Lecture 6: Haploinsufficiency, Imprinting, and PWS/AS

What is haploinsufficiency?

• One gene copy is inactivated/deleted and the remaining copy is NOT adequate for normal function.

• Caused by deletion or loss-of-function mutation.

• Contrast: haplosufficiency = one copy IS adequate.

How common are autosomal deletions?

• 1/7000 live births.

• Usually part of a chromosome or a few genes.

• Most not clinically recognized.

  • Often small.

  • Remove non–dosage-sensitive genes.

    • The delete genes do not require two copies to function normally.

  • One copy is sufficient (no haploinsufficiency).

  • Cause mild or no phenotype → no testing.

What is a contiguous gene syndrome?

• Multiple neighboring genes from same chromosome are lost together.

• Caused by deletions at low copy repeat sequences.

  • Low copy repeats = similar DNA sequences that can misalign during meiosis → unequal crossover → deletion of neighboring genes.

• Results in variable clinical phenotypes.

What causes genomic disorders?

• Centromeric and telomeric breakpoints cluster.

• Homologous recombination between low copy repeat sequences.

• Altered gene dosage causes phenotype.

• Phenotypic variability even with same dosage imbalance.

What is epigenetics?

• Study of gene expression and phenotype.

• Heritable gene expression changes that do NOT depend on DNA sequence.

• Mechanisms: DNA methylation and histone modification.

  • DNA methylation: addition of methyl groups to DNA that usually silences gene expression.

  • Histone modification: chemical changes to histone proteins that loosen or tighten chromatin to regulate transcription.

What is genomic imprinting?

• Monoallelic gene expression from only ONE parental chromosome.

• Affects ~1% of mammalian genes (several dozen to few hundred).

• Normal, reversible process using DNA methylation.

  • DNA methylation turns one parental allele on or off. This mark is not permanent and is reset in eggs/sperm.

• Marks parental origin of chromosome/subregion.

When does genomic imprinting occur?

• The cell is erased and reset during gametogenesis (before fertilization).

• Marks genes as paternal or maternal using DNA methylation.

• Imprinted genes retain memory even during paternal genome demethylation at fertilization.

• Persists postnatally through hundreds of cell divisions.

Why must imprinting be reversible?

• Methylation at CpG dinucleotides (differentially methylated regions - DMRs).

• A paternal allele inherited by a female must have its male-specific methylation removed and be re-marked as maternal in her eggs.

• Similarly, a maternal allele inherited by a male must be re-marked as paternal in his sperm.

• Governed by imprinting centers.

  • Ensures proper parent-of-origin gene expression in the next generation.

What is the parental gene growth rule?

• Paternal genes generally ENHANCE growth.

• Maternal genes generally SUPPRESS growth.

• Expression depends on whether mutant allele inherited from father or mother.

Describe germ cell methylation during development.

• Primordial germ cells: start highly methylated.

• Demethylation occurs during development.

• After gonadal differentiation: de novo methylation occurs.

  • Gonadal differentiation: the stage in development when the primitive gonads in the embryo develop into testes or ovaries.

What happens during embryo reprogramming?

• Fertilization: sperm & egg highly methylated (maternal/paternal differences).

• Early embryo (morula/blastula): most DNA demethylated.

• Pregastrulation: de novo methylation begins.

  • Pregastrulation = the stage just before gastrulation, which is when the embryo begins forming the three primary germ layers: ectoderm, mesoderm, and endoderm.

• Somatic cells: heavily methylated.

• Placenta/trophoblast: less methylated.

• Primordial germ cells: mostly unmethylated → will get sex-specific methylation at gonadal differentiation.

What are the three causes of PWS and AS?

• Cytogenetic deletion (70%): information from only one parent.

  • Can do array CGH to see the deletion!

• Uniparental disomy (30%): both chromosomes from one parent.

  • Isodisomy = two copies of the same chromosome from one parent.

  • Heterodisomy = both homologs from one parent.

• Imprinting center defect (5%): gametogenesis switches do not occur.

What is Prader-Willi syndrome?

• First human genomic imprinting disorder

• Loss of PATERNAL 15q11-q13 → only maternal genes expressed.

• Long arm of chromosome 15; contiguous gene syndrome.

• 1/10,000-15,000; both sexes and all ethnicities.

What are infant PWS phenotypes?

• Hypotonia.

• Feeding difficulties.

• Almond-shaped eyes.

• Small hands and feet.

• Hypogonadism and genital hypoplasia.

• Respiratory problems.

What are childhood PWS phenotypes (2-4 years)?

• Obesity (leading genetic cause).

• Excessive and indiscriminate eating.

• Food-seeking behavior and increased appetite.

• Reduced metabolic rate.

• Abnormal body composition.

• Moderate cognitive impairment.

What are PWS obesity complications?

• Cardiorespiratory insufficiency.

• Diabetes.

• Obstructive sleep apnea.

How is PWS diagnosed prenatally?

• Prenatal onset hypotonia

• Decreased fetal movement

• Abnormal fetal heart rhythm

• Children born full term and normal size.

  • The other phenotypes do not affect overall growth or cause early delivery.

