Atypical (Non-Traditional) Inheritance II

Anticipation

The apparent worsening of a disorder with subsequent generations First known molecular mechanism for anticipation was expanding trinucleotide repeat regions Other mechanisms possible

Fragile - X

X-linked Mental Retardation Autistic like behaviors Dysmorphic facies (thin, elongated face), hypotonia, joint laxity, macro-orchidism Demonstrates anticipation in unusual X- linked pattern - “Sherman Paradox”

1 Fragile - X

Trinucleotide repeat = CGG Expansion of repeat only when transmitted maternally Repeat in 5’ untranslated region of FMR- 1 gene Repeat enlargement blocks transcription Number of repeats correlates with disease severity

Trinucleotide Repeats in FraX

Mean number of repeats (CGG) = 29-30 Transcription impaired with increasing size of repeat With full expression (repeats > 200): gene methylated transcription completely turned off Transcription potentially less effective with < 29 repeats

Fragile X

Clinical Status CGG Repeats Normal 6 – 46 Transmitting male 52 – 200 Carrier female 52 – 200 Affected males >200 Affected females > 200

2 Myotonic Dystrophy

Autosomal dominant multi-system disorder characterized by myotonia, weakness in face and distal limbs, cataracts, frontal baldness, and multiple endocrinopathies Myotonia = the impaired ability to relax muscle after contraction

Myotonic Dystrophy

Anticipation long debated - clearly confirmed Ancestors with AD cataracts Common ancestors in extended French Canadian pedigrees suggest transmission of abnormal gene for 15 generations with little to no clinical sequelae

Myotonic Dystrophy

Trinucleotide repeat = CTG Repeat up to 5 kB Repeat is transcribed Repeat is in 3’ untranslated region of gene Gene product is protein kinase Repeat enlarges with maternal and paternal transmission

3 Myotonic Dystrophy

Severe infantile form seen with affected mothers.

Trinucleotide repeat does not adequately explain severe congenital form

Trinucleotide Repeat Disorders

Fragile - X syndrome (FRAXA) Other “Fragile” Mental Retardation Syndromes (FRAXE, FRAXF, FRAX16A) Myotonic Dystrophy Huntington Disease Kennedy Disease Spinocerebellar Ataxia types 1 - 10 Oculopharyngeal Dystrophy Dentatorubral Pallidoluysian Atrophy Friedreich Ataxia

How do repeats cause disease?

Loss of function Disrupts gene transcription, translation, etc. [Fragile-X, Friedreich ataxia]

4 How do repeats cause disease?

Gain of function Excess metabolite(s) inhibit other enzyme and/or regulatory systems (direct toxicity ) [Huntington Disease, DRPLA] ⌧GADPH binds to stretches of glutamine. Excess glutamine (>760 repeats) inhibit enzyme ⌧Better binding of huntingtin associated protein with increased regulation of protein

How do repeats cause disease?

Dominant negative effect Abnormal product interferes with normal physiologic function of product [Myotonic dystrophy]

Contiguous Gene Disorders (microdeletion syndromes)

Well described phenotype Typically variable expression break points / size of deletion may explain Segregation looks Mendelian Deletion often detectable only by FISH studies

5 DiGeorge Anomaly

Developmental field defect of 3rd and 4th branchial arches Lower face hypoplasia Thymic hypoplasia (T- cell dysfunction) Hypoparathyroidism (hypocalcemia) Conotruncal heart defects

Shprintzen Syndrome (Velo-cardio-facial syndrome)

Velo = palate Cleft or insufficiency Conotruncal heart defects Characteristic facial appearance Learning disabilities Neurobehavioral changes frequent

Spectrum of 22 q Deletions Associated with multiple syndromes, sequences and associations (DiGeorge, Shprintzen, CHARGE, Opitz) Associated with over 100 types of anomalies Major organs involved are heart, palate, brain Inter- and intra familial variability Incomplete penetrance

6 Mitochondria : General Features

Human mtDNA: Circular, Double-Stranded 16,569 base pairs No introns Contained within mitochondria located in cytoplasm (i.e. non-nuclear) DNA code differs UGA read as tryptophan rather than ‘stop’ AGA and AGG read as ‘stop’ rather than arginine AUA and AUU are sometimes initiation codes instead of AUG

mtDNA: General Features (Cont)

mtDNA Codes for: 13 Oxidative Phosphorylation Enzyme Complex Subunits (of 67 or so subunits) 2 Ribosomal RNAs & 22 transfer RNA Relative reliance of tissue on mitochondrial ATP correlates best with mtDNA content mtDNA sequence is 0.0006% of the nuclear sequence but amounts to 1% of the total mass of cellular DNA

mtDNA: General Features (Con’t)

