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Of Mice and Men and MCM4: A Novel Cause of Multisystem Dysfunction. Jason Klein, M.D. Fellow, Pediatric Endocrinology Fellows Journal Club, CCMC. Goals and Objectives. Appreciate the importance of investigating interesting phenotypes

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of mice and men and mcm4 a novel cause of multisystem dysfunction

Of Mice and Men and MCM4: A Novel Cause of Multisystem Dysfunction

Jason Klein, M.D.

Fellow, Pediatric Endocrinology

Fellows Journal Club, CCMC

goals and objectives
Goals and Objectives
  • Appreciate the importance of investigating interesting phenotypes
  • Understand the basic principles of mapping genes in pedigrees affected by a common phenotype
  • Understand how specific gene defects are mapped
  • Understand how to prove causality (that gene defects cause observable disease)
  • Understand the typical presentation and molecular causes of Familial Glucocorticoid Deficiency (FGD)
  • Understand the role of the natural killer (NK) cell in human immunity
  • Recognize the typical phenotype of mini-chromosome maintenance complex component 4 (MCM4) deficiency
  • Recognize the phenotypic differences between the MCM4 knockout mouse model and humans
  • Know the role of MCM4 in cell cycling and appreciate the effects of MCM4 loss
origins of research
Origins of Research
  • 2004 – Siblings with NK deficiency
    • Elder deceased from CMV
    • Younger developed osteosarcoma
    • Both IUGR, neutropenic, transient lymphopenia
  • 2006 – 4 Irish Travelers, consanguineous kindred
    • NK deficiency
    • Viral infections (EBV, respiratory illnesses)
    • Adrenal insufficiency
    • Ante- and post-natal growth retardation/microcephaly
phenotype
Phenotype
  • Hypocortisolemia with elevated ACTH
    • Milder that other forms of glucocorticoid deficiency, including FGD
    • Typically appeared after period of normal function
  • Normal plasma renin and aldosterone
    • Normal mineralocorticoid function
  • Short stature
  • NK deficiency/Frequent infections
adrenal insufficiency
Adrenal Insufficiency
  • Glucocorticoid (Cortisol)
    • Hypoglycemia
    • Muscle weakenss
    • “Tanning” of non-exposed areas AKA hyperpigmentation (due to ACTH effect in PRIMARY DISEASE, NOT CENTRAL DISEASE)
  • Mineralocorticoid (Aldosterone)
    • Hyponatremia, Hyperkalemia
    • Salt-craving behavior
    • Dehydration
    • Dizziness
    • Weight loss
    • Hypotension
  • Adrenal Androgens
  • Adrenal Crisis
familial glucocorticoid deficiency fcd
Familial Glucocorticoid Deficiency (FCD)
  • Autosomal recessive disease
  • Primary isolated glucocorticoid deficiency
    • Low/undetectable serum cortisol
    • Elevated plasma ACTH
  • Childhood presentation
    • Fasting hypoglycemia  seizure, learning disabilities
    • FTT
    • Recurrent infections
    • Hyperpigmentation (melanocortin 1 receptor)
    • Death (if untreated)
familial glucocorticoid deficiency fcd1
Familial Glucocorticoid Deficiency (FCD)
  • ~50% due to:
    • Defects in ACTH receptor (MC2R)
    • Defects in MC2R-accessory protein (MRAP)
    • Defects in steroidogenic acute regulatory protein (STAR)
mc2r and mrap
MC2R and MRAP
  • Melanocortin 2 Receptor (ACTH Receptor)
    • 21q22.1
    • 7 transmembrane G-coupled receptor on cell surface
    • Mutations first discovered in 1993
    • Over 40 pathogenic mutations
    • Most common of known mutations
  • Melanocortin 2 Receptor Accessory Protein
    • Small single transmembrane accessory protein
    • Facilitates MC2R trafficking from ER to cell surface
slide12

FCD due to failure of ACTH signalling due to:

INEFFECTIVE LIGAND BINDING

INEFFECTIVE SIGNAL TRANSDUCTION

FAILURE OF RECEPTOR TRAFFICKING

slide14
STAR
  • Steroidogenic acute regulatory protein
  • Chromosome 8
  • Transport of cholesterol across mitochondrial membrane
  • More severe than FGD patients
    • Adrenal + Gonadal insufficiency
    • Elevated ACTH and Renin
    • Low cortisol and aldosterone
    • Failure of androgenization in males
  • Lipoid congenital adrenal hyperplasia
familial glucocorticoid deficiency fcd2
~50% due to:

Defects in ACTH receptor (MC2R)

Defects in MC2R-accessory protein (MRAP)

Defects in steroidogenic acute regulatory protein (STAR)

~50% due to:

???

