Lecture 16: Molecular Cytogenetics DNA structural variation: Copy Number Variants (CNVs) Flashcards
Abnormal whole chromosome copy
number (chromosome aneuploidy)
MONOSOMY = 5
1 ➢Loss of a single chromosome
2 ➢Autosomal monosomy
3. ➢ Generally not compatible with life
- ➢Sex chromosomal monosomy
- ➢ 45,X compatible
Mechanism for aneuploidy: Error at cell division…Chromosomal nondisjunction
MEIOSIS ONE
- nondisjunction during meiosis I
- HOMOLOGOUS CHROMOSOME
MEIOSIS 2
- Sister chromatids
- nondisjunction during meiosis II
Whole chromosome aneuploidy = 3
1 *Major cause of fetal death as most aneuploidies are incompatible with life
2 *Accounts for ~50% of pregnancy loss in the first trimester
- *However, some aneuploidies do result in viable offspring with a spectrum of
developmental disorders
What is Trisomy 21 = Down syndrome (
*Most individuals (95%) have 3 free copies of chromosome 21
* ~5% case - 1 copy is translocated to another acrocentric chromosome (often 14 or 21)
*Most cases arise from error at maternal meiosis I
*Minority cases ‘from error during spermatogenesis’
- ~5% cases error mitosis (postzygotic nondisjunction)
*Clinical features may include:
◦ Intellectual disabilities
◦ Congenital heart disease
◦ Thyroid disorders
◦ Single palmar crease
◦ Characteristic facial features
◦ Poor immune function
◦ Increased risk leukemia (both ALL and AML)
Trisomy 18 = Edwards Syndrome
- Severe - majority of infants die in 1st year of life
- Dysmorphic facial features (micrognathia, small ears)
- Rocker-bottom feet and clenched hands
- Many organ malformations including:
◦ Severe intellectual disability
◦ Failure to thrive
◦ Cardiovascular( >95%)
◦ Gastrointestinal
◦ Genitourinary
Trisomy 13 = Patau syndrome
- Severe - high postnatal mortality
- Only 5-10% of children live past 1st year
- Clinical features include:
◦ Heart defects
◦ brain or spinal cord abnormalities
(holoprosencephaly)
◦ Urogenital abnormalities
◦ Rocker bottom feet
◦ Small or poorly developed eyes (microphthalmia)
◦ Polydactyly (Extra fingers or toes)
◦ Cleft lip with or without cleft palate
Klinefelter syndrome 47,XXY
- Variable symptoms of Klinefelter syndrome
- Males with 47,XXY are invariably infertile
- Some have poor development of secondary sexual characteristics and female fat distribution
- Other clinical features may include:
◦ Noticeable breast tissue (gynecomastia)
◦ Long legs/arms
◦ Developmental delay
◦ Speech/language deficits
◦ Learning disabilities/academic issues
◦ Behavioural issues
45,X Turner syndrome
- Variable phenotype in females
- Common abnormalities include:
◦ Short stature
◦ Swelling of the hands and feet
◦ low-set ears
◦ webbed neck
◦ Reproductive sterility (No menstrual periods)
◦ congenital heart disease
◦ Low thyroid hormone
◦ Diabetes
◦ Vision problems and hearing problems
◦ Possible cognitive deficits
Diagnosis of whole chromosome copy
number changes = 5
- QF-PCR - most commonly used for prenatal detection. Cheapest and fastest.
- NIPT - non-invasive prenatal detection used as a screening test.
- FISH – good if mosaicism is suspected.
