonsense mutation − Hb-Lepore o Genetic mutation: Unequal crossing over ! Hb-Lepore o Context1: In Hb-Lepore, transcription of β-globin is under the control of the δ-globin promoter, not the normal β promoter - so the hybrid gene is expressed at the same low level as δ - Functionally equivalent to having null mutation in β-globin gene

Disease: Phenylketonuria (PKU)

Mode of Inheritance: Autosomal recessive Genetic mutation: Phenylalanine hydroxylase deficiency - unable to breakdown phenylalanine Characteristics: Fair skinned, blue eyes; can be treated with low-Phe diet if caught early enough; no abiding by diet = decreased IQ due to toxic effects of phenylpyruvate & other metabolites − A pregnant female with PKU who does not stick to low-Phe diet will cause her child to be mentally retarded despite the child's Aa genotype - this isn't treatable because brain damage already done Context1: Enzyme deficiency = loss of function = recessive Context2: Allelic heterogeneity - different mutations in the same gene cause the same disease

Disease: Spider Leg Syndrome

Mode of Inheritance: Autosomal recessive (homozygous LOF) Genetic mutation: Null mutation in FGFR3 [T546K] ! lack of inhibition of bone growth Characteristics: Tall stature, severe lateral tibial deviation, scoliosis, campodactylyl Context1: Homozygous loss of function

Disease: Oculocutaneous albinism I (OCA1)

Mode of Inheritance: Autosomal recessive (mostly) Genetic mutation: Tyrosinase deficiency: inability to convert tyrosine ! melanin Characteristics: Lack of eye pigment (red eyes) affects visual acuity and projection of visual pathways to the optic cortex Context1: Enzyme deficiency = loss of function = recessive Context2: Locus heterogeneity - mutations in different genes lead to same phenotype

Disease: Alkaptonuria

Mode of Inheritance: Autosomal recessive (rare) Genetic mutation: Deficiency of homogentistic acid oxidase ! accumulation of homogentistic acid Characteristics: Rare; bluish-black discoloration of the sclera & the auriculum, black urine (after O2 exposure), severe calcification of aortic valves, extensive calcification & blackening of the tricuspid aortic valve and discoloration of the aortic intima - anything that touches oxygen will turn black Context: Enzyme deficiency = loss of function = recessive

Disease: Down Syndrome/Trisomy 21 [Case 8]

Mode of Inheritance: Maternal NDJ; aneuoploidy Genetic Mutation: Trisomy 21 Characteristics: Hypotonia, excess nucal skin, bilateral single palmer (simian) creases Context: Use FISH with BAC probes to see # of chromosome 21 an individual has after suspected diagnosis Genetic Counseling: First screening done at 10-14 weeks to assess hCG, PAP, nuchal measurements, & maternal age. If at risk, do CVS at 10-14 weeks. Second screen done at 12-16 weeks.

Disease: LHON [Case 6]

Mode of Inheritance: Mitochondrial inheritance Genetic mutation: G!A mutation in mtDNA (creates new restriction site & destroys an old one) Characteristics: Progressive worsening of central vision Context: Single base change in mtDNA creating a new restriction site ! PCR & restriction digest/ASO

Disease: Turner Syndrome [Case 9]

Mode of Inheritance: Mosaic - loss of X in women Genetic Mutation: 45X Context: Diagnosed by karyotyping (45X); PCR for Y-specific sequence to confirm or rule out presence of 46XY cells (residual Y cells ! malignancies)

Disease: Hemophilia [Case 12]

Mode of Inheritance: X-linked Genetic Mutation: Defect in Factor VIII (Hem A) or Factor IX (Hem B) Characteristics: Bleeding around elbow, retinal bleed, repeated bleeds into joints produce severe arthritis; severity of disease is relatedly to how much function is left with the protein − Milder disease: 6-30% residual F8 activity of ~40% − Severe disease: little/no F8 activity (<1%) in ~50% Context1: Severe Hemophilia A is often the result of an intrachromosomal rearrangement (inversion) of the FVIII gene. The rearrangement changes the length of DNA between restriction sites and can be seen on Southern blot. − Depending on whether F8A1 lines up with F8A2 or F8A3 will cause different outcomes/different banding − *If the patient has hemophilia but does not show on Southern blot ! point mutation (not the inversion) Context2: Variation in severity of disease due to making no protein vs making some protein with residual function

Disease: Duchenne Muscular Dystrophy [Case 4]

