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!