Microarray-based comparative genomic hybridization (array CGH) is a newly emerged molecular cytogenetic technique for rapid evaluation of the entire genome with sub-megabase resolution. It allows for the comprehensive investigation of thousands and millions of genomic loci at once and therefore enables the efficient detection of DNA copy number variations (a.k.a, cryptic genomic imbalances). The development and the clinical application of array CGH have revolutionized the diagnostic process in patients and has provided a clue to many unidentified or unexplained diseases which are suspected to have a genetic cause. In this paper, we present three clinical cases in both prenatal and postnatal settings. Among all, array CGH played a major discovery role to reveal the cryptic and/or complex nature of chromosome arrangements. By identifying the genetic causes responsible for the clinical observation in patients, array CGH has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner.
Genomic disorders, resulting from DNA rearrangements involving region-specific repeat sequences, are caused by abnormal dosage of one or more genes located within the rearranged genomic fragments. Cytogenetic analysis has been a useful diagnostic tool for this disease category especially in idiopathic developmental delay/mental retardation, multiple congenital anomalies, dysmorphism, and pregnancy at risk for chromosomal abnormalities. However, the limitation of band resolution in the conventional cytogenetic methodology karyotype (5–10 Mb) has prompted the development of technologies which can identify previously unrecognized chromosomal anomalies. Since Solinas-Toldo et al. published the first article on array-based comparative genome hybridization (array CGH) in 1997 [
In pediatric patients with idiopathic developmental delay and dysmorphic features, it is difficult to come up with a specific diagnosis due to the lack of cardinal features of a syndrome. Similarly, in the prenatal setting when congenital anomalies are seen which do not suggest a specific syndrome, it is especially difficult to make the diagnosis within a limited time frame so that appropriate management can be performed. In these cases, the use of array CGH has demonstrated great advantages to both patients and physicians. First of all, the multiplex format of array enables the simultaneous screening of hundreds of well-characterized disease loci and the subtelomeric regions, leading to the shortened diagnostic process and reduced cost compared to ordering sequential tests for each individual locus. Second, array CGH is a discovery-based approach. When a CNV with unknown clinical significance is identified, the genes located within it become the potential candidates, which facilitate further studies leading to disease gene discovery. Besides, identifying novel CNVs may also help to characterize a new genetic disorder. Third, the technical developments from arrays based on BAC (bacteria artificial chromosome) to oligonucleotide probes and the much improved array resolution enable the potential to better define the boundary of genomic gains and losses [
We have chosen to focus on patients in whom the complete cytogenetic abnormalities were principally discovered by array CGH, rather than by conventional karyotyping or fluorescence insitu hybridization. By presenting the three cases here, we would like to emphasize the power of array CGH in identifying the genetic causes responsible for the clinical presentations in the patients so that accurate diagnosis, prognosis, and clinical management can be provided and achieved.
The patients and their families were recruited through the Genetics Clinic at the Alberta Children’s Hospital. Informed consents were obtained from the patients or their parents.
Genomic DNA was extracted from the whole blood of patients using Gentra Puregene Cell Kit (Qiagen) according to the manufacturer's protocol. Genomic DNA from each patient was labeled with red fluorescent dye cyanine-5 (Cy-5) and hybridized with same-sex normal reference DNA (Promega) labeled with green fluorescent dye Cy-3. Array CGH was performed using two platforms. The first platform, CytoChip ISCA 8 × 60 K v2.0 Oligonusleotide array (BlueGnome), consist of 60,000 oligonusleotides and evaluates the whole genome with an effective backbone resolution of 170 Kb. 137 OMIM genes are represented with average oligonucleotide spacing of 3.5 Kb and at centromeres and subtelomeres of 4.5 Kb. The second platform, Cytosurs Syndrome Plus V2 2 × 105 K Oligonucleotide array (OGT), consists of 105,000 oligonucleotides with a backbone resolution of 40 Kb. 410 genes in disease associated areas are targeted with oligonucleotide distributing every 3 Kb. Subtelomeric regions are represented with an average resolution of 40 Kb.
Hybridization of highly repetitive sequences was suppressed by addition of unlabeled
The copy number variations identified by a CGH, which we considered for further validation, were analyzed using fluorescence in situ hybridization (FISH) analysis. FISH probes were either chosen from the eFISH (build 36) and ordered from the Toronto Centre for Applied Genomics or purchased commercially. FISH was performed on metaphase chromosomes or interphase nuclei using the standard FISH clinical protocol preestablished in the Cytogenetic Laboratory in the Alberta Children’s Hospital. Parental blood samples were examined to determine the inheritance pattern of the variations.