- Karyotype – confirms QF-PCR result, valuable as can detect any translocations
which is helpful for recurrence risk - Microarray – good for pregnancy loss as no cell culture required
Microdeletion and microduplication
syndromes = 6
1 *Hundreds of syndromes are known to be caused by small deletions or
duplications that cannot be seen by conventional cytogenetics (karyotype)
2 *Mostly detected by microarray, MLPA or FISH
3 *Many are recurrent – occur in same location in the genome in many different
patients
4 *The DNA sequences in these regions of the genome are often responsible for the
rearrangements
5 *Low copy repeat (LCR) sequences can cause DNA to misalign at meiosis or mitosis and result in deletions or duplications
- LCRs predispose the region to recurrent genomic rearrangements by non-allelic homologous recombination
Non allelic homologous recombination
(NAHR) = 2
- Misalignment and recombination between Low Copy Repeat (LCR) sequence
- Gives rise to reciprocal duplication & deletion
diagram on slide 15
Examples of recurrent microdeletion and
microduplication syndromes = 5
- 22q11.2 deletion syndrome
- 22q11.2 duplication syndrome
- Williams syndrome (7q11.23 deletion)
- Prader Willi / Angelman syndrome (15q11.2 deletion)
- 15q11.2 duplication syndrome
Case 1: 22q11.2 deletion syndrome = 8
1 *The most common microdeletion syndrome (~1:4,000 live births)
2 *Patients with 22q11.2 deletion have characteristic clinical features
3 *The acronym CATCH 22 has been used:
…4 - C: Cardiac defects
…5 - A: Abnormal facies
…6 - T: Thymic hypoplasia (underdeveloped thymus), Immunodeficiency
…7 - C: Cleft Palate
…8 - H: Hypocalcemia (low calcium), secondary to hypoparathyroidism
also neonatal seizures, developmental delay, behavioural issues, poor muscle tone
Microarray Result : 22q11.21 deletion
~2.6Mb deletion
TBX1 is a critical gene
~45 genes
diagram on slide 18
Sub-microscopic 22q11.2 deletions occur within a region of complex genomic architecture
8 LCRs (A-H)
diagram on slide 19
22q11.2 deletion identified by FISH
NORMAL INDIVIDUAL vs. 22q11.2 DELETION PATIENT
NORMAL INDIVIDUAL:
- 2 normal chromosome 22s
with a red and green signal
Red – control probe 22q telomere
Green – 22q11.2 probe
22q11.2 DELETION PATIENT
One normal chromosome 22
Two red control probe signals
One green 22q11.2 probe signal
Indicates a 22q11.2 deletion
Case 2: 22q11.2 duplication syndrome
1 *This duplication is the same region as the deletion associated with 22q11.2 deletion syndrome
2 *Highly variable phenotype – duplication is milder than deletion
3 *Features include:
◦ mild to moderate intellectual disability
◦ delayed psychomotor development
◦ growth retardation
◦ hypotonia
◦ some abnormalities associated with 22q11.2 including heart defects
4 *This duplication is also found in unaffected individuals including parents
5 *Variable expressivity and incomplete penetrance
Microarray Result: 22q11.21 duplication
Duplication of 22q11.21
~3 million base pairs
(3Mb) in size
Approx 45 genes
duplicated i.e. 3 copies
Including TBX1 gene
Case 3: Williams syndrome (7q11.23 deletion)
Disease characteristics include:
Distinctive facial features
Developmental delay, speech
Intellectual disability, ADHD
Failure to thrive
Heart defects
Recurrent 7q11.23 deletion associated
with Williams Syndrome
diagram on slide 24
ELN – cardiovascular aspect
RFC2 – growth deficiency and developmental disturbances
LIMK1 - impaired visuospatial constructive cognition
GTF2RD1 - craniofacial and cognitive development
LCRs at breakpoints
~1.5Mb in size
LCRs at breakpoints
Case 4: Prader Willi (PWS) / Angelman (AS) syndrome (15q11q13 deletion)
diagram on slide 25
~5Mb deletion
Many genes included.
Inprinted region
Maternal deletion causes AS
Paternal deletion causes PWS
Prader Willi syndrome (PWS)
Floppy at birth – developmental delay, failure to thrive, poor feeding.
After 2 years usually develop an obsession with eating and obesity.
Intellectual disability and other behavioral problems.
Angelman syndrome (AS)
Developmental delay, functionally severe. no or minimal use of words;
- Movement or balance disorder, usually ataxia of gait frequent laughter/smiling; apparent happy demeanor;
- easily excitable personality, often with
hand flapping movements; short attention span
Delayed, disproportionate growth in head circumference, usually resulting in microcephaly (absolute or relative) by age 2
Seizures, onset usually < 3 years of age
Some genes in the PWS/AS region are imprinted and only expressed on the maternal or paternal chromosome 15
Maternal deletion causes most cases of AS.
Also caused by paternal UPD i.e. no contribution from mum so no maternally expressed genes
Also caused by UBE3A mutation in the gene
Paternal deletion causes most cases of PWS.