Each human cell has hundreds of mitochondria and thousands of mtDNA’s 2 to 10 copies per mitochondria in non-replicating cells Human eggs have ~ 100,000 mtDNA Human sperm have ~ 100 – 1500 mtDNA

7 Mitochondrial Inheritance: Basic Principles

Semi-autonomous inheritance Maternal inheritance Replicative segregation “Bottleneck” phenomenon Threshold expression of phenotype High mutation rate Genotype / phenotype correlation Accumulation of mutations

1. Semi-autonomous Inheritance

Mitochondrial DNA segregates in daughter cells independent of nuclear chromosomes

2. Maternal Inheritance

In humans mtDNA transmission is exclusively maternal mtDNA transmitted via oocyte cytoplasm from mother to all children mtDNA from spermatozoa lost at 2- 4 cell stage

8 3. Replicative Segregation

Homoplasmic: Single mtDNA sequence in a given cell Heteroplasmic: More than one mtDNA sequence in a cell Cytokinesis of heteroplasmic cells partitions different mtDNAs into daughter cells --> replicative drift Over time may return to homoplasmy

4. Bottleneck Phenomenon During replication, the number of mtDNA copies in oogonia decreases to 1-30/mitochondrion Oogenesis: number of mitochondria increases by about 100x After fertilization: rapid nuclear DNA replication and cell cleavage without much increase in number of mitochondria or mtDNA

4. Bottleneck phenomenon

This restriction and subsequent amplification during oogenesis creates a genomic ‘bottle-neck’ The consequence of this bottleneck is a reduction in diversity

9 5. Threshold Expression of Phenotype

Phenotypic expression is a product of: The nature of the mutation The percentage of the mutant mtDNA The relative reliance of each organ system on OXPHOS processes Once the cumulative liabilities of the above 3 factors exceeds the cell’s need for energy, expression begins

6. Mutation Rate

Much higher mutation rate than occurs in nuclear DNA Nucleotide substitutions and deletions up to 17X more common mtDNA has no protective mechanisms (e.g. histones) mtDNA has little ability to repair mutations (no repair mechanisms) Substitutions may not (“synonymous”) or may (“replacement”) alter the resulting amino acid sequence

7. Genotype / Phenotype Correlation

mtDNA mutations: highly polymorphic and dissociation of genotype with phenotype (different mutations produce the same phenotype and the same mutation may have different phenotypes associated with it) Different family members can inherit different percentages of mutant mtDNAs, and thus present with different clinical manifestations (wide intrafamilial variability)

10 8. Accumulation of Mutations

Somatic mutations accumulate in post- mitotic tissues with age Progressive disorders Different than the restriction of mitochondrial mutations in germ cells

Clinical Features of Mitochondrial Disorders Primary Tissues Nerve (CNS & PNS) Eyes Muscle Heart Liver

Clinical Features of Mitochondrial Disorders (con’t)

“Ragged Red” muscle fibers abnormally shaped cristae, paracrystalline inclusions, clumped oxidative enzymes, increased neutral lipids, in muscles Pathognomonic for mitochondrial disorder

11 Common Mitochondrial Syndromes

Examples : Leigh disease (Pyruvate carboxylase and dehydrogenase respiratory chain deficiency) MERRF (Myoclonic Epilepsy with Ragged- Red Fibers) MELAS (Mitochondrial Encephalomyopathies, Lactic Acidosis, and Stroke-like episodes) Kearns-Sayre Syndrome LHON (Leber Hereditary Optic Neuropathy) Mitochondrial “syndromes” actually artificial distinctions

Mitochondrial Disorders

Mitochondrial DNA (mtDNA) has been shown to be directly involved in a number of diseases and disease processes Examples include: Diabetes Mellitus (its most common molecularly defined form) Hearing loss Alzheimer’s, Parkinson’s, Multiple Sclerosis AZT use (often ceases some time after discontinued)

Isolated Mitochondrial Hearing Loss

12S rRNA gene mutation A1555G confers a sensitivity to aminoglycosides (makes the RNA more similar to bacterial RNA) A1555G also can be seen in maternally transmitted hearing loss

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