The couple came to our attention due to the positive first trimester screen. The woman is a healthy 25-year-old, G1P0. The first trimester nuchal translucency screen at 13 weeks was positive, and the couple decided to proceed with chorionic villus sampling. To rule out common chromosomal aneuploidies, fluorescence in situ hybridization (FISH) studies were performed using specific probes targeting chromosome 13, 18, 21, X, and Y (AneuVysion kit, Abbott Molecular Inc.), and the results were normal. The followup G-banded chromosome study revealed a reciprocal translocation involving the short arm of one chromosome 11 and the short arm of one chromosome 12 – 46,XY,t(11;12)(p14;p13.2) (Figure
Apparently balanced translocation is accompanied by a cryptic genomic deletion (a) Conventional karyotyping revealed a reciprocal translocation involving the short arm of one chromosome 11 (11p14) and the short arm of one chromosome 12 (12p13.2), which appears to be balanced. Chromosomes 16 appear normal. (b) Array CGH study detected a significant copy number loss on chromosome 16 (q23.2-q24.1) (c) This deletion was confirmed by FISH studies using a BAC probe RP11-625B13 (red) mapped to 16q23.3 and a control probe RP11-6C20 (green) mapped to 16p13.3. (d) Genes located within the deleted region 16q23.2-q24.1 (nucleotide 79,945,820 to 85,430,304, NCBI 36/HG 18) as shown by the UCSC genome browser. These include FOX family cluster
The Database of Genomic Variants (DGV, NCBI 36/HG 18) is a useful tool to exclude benign CNVs. DGV revealed that there are no reported benign CNVs encompassing this deleted region, suggesting that it is not likely to be a benign change. This
The majority of apparently balanced structural rearrangements are not associated with an abnormal phenotype. After the identification of such rearrangements, it is important to test the parents and determine whether it is inherited or
A 2-year boy was referred to the genetics clinic for assessment of his recurrent thrombocytopenia, short stature, and dysmorphic features. He was found to have isolated thrombocytopenia during acute illnesses which would resolve spontaneously with resolution of the intercurrent illness. Platelet count was below normal when well. Hematologic investigations included a normal peripheral blood smear, bone marrow aspirate and biopsy, and a normal immune workup. His past medical history was significant for vesicoureteral reflux, hypospadias, bilateral cryptorchidism, laryngomalacia, eczema, and constipation. He has mild fine motor and speech delays as well as behavioural issues including poor socialization, aggressive behaviour, and decreased attention. On physical examination at 6 years of age his growth parameters were all below the 3rd percentile. He had coarse hair, hypertelorism, epicanthal folds, short palpebral fissures, a depressed nasal root with a bulbous nasal tip, small teeth, low-set ears which were posteriorly angulated as well as short 5th fingers bilaterally with clinodactyly of the 5th digit on the left hand. Review of the family history revealed a younger brother who was small for age but was proportionate. He was nondysmorphic and had no other clinical concerns. Considering that he has a normal karyotype and his parents are small in stature, no further cytogenetic investigations (e.g., array CGH) were performed.
Cytogenetic investigations in the patient showed a chromosome abnormality with additional satellites on the distal long arm of one chromosome 21 (46,XY,21qs), which was confirmed by FISH studies using an Acro-p probe (Abbott Molecular Inc.) (Figure
Complex rearrangement is revealed by array CGH. (a) FISH studies using acro-p probe targeting the short arms of all acrocentric chromosomes (red) were used to investigate the additional satellite on the long arm of 21qs. Insert shows the G-banded 21 chromosome with additional satellite on 21q (as indicated by
The first and most proximal copy number variation (CNV) is a gain which encompasses a region of 1.1 Mb from nucleotide 23,449,744 to 24,557,710. DGV revealed there are no reported benign CNVs fully encompassing this duplicated region. However, there are no known genes within this region (by UCSC genome browser), suggesting that this duplicated region is of no clinical significance. The second CNV is a loss which encompasses 1.81 Mb from nucleotide 34,965,815 to 36,781,907 and includes the gene
