RETand HSCR

Question 1

What is Harald Hirschsprung
(1830-1916) famous for?

Answer 1

Constipation associated with dilation and hypertrophy of the colon

Question 2

Describe Hirschsprung’s Disease (HSCR)

Answer 2

• HSCR is characterized by the ABSENCE of enteric ganglia belonging to the hindgut. Usually the abnormalities are restricted to the last few centimeters of the distal colon, but they can extend to the proximal colon or the distal small intestine.
• The aganglionic gut is tonically contracted. Symptoms range from life threatening total obstruction to chronic constipation or fecal incontinence.

Question 3

Describe enteric ganglia distribution

Answer 3

The enteric ganglia are distributed throughout the gut wall 500 million neurons.

Question 4

TRUE or FALSE: Both the submucosal and myenteric ganglia
are missing in HSCR

Answer 4

TRUE

Question 5

What was the first clue that RET was involved in HSCR?

Answer 5

• The neuronal phenotype of RET-/- mice resembles that of humans suffering HSCR.
• The phenotype also resembles that of mice lacking GDNF and mice lacking GFRa

Question 6

RET -/- Mice

Answer 6

1) Die soon after birth
2) Lack enteric neurons throughout the digestive

tract

3) Abnormal kidney development

(A study of human stillborn foetuses in which the kidneys

failed to form showed an association with RET mutations)

Question 7

The Genetics of RET in HSCR

Answer 7

• At least 24 different genes have been strongly associated with HSCR
• Heterozygous germline mutations in RET account for 50%of familial HSCR patients (about 20% of sporadic).
• Functional loss of a single copy of the gene is sufficient tocause pathology in some patients suffering from HSCR.(inheritance is dominant but shows incomplete penetrance).
• A large number of “gene modifiers” contribute to the severity/lack of severity of the phenotype.
• Gene modifiers include the genes for the neuropeptide endothelin-3 (ET-3) and its G-protein coupled receptor, endothelin receptor (EDNRB) as well as non-coding single nucleotidepolymorphism variants in the vicinity of RET and SEMA3 (semaphorin 3).

Question 8

A FAMILY WITH HSCR

Answer 8

All subjects with HSCR carry the m (mutant) allele

Question 9

Interpretation of Affected Heterozygotes

Answer 9

• Two wild-type copies of RET may be required for normal RET function. Therefore receptor number or amount of signal is crucial for at least some RET functions.
• You can’t always get by with one good allele

Question 10

RET mutations in HSCR

Answer 10

• Deletions of the whole gene Intragenic deletions and insertions Nonsense mutations (also called STOP/GAIN) Splicing errors
• Missense mutations throughout the coding region
• SNPs in non-coding regions (promoters and introns) which decrease expression

Question 11

The Location of the HSCR Mutation Determines the
Functional Consequence of the Mutation

Answer 11

Question 12

Why do we get MEN2A and HSCR in the same patient?

Answer 12

• Constitutively active receptors that don’t bind ligand may not signal strongly enough in the enteric neurons.
• Mutations in RET which cause constitutive activation often reduce the expression of RET at the plasma Membrane.

Question 13

Describe RET Expression in Normal Mouse Development

Answer 13

1) Central nervous system (motor and catecholaminergic neurons)
2) Cells of the peripheral nervous system

derived from the neural crest including sensory, autonomic, and enteric ganglia

3) C cells of the thyroid
4) Kidney organogenesis

Question 14

RET is Expressed in Cells Derived from the
Neural Crest including the enteric ganglia

Answer 14

Question 15

What is GDNF (Glial Derived Neurotrophic Factor?

Answer 15

• GDNF was discovered in the search for factors that promote the survival of midbrain dopaminergic neurons, in the hopes of finding a treatment for Parkinsons
• GDNF supports the survival in vitro of sensory, sympathetic, and motor neurons
• In vitro GDNF is a potent chemotrophic, mitogenic and trophic factor for neural-crest-derived enteric progenitors

(progenitors can both divide and give rise to cells which differentiate)

Question 16

Describe RET and GDNF in the Enteric Nervous System

Answer 16

• Vagal neural crest cells (NCC—shown as neuroblast) express RET and GFRa1 during their migration through the intestinal mesenchyme and subsequently in the myenteric and submucosal plexuses. Most ENS progenitors are Vagal NCC.
• GDNF is expressed in the mesenchyme of the gut wall.

Question 17

Migration of NCC in the Gut

Answer 17

Enteric Nervous System progenitors originate in the Vagal neural crest (NC).

The NCC migrate through the foregut and midgut to reach the hindgut. In a mouse, the process takes several days.

Question 18

Evidence that NCC Migrating through the Gut Express RET

Answer 18

• NCC migration in transgenic mice in which one allele of RET is driving GFP. (confocal microscopy of a whole mount)
• Phox2b is a transcription factor expressed in NCC

Question 19

Migration of NCC

Answer 19

Explants of E12.5 hindgut in the mouse. Time Lapse of GFP-labelled NCC.

Question 20

Timing of NCC Migration in the Gut

Answer 20

• In humans, NCC enter the foregut during the 4th week of gestation.
• When the NCC enter the foregut, they rapidly proliferate
• By week 7.5, they have reached the hindgut.

Question 21

Hypothesis

Answer 21

• The absence of enteric neurons in HSCR patients with mutations in RET results from the failure of neural crest cells destined for the gut to survive, proliferate, migrate, or differentiate.
• There is evidence to support all of these.

Question 23

Describe current and future therapies

Answer 23

• Current Therapy: Surgery to remove aganglionic bowel
• Refractory constipation and fecal incontinence affecting between 10% and 30% of patients,
• Future Therapy: Stem cell replacement of missing neurons

Question 24

Proposed Steps involved in Stem Cell Therapy for HSCR

Answer 24

Question 25

Alternate Approach: IPSC and CRISPR/CAS9

Answer 25

Correction of Hirschsprung-Associated Mutations in HumanInduced Pluripotent Stem Cells Via Clustered RegularlyInterspaced Short Palindromic Repeats/Cas9, RestoresNeural Crest Cell