Genet 302 lec 29 & 30
What gene is emphasized in the genetics of muscular dystrophy lecture?
DMD- the longest human gene
Where is the human DMD gene located?
Xp21
What protein does the DMD gene encode?
Dystrophin
What is a key function of dystrophin in muscle cells?
It links the intracellular actin cytoskeleton to the membrane-associated complex and extracellular matrix
Why is dystrophin important for skeletal muscle cells?
It helps stabilize muscle fibers during contraction
What is muscular dystrophy in this lecture?
Progressive degeneration and loss of skeletal muscle cells caused by mutations in DMD
What does dystrophy mean?
Degeneration of a tissue
What is the inheritance pattern of DMD-related muscular dystrophy?
X-linked
What genotype typically gives an affected male with severe early disease?
DMD−/Y
Why are males usually more severely affected by DMD mutations?
Because they have only one X chromosome, so a mutant DMD allele leaves them with no normal copy
What genotype can give an affected female?
DMD+/DMD−
Why can DMD+/DMD− females vary in severity?
Because severity depends on the pattern of random X inactivation
How do in-frame deletions usually affect the DMD phenotype?
They usually cause Becker muscular dystrophy, which is milder
How do out-of-frame deletions usually affect the DMD phenotype?
They usually cause Duchenne muscular dystrophy, which is more severe
Why are in-frame deletions usually milder?
Because the reading frame is preserved, so a shorter but partly functional dystrophin protein can still be made
Why are out-of-frame deletions usually more severe?
Because the reading frame is disrupted, so little or no functional dystrophin is produced
What is the difference between Becker and Duchenne muscular dystrophy at the protein level?
Becker usually has a shorter hypomorphic dystrophin protein, while Duchenne usually has no functional dystrophin
Why is the DMD gene especially prone to deletion mutations?
It is the longest human gene
What big genetics rule from this lecture links mutation type to severity in muscular dystrophy?
In-frame deletion tends to give Becker MD, while out-of-frame deletion tends to give Duchenne MD
What is the mdx mouse strain?
A mouse model of muscular dystrophy caused by a mutation in the X-linked Dmd gene
What does mdx stand for?
Muscular dystrophy X chromosome
What type of mutation causes the mdx allele?
A spontaneous nonsense mutation
What is the molecular problem in mdx mice?
A premature stop codon in Dmd prevents normal dystrophin production
Why is mdx a good model for Duchenne muscular dystrophy?
Because Duchenne MD is caused by severe loss of functional dystrophin
How can Sanger sequencing look distorted in a heterozygote or somatic mosaic?
Mixed templates produce overlapping peaks at the variable site
How do you deconvolute mixed Sanger sequencing results in this lecture?
PCR the target region, clone individual PCR products into plasmids, transform E. coli, then sequence separate clones
Why does cloning PCR products help deconvolute a mixed sequence?
Each bacterial colony usually contains one recombinant plasmid with one PCR product, so each sequence is read separately
Can one E. coli cell usually take up more than one recombinant plasmid in this method?
No
What was the goal of the CRISPR experiment in mdx zygotes?
To correct the mdx Dmd mutation and restore dystrophin
What reagents were injected into mdx zygotes in the HDR strategy?
Cas9, sgRNA, and an ssODN donor DNA
What type of repair was the main intended strategy for correcting mdx in zygotes?
CRISPR plus HDR
What kind of reverse mutation were they trying to make in mdx?
An equivalent reverse mutation
What is an equivalent reverse mutation?
A repaired sequence that restores function but is not necessarily the exact original wild-type DNA sequence
Why was the donor DNA not an exact match to the target sequence?
To prove the repair used the donor and to prevent the repaired gene from being cut again by CRISPR
What 2 gene-level outcomes were observed after CRISPR editing of mdx?
HDR-mediated correction and NHEJ-mediated correction
How can NHEJ correct the mdx mutation without exact HDR repair?
By making an in-frame deletion that removes the premature stop codon
What kind of NHEJ corrections were shown in the lecture?
In-frame deletions of 12 bp and 3 bp
Why can an in-frame deletion rescue the mdx allele?
Because the reading frame is preserved and the stop codon is removed, allowing dystrophin production
Why was deconvolution needed when sequencing a corrected mouse?
Because the mouse was mosaic and contained a mixture of corrected and uncorrected DNA
What did deconvolution reveal for the example HDR-corrected mouse?
About 41% of its DNA was repaired
How was correction measured at the tissue level?
Muscle biopsies were stained with anti-dystrophin antibodies
What did dystrophin staining show in corrected mice?
Restored dystrophin expression in muscle fibers
How was correction measured at the animal level?
With a forelimb grip strength test
How did the corrected mdx mouse perform in grip strength compared with mdx and wild type?
It was much stronger than mdx and close to wild type
What is the full mdx plus CRISPR workflow you need to know?
Start with mdx zygotes, inject Cas9 plus sgRNA plus donor DNA, reimplant embryos, identify corrected offspring, verify repair by sequencing and deconvolution, check dystrophin restoration in muscle, then test improvement in whole-animal muscle strength
What was the big takeaway from the 2014 mdx CRISPR experiment?
It was proof of concept that CRISPR editing of germline DNA could prevent muscular dystrophy in mice
How did the lecture distinguish the 2014 work from later CRISPR muscular dystrophy work?
2014 was proof of concept in mouse zygotes with a mouse-specific mutation, while later work moved toward prototype treatment of human-specific mutations in muscle of newborn animals