This patient is the firstborn child to a healthy unrelated couple. At the time of birth the father and the mother’s ages were 30 and 29, respectively. No anomalies were seen on the prenatal ultrasound, and the newborn was healthy. By nine months, the weight and length were at the 3rd percentile, while the head circumference remained at the 50th percentile. Developmental delay (delay in sitting, standing, and walking) was evident in the first year. At 16 months, severe expressive language delay was noted but receptive language was within normal limits. At 14 months, he had five seizures associated with fever and some focal signs. The patient was first seen at the genetics clinic at 20 months. No precise diagnosis had been made despite many investigations. The karyotype showed a normal male chromosome complement. Subtelomere FISH studies (TelVysion kit, Abbott Molecular Inc.) also showed a normal result. Other studies included UPD7, multiple biochemical tests, CT, brain MRI, skeletal survey, and thyroid studies which were all normal. At seven years nine months, the patient’s height and weight were less then the 3rd percentile and head circumference remained at the 50th percentile. Now at the age of nine, he speaks intelligibly and in sentences. There are no behavior problems, no further seizures, and no sleep problems. He remains small despite a good appetite. His physical features demonstrate a relative macrocephaly with a prominent forehead, sparse hair and eyebrows, prominent ears which are low set, and cup-shaped, deep set eyes with esotropia, astigmatism, and possible congenital anomaly of the left optic disc (Figure
Array CGH identified a genomic loss and gain responsible for the patient’s phenotype. (a) Patient’s photo at the age of nine. His physical features demonstrate a relative macrocephaly with a prominent forehead, sparse hair and eyebrows, prominent ears which are low set, and cup-shaped, deep set eyes with esotropia, astigmatism, and possible congenital anomaly of the left optic disc. (b) Array CGH study detected a significant copy number loss on chromosome 4 (q13.2-q21.1). (c) Array CGH study also identified a significant copy number gain on chromosome 6 (q24.3). (d) The deletion was confirmed by FISH studies using a BAC probe RP11-165D10 (red) mapped to 4q13.2 and a control probe RP11-483A2 (green) mapped to 4q25. (e) The gain was confirmed by FISH studies using a BAC probe RP11-1077K2 (red) mapped to 6q24.3 and a control probe RP11-346K8 (green) mapped to 6p21.33. Of note, D and E are the same metaphase with different probe sets to visualize both chromosomes 4 and 6. The extra red signal appears to locate on the derivative chromosome 4. (f) Genes located within the deleted region 4q13.2-q21.1 (nucleotide 67,133,352 to 77,615,947, NCBI 36/HG 18) as shown by the UCSC genome browser. (g) Genes located within the duplicated region 6q24.3 (nucleotide 146,155,718 to 148,055,364, NCBI 36/HG 18) as shown by the UCSC genome browser.
Once the technology was developed locally, array CGH was performed on the peripheral blood of the patient using the CytoChip ISCA 8 × 60 K v2.0 Oligonucleotide array platform (BlueGnome). The array analysis identified two chromosomal abnormalities. The first one is a copy number loss of a 10.5 Mb region on the long arm of chromosome 4 (q13.2-q21.1) from nucleotide 67,133,352 to 77,615,947 (Figure
FISH studies revealed a four-break balanced complex rearrangement in the patient’s father. (a) By using BAC probe RP11-165D10 (red) and control probe RP11-483A2 (green), FISH studies identified that the 4q13.2 segment has moved from derivative chromosome 4 to derivative chromosome 6. (b) By using BAC probe RP11-1077K2 (red) and control probe RP11-346K8 (green), FISH studies identified that the 6q24.3 segment has moved from derivative chromosome 6 to derivative chromosome 4. (c) A simplified diagram to show the increased risk of having an unbalanced offspring in this family. Green represents the 4q13.2-q21.1 segment and the red represents the 6q24.3 segments. In the father, the green and red segment, switch locations leading to unbalanced offspring with either 3 copies of the red segment and 1 copy of the green segment (as in the patient) or 3 copies of the green segment and 1 copy of the red segment.
The patient’s clinical presentation is most likely due to the deletion and duplication of many genes involved in the imbalanced regions (Figures
Through combining array CGH and FISH techniques, we successfully identified the genomic imbalances responsible for the patient’s clinical manifestation and further a familial chromosomal rearrangement which changes the predicted outcome and clinical management in this family. The father now carries a significant risk of having another abnormal liveborn with either der(4)t(4;6) as in the patient or der(6)t(4;6) (Figure
The wide application of array CGH has remarkably improved the detection of DNA copy number variation and complex chromosome rearrangement. It has revolutionized the diagnostic process of patients with global developmental delay, dysmorphic features, multiple congenital anomalies, as well as complicated pregnancy at risk for chromosomal aberrations. By identifying the genomic imbalances responsible for the clinical presentation in patients, clinicians can provide an accurate diagnosis, predict the potential risk in the future and alter the clinical management in the patients and/or their families.
The authors would like to thank patients and their families for their support. Informed consent was obtained from the patients or their parents for publication of the patients clinical history and lab test results. They would also like to thank the Cytogenetics laboratory at Alberta Children’s Hospital for the karyotype, array CGH, and FISH analysis.