Transcript No Slide Title - ARUP Laboratories

Identification of carrier status as a consequence of
whole-genome microarray analysis: dilemmas regarding
clinical obligation, confirmation and reporting
L.R. Rowe1, A. Millson1, J. Swenson2,3, E. Lyon1,2,3, E. Aston3, D. LaGrave3,
S.Shetty1,2,3, A.R. Brothman1,2,3,4,5, S.T. South1,2,3,4
1Institute
for Clinical and Experimental Pathology , ARUP Laboratories, Salt Lake City, UT
2Department of Pathology, University of Utah, Salt Lake City UT ; 3ARUP Laboratories, Salt Lake City, UT
4,5Departments of Pediatrics, Human Genetics, University of Utah School of Medicine, Salt Lake City, UT
Abstract
Although carriers of mutations resulting in autosomal recessive disorders are not usually affected phenotypically, nor are they symptomatic, identifying heterozygous deletions for genes in which homozygous deletions have
clinical consequences has merit. For example, identification of carrier status allows an individual to make informed decisions regarding child bearing. We discuss heterozygous findings involving three genes in which
homozygotes are clinically affected. Nephronophthisis (NPH) is an autosomal recessive nephropathy with chronic tubulointerstitial involvement which represents the leading cause of end-stage renal disease in children
and adolescents. The most frequent genetic abnormality found in NPH is a large homozygous deletion of the NPHP1 gene. Homozygous deletions of NPHP1 have also been identified in a subset of patients with Joubert
syndrome. The most common cause of renal tubular Fanconi syndrome, cystinosis, is a lysosomal storage disorder which can be caused by homozygous deletion of the CTNS gene which encodes the lysosomal cystine
carrier cystinosin. Alpha thalassemia, the most prevalent worldwide autosomal recessive disorder, is a hereditary anemia. Homozygous deletion of both HBA1and HBA2 genes results in prenatal or early neonatal death.
We have identified heterozygous deletion of NPHP1, CTNS, or HBA 1 and HBA2 genes in routine clinical samples submitted for array comparative genomic hybridization (aCGH) analysis in pediatric patients referred for
developmental delays and/or multiple congenital anomalies. Although these findings are likely not pertinent to the patient's indication for testing, we aim to perform confirmatory testing and include the findings of such
testing in the clinical report. Using a combination of molecular tests, we have confirmed heterozygous deletions in each of these cases. While it is important to develop a robust test for confirmation and carrier status
detection in heterozygous cases such as these, conveying of this information, and how it is done requires careful education and explanation. We believe these three examples are likely to be representative of multiple
additional genes where clinical interpretation of aCGH results needs to be carefully presented through a health care provider such as a genetic counselor.
Introduction
Array comparative genomic hybridization (aCGH) has become an accepted method for detecting genomic
copy number variation in patients with developmental delay, dysmorphic features, and multiple congenital
abnormalities. Recent advances in aCGH technology allow for whole-genome analysis at a resolution that is
impossible using standard cytogenetic techniques. However, while the resolution of whole-genome aCGH
has the potential to improve diagnosis and prognosis, unexpected findings arising from the information this
technology produces may result in dilemmas regarding clinical obligation, confirmation and reporting of these
findings. We have identified small heterozygous deletions of the NPHP1, CTNS, HBA1 or HBA2 genes from
routine clinical samples submitted for aCGH analysis in pediatric patients referred for developmental delays
and/or multiple congenital abnormalities. We describe the molecular tests used to confirm the presence of
these heterozygous deletions and discuss the dilemmas associated with interpretation and reporting.
Figure 1. Left: Log ratio plot showing a 15.6kb deletion of HBA1 and
HBA2 genes. Below: Alpha thalassemia gene deletion determined
by comparing the amplicon size (arrow) to a DNA size standard run
concurrently on an agarose gel. Band size indicates a Filipino –type
alpha thalassemia deletion.
Methods
Microarray testing was performed using an Agilent 44k oligonucleotide array with the International
Standard Cytogenomic Array (ISCA) design. The array is designed to detect gains or losses at a minimum
of 500 kb across the genome, or smaller imbalances in regions of known microdeletion/duplication
syndromes or targeted genes.
The alpha thalassemia assay is based on PCR amplification of the alpha-globin gene cluster in genomic
DNA isolated from peripheral blood and is designed to interrogate for 7 common deletion forms and HBA2
gene in a single multiplexed reaction (Figure 1). The inclusion of the HBA2 primer set within the multiplex
reaction acts as a secondary control for PCR in the deletion detection reaction in the event that no
deletions are present in the sample. The products of PCR are detected by gel electrophoresis and size
determination is made by comparison to a DNA size ladder.
b2m
Control Ct: 28.05
Patient Ct: 26.82
NPHP1
Control Ct: 25.98
Patient Ct: 25.83
Quantitative real-time PCR was used to amplify targets contained within the NPHP1 gene (Figure 2). Two
amplicons of similar size (100 bp) were used as reference controls (one contained in the β-2microglobulin
gene and one in the β-globulin gene). The target and reference amplicons were amplified using the patient
sample and a two-copy control sample. The LightCycler® was used for amplification and real-time
quantification. The PCR efficiency was assumed to be 2 and the gene dosage ratio was calculated using
the comparative Ct (2-DDCt method) and the equation 2-[DCt(target) - DCt(ref)] where :
DCt(target) = Patient Ct- Control DNA Ct of target amplicon
and DCt(ref) = Patient Ct - Control DNA Ct of reference amplicon
DExon 7
Db2m
Ratio
-0.15
-1.23
0.47
Figure 2. Left: Log ratio plot showing 97 kb NPHP1 gene deletion.
Above: Gene dosage analysis of PCR amplicon using comparative
2-DDCt method resulted in a value of 0.47 (deletion of the NPHP1
gene).
Deletion values fall within the 0.4-0.7 range. No change in copy number ranges from 0.8-1.2. Duplications
fall in the range of 1.3-1.7
To confirm the CTNS gene deletion, a 25 kb oligonucleotide FISH (oFISH) probe was generated using PCR
amplification followed by fluorescent labeling (Figure 3).
Conclusion
The use of aCGH has greatly increased the amount and resolution of data we can collect on a
patient. Consequently, the potential for unveiling incidental findings in the course of the analysis
has also increased. The reporting of these findings, however, is not a novel concept. Clinical
cytogeneticists have reported what are believed to be incidental but clinically significant
chromosomal aberrations identified in metaphase chromosomes for decades. Examples include
the finding and reporting of a constitutional balanced rearrangement in a leukemic bone marrow
chromosome analysis or the finding of an inherited balanced rearrangement in a prenatal
chromosome analysis.
Reporting incidental information which is not pertinent to the patient’s indication for testing has
potential benefit both child and parent. If the child presents with symptoms suggestive of the
recessive condition, it may be that the identified deletion is in trans with an as of yet unidentified
mutation in the non-deleted allele. Carrier testing of parents can help identify a couple at risk for
conceiving an affected child
Figure 3. Left: Log ratio plot of 15.3 kb CTNS gene deletion .
Above: CTNS gene deletion confirmed using oFISH (see poster # 1447/W)
We encourage discussion among cytogeneticists to come to a consensus regarding reporting of
copy number changes such as those shown in this study. These findings clearly indicate the
need for patient education and careful interpretation through a genetic counselor or wellinformed health-care provider.