Microdeletions

Question 1

In each syndrome caused by a micro deletion, the genomic imbalance affects approximately the same region of the genome and, therefore, a relatively well-defined set of genes. What is the name of these affected genes?

Answer 1

critical genes (in the ‘critical region’)

Question 2

Why are micro deletion syndromes often referred as “contiguous gene syndromes”?

Answer 2

Because these disorders remove a number of adjacent genes, and because multiple genes within the critical region contribute to the phenotype

Question 3

46,XY,del(22)(q11.21q11.23) indicates what?

Answer 3

a microdeletion of chromosome 22 at q11.22

Question 4

What is the normal human chromosomes range in size?

Answer 4

50Mb (chromosome 21) to 250 Mb (chromosome 1)

Question 5

As a general rule, genomic imbalance smaller than __ Mb will be below the detection limit for routine chromosome analysis (karyotype)

Answer 5

5 Mb

Question 6

Where are microdeletions particularly common?

Answer 6

at the ends of the chromosome (although they can occur anywhere)

Question 7

Which are typically more viable, micro deletions or macrodeletions?

Answer 7

microdeletions. In fact, many are so small that their phenotypic effects are mild enough to go undiagnosed (or even unnoticed on some occasions)

Question 8

What is a common feature that ALL micro deletions share?

Answer 8

some form of development delay and intellectual disability (the brain is the most susceptible organ)

Question 9

What is Prader-Willi syndrome?

Answer 9

Microdeletion syndrome with partial monosomy for a proximal segment of 15q (specifically 15q12

Question 10

What is Prader-Willi exhibited by clinically?

Answer 10

In infancy, affected individuals have hypotonia, feeding difficulties, poor growth, developmental delay, childhood hyperphagia & obesity

Question 11

What are the potential breakpoints in chromosome 15 for PWS patients?

Answer 11

BP1- near the top of the gene segment,
BP2- about 5 genes into the segment
BP3- near the bottom of the gene segment

Question 12

What is the difference between a Type 1 and a Type 2 deletion causing PWS?

Answer 12

The Type I deletion extends from BP1 to BP3. The Type II deletion extends from BP2 to BP3

Question 13

Do Type I and II deletions manifest themselves differently?

Answer 13

No, they both result in PWS with similar phenotypic results, which defines the minimal critical region underlying PWS as the segment between BP2 and BP3

Question 14

What is the primary mechanism giving rise to micro deletions (or microduplications)?

Answer 14

unequal crossing over between misaligned low copy repeats (LCRs). LCRs also are called segmental duplications

Question 15

What are LCRs?

Answer 15

They are segments of DNA (typically a couple hundred thousand basepairs in length) that exist in multiple copies, either dispersed at different genomic locations or clustered in a region of a chromosome

Question 16

Generally, are the effects of duplication are significantly milder or more severe than the effects of deletion?

Answer 16

milder (consequences of monosomy are more severe than the effects of the corresponding trisomy)

Question 17

What does 22q11.2 Deletion Syndrome (22q11DS) manifest as clinically?

Answer 17

heart defect (conotruncal abnormalities, such tetralogy of Fallot and VSD, caused by abnormal development of the pharyngeal pouches), hypoplastic thymus (leading to immune deficiencies), and hypocalcemia (which can be life-threatening in the newborn period). Developmental challenges and cleft abnormalities also are common

**What is more striking than the “classic” features is the extreme phenotypic variability seen in individuals with a 22q11 deletion. **

Question 18

Why does 22q11.2 deletion syndrome have other names such as DiGeorge syndrome and Velocardiofacial syndrome (as well as others)?

Answer 18

Because there is often a lot of phenotypic diversity that led to different diagnoses being made when patient’s presenting with differing clinical signs

We now understand that individuals who have a partial monosomy in this region can present with essentially any combination of common (and rare) signs and symptoms

Question 19

Do most microdeletions arise from inheritance or de novo?

Answer 19

de novo (~90%)

Question 20

Are most cases of 22q11.2 deletion syndrome de novo?

