Cytogenetics, FISH and MCA Flashcards
Down Syndrome causes
-mostly meiotic errors (95%) \+increased risk with advanced maternal age -robertsonians (3-4%) -mitotic errors (1-2%) * ~1% recurrence risk
life expectancy in Down Syndrome
now about 60y, greatly increased from 9y
-can see early death in 10-20% affected individuals related to severe cardiac anomalies
complications associated with Down syndrome
-cardiac defects (40%) \+mostly A-V canal -GI anomalies (~5%) \+duodenal atresia/stenosis most common -increased leukemia risk (15-20 fold) -Alzheimer's disease for most affected adults
Down syndrome incidence
- 1 in 700 live births
- 75% of SABs
Trisomy 18 phenotype
- SGA
- overlapping clenched fists and rocker bottom feet
- micrognathia and severe ID
- hyper or hypotonia
- severe renal, GI or cardiac anomalies (95%)
Trisomy 18 incidence
- 1 in 5000-1 in 8000 live births
- 4:1 F:M sex ratio
- extremely low survival, 90% affected infants die within the first year of life, 95% result in SAB
Trisomy 13 phenotype
-FTT + hypotonia
-holoprosencephaly
+micro or anopthalmia
+severe ID
+clefting
-cardiac defects
-polydactly
Trisomy 13 incidence
- 1 in 10000-1 in 15000 live births
- 90% die within first year of life, 95% results in SAB
- 20% caused by robertsonian translocation
X inactivation
- reversible in germ cell development
- 15% of X-linked genes escape this
Turner syndrome incidence and causes
-incidence 1 in 1500-1 in 5000 live births-variable by study
+1 in 30-1 in 50 female conceptuses (3%) w/94% resulting in SAB
-extremely low recurrence risk & several chromosome and mutation arrangements that cause it (50% 45, X)
+2/3 retain maternal X chromosome
Klinefelter incidence
- 1 in 500- 1 in 600 newborns
- no recurrence risk
- 50% due to paternal meiosis I errors
48 XXYY
similar to Klinefelter but with more severe ID and 90% risk for dicentric chromosome w/Robertsonian
Klinefelter phenotype
- small testes after puberty, gynecomastia, sparse facial hair, low fertility d/t fibrosis of seminiferous tubes
- hypotonia
- scoliosis + “Euchenoid habitus”
- low to normal IQ, rarely ID, LD/reading disability related to language processing and verbal memory
Turner phenotype
-short stature, broad chest, webbed neck
-primary amenorrhea, gonadal dysgenesis (90%)
-cardiac anomalies (40-60%)
+coarctation of the aorta
-renal anomalies (greater than 60%)
-hand and foot edema
robertsonian translocations
- fusion between acrocentric chromosomes (13, 14, 15, 21, and 22)
- 90% dicentric, p arms usually not involved
- usually get homologous chromosomes from one parent-risk for UPD higher
- usually associated with trisomy
paracentric inversion
-no centromere involvement
-usually only in part of long arm or short arm
-usually only gives acentric or dicentric fragments
+lower risk to have unbalanced offspring
reciprocal translocation
- mutual exchange of material between two non-homologous chromosomes
- leads to increased risk for fetal loss and children with problems
- occurs in 1 in 500 individuals
pericentric inversion
-centromeric involvment
-involves material on both long arm and short arm
-can result in recombination and unbalanced offspring
+can result in duplication and deletion of material
meiotic segregation with reciprocal translocations
- not like normal homologous chromosome line-up
- all chromosomes interconnect and create a 4-way junction
alternate segregation
always results in normal outcome because two normal and two translocated chromosomes go together
-one of the most common types of segregation
adjacent 1 segregation
chromosomes on same side of horizontal axis pair
-another of the most common types of segregation, can result in normal outcomes depending on chiasma placement
adjacent 2 segregation
chromosomes on same side of vertical axis pair
-always gives abnormal outcomes
3:1 segregation
always causes abnormalities because 3 chromosomes pair together and one is alone
risk of unbalanced segregates
~11-12%, but depends upon the ascertainment of the translocation
- if a previous child is affected, recurrence risk is about 20%
- if multiple SABs, risk might be lower because affected babies can’t survive
robertsonian incidence
- relatively common-1 in 1000 individuals
- rate of inheritance depends on the translocation and the parent it was inherited from
rob(13q14q) and rob(14q21q)
most common robertsonian translocations
risk of unbalanced segregates in robertsonian carriers
based on empiric, not theoretical risk
female carrier of rob(14q21q)
15-20% empiric risk for unbalanced segregants
male carrier of rob(14q21q)
2% empiric risk for unbalanced segregants
female carrier of rob(13q14q)
1% empiric risk for unbalanced segregants
male carrier of rob(13q14q)
<1% empiric risk for unbalanced segregants
X-inactivation mechanisms
- altered chromatin structure by CpG island methylation
- X inactivation center at Xq13.2
XIST
RNA specifically produced by XIC from the initially inactivated X chromosome for initiation and continuation of X inactivation
de novo translocations
related to increased risk for ID and congenital anomalies
+20% newborns with these
+55% individuals with ID with these
+3 in 1000 individuals with ID have these
clinical effects of de novo translocations
- position effects; genes turned off or on
- breakage within a gene; ex: DMD
- del/dup requiring FISH or MCA
- UPD
risk of phenotypic abnormality in de novo translocation