Mode of Inheritance: X-linked recessive Genetic Mutation: Deletion of 1 or more exons from dystrophin gene due to deletions, frameshift, nonsense mutations! near or complete absence of dystrophin; OR "non-random" X inactivation - translocation with an autosome disrupts the gene structure [*see X-chromosome section] Characteristics: Onset < 5 years old; progressive symmetrical muscle weakness (proximal>distal), calf hypertrophy, wheelchair dependent by 13 years old; 1/5000 males Context1: Use PCR amplifications to detect deletions in patients looking at the entire dystrophin gene Context2: Variation in severity of disease due to making no protein vs making some protein with residual function Context3: Deletions, Frameshifts, Nonsense = No protein production

Disease: Becker Muscular Dystrophy

Mode of Inheritance: X-linked recessive Genetic Mutation: In-frame deletions ! residual function of dystrophin gene Characteristics: Onset late childhood; progressive symmetrical muscle weakness (proximal>distal), calf hypertrophy, wheelchair dependent by 16 years old, activity-induced cramping, preservation of neck flexor muscle strength, 1/18000 males Context1: Use PCR amplifications to detect deletions in patients looking at the entire dystrophin gene Context2: Variation in severity of disease due to making no protein vs making some protein with residual function Context3: In-frame deletion = deletions in multiples of 3 = maintains reading frame = protein is produced (shorter protein with residual function)

Disease: Campomelic dysplasia ("bent bones") Genetic mutation:

Mutation in distal elements of SOX9 Characteristics: Thin bones and/or sex reversal − 46,XY individual ! sex reversal because SOX9 affects sex differentiation, as well as skeletal phenotype − 46,XX individual ! SOX9 not used for sex differentiation in females; will only have skeletal phenotype

Disease: Smith Magenis Syndrome Genetic mutation:

Recombination after misalignment ! 3700kb deletion 17p11.2

Disease: Lobar Holoprosencephaly (HPE) Genetic mutation:

Single base change within enhancer (SBE) controlling SHH during brain development Characteristics: Midline defect in which the brain does not separate

DYNAMIC MUTATION DISORDERS − Genetic anticipation:

a phenomenon in which disease severity increases and/or age of onset of disease decreases from one generation to the next o Triple repeat expansion is the molecular explanation of the phenomenon of anticipation

DYNAMIC MUTATION DISORDERS − Phenotype can be result of

absence of gene product OR can be result of expansion of repeat itself

DYNAMIC MUTATION DISORDERS o Expansion can occur where? Where do the repeats get larger?

can occur in 5' UTR, intron, coding region, or 3' UTR o Repeats get much larger in non-coding regions

GENOMIC DISORDERS o [Contiguous gene deletion:

chromosomal abnormalities, i.e. deletion/duplication removing several genes in close proximity to each other on the chromosome]

REMEMBER: for every gene deletion there must be a?

gene duplication o Deletion seems to result in a phenotype more often; o Deletion & duplication phenotypes may be approx. opposite of each other

o Central pathological feature of polyQ diseases:

insoluble aggregates or inclusions

What is a gene conversion?

is the product of recombination & does not involve loss or gain of DNA

DYNAMIC MUTATION DISORDERS Do you see anticipation in AD or AR?

o Do not see anticipation in autosomal recessively inherited disorders because there are not successive generations with the phenotype

− Haploinsufficiency ("half is NOT enough")

o Loss of function o 50% reduction in the amount of functional protein results in phenotype " Heterozygous LOF resulting in dominant phenotype " Disease is a result of lack of normal protein, not the result of abnormal protein " Remember: When half IS enough = recessive phenotype When half is NOT enough = dominant phenotype o Almost always see allelic heterogeneity: different mutation - same gene - same phenotype " *Type of mutation cannot be predicted by range of symptoms/severity of disease! o Null mutations (i.e. missense, nonsense, splice, frameshift, deletion, translocation - anything stopping protein from being made)

DYNAMIC MUTATION DISORDERS − Germline instability What are the maternal expansions disorder?

o Maternal expansion: most CGG expansion [Fragile X] & CTG expansion [Myotonic Dystrophy]

− Dominant-negative

o Mutant gene product interferes with normal gene product & takes it out of circulation (much less normal available than if it was just haploinsufficient) o Mutant protein is synthesized! o Missense & splice mutations o If a missense mutation acts in DN manner, a NULL (LOF) mutation in the same gene should result a milder phenotype " Severe OI vs Mild OI

− what are the tests for tandem repeat expansions

o PCR & gel electrophoresis for tandem repeats (<1000bp or 300 rpts THIS WAS A PAST TEST QUESTION) - usually coding region o Southern blotting of restricted genomic DNA for greatly expanded repeats

DYNAMIC MUTATION DISORDERS − Germline instability What are the paternal expansion disorders and what region do they occur in?

