jason klein, m.d. fellow, pediatric endocrinology fellows journal club, ccmc of mice and men and...
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Jason Klein, M.D.Fellow, Pediatric Endocrinology
Fellows Journal Club, CCMC
Of Mice and Men and MCM4: A Novel Cause of
Multisystem Dysfunction
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• 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• 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 Gland
• 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
Adrenal Insufficiency
ACTH-related Hyperpigmentation
• 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 (FCD)
• ~50% due to:– Defects in ACTH receptor (MC2R)– Defects in MC2R-accessory protein (MRAP)– Defects in steroidogenic acute regulatory
protein (STAR)
Familial Glucocorticoid Deficiency (FCD)
Adrenal Steroidogenesis
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
FCD due to failure of ACTH signalling due to:
INEFFECTIVE LIGAND BINDING
FAILURE OF RECEPTOR
TRAFFICKING
INEFFECTIVE SIGNAL TRANSDUCTION
Adrenal Steroidogenesis
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
Adrenal Steroidogenesis
• ~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)
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
• 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
Natural Killer (NK) Cells
NK Cell Function
NK Cell Activity
NK Cell Activity
Hypothesis?• Link between
– Adrenal insufficiency– Selective NK cell deficiency– Predisposition to viral infections– Growth retardation– Predisposition to cancers/malignancy
Pedigrees
Absolute numbers (per mm3 of whole blood) and percentages (% lymphocytes) of NK cells
Study Patients
STEP 1: Where is the genetic mutation?
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 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? • 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
MCM4 - Substitution
MCM4 – Frameshift in cDNA
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• 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?
Pos 51 Pos 75
Missing the N-terminal
domain
STEP 4 – Why are these short proteins made?
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
Pre-replication complex (pre-RC)
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?
• 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• Fanconi anemia – short stature, bone
marrow disease/failure, neurodevelopmental problems, renal disease When WT
MCM4 transfecte
d into affected
cells, reduction
of breakage back to baseline
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)• 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?NKCD56dim – 90%,
cytolyticNKCD56bright – 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 phenotype?
CD56bright does not
proliferate in response to IL-
2
Increased spontaneous
apoptosis
Patient Outcomes?• Steroid replacement• Frequent infections• Growth hormone?• Risk of cancer?• Possibility of gene therapy?
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
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
Thank you VERY much for listening!