• Hypotonia also occurs in Down syndrome, Fragile X, other chromosomal disorders

What are the genetic causes of PWS?

• ~70% paternal deletion:

  • Missing part of chromosome 15 (15q11–q13) from dad

  • Region is ~4 Mb with ~50 genes

• ~25% maternal uniparental disomy (UPD):

  • Both chromosome 15s come from mom, none from dad

  • Often due to an embryonic trisomy that “loses” the paternal chromosome

  • Risk increases with maternal age

• 2–3% chromosome rearrangements:

  • Translocations or other structural changes

• 2–3% imprinting center defects:

  • Tiny deletion or mutation that prevents normal imprinting

How do deletion vs UPD differ in PWS?

• Maternal UPD less likely:

  • Almond-shaped eyes.

  • Thin upper lip.

  • Hypopigmentation.

• Maternal UPD more likely:

  • Higher IQ.

  • Milder behavioral problems.

  • Psychosis and ASD.

What is SNRPN and its role in PWS?

• Small nuclear ribonucleoprotein N.

• Controls gene splicing.

• Expressed from paternal chromosome.

• Highest expression in heart and brain.

What are MAGEL2, MRKN3, and IPW in PWS?

• MAGEL2: Highest expression in hypothalamus and brain (late development).

• MRKN3: Zinc finger gene, function unknown.

  • Zinc finger = DNA-binding protein that can control gene activity.

• IPW: Non-coding gene, function unknown.

What is Necdin (NDN) in PWS?

• Maternally imprinted gene.

• Involved in cell cycle control and apoptosis.

• Highest expression in hypothalamus.

What are P gene and GABRB3 in PWS?

• P gene (nonimprinted): Tyrosinase positive albinism; causes hypopigmentation in 1/3-1/2 patients.

  • Not fully albino.

• GABRB3: Implicated in seizure susceptibility.

How is PWS molecularly diagnosed?

• DNA methylation test: looks at the 5′ CpG island of the SNURF-SNRPN gene.

  • Detects >99% of PWS cases.

  • Shows if the paternal copy is missing or silenced.

  • Cannot tell whether it’s due to deletion, UPD, or imprinting defect.

• Other tests to clarify the cause:

  • Cytogenetics (chromosome analysis).

  • FISH using SNRPN probe.

  • Array CGH (aCGH).

What is Angelman syndrome?

• Loss of MATERNAL 15q11-q13 → only paternal genes expressed.

• 1/12,000-20,000.

• “Flat heads, jerky movements, protruding tongues, bouts of laughter.”

What is the early presentation of AS?

• Normal at birth.

• Feeding problems in first months.

• Developmental delays by 6-12 months.

• Seizures begin 2-3 years.

• Severe speech impairment (little to no words).

What are characteristic AS features?

• Microcephaly (decreased brain growth).

• Severe cognitive impairment.

• “Puppet-like” ataxic gait with jerky movements.

• Seizures.

• Distinct facial appearance.

• Short stature.

What are additional AS features?

• EEG abnormalities.

• Hyperactivity.

• Bouts of laughter (happy demeanor).

• Severe speech and language limitations.

What are the AS genetic classes and severities?

• Class 1 (70%): 15q11-q13 deletion → most severe.

• Class 2 (5%): Uniparental disomy → less severe.

• Class 3 (5%): Imprinting defect → less severe.

• Class 4 (10%): UBE3A mutation → intermediate, displays obesity.

• Class 5 (10%): Unknown → most severe.

• Classes 4 & 5 have normal methylation and PWS-imprinting center (PWS-IC).

  • Classes 4 & 5 can’t be diagnosed by standard methylation testing; they need gene sequencing or other approaches.

What is the AS imprinting center?

• 880 bp exon.

• Mediates switch from paternal to maternal imprint during oogenesis.

• AS-IC methylates PWS-IC and silences expression.

• Most class 3 AS cases have NO defect in AS-IC → suggests epimutations.

  • Epimutations: abnormal changes in gene expression caused by faulty epigenetic marks (like DNA methylation or histone modification) without changing the DNA sequence itself.

What is UBE3A and its function?

• A protein that tags other proteins with ubiquitin → marks them for degradation or regulation.

• The E3 ligase part decides which proteins to tag.

• The HECT domain at the end is needed to actually transfer ubiquitin.

• Can also interact with nuclear receptors and help turn certain genes on.

How do UBE3A mutations cause AS?

• Mutations cause premature termination and HECT domain disruption.

• Loss causes substrate protein accumulation → neurological abnormalities.

• Inherited through MATERNAL line (maternally expressed).

• Expressed in hippocampal neurons and cerebellar Purkinje cells.

  • Purkinje cells = large neurons in the cerebellum (the brain region that controls movement and coordination).

What are neurological effects of UBE3A loss?

• Microcephaly = abnormally small head size, usually due to reduced brain growth.

• Impaired motor function and long-term potentiation.

• Defects in contextual and spatial learning.

• May regulate estrogen receptor stability (decrease linked to cancer).

What’s the key PWS vs AS difference?

• PWS: Loss of PATERNAL 15q11-q13.

• AS: Loss of MATERNAL 15q11-q13.

• Same region, opposite inheritance, completely different phenotypes!