Answer 20

Yes. However, , familial cases of 22q11DS do occur – in such families, we would expect to see parents who are very mildly affected and, often, previously undiagnosed, along with children who illustrate the extreme variable expressivity that characterizes the disorder.

***In other words, variable expressivity is expected even in those with exactly the same genomic lesion.

Question 21

T or F. The vast majority of our CNVs are normal variants.

Answer 21

T. However, pathogenic CNVs that cause microdeletion syndromes. Also note that a growing number of normal CNVs are being associated with some elevated risk for one thing or another (for example, adverse drug reaction)

Question 22

Under what circumstances may chromosome testing be warranted (or even just requested) during pregnancy?

Answer 22

Screening for chromosome abnormalities in a presumably healthy pregnancy (typically older women request this)

Seeking a diagnosis in an abnormal pregnancy, e.g. ultrasound anomalies

Question 23

Under what circumstances may chromosome testing be warranted (or even just requested) during early developmental years (post-natal)?

Answer 23

Congenital anomalies
Developmental problems in childhood
Delayed or abnormal development of secondary sexual characteristics

Question 24

Under what circumstances may chromosome testing be warranted (or even just requested) during adulthood?

Answer 24

Infertility (failure to conceive or multiple miscarriage)

Birth of a child with a chromosome abnormality

Question 25

What is the difference between screening vs. diagnostic testing?

Answer 25

SCREENING – population-level testing to identify individuals who are at elevated risk so that they may be referred for definitive diagnosis and intervention.
Preliminary result that requires confirmation

TESTING – testing at the level of an individual to identify or rule out a genetic abnormality or variant.
Confirmatory result

Question 26

In postnatal life (birth → adulthood) what is the preferred source of specimen for testing?

Answer 26

peripheral blood

Question 27

In prenatal life, what is the preferred source of specimen for testing?

Answer 27

amniocentesis and chorionic villus sampling (CVS) are established procedures for the collection of cells of fetal or extraembryonic origin

Question 28

For karyotype testing, how are chromosomes obtained?

Answer 28

by culturing lymphocytes and arresting them in mitosis

Question 29

What is the traditional gold standard for diagnosis of aneuploidy?

Answer 29

karyotyping (although molecular approaches play an increasingly important role.)

Question 30

How does karyotyping work?

Answer 30

A patient’s cells are grown in culture, and then mitotic cells are examined by light microscopy. Numerical abnormalities are easily identified by counting and identifying the chromosomes present in a single cell and doing the same in multiple cells to confirm consistency of the result. Similarly, trained technologists can identify structural abnormalities by systematic identification of deviations from the normal banding pattern.

Karyotyping was the original genomic test, in that it provides a low resolution scan of the entire genome.

Question 31

When is karyotyping the preferred test?

Answer 31

Cases of a suspected chromosome abnormality
E.g., Patient presenting with presumed Down syndrome or Klinefelter syndrome

Suspicion of a balanced rearrangement
E.g., Possible reciprocal translocation in a couple with recurrent miscarriages or with a child who has a chromosome abnormality

Invasive procedure (e.g., amniocentesis) for routine prenatal diagnosis (e.g., maternal age)

Question 32

What are the main strengths of karyotyping?

Answer 32

Widely available, established test

Scans the entire genome

Best option for balanced structural abnormalities

Question 33

What are the main weaknesses of karyotyping?

Answer 33

Limited resolution (abnormalities smaller than a few Mb likely to be missed)

Relatively slow turnaround time since cell culture is required.

Labor intensive; less amenable to high-throughput automation.

Question 34

How does Fluorescence In Situ Hybridization (FISH) work?

Answer 34

a probe of known sequence and specificity is hybridized to genetic material from a patient. Since the probe is fluorescent, each site of hybridization is seen as a “dot” when viewed in a fluorescence microscope.

Question 35

What is William’s syndrome?

