Single Gene Mutations and Inheritance I

April 3, 2008

Lisa Schimmenti, M.D.

Objectives

• Understand the basic types of single gene mutations

• Understand how mutations lead to abnormal protein expression

• Understand how a dominant mutation is passed from parent to child (inherited)

• Know retinoblastoma genetics: dominant inheritance, but recessive on the cellular level.

Mutations are a change in DNA sequence that leads to a change in protein expression

ASCO

The genetic code

A codon is made of 3 base pairs

64 codons total

1 codon (AUG) encodes 1 codon (AUG) encodes methionine methionine andand starts starts

translation of all proteinstranslation of all proteins

3 codons stop 3 codons stop protein translationprotein translation

60 codons encode 20 60 codons encode 20 amino acidsamino acids

(redundant code)(redundant code)

U A A

A U G

Met

G C A

Ala

Genetics Vocabulary

• Allele: refers to different forms of the same gene

• Example: – wildtype allele– mutant allele– null allele

More new words

• Phenotype: the physical appearance of a trait

• Genotype: the allele associated with a trait

ASCO

Disease-Associated Mutations

A mutation is a change in the normal base pair sequence that has been proven to associate with a disease state

Commonly used to define DNA sequence Commonly used to define DNA sequence changes that alter protein functionchanges that alter protein function

ASCO

Disease-Associated Mutations Alter Protein

Function

Functional proteinFunctional protein Nonfunctional or Nonfunctional or missing proteinmissing protein

Classes of Mutations

Type of mutations

• Missense: nucleotide change leads to amino acid change

• Nonsense: change leads to stop codon• Insertion: addition of nucleotides that

lead to frameshift• Deletions: deletion of nucleotides• Splice site: changes RNA splicing• Expansion of repeat units

Nucleotide substitutionscan occur anywhere in the

genome

A

TG

C

Transversions Pyr -> PurPur -> Pyr

Transitions Pyr -> PyrPur -> Pur

Missense mutation in coding sequence

Normal sequence

ATG CCG TTT

Met Pro Phe

Mutant Sequence

ATG CTG TTT

Met Leu Phe

Nucleotide changes favor certain spots

• CpG dinucleotide– C frequently methylated– Spontaneous deamidation to T

• 8.5 x more likely to change than any other dinucleotide pair

Insertions/Deletions

• Insertions (1 bp or >1 bp)

• Deletions (1 bp or >1 bp)

≤ 3 bp change (coding)• ins or del

– 3 bp - ins or del one AA (most common mutation in CF delF508)– 2 bp ins or del - frame shift– 1 bp ins or del - frame shift

• 1 bp substitution– Silent - “wobble”– Nonsense - AA to “stop”– Missense

• Conservative - changes AA to same or similar AA• Non-conservative - changes AA by function or charge• May not change a basepair but may change the RNA splice recognition

site

Changes in > 3 bp

• Whole gene deletions or duplications– Often due to repeated sequences and

mispairing in replications

• Portion of gene deleted or fragment inserted– Often due to aberrant splicing

What does it look like?

THE BIG RED DOG RAN OUT.

THE BIG RAD DOG RAN OUT.

THE BIG RED.

THE BGR EDD OGR ANO….

THE BIG RED DOO GRA NOU

THE BIG DOG RAN OUT.

THE BIG BIG BIG BIG BIG RED DOG RAN OUT.

Normal

Missense

Nonsense

Frameshift (1 bp deletion)

Frameshift (1 bp insertion)

Frameshift (3bp deletion)

Triplet repeat expansion

Modifed from ASCO

Non-coding mutations that affect gene function

• Promoter/enhancer element Trinucleotide repeats in 5 and 3’UTRs

• Splice sites

Factors that influence how mutations alter gene function

• Increase or decrease expression of gene product

• Is one altered allele sufficient to cause disease?

• Modifiers • Proportion of cells affected• Parent of origin

Effects of mutation on gene product

• Null allele (loss of function) - no gene product• Hypomorph - decreased amt/activity

• Gain of function - increased amt/activity• Dominant negative - antagonizes normal product• Neomorph - novel activity of product

Predicting that a gene product won’t do the job

• Deletion, nonsense, frameshift of sequence is deleterious

• Mutation in splice site usually bad

• Missense mutations– Depends on location in protein– Is it non-conservative?

• Is the AA conserved in evolution?

Which ones will cause disease?

• All but silent or conservative missense sequence changes are likely to significantly alter product function

• Among frameshifts, location of mutation alters likelihood of severity

• Mutations in coding sequence are identified most frequently…but this may change

You only see the ones that hurt...

• Sequence changes or variants can be – Silent (no change in product): Polymorphism

• We have thousands of these

– Manifesting (changing RNA or protein product): Mutation

We all carry 7-10 gene mutations

AHHHH!

