Chromosomal microarray analysis as the first-tier test for the identification of pathogenic copy number variants in chromosome 9 pericentric regions and its challenge
Chromosomal microarray analysis (CMA) has been recommended and practiced routinely in the large reference laboratories of U.S.A. as the first-tier test for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Using CMA as a diagnostic tool and without a routine setting of fluorescence in situ hybridization with labeled bacterial artificial chromosome probes (BAC-FISH) in the large reference laboratories becomes a challenge in the characterization of chromosome 9 pericentric region. This region has a very complex genomic structure and contains a variety of heterochromatic and euchromatic polymorphic variants. These variants were usually studied by G-banding, C-banding and BAC-FISH analysis. Chromosomal microarray analysis (CMA) was not recommended since it may lead to false positive results. Here, we presented a cohort of four cases, in which high-resolution CMA was used as the first-tier test or simultaneously with G-banding analysis on the proband to identify pathogenic copy number variants (CNVs) in the whole genome. CMA revealed large pathogenic CNVs from chromosome 9 in 3 cases which also revealed different G-banding patterns between the two chromosome 9 homologues. Although we demonstrated that high-resolution CMA played an important role in the identification of pathogenic copy number variants in chromosome 9 pericentric regions, the lack of BAC-FISH analysis or other useful tools renders significant challenges in the characterization of chromosome 9 pericentric regions.
Trial registration: None; it is not a clinical trial, and the cases were retrospectively collected and analyzed.
KeywordsChromosome 9 Euchromatic variants First-tier test Heterochromatic variants Interstitial deletion Interstitial duplication Pericentric inversion
CMA as the first-tier test for the identification of pathogenic CNVs in chromosome 9 pericentric regions and its challenge.
Letter to the Editors
Chromosomal microarray analysis (CMA) has been recommended as the first-tier test for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies since 2010 [1, 2], and was later confirmed by other group . This practice has become a standard for the large reference laboratories in U.S.A after the American College of Medical Genetics (ACMG) published the professional guidelines . In these laboratories, CMA has largely replaced fluorescence in situ hybridization (FISH) analysis for the identification of pathogenic copy number variants (CNVs) across the whole genome.
Chromosome 9 pericentric regions have been known to contain a very complex genomic structure because of the presence of a huge amount of heterochromatin and large tracks of segmentally duplicated euchromatin [5, 6]. The segmentally duplicated sequences predispose and mediate the generation of deletion, duplication, insertion, triplication and amplification variants within the pericentric regions of chromosome 9 [5, 7]. In a cohort of 334 carriers studied by using FISH analysis with different sets of labeled bacterial artificial chromosome (BAC) probes (BAC-FISH), 17 different types of heterochromatic variants of chromosome 9 have been identified , with pericentric inversions being the most frequent variant (50 %) followed by 9qh-variants (24 %) and 9ph-variants (11 %). Additionally, four different types of euchromatic variants have been detected: (1) 9p12 amplification variant [8, 9], (2) 9q12 insertion variant , (3) 9q21 deletion variant , and (4) 9q21 amplification variant . Because of the presence of repetitive sequences, chromosomal microarray analysis (CMA) was not recommended to be utilized since it may lead to false positive results [6, 7].
For this reason, we retrospectively reviewed our postnatal database to search interesting cases with CNVs in chromosome 9. High-resolution CMA was either used as the first-tier test or simultaneously ordered with G-banding analysis. For G-banding, peripheral blood was cultured for 72 h in RPMI-1640 medium. Metaphase chromosomes were analyzed using standard G-banding techniques. CMA was performed using genomic DNA extracted from uncultured whole blood on the oligo-SNP array (CytoScan HD®, Affymetrix). Hybridization, data extraction and analysis were performed as per manufacturer’s protocols. The Affymetrix® Chromosome Analysis Suite (ChAS) Software version 2.0 was used for data analysis, review and reporting. For chromosome 9, the probes from CytoScan HD® covered the following genomic regions: 9p (chr9:192,129–40,784,142, 43,400,082–44,900,526) and 9q (chr9:66,837,485–141,025,328). Thresholds are set at >200 kb for gains, >50 kb for losses for genome-wide region, and at >100 kb for gains, >20 kb for losses for cytogenetic relevant regions. Genomic coordinates are based upon genome build 37/hg19 (2009).
Patient 2 was a 2-year-old girl with developmental delay, lack of coordination, mixed receptive expressive language disorder and convulsion. It was initially considered as a chromosome 9 euchromatic variant with an extra G-dark band within the proximal 9q heterochromatin region or it could be an insertion or duplication from 9q13–q21.1 (Fig. 1c, arrow). The C-banding was negative. This type of euchromatic variant has been reported in several cases [5, 15, 16] and classified as one of the four major categories of euchromatic variant . The high-resolution CAM that was simultaneously performed, however, showed a genomic gain of 6.3 Mb from 9q21.33–q22.31 (chr9:90,118,500–96,395,801; hg19; Fig. 1d; genes involved listed in the Additional file 1: Table S1). This case was an excellent example of how CMA analysis showed that an extra G-dark band in proximal 9q was a pathogenic duplication of proximal 9q rather than a euchromatic variant. This gain did not include the PTCH1 gene (located at chr9: 98,205,265–98,279,247) and, thus the phenotype was different from the previously reported familial cases with 9q22.3 microduplication spanning PTCH1 , and without microcephaly or holoprosencephaly that were often seen in patients with gain of PTCH1 .
Patient 3 was a 24-year-old woman referred to rule out a chromosomal disorder with reported family history of a chromosome 9 abnormality. G-banding analysis considered it was an insertion of an extra band to the 9q22 region (Fig. 1e). CMA revealed it was a duplication of 10.4 Mb from 9q21.31-q22.2 (chr9:82,745,056–93,173,691; Fig. 1f; genes involved listed in the Additional file 1: Table S1). Duplication of 9q21.2–q22.3 was reported in a 2-year-old female with growth and motor retardation and in her aunt without apparent phenotypic anomalies , and a more distal duplication of 9q22.31–q22.32 was associated with congenital diaphragmatic hernia . Our patient was not known to have these anomalies.
Patient 4 was a 10-year-old boy with failure to thrive and cognitive deficits. G-banding analysis revealed an extra band at 9q12 region (Fig. 1g), and C-banding was positive (Additional file 2: Figure S1). CMA did not reveal copy number variants (Fig. 1h) since the probes from the CMA apparently did not cover the repetitive sequences. The G-positive and C-positive extra band in this case was concluded as a rare heterochromatic variant , instead of a G-positive and C-negative euchromatic variant .
The four cases described here demonstrate how CMA characterize the chromosome 9 pericentric regions along with G-banding and/or C-banding without access to BAC-FISH analysis. Patient 1 was a very rare case with the presence of an interstitial deletion which coexisted with a polymorphic pericentric inversion. Patient 2 was an excellent example of how CMA differentiated a pathogenic gain from a heterochromatic variant as observed in patient 4. In patient 3, CMA revealed it was a likely pathogenic duplication although previous report showed a reduced penetrance in one family study .
Although we demonstrated that high-resolution CMA played an important role in the identification of pathogenic copy number variants in chromosome 9 pericentric regions, the lack of BAC-FISH analysis as a routine setting in the large reference laboratory renders significant challenges in further characterization of chromosome 9 variants. The major advantage of using CMA as the first-tier test is to detect pathogenic CNVs across the whole genome including chromosome 9. However, when a huge amount of heterochromatin and large tracks of segmentally duplicated euchromatin are present in chromosome 9 pericentric regions, CMA was unable to differentiate tandem, inverted and insertional duplication, or to confirm pericentric inversion. Our cohort supported using CMA as first-tier test when chromosomal disorders were indicated to rule out the presence of pathogenic copy number variants, and using BAC-FISH analysis to further evaluate heterochromatic and euchromatic variants of chromosome 9.
BAC, bacterial artificial chromosome; CMA, chromosomal microarray analysis; FISH, fluorescence in situ hybridization
The authors acknowledge that Andrew Hellman and Jeff Radcliff provided valuable assistance in manuscript revisions.
Availability of data and materials
The raw data and materials used for this study have been de-identified and were freely available to any scientist wishing to use them without breaching participant confidentiality.
JCW- reviewing, analysis and interpretation of the data, drafting and revising the article, final approval of the version to be published. FZB - interpretation of data. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
This manuscript is a retrospective case report that does not require ethics committee approval at our institution. Consent was considered not required because the cases reported here were anonymous and de-identified.
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