Answer 35

caused by a microdeletion of a region of chromosome 7 containing the elastin gene

Mild to moderate intellectual disability
Cardiac anomaly (supravalvular aortic stenosis)
Distinctive facial appearance

Question 36

What are the main strengths of FISH?

Answer 36

Results are specific and easy to interpret

Versatile approach in numerous clinical applications

Question 37

What are the main weaknesses of FISH?

Answer 37

The specificity also is a weakness, since the information provided is strictly limited to the probe chosen for the analysis

Question 38

What is the main downside of invasive pre-natal procedures like Amniocentesis and CVS?

Answer 38

The carry a small risk of miscarriage (0.5-1.0%)

Question 39

Amniocentesis and CVS are the most common pre-natal procedures to elucidate chromosome abnormalities before birth. How do these tests work?

Answer 39

Amniocentesis – amniotic fluid drawn from the uterus in early second trimester. Used to diagnose chromosome abnormalities (karyotype) and neural tube defects.

Chorionic villus sampling (CVS) – chorionic villi
taken from the placenta in late first trimester. Used to diagnose chromosome abnormalities (karyotype).

Invasive procedures to obtain a fetal karyotype still are the gold standard for a definitive prenatal diagnosis of aneuploidy

Question 40

What are some common non-invasive pre-natal screening procedures?

Answer 40

Maternal serum screening & ultrasound in the second trimester

Non-Invasive Prenatal Testing (NIPT) – fetal DNA in maternal circulation provides a substrate for screening in the late first or early second trimester

NOTE: these are just screening procedures, and would need to be confirmed by an invasive diagnostic procedure like amniocentesis or CVS

Question 41

What is Maternal serum screening used for?

Answer 41

The levels of specific hormones and proteins in maternal serum, in combination with fetal measurements by ultrasound, are used to estimate the risk of a chromosome abnormality relative to a woman’s age-related risk

Question 42

What is a major downside of maternal serum screening?

Answer 42

maternal serum screening has a significant false positive rate, as well as a risk of false negative results

Question 43

T or F. Most of Down Syndrome babies are born to younger mothers

Answer 43

T. the chance of nondisjunction and conceiving a child with a numerical abnormality, such as trisomy 21, increases sharply with maternal age. However, since there is some risk of aneuploidy in every pregnancy, and since the majority of babies are born to younger mothers, the fact is that most Down syndrome infants are born to younger mothers.

75% of Down syndrome babies are born to mothers younger than 35

Mothers >35 account for 9% of pregnancies & 25% of Down syndrome babies

Question 44

Who carries a higher risk for having a baby with DS, younger or older women?

Answer 44

older (i.e over 40 y/o -> 1 in 100) vs. younger (i.e less than 30 y/o -> 1 in 1000)

Question 45

Are invasive testing procedures for Down Syndrome limited to older women?

Answer 45

No.

Historically, invasive prenatal procedures have been reserved for older mothers (>35), since elevated risk for an individual helped to justify risk associated with the procedure. However, in recent years, changing attitudes have led to the elimination of age-based recommendations. With the introduction of improved non-invasive testing, prenatal testing is becoming more accessible, regardless of maternal age.

Question 46

How does Non-Invasive Prenatal Testing work?

Answer 46

Normal blood contains low levels of genomic DNA from cellular breakdown. In a pregnant woman, ~10% of the cell-free DNA is of fetal origin. Sophisticated molecular approaches make it possible to identify this “fetal fraction”, typically by focusing on alleles that are of paternal origin (alleles that differ from the majority which must be maternal). The fetal fraction can be analyzed for genomic abnormalities, such as aneuploidy.

Validated tests target specific genomic regions to detect trisomy 21, 18, 13, and sex chromosome aneuploidies.

Question 47

What are the major applications of NIPT?

Answer 47

Prenatal screening for aneuploidy in pregnancies >10 weeks.

Rapidly emerging technology with potential to target diverse disorders.

Screening test; an invasive procedure (amniocentesis) is required to confirm an abnormal result.

