Dr KW Choy, Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR. Email firstname.lastname@example.org
Objective We investigated the application of high-resolution microarray-based comparative genomