Tegay Lecture #4 - Molecular and Mendelian Basis of Disease

Genomic Variation

• Copy number varitiaons

  • genomic region presnet in the normal number of cpies

    • increased gene expression with increaed gene ocpy number (Trisomes, duplciaitons, mcirocudplication)

    • Decreased gene expression with decreased gene copy number (Monosomie, Deletions, Microdeltions)

• Epigenetic Variaitons

  • DN modifcaitons outdisoe of the chomse strucutre of nucletodie sqeucne that affect gene expression

    • Increased DNa methlyation = decreased expression

    • Decread DNA methylation = increased gene expression

• Seqeunce Variations

  • Changes in nucleotide sequence from the population norm

    • ExL single nucleoide dubsition, insertion/Deltions (InDels)

    • inter genic (between genes)

    • intra genic (within a gene)

Mechanisms of Disease

Major mutation categories

• Loss of function

  • Decrease amount or activity of a gene product

    • Null: comeplte los of function

    • Hypomorphic: Partial loss of function

• Gain of function

  • Increased amoun or activt of a gene product

• Change of function

  • Altered/novel funciton of a gene product

Sequence Variation

• Point mutations in Coding regions

  • synonymous (silent): mutation does not change aminoacid/codon

  • nonsynomous (MissensE) mutaiton changes a amino acid/codoon

  • Nonsense (truncating): Mutation creates stop codon

  • Readthrough: Mutation abolishes stop codon

• Insertion/Deltion in Coding Regions

  • In Frame InDel: codon insertion without changing reading frame (nucleotide insertion an mutliple of 3)

  • Frameshift InDel: Changes cododn reading frame (Nucleotide insertion/dletion not an multiple of 3)

• Non-coding Mutations

  • Can still effect gene epxression

    • ExL splice site, promoter, enhancer, evne occaltionl intorinc

Polymorphism vs Mutation

• Polymorphic

  • something that exist in more than one form

  • Ex: single nucleotide polymorphism

  • common SNP: seen in >1% of the population

• Mutation

  • A process by which genomic changes occur

  • Ex: Deleterious or Pathogenic mutation

  • Genomic change predicted to cause disease

• Both Polymorphism and Mutations

  • Can be:

    • intragenic or intergenic

    • intronic or exoni

    • inherited or de novo

    • Can apply to SNPs, InDels, or SNVs

Locus vs. Allele

• Locus

  • Chromosomal location for a given Assocation

  • Ex: Beta-Globin gene locus is 11p15

• Allele

  • Form of a gene present at a locus

    • Wild Type: Predominant gene form (wt)

       Mutant: Variation from wild type allele (mt)

       Homozygosity: Identical alleles at a locus (wt/wt or mt/mt)

       Heterozygosity: One variant allele at a locus (wt/mt)

       Compound Heterozygosity: Different variant alleles at locus (mt1/mt2)

       Hemizygosity- When only one allele is present for a given locus

       Ex, Males are hemizygous for genes on their X-chromosome

       Ex, Hemizygosity can be created by heterozygous autosomal deletions

Genotype/Phenotype

• Genotype:

  • Genetic make-up

    • Ex. Trisomy 21 or CFTR gene ∆F508 homozygote

• Phenotype:

  • Observable product of that individuals genotype + environment

    • Ex. Features of Down syndrome or Cystic Fibrosis (CF)

• Allelic Heterogeneity:

  • Multiple mutant alleles exist for a given locus

    • Ex, CF- ∆F508 most common mt allele but >1,000 mt CF alleles

• Locus Heterogeneity:

  • Multiple loci exist for a given phenotype

    • Ex, Now >12 Parkinson’s disease associated loci

• Phenotypic Heterogeneity:

  • When different phenotypes result from different alleles at one locus

    • Ex. Mutant Lamin A/C alleles cause >9 different phenotypes

Sanger Sequencing

• Dideoxynucleotide chain termination

  sequencing

  • Requires a DNA fragment template, an oligonucleotide primer, DNA polymerase, dNTP’s and ddNTP’s

Next-Generation Sequencing

• Massively parallel sequencing

  • Millions of short reads on single chip

  • Massive sequence data generation at low cost

• Enables clinical whole-exome and even whole-genome sequencing

• Ushering in age of personalized/precision medicine

Next-Generation Sequencing: Multigene Panels

• Some Advantages of Gene Panels vs WES/WGS

  • Broad but phenotype-guided

    • No secondary findings

    • Greater mutation sensitivity

    • Often don’t need to send Trio

    • Faster result and lower cost

• Some Disadvantages of Gene Panels vs WES/WGS

  • Genes on panel vary by lab

    • Newly discovered/rare genes may not yet be on panel

• Cannot detect larger CNV’s (WES/WGS often can)

• Often can’t resolve pseudogenes, find intronic variants, count nucleotide repeats (WES also may not, WGS often can)

Whole Exosome/ Genome Sequencing

• 2021 American College of Medical Genetics and Genomics (ACMG) practice guidelines:

• Recommend exome or genome sequencing as 1st or 2ndtier testing for pediatric patients with unexplained congenital anomalies, dev delay, or intellectual disability

  • WES/WGS has higher diagnostic yield vs other genetic testing (ex. single gene, panels, CMA)

  • WES/WGS shows increased evidence of therapeutic impact

  • WES/WGS imparts lower diagnostic cost when used as a 1st or 2nd tier test in a diagnostic work-up

• Not all insurance plans agree!

  • Need to pre-authorize

  • Can be high cost to patient

Penetrance

Probability that a mutant allele causes any phenotypic effect:

• Complete vs Incomplete Penetrance

  • Complete Penetrance: 100% of individuals with a mutant allele will develop some features of a phenotype

• Incomplete Penetrance: Some individuals with a mutant allele will not develop any signs of that phenotype

Expressivity

Degree/severity of expression of a phenotype in affected individuals

• Variable Expressivity: Affected individuals differ significantly in degree/severity of phenotypic manifestations

• Non-Variable Expressivity: Affected individuals do not differ significantly in degree/severity of phenotypic manifestations

Pleiotropy

• When one gene has phenotypic effects on multiple organ systems

Incomplete Dominance

Heterogenity produce intermediat phenotype

Co-Dominance

• Heterozygosity produces a mixed phenotype (ABO Blood Groups: “A” allele and “B” allele produces blood type “AB”)

Autosomal Domain Conditions

Males and females equally likely t0be affected

 Phenotype present in heterozygous individual (homozygosity rare-usually more severe)

 Male to male transmission is seen

 Roughly 50% of offspring of an affected individual are affected (recurrence risk = 50%)

 Often de novo (new) mutation, especially if low reproductive fitness

Common Autosomal Dominant conditions

• Marfan’s Syndrome

• Achondroplasia

• Neurofibromatosis I & II

• Tuberous Sclerosis

• Autosomal Dominant Polycystic Kidney Disease (ADPKD)

• BRCA, HNPCC, FAP, Li-Fraumeni Syndrome

Marfan Syndrome

• Autosomal Dominant pleiotropic Disorder

  • Fibrillin 1 (FBN1) gene mutation

  • Iniceinde 1:5-10,000

  • 25% new mutations (De Novo)

• Fibrillin 1 Protein

  • Abundant connective tissue protein (aOrtam ligaments, ciliry zonules, other tissues)

  • Inverse assocation with TGF-B epression and inflamation

    • FBN1 down, TGF-B up, Cytokine up

• FBN1 mutation—> pleiotropi Multi-sytem effects

  • skeletal (Dolichostenomelia, Arachnodactyly, Thumb/Wrist Sign)

  • occular (Ectopia lentis)

  • pulmoanry (Pectorus/Aortic dlaiton)

  • skin

  • dural features

• comeple penerence

  • variable epxressvity (Intra and Inter-familial)

Marfan Sydnrome Inehrtiance

Autosomal Dominant

Complete Penetrance

Variable Expressivity

Pleiotropy

Achondroplasia

• an Autosomal Domainat disoer

  • FGFR3 gene mutation

    • 80% new (de novo) mutaitons (a mutaiton hotspot

  • Homozygosity lead to neonatal lethalit (incomeplte dominance)

    • recurrence risk 50% for offspring of one affected individual

    • Recurrence risk 66% for live offspring of two affected

• Non-Pleitropic

  • Dwarfism with disproportionare short stature

    • short armes and legs

    • large head

    • frontal bossing and mid-face hypoplasia

  • Intellegence and life span usually normal

• Comepltle penetrance

  • non-variable epxressvity

Autosomal Receisive Codntions

• Males and females equally affected

• Phenotype in homozygotes (sometimes in hemizygotes)

• Parents usually unaffected carriers

• Disease not in every generation

• Consanguinity increases risk

• Recurrence risk for parents: 25%

  • Carrier frequency affected by phenomenon like heterozygote advantage, population bottlenecks, and founder effects

Mechanisms for Increased Carrier Frequenceis

• Heterozygote advantage

  • selective advantage to heterozygosity: Hemoglobinopathy and Malaria resistance

• Population bottleneck

  • Population decrease and subsequent expansion: Ashkenazi Jewish genetic disorder

• Founder Effects

  • Large percent of population descends from number of individuals

    • CFTR Delta F508 mutation i ncauscaisn

Common Automsoal Recessive sydnromes

• Alpha-1-Antitrypsin Deficiency

• Cystic Fibrosis

• Hemochromatosis

• Sickle-Cell Disease

• Tay-Sachs Disease

• Many Metabolic Disease

Tay-Sach Disease

• Autosomal Recessive Lysosomal Storage Disorder

  • Neurodegrnetive infantile onset (most often)