6-8% of all fetuses based on empiric data
Fluorescent In-Situ Hybridization (FISH)
-metaphase cells, interphase nuclei
-prepared like karyotype, but tagged probes can hybridize to region of interest in interphase cells
+have to know what is being looked for
contiguous gene syndrome/segmental aneusomy/microdeletion syndromes
deletion of chunk of DNA, including multiple genes on a chromosome physically close to one another of unrelated function
detection of CGS
- if 5-10Mb del-400-500 band resolution
- if 2-5Mb del-1000 band resolution
- if <2Mb need array or FISH
3Mb deletion
equivalent to loss of 10-100 genes
percutaneous umbilical blood (PUBs)
sampling of fetal blood from umbilical cord
FISH limitations
- labor and time intensive to design multiple probes
- expensive to design new probes
- requires specified probe and knowledge of specific genome site of interest
traditional cytogenetic testing limitations
lower resolution cannot indicate the size of abnormality, nor if microdel/dups present
traditional cytoogenetic testing benefits
- detects balanced and unbalanced rearrangements
- provides origins of derivative and marker chromosomes
FISH benefits
- higher resolution
- quick results for known/suspected alterations
protein-coding DNA
0.8%
total DNA
-3B bp
+40-50% repetitive
-~25000 functional genes
average gene size and density
- one gene per 45kb
- 20kb average size with significant variation
unbanded chromosome resolution
20Mb
banded chromosome resolution
10Mb
high resolution banding
3-5Mb
FISH resolution
150 kb (40-250kb)
microarray resolution
50-150 kb, ~30kb
g-banding
-35y SoC
-detects structural and numerical anomalies
+4% detection rate for nonspecific issues like ID and MCAs
-3-10Mb resolution
expression array
- RNA based array
- looks at series of genes and how they are expressed
- ex: cancer profiling
array CGH
- control DNA red, test DNA green
- two DNA types mixed in equal parts and hybridized
- ratio of red: green analyzed electronically to detect any gains or losses of info
BAC array
- looks for small CNVs
- can only study up to 1Mb DNA at a time
- uses CGH
oligo array
-uses current + high throughput technology
+uses CGH
+smaller probes ~60mers
-uses HGP data
SNP arrays
-uses high throughput tech + HGP data
-polymorphic + structural probes
-detects quantitative change, not CGH
+now most use SNP backbone+oligo
-allows for UPD, IBD and areas of contiguous homozygosity detection
-only array that can detect triploidy and maternal or twin sample contamination
identity by descent (IBD)
-detection of small haplotypes inherited from common ancestor
+allows for identifying difference between increase in homozygozity related to this versus consanguinity (determines degree of relation)
-greater shared homozygosity increases risk of recessive disorder
CMA and ACMG guidelines
- used for cases of MCA w/o known syndrome, apparently non-syndromic ID/DD and ASDs
- detection requires follow-up, parental studies and clinical evaluation + counseling
CMA detection rates
-detection rate of ~3.7% in individuals with ID and DD
+Hostenbach study states 19% in their population, another says 17% in selected population versus 12% in non-selected
*generally 15-20%
risk of anomaly in consanguinity
2-2.5% above general population if parents are first cousins
importance of POC
-15% pregnancies result in SAB in first tri
+50% are chromosomally abnormal
-some of these tissues fail to grow in culture, but reflex study by CMA can identify results successfully in 95% of cases
challenges with POC use
- low viability of cells with high culture failure
- culture contamination
- extended culture time
- can get overgrowth of maternal cells and get failure of ruling out MCC or molar pregnancy
- low resolution of g-banding
Law of Independent Assortment
chromosomes and genes are inherited independently from one another
Law of Segregation
gametes contain only one homologous chromosome and therefore only one gene for each trait
maternal meiosis I
most non-disjunction occurs here
interchange
3:1 segregation involving both translocated chromosomes
tertiary
3:1 segregation involving only one translocated chromosomes
percent of fetuses with DS that result in SAB
75%
empiric risk of female 14:21 rob to have a child affected with Down Syndrome
5-10%
Mosaicism testing
Best with karyotype, rarely picked up on oligo or SNP arrays
Targeted arrays
-probes localized around known disease causing genes & platforms
+often used as part of phenotype-specific panels because sequencing misses del/dup
-limits unknown findings/VUSs
Genome wide array
-probes scattered throughout genome
+helpful in determining del/dup breakpoints
-more likely to have CNVs returned with unknown significance
targeted array
-probes localized to specific regions of the genome (often exons associated with disease)
+often used as part of panel tests, as sequencing can miss del/dups
-less likely to identify CNVs of unknown significance
genome wide array
- probes spread across genome
- helps delineate specific break points
- more likely to identify CNVs of unknown significance
CNVs
-micro del/dups of ~400bp
+often benign if <500bp so most labs won’t call below this
-common in healthy individuals, though others are well-characterized in association with phenotypes
+most individuals are thought to have about 20
SNP
single variable base pair in the general population