Question 48

Is NIPT a diagnostic test?

Answer 48

No. it is a screening test. (Although It has been described as a screening test with “near-diagnostic capabilities”)

Question 49

What is a chromosome microarray (CMA)?

Answer 49

CMA is a very high-resolution view of the entire genome. (in contrast to karyotypes, which are low-resolution views of the entire genome)

Question 50

T or F. Karyoptying can detect microdeletions

Answer 50

F

Question 51

Can CMA detect micro deletions or microduplications?

Answer 51

Si. And it will detect aneuploidy

Question 52

What is the big difference between something like FISH and CMA?

Answer 52

FISH is a diagnostic tool that uses a specific gene-targeting probe to see if a certain suspected micro deletion is present (which is why its used post-natal), while CMA will tell you if there is genomic imbalance anywhere in the genome but is not specific

Question 53

How does CMA work?

Answer 53

In simple terms, the test involves hybridization of patient DNA to a silica chip. The silica chip contains an ordered array of immobilized DNA sequences that span almost all of the genome. To give you an idea of the complexity of the data generated, an array can contain more than a million unique sequences or “features”. The amount of hybridization of patient DNA to each feature on the array is measured by fluorescent detection. The normal diploid amount of DNA is reported as normal baseline, as a yellow fluorescent . Any region of the patient genome that is deleted will have a quantitative decrease in hybridization and will be reported as red fluorescence (corresponding to half the normal amount). If a duplication (partial trisomy) is present, excess hybridization (green) will be reported.

As mentioned, this technology is very sensitive and can detect subtle genomic imbalance that would never be detected by karyotype. Typically, imbalances down to ~500,000 basepairs (0.5 Mb) are reported. Although the technology is capable of detecting much smaller imbalances, these are ignored since they are most likely normal variants (normal CNVs that everyone has)

Question 54

T or F. CMA is that it reports the precise size of a lesion and what genes are involved

Answer 54

T. This is a major advantage

Question 55

What are the major applications of CMA?

Answer 55

Recommended diagnostic test for individuals with developmental disabilities or congenital anomalies
Higher yield than karyotype (15-20% vs. 3%)
More precise characterization of abnormalities
Better detection of duplications than FISH

Recommended test after fetal anomalies have been detected by ultrasound.** HOWEVER, at this time, it is not recommended for routine prenatal diagnosis since the significance of some variants may be difficult to interpret. (In other words, when the fetal phenotype appears normal, a variant of uncertain significance may raise issues that cannot be resolved.)

Question 56

What are the main strengths of CMA?

Answer 56

Scans the entire genome

High sensitivity & precise description of abnormality

Question 57

What are the main weaknesses of CMA?

Answer 57

Interpretation may be complicated by CNVs of uncertain clinical significance (benign or pathogenic?)

Cannot detect balanced rearrangements

Cannot detect point mutations

Poor at detecting polyploidy (e.g. triploidy)

Question 58

Summary of Karyotype testing

Answer 58

Requires viable cells grown in culture
Diagnostic test with established track record
Scans the whole genome, but at low resolution
Good for numerical abnormalities, structural abnormalities, balanced or unbalanced abnormalities UNLESS they are too small to be seen

Question 59

Summary of FISH testing

Answer 59

Requires cells (interphase or mitotic)
Highly accurate for probe(s) used, but informative results restricted to probe target (NOT whole genome)
Versatile applications; can be used for screening or for confirmation of diagnosis in different contexts

Question 60

Summary of NIPS testing

Answer 60

Uses cell-free fetal DNA in maternal circulation
Noninvasive with very good accuracy; rapid acceptance of new test with expanding utility
Screening test; diagnostic confirmation required

Question 61

Summary of CMA testing

Answer 61

Uses genomic DNA
Scans the whole genome; capable of detecting genomic imbalance of any size
Interpretation may be complicated by CNVs of uncertain clinical significance (benign vs. pathogenic).

CANNOT detect balanced rearrangements or point mutations