Also caused by maternal UPD i.e. no contribution from dad so no paternally expressed genes
diagram on slide 28
Case 5: 15q11q13 duplication syndrome
1 *Duplications also exist for the common deletion region seen in Angelman and Prader-Willi syndromes OMIM # 608636
2 *Variable clinical features may include:
* Autism
* Intellectual disability
* Seizures
* Developmental delays (including speech)
* Hypotonia
* Behavioral problems
* Mild facial anomalies
3*Duplications are mostly maternally inherited
4 *Paternally inherited duplication have a mild or no abnormal phenotype
Microarray result – 15q11q13 duplication
diagram on slide 30
Duplication
15q11.2q13.1
5.Mb in size
Deletion of this region causes Prader-
Willi/Angelman (PWS/AS) syndrome
Imprinted region – paternal deletion gives PWS / Maternal deletion gives AS
MS-MLPA of the 15q11q13
PWS/AS region = 4
1 *Methylation-specific MLPA can identify the deletions and duplications of 15q11q13
2 *It will also reveal the inheritance pattern
3 *Did the CNV come from mum or dad?
4 *Paternal duplications of 15q11q13 are usually associated with incomplete penetrance, and a milder clinical presentation
Case 6: Two terminal CNVs - deletion and duplication
the diagram on slide 33
A karyotype is required to determine the structure of the rearrangement
Need to test parents as could be inherited from a parent with a balanced rearrangement
diagram on slide 34
the diagram on slide 35
parent 1 normal karyotype vs parent 2 balanced tranalocation
PMP22 gene duplication
1.3Mb duplication containing the PMP22 gene
SLIDE 37 DIAGRAM
PMP22 gene on 17p12
= 7
1 *17p12 duplication including the PMP22 gene (OMIM 601097) causes Charcot Marie Tooth Type 1A (CMT1A).
2 *Adult-onset progressive peripheral neuropathy.
3 *CMT1A is characterised by distal symmetrical muscle weakness
4 *PMP22 protein is produced predominantly by Schwann cells.
5 *It is critical to myelination in the peripheral nervous system (PNS)
6 *PMP-22 duplication gives an increase in the protein which causes CMT1A
7 *PMP-22 deletion gives a decrease in the protein which causes a milder peripheral
neuropathy called hereditary neuropathy with liability to pressure palsies (HNPP)
FISH confirming a PMP-22 duplication
= 2
- Duplication of the region spanning PMP22 (red) in an individual with Charcot-Marie-Tooth disease type 1A (CMT1A).
- Detection can be by microarray, FISH or MLPA
PLP1 gene on Xq22.2 =
1 *Duplication of the PLP1 gene causes a myelin disorder called Pelizaeus Merzbacher disease
2. * PLP1 proteolipid protein gene.
3. Major myelin protein in the central nervous system (CNS)
4 *Boys with a duplication have dysmyelination
5 *Deletion of PLP1 gene causes a mild myelin disorder
6 *No production of PLP1 protein is less severe than over production.
7 *Another example of a dosage sensitive gene
8* ‘Detection by FISH, microarray or MLPA’
PLP1 gene duplication
Male patient vs. female carrier
Yellow signal – PLP cosmid
Red signal – X centromere probe
Exons (part of a gene):
Heterozygous deletion of chromosome
Xp21 (female patient)
- Intragenic deletion of the DMD gene
encompassing exons 49-51
~ 90Kb in size
- Female carrier
- Risk for male offspring –
- Becker/Duchenne Muscular
Dystrophy - Mostly detected by MLP
DIAGRAM ON SLIDE 43
Hemizygous deletion on chromosome
Xp21.2 (male patient)
DIAGRAM ON SLIDE 44
Intragenic deletion of the
IL1RAPL1 gene encompassing exons 4,5 and 6.
Approximately 300Kb in size
Pathogenic partial deletion of IL1RAPL1
gene
1 *Intragenic deletion - Exons 4, 5 and 6.
2 *IL1RAPL1, expressed in brain that is responsible for a nonspecific form of X linked intellectual disability
3 *Similar deletions, as well as truncating mutations and cytogenetic rearrangements disrupting this gene, have a reported association with intellectual disability and possible mild dysmorphism
4 *‘Loss of function of the IL1RAPL1 protein’ causes intellectual disability
5 *Common facial features in males with a deletion include prominent and wide nasal root, ptosis, synophrys and broad face
6 ‘*Detected by microarray but could also be detected by MLPA or gene sequencing’
Summary
1 *The importance of CNVs in the genome
2 *Methods of CNV detection and their applications, advantages and disadvantages
3 *The range of different copy number variations and examples of clinical scenarios