CMA limitations
- cannot detect balanced arrangements (balanced translocations, inversions)
- may not detect low-level mosaicism
- can detect CNVs of unknown significance
- no info on structural nature possibly requiring further analysis
runs of homozygosity
- called LSCH or LOH
- stretches <3Mb common
- longer stretches identified on one chromosome may suggest UPD, across genome may suggest consanguinity based on IBD info
coefficient of inbreeding (F)
avg proportion of autosomal genome that is IBD in the offspring of related parents
percent IBD
estimated by sum of homozygous segments divided by total autosomal genomic length
-usually reported when about 2-5Mb
10% IBD
ROH of 2-5Mb with combined effect above this cut-off are suggestive of first or second degree parental relationship
-brings up possible psychosocial concerns of rape or abuse
interpretation confounds
- variable expressivity
- reduced penetrance
- unknown prognosis and clinical info for rare CNVs
- ambiguous results (CNVs of unknown significance)
- often requires parental studies for more accurate risk assessment
WES detection rates
25-51% (quote ~30% clinically) -dependent upon \+indication of patient and phenotyping \+ethnic background of patient \+availability of family members for testing that can aid in interpretation
ACMG WES recommendations
- non-specific phenotype in patient or family history that suggests undiagnosed genetic etiology with overlap in symptoms that are not consistent with a single condition (ex: pt with epilepsy and MCAs)
- presenting phenotype has a high degree of genetic heterogeneity
- prior specific testing has not provided an answer for the phenotype
- fetus with suspected syndrome not identified by targeted approaches
Coverage
Number of reads at a particular site in genome
Variant pipeline sorting
- allele frequency
- conservation
- inheritance pattern
- phenotype of patient
- in silico predictors
Human Genome Mutation Database
- collection of clinically meaningful variants implicated in human inherited disease
- contains approximately 4000 genes
Informatics pipeline
-common variants filtered out
-uncommon variants (~400-500) studied in association with relationship to SGDs
+phenotypic info helps specify candidates
-study filtered variants into those that are or are not thought to be deleterious
-deleterious variants confirmed via Sanger in individual and tested family members to confirm a diagnosis
Secondary/incidental findings
-deleterious, clinically actionable mutations identified that do not relate to the phenotype of the patient
+typically opt in/opt out
+VUSs not reported
-includes hereditary cancer, hereditary cardiac and connective tissue disorders
Interpretation challenges
-reduced penetrance and variable expressivity
-multifactorial disease
-non-detectable epigenetic factors
-phenocopies
+ex teratogens mimicking syndrome
-UPD
-XL mutations in females
+related to skewed X inactivation?
benefits of WES
-more cost effective than individual panels and single gene tests
+best approach for non-specific phenotypes and high heterogeneity conditions
-identifies atypical presentations of known syndromes
-novel gene discovery
+this is important, as most monogenic disorders are not thought to have been discovered yet
limitations of WES
-limited detection of large rearrangements and CNVs, mtDNA mutations, trinucleotide repeat expansions
-only 85-92% exons targeted
+plus all targeted exons aren’t well covered (~90%)-if requested labs can provide specific targeting
-cannot detect regulatory and intronic regions
-many labs will not report adult-onset Alzheimer’s and dementia genes
WES risks
- discovery of undisclosed non-paternity, family secrets and relationships
- incidental findings
- greater numbers of VUSs and possibly ambiguous results
- may provide unwanted insight into health of other family members or have implications for their health
- discovery of a novel gene can limit counseling regarding long term outcomes
- possible risks for future disability and life insurance coverage
MUTYH secondary finding
only homozygotes reported, despite slight increased risk for heterozygotes
chromosome 18p deletions
- ID, DD, SD, behavioral problems
- brachydactly
- hypodontia, frequent cavities, cleft palate
- antihelix abnormalities with protruding ears
- characteristic facies: ocular hypotelorism, “carp” mouth, brachycephaly, sometimes cyclopia
- GU anomalies
- neck webbing
- hypotonia and growth restriction
triploidy
- low hCG is a sign, often causes FT loss
- sporadic with minimal RR
- 69, XXY>69XXX»>69XYY
maternal triploidy
- small placenta
- severe asymmetric IUGR with large head
paternal triploidy
- large cystic placenta
- IUGR
- normal to microcephalic
Sotos syndrome
- 5q del of NSD1
- more common in Japanese pop
- overgrowth, macrocephaly
- hypotonia, ID, DD, LD
- dysmorphic features-flushed cheeks, long face, high forehead, pointed chin
- poor balance
complete hydatidaform mole
-usually diploid, 46,XX pregnancy completely of paternal decent
+23, X sperm fertilizes an egg lacking a nucleus
-trophoblastic hyperplasia with grossly disorganized or absent fetal tissue
partial hydatidiform mole
- usually diandric triploid
- fetus will be present early on, then will demise
- hydropic villi
ovarian teratoma
- usually 46, XX completely of maternal origin
- growth that can contain multiple tissues including hair and teeth
