Download imbalances within regions containing large

IMBALANCES WITHIN REGIONS CONTAINING LARGE-SCALE COPY-NUMBER VARIATIONS IN
INDIVIDUALS WITH DEVELOPMENTAL ABNORMALITIES:
WHEN DOES NORMAL BECOME ABNORMAL?
Nelson, M., Pickering, D., Golden, D., Haskins Olney, A., Schaefer, G.B., Dave, B.J., Sanger, W.G.
Munroe-Meyer Institute, University of Nebraska Medical Center, Omaha, NE
Case 1: Clinical Report and Molecular
Cytogenetic Findings
Large-scale Copy-number Variations (LCV’s): What do we know so far?
¾ 3 ½ month old female previously seen in the newborn
period for multiple congenital anomalies.
¾ Patient findings are strongly suggestive of CHARGE
Association including congenital heart defects, coloboma
and growth retardation.
¾ What are they? LCV’s are large segments of DNA, several to hundreds of kilobases in size, that vary
severalfold in copy number between phenotypically normal individuals.
¾ How are they detected? LCV’s manifest themselves as duplication/loss in DNA microarray studies
and/or as deletions in FISH studies. They may also be seen to a lesser extent as duplications in
interphase FISH studies.
Case 2: Clinical Report and Molecular
Cytogenetic Findings
¾ Six year old male with developmental delays and moderate
to severe bilateral neurosensory hearing loss.
¾ Family history negative for hearing loss or neurodevelopmental
delays.
¾ High-resolution G-band studies revealed a 46,XY karyotype.
¾ Family history of a full brother who was stillborn with
unknown etiology.
¾ How many are there? Estimates of greater than 200 LCV’s are present in the human population.
This is based on limited population studies and may be an underestimation.
¾ High-resolution G-band studies revealed a 46,XX karyotype
¾ How common are they? On average, two individuals vary by 11 or more LCV’s, however less than
50% of the LCV’s currently described were identified in more than 1individual.
¾ Microarray analysis with the Spectral Genomics™ Constitutional
Human BAC Array was normal.
¾ Where are they located? Greater than ½ of the polymorphic clones may overlap known coding regions
and ¼ may encompass one or more complete genes. Several LCV’s have been identified near loci
associated with cancer or genetic syndromes and are frequently in regions susceptible to
rearrangement.
¾ Microarray analysis utilizing the Spectral Genomics™ 2600 BAC
1Mb Array confirmed a deletion of BAC clone CTB-31J3 at
4q35.2 (Figure 4) along with deletions at 1p36.33* and 22q13.33*
and a duplication at 1p21.
¾ Subtelomeric FISH studies were normal.
¾ FISH studies utilizing a homebrew locus specific DNA
probe revealed a deletion of the CHD7 gene at 8q12.2.
Deletions of this gene have been associated with CHARGE.
¾ Microarray analysis utilizing the Spectral Genomics™
Constitutional Human BAC Array was normal.
¾ Microarray analysis with the Spectral Genomics™ 2600
BAC 1Mb Array confirmed a deletion at 8q12.2 but also
revealed a duplication at 1q44 and 1p36.32-33* (Figure 1).
A deletion at 5p14.3 and duplications at 14q12 and
22q13.33* were also present
¾ FISH studies utilizing homebrew loci specific DNA probes
derived from the same clones found to be duplicated at
1q44 (RP11-407H12 and RP11-438F14) in the microarray
study were positive for a duplication in approximately 1/3 of
the interphase cells scored (Figure 2a and 2b).
¾ Microarray and FISH studies of the parental chromosomes
revealed that the same 4qter deletion was present in the
phenotypically unaffected father implying that this aberration has
no pathogenic affect (Figure 3b) and revealed inheritance pattern of
other suspected LCV regions.
Database of Genomic Variants
Supported by:
¾ Microaray and FISH studies of the parental chromosomes
revealed the inheritance pattern of the suspected LCVs.
¾ Further investigation revealed that the clones duplicated at
1q44 (RP11-407H12 and RP11-438F14) and 14q12 (RP11125A5) have previously been reported as polymorphic
clones located within a LCV region. The clone showing gain
at 5p14.3 (RP11-88L18) to the best of our knowledge,
however, is not within a reported LCV.
Chromosome 1 profile
from 1 Mb microarray
Legend: A blue dot indicates a reported gain;
A red dot indicates a reported loss; An orange
dot indicates a reported inversion breakpoint;
A green dot to the left of a region indicates that
it overlaps with segmental duplication and/or is
within 100k of a gap
¾ Mapping of the region with information gained by FISH and
microarray has revealed a possible deletion size ranging from
<400 bp to 40 kb. This region contains an autosomal dominant
deafness gene, DFN24. The possibility of further molecular studies
to determine if mutations/deletions of DFN24 are present in the
proband are being considered (Figure 5).
* The duplications or deletions at 1p36.33 (RP11-703E10) and
22q13.33 (CTD3018K1) observed in multiple individuals could not be
resolved due to conflicting results.
Chromosome 4 profile
from 1 MB microarray
LCV’s: What does it mean when they are observed in affected individuals?
703E10
Although LCV’s are considered normal genomic variants, the possible phenotypic consequences of
all LCV’s has yet to be determined .
¾ Some LCV’s resulting in deletion may uncover recessive mutations resulting in phenotypic
changes.
¾ Some LCV’s may have yet to manifest themselves in age-related susceptibilities.
¾ Some LCV’s may lead to further genetic alterations within adjacent regions.
Figure 2a
407H12
438F14
Figure 1
¾ Subtelomeric FISH studies with Vysis ToTel DNA FISH probe
panel revealed a deletion of 4qter (Figure 3a).
Figure 2b
List of significant clones from 1 Mb microarray
Clone
Location DNA Copy Reported LCV
RP11-407H12 1q44
Gain
Yes
RP11-438F14 1q44
Gain
Yes
RP11-88L18
5p14.3
Loss
No
RP11-91I20
8q12.1
Loss
N/A
RP11-125A5
14q12
Gain
Yes
Inherited
Mother
Mother
Mother
No
Mother
Conclusions about associations between LCV’s and phenotypic abnormalities should be approached
cautiously.
¾ An aberration in a LCV region should be considered as a possibility for the patients’ malformation
only if it has been observed in other patients with similar phenotypes or is associated with regions
containing genes which may have contributed to the abnormal phenotype.
¾ Aberrations observed by either DNA microarray or subtelomeric FISH studies that have not been
associated with a specific phenotype in other case studies or cannot be tied to a possible
causative gene within that region may represent a previously unreported LCV region.
¾ Parental studies are necessary to help classify all possible LCV regions.
To achieve optimal utilization of molecular cytogenetic techniques and to enhance our basic
understanding of LCV’s it is of great importance to report cryptic aberrations thought to be genomic
polymorphisms and this must be accumulated in a national database to allow clinical laboratories to
accurately determine whether duplications or deletions, by these methods are clinically relevant.
Figure 3a
CTB-31J3
Figure 4
Figure 3b
List of significant clones from 1 Mb and Constitutional microarray
Clone
Location
DNA Copy Reported LCV
Inherited
RP11-259N12 1p21
Gain
Yes
Father
RP11-354H17 4q35.2
Normal
N/A
N/A
CTB-31J3
4q35.2
Loss
Yes
Father
RP11-45F23
4q35.2
Normal
N/A
N/A
354H17
Figure 5
CTB-31J3
45F23
DFN24 (deafness gene)