Polymorphism

• A change in DNA sequence that is not disease causing

• Occurs usually in greater than 1% of population

• Usually does not change an amino acid sequence or produces a significant change (ie: valine for isoleucine)

Autosomal Dominant

Autosomal Dominant Inheritance

• males and females equally affected• 1 in 2 chance of affected offspring

from an affected parent• male to male transmission• structural genes, transcription

factors• only one abnormal copy of the

gene (allele) to have the phenotype

Mechanisms yielding dominant alleles

• Haploinsufficiency: if one copy isn’t enough for function

• Dominant negative: when a mutant product dimerizes or oligomerizes

• Gain of Function: FGFR mutations causing activation of the receptor without a ligand present

Autosomal Dominant probabilities

D

d

d

d

Dd dd

Dd dd

Daughters 50% normal50% affected

Sons 50% normal50% affected

Mother

Fat

her

Variations in the AD “rules”

• Co-dominant expression• New mutation• Homozygosity for an AD trait

– May be a more severe phenotype

• Variable expression• Penetrance• Sex limited• Variation in age of onset

Variations:Co-dominant expression

Definition: alleles that are both expressed when they occur in the heterozygous state.

Example: ABO blood group antigens

ABO Blood Groups

Defined by the presence or absence of two antigens on the surface of the red cell

A and B• if you have A, you have anti-B antibodies • if you have B you have anti-A antibodies • if you have neither (O) you have both anti- A and -B• Absence of A or B = O• Three allele states: Ia Ib Inull

ABO blood typing: co-dominant alleles

A/- B/- B/- BB

BBB/-ABA/--/-

AB BBA/-

Variations: New Mutations

• Occur at a rate of 10-4 to 10-7 per locus per cell division

• “Hot spots” occur: CG pairs (8.5 x higher rate)

• More easily seen in a very large gene

• Paternal age effects

Autosomal dominant: new mutation

dd dd dd dd

dddddddddd

dd ddDd

Achondroplasia• Autosomal dominant

• 100% penetrance

• 1/26000: most common genetic dwarfism

• 80% new mutations

• Features– rhizomelic dwarfism (upper arms and thighs are

short) – lumbar lordosis (lower spine curves out)– large head– small foramen magnum – intact intellect– Mutation in FGFR3: activating mutation

Fibroblast Growth Factor Receptors

• Receptor tyrosine kinases

• Four distinct genes: 1 through 4

• Critical in coordination of proportionate bone growth and development

• Signaling regulates cellular processes

Achondroplasia and FGFR

• Mutations in FGFR3 cause achondroplasia

• Despite frequent new mutations almost all are caused by the same mutation (G -> A at nt 1138, causes gly 380 arg)

Effects of homozygosity

dd dd dd dd

ddDdDddd

dd DdDD

Homozygosity for some dominant traits is lethal

Homozygosity for achondroplasia causes very severe phenotype

http://www.echt.chm.msu.edu/courseware/blockII/Pathology/382131.jpeg

AD and variable expression

• Pleiotropy: one gene, many effects on different systems

• Random chance

• Modifier genes

Marfan Syndrome: variable expression

• Skeletal findings- disproportionate long bone growth

• Tall Stature• Eyes- dislocated

lenses• Heart-dilation of

aortic arch http://medgen.genetics.utah.edu/

AD traits are often variable in expression

Dd dd dd dd

ddddDdDd

dd DdDd

dd

Normal

TallDislocated lensesDilated Ao

TallDilated Ao

Dislocated lensesDilated Ao

Variations: reduced penetrance

• An individual may carry an altered allele and never manifest phenotypic evidence of this.

• Penetrance: an “all or nothing” phenomenon in expression of a trait in a population–phenotype, NOT genotype

Tumor Suppressor GenesClinical Effects of Mutations

• Cancers due to mutations are early in onset• Cancers due to mutations are often bilateral• Phenotype is inherited as a dominant trait BUT at the

cellular level these are manifest as recessive: two abnormal alleles are required for manifestation

• Genotype is that of one germline abnormal allele: malignancy occurs with environmental damage of second normal allele (Knudsen two hit hypothesis)

• Penetrance reduced: no 2nd hit, no malignancy occurs

Variation: reduced penetrance

dd Dd dd dd

ddddDddd

dd Dddd

Familial Retinoblastoma: a recessive state in the cell

inherited as AD trait

http://www.retinoblastoma.com/Images/CT%20Scan.jpg

http://www.people.virginia.edu/~rjh9u/retblst.html

Familial RetinoblastomaCase history:6 month old child found to have leucocoria (white pupil) On exam

there is a large mass in the eye. The other eye has a small lesion

Father gives the history that his mother had only one eye because the other was removed when she was a baby. He and his 2 sibs were healthy and have both eyes

Explanation: REDUCED PENETRANCE of this trait

Autosomal Dominant: sex limited trait

Dd dd dd dd

ddddDddddd

dd ddDdDd

Dd

A sex-limited AD trait

• Familial male precocious puberty • Pubertal onset in males by age 4.• Due to gain of function mutation

that keeps the leutinizing hormone (LH) receptor in an “on” state

Autosomal Dominant: variation in age of

onsetDd dd dd dd

ddddDdDddd

dd ddDd

Dd dd

dd Dd

Variation:Altered age of onset

• Huntington’s Chorea– Progressive dementia

– Choreiform movements

• Autosomal Dominant pattern

• Age of onset usually after 30

• Triplet repeat expansion in the Huntington gene that can expand

What to expect for the next lecture:

• Recessive inheritance

• X-linked inheritance– Dominant– Recessive

•Population genetics for docs

Example Question• Find the one true statement about dominant

inheritance :– A. Male are more commonly affected than

females.– B. Two copies of the gene carry mutations to have

the phenotype– C. Only gain of function mutations are dominant.– D. Haploinsufficiency mutations can only cause

dominant disorders.– E. Mutations can be passed from fathers to sons.