Familial Glucocorticoid Deficiency (FCD)
phenotype1
Phenotype

Hypocortisolemia with elevated ACTH

Milder that other forms of glucocorticoid deficiency, including FGD

Typically appeared after period of normal function

Normal plasma renin and aldosterone

Normal mineralocorticoid function

Short stature

NK deficiency/Frequent infections

natural killer nk cells
Natural Killer (NK) Cells
  • Lymphocytes of innate immune system
  • Essential role in host defense
  • Deficiency of MHC class 1 on cell triggers attack
  • Activation results in
    • Release of cytokines
    • Stimulation of other immune cells
    • Contact-dependent cytotoxicity of targeted cells
      • Perforin, proteases
  • Susceptibility to infection with viruses
    • Herpesvirus
    • Papillomavirus
hypothesis
Hypothesis?
  • Link between
    • Adrenal insufficiency
    • Selective NK cell deficiency
    • Predisposition to viral infections
    • Growth retardation
    • Predisposition to cancers/malignancy
pedigrees
Pedigrees

Absolute numbers (per mm3 of whole blood) and percentages (% lymphocytes) of NK cells

multipoint linkage analysis
Multipoint Linkage Analysis
  • Genetic linkage – tendency of genes located proximal to each other on a chromosome to be inherited together
    • Less likely to be separated onto different chromatids during chromosome crossover
  • LOD Score – statistical test that compares the likelihood of obtaining the test data if the two loci are indeed linked to the likelihood of observing the same data purely by chance
    • Higher LOD, more likely to be genetically linked
    • +3 = 1000:1 odds that the linkage did not occur by chance
step 1 where is the genetic mutation1
STEP 1: Where is the genetic mutation?
  • Previously known area at centromeric region of chr 8
  • LOD 6.45 at marker D8S532
  • Confirmed in second kindred (founder effect)
  • Fine mapping with narrower region of interest (8p11.23-q11.21)
  • No obvious gene candidates
  • Complete sequencing of region
step 2 what is the genetic mutation
STEP 2: What is the genetic mutation?
  • Homozygous substitution (c.71-2A  G) in MCM4
  • Frameshift mutation (insertion of c.70_71insG)
  • Premature stop codon
  • Truncated protein
  • FOUND IN ALL 6 PATIENTS
  • PARENTS ALL HETEROZYGOUS
step 3 what is the result of this mutation
STEP 3: What is the result of this mutation?
  • B-cells and fibroblasts of patients and controls
    • Similar amounts of MCM4 mRNA
    • Mutation does not destabilize mRNA
  • Protein
    • NO FULL LENGTH MCM4 protein (~100 kDa) using Western blot
    • 2 new smaller proteins (~95 kDa and ~90 kDa) in patients
double check
DOUBLE CHECK
  • Transfect affected fibroblasts with WT MCM4 allele  normal MCM4 protein
  • Transfect control cells with abnormal MCM4 cDNA  presence of the two more rapidly migrating bands
step 4 why are these short proteins made
STEP 4 - Why are these short proteins made?

Pos 51

Pos 75

Missing the N-terminal domain

hold on what the heck is mcm4
HOLD ON! WHAT THE HECK IS MCM4?
  • Mini-chromosome maintenance complex component 4
  • Part of a heterohexameric complex (MCM2-7)
  • Responsible for normal initiation and elongation phases of DNA replication
step 5 what are these short proteins
STEP 5 – What are these short proteins?
  • Are they pseudo-MCM4?
  • Able to form MCM replication complex in fibroblasts of affected patients
step 6 what happens at the cellular level
STEP 6 – What happens at the cellular level?
  • Cell cycle in affected fibroblasts
    • Low G1
    • Low S
    • High G2/M
  • DNA content of affected fibroblasts
    • Decreased
    • Amplified with aphidicolin (inhibitor of replication)

DISRUPTION OF DNA REPLICATION BY DISRUPTING PREVENTION OF RE-REPLICATION AND IMPACTING MITOTIC PHASE

chromosome fragility
Chromosome Fragility
  • Fanconi anemia – short stature, bone marrow disease/failure, neurodevelopmental problems, renal disease

When WT MCM4 transfected into affected cells, reduction of breakage back to baseline

step 7 how does this create the adrenal phenotype
STEP 7 – How does this create the adrenal phenotype?
  • Adrenal glands of MCM4-depletion mouse model show abnormal morphology
    • Small, tightly packed, intensely stained spindle-shaped cells in the cortex
    • Negative for CYP11A1 and CYP11B1
    • Unable to produce glucocorticoid
of mice and men and mcm4
Of Mice and Men (and MCM4)
  • Cancer
  • Developmental Delay
  • Embryonic Death
  • Why is this so severe in mice and not in humans???
step 7 how does this create the nk phenotype
STEP 7 – How does this create the NK phenotype?

NKCD56dim – 90%, cytolytic

NKCD56bright – 10%, produce IFN-γ in response to cytokines

Blocking of differentiation of NK cells into cytolytic CD56dim

step 7 how does this create the nk phenotype1
STEP 7 – How does this create the NK phenotype?

CD56bright does not proliferate in response to IL-2

Increased spontaneous apoptosis

patient outcomes
Patient Outcomes?
  • Steroid replacement
  • Frequent infections
  • Growth hormone?
  • Risk of cancer?
  • Possibility of gene therapy?
summary
Summary
  • MCM4 is a ubiquitous gene product for a universally required cellular function
  • Autosomal recessive inheritance
  • Phenotype - glucocorticoid deficiency, growth retardation, NK deficiency, predisposition to viral infections, cancers (?)
  • Necessary to form MCM2-7 complex in DNA replication and cell proliferation
summary1
Summary
  • Frameshift mutation causing premature stop codon
  • Short protein product fragments with function
  • Adrenal pathology due to structural defects and loss of glucocorticoid production
  • NK cell pathology due to loss of differentiation and early apoptosis
  • Growth pathology due to fibroblast dysfunction? Other means?
  • Pizza will get fellows to come to most anything