o Paternal expansion: most CAG expansions (poly-glutamine) disorders - coding region

What is the pathogenesis of polyQ diseases?

o Pathogenesis of polyQ diseases is not due to cellular inclusion formation, but through interference with transcriptional regulation of key cell cycle regulator genes

DYNAMIC MUTATION DISORDERS Triplet repeat disorder?

o There are approx. 40 of these disorders in humans & all are neurological diseases

− Gain of function

o Up-regulation or novel function of an otherwise normal gene o Mutant protein is synthesized! o Mutation specific: specific missense mutations result in specific phenotypes " Different mutations in the same gene may result in different phenotype o If a missense mutation acts to cause a GOF, a NULL (LOF) mutation in the same gene should result in the opposite phenotype " Knock in FGFR3 mutation in mice ! shorter bones [gain of function] " Knock out FGFR3 in mice ! longer bones [LOF: haploinsufficiency]

− What are the polyglutamine Diseases

o i.e. Huntington & many SCAs

DYNAMIC MUTATION DISORDERS

products that continue to mutate within tissues & across generations (dependent on type of repeat & the gene involved)

MUTATIONS OUTSIDE CODING REGION o Blue/brown eye color variation -

single base change in intron of gene upstream from OCA2

GENOMIC DISORDERS How do you diagnose contiguous gene deletions/duplication?

use FISH as diagnostic tool

HEMOGLOBINOPATHIES

− 65-700 new cases born everyday! − Almost ALL exhibit autosomal recessive inheritance − Different hemoglobins formed from different combinations of globin gene products o Embryonic: (ζ2, ε2) o Fetal: (α2, γ2) o Adult: (HbA: α2, β2) & (HbA2: α2, δ2) " HbA2 >4% usually indicates β-thalassemia − LCR controls expression of the different Hb types in different tissues & at different times of development o When there is deletion of certain genes, there is no promoter target of that gene for LCR to interact with - it will continue to interact with/synthesis a different protein − Thalassemias: imbalance of globin chain production ! accumulation of free globin chains in the RBC precursors ! hemolysis of RBCs ! hemolytic anemia with consequent compensatory hyperplasia of the bone marrow o α-thalassemias: deletion o β-thalassemias: null mutations = complete loss of function

What are GENOMIC DISORDERS caused by?

− Caused by recombination between non-homologous repeat sequences (misalignment during meiosis) ! one gamete with deletion & one gamete with duplication of same sequence

How do different enhancers direct expression of tissues?

− Different enhancers can direct expression of the same gene in different tissues o When multiple enhancers control the expression of a gene in different parts of the body, a change to one enhancer can alter the gene's activity in a specific place without affecting it elsewhere

DISORDERS OF AMINO ACID METABOLISM: Recessive

− Enzyme deficiencies = AR (mostly) − Locus heterogeneity: different mutation - different gene - same phenotype − Allelic heterogeneity: different mutation - same gene - same phenotype

RECESSIVELY INHERITED DISEASES

− If mutation affects enzymes or transporters ! usually recessive o Half of the normal amount of a functional enzyme/transporter is usually sufficient for healthy function; therefore, one functional gene is enough − Recessive = LOSS OF FUNCTION (reduced or abolished protein function) − Variation in severity due to making no protein vs making some protein with residual function o *For mild presentation of a recessive disorder, there must be some protein made! − Missense, nonsense, splicing mutations, frameshift, chromosomal, deletions

DOMINANTLY INHERITED DISEASES

− If mutation affects structural proteins or transcription factors ! dominant o Control of differentiation or development is tightly regulated so usually both copies of the gene are necessary for healthy function

Disease: Congenital Adrenal Hyperplasia (CAH) [Case 19] Is there a gain or loss of material? What goes on?

− No gain/loss of material but sequence is copied from ψ-gene into functional gene ! CAH allele/ nonfunctional CYP21 gene − It is equally likely that sequence is copied from functional gene into ψ-gene ! no change in CYP21 activity

Disease: Congenital Adrenal Hyperplasia (CAH) [Case 19] Detectable by?

− Unequal recombination results in a 30kb deletion (detectable by SOUTHERN BLOT)

MUTATIONS OUTSIDE CODING REGION What happens when you have variation in (conserved, noncoding) regions?

− Variation in (conserved, noncoding) sequences controlling gene expression are important causes of human variation and disease

MUTATIONS OUTSIDE CODING REGION What are enhancers

−key components of the "switches" that control gene expression o Activation - TFs attach to binding sites within enhancer sequence triggering polymerase to begin transcribing RNA copy of the gene o Lactase persistence