  • Hexosaminidase A (HEXA) Enzyme Deficiency

    • GM2 Ganglioside accumulation in CNS→ neurodegeneration in retina→ cherry red spot

• Incidence

  • Worldwide: 1 to 360,000

  • Ashkenazi: 1 to 3,600 (carrier frequency 1:30)

  • also in Quebec French Candian, Cajun, Amish groups

X-Linked Recessive

Skewed male:female ratio

 Males affected; M>F ratio

 No male-male transmission

 Fathers only pass Y chromosome

to their sons

 Females may be carriers

 Usually unaffected (lyonization)

 De novo mutations usually in male meiosis

 Carrier female, recurrence risk=25%

 50% of sons affected, 50% unaffected

 50% of daughters carriers, 50% not carriers

X-Linked Dominant

No male-male transmission (sex-linked)

 Disorder is manifest in the heterozygous

female

 Often more severe in males (even lethal)

 Less common than X-Linked Recessives

X-Linked Disorders

• X-Linked Recessive

  • Hemophilia A & B

  • Duchenne Muscular Dystrophy

  • Red-Green Color Blindness

  • Glucose-6-Phosphate Dehydrogenase Deficiency

• X-Linked Dominant

  • Rett Syndrome

  • Incontinentia Pigmenti

Hemophilia

• X-Linked recessive inheritance

  • 1/3 of cases have no fam hx of hemophilia (De novo)

  • Males with life threating bleeding episode

  • Females carriers, usually asymptomatic

• Coagulation Factor Deficiency

  • Hemophilia A: Factor VII Deficiency

    • 1:10,000 males

  • Hemophilia B: Factor IX Deficiency

    • 1:20,000 males

• Treatment

  • Factor replacement therapy

Incontinentia Pigmenti

• X-Linked Domaint Inheritance

  • Females only viable, lethal in males

  • Almost always De Novo mutation

• Genetic Defect

  • IKBKG (NEMO) gene

• Clinical Feaures

  • Dermatologic

    • newborn-blisterning rash laong line of Blaschko

    • Follwed by chronic weirled hyper and hypogpigmenation

• Neurologic

  • Epilsopu and mental reatrdation

• Ectodermal dysplasia

Y-LINKED INHERITANCE

Only affects males

Male to male transmission only

 Few genes on the Y Chromosome

Most code for spermatogenesis and male gender

Mutations result in female gender or male infertility

 Frequency may change with Intra-Cytoplasmic

Sperm Injection (ICSI).

Mitochondrial (Maternal) Inheritance

mtDNA mutations: Inherited only through the motherInherited only through the mother

 All mitochondria provided by the OvumAll mitochondria provided by the Ovum

 No mitochondria provided by spermNo mitochondria provided by sperm

 Transmission only through femalesTransmission only through females

 No transmission through malesNo transmission through males

(mtDNA or nDNA mutations can cause mitochondrial dysfunction)(mtDNA or nDNA mutations can cause mitochondrial dysfunction

Heteroplasmy and Threshold Effect

• Heteroplasmy: The presence of both normal and mutated mtDNA within a cell or individual.

• Threshold Effect: The concept that a certain proportion of mutated mtDNA must be present before mitochondrial dysfunction and disease symptoms occur.

Nucelotide Repeat Disorders

• Genome contain mutliple areas with repative nucleotide squeqnces, some within/near genes

  • ex: trinucleotide repeats

• Can become unstable and epxand in meisoiis

  • norma→Greyzone→Premutaiton→Full Expansion

• Shoe “anticpation”

  • incresing repeat size correlating with increing severity/earlier onset in sucessib gnerations

• Exmaples

  • Huntingtons diease

  • Fragile X syndrome

  • Spinocerebrellar Ataxia

  • Myotonic Dystrpy

Fragile X Syndrome

• #1 Genetic Cause of Mdoerate Menatal etardation

• X-Linked nucleorid repat disoer

  • CGG expaions effect FMR1 gene epxression—> cause mthylation

  • Exapsion occurs through premutation carrier female meisoses

    • 60-200 repeat : PRemuation

    • >200 repeats: Full mutation, no FMR1 expression

• Diagonsis:

  • FMR1 Gene Trinucleotide Repeat Testing

• Disease Prevalence:

  • 1:3,600 male

  • Premutation carriersL 1:260 female, 1:8000 males

  • Prenatal FMR1 Repeat screening in females

Bardet-Biedl Sydnrome

• Cardinal Features

  • Obeisty >75%

  • Retinitis Pigmentosa>75%

  • Mild Mental Retardation >75%

  • Hypogonadism >75%

  • Postaxial polydactyly 66%

  • Renal abnromalties 40%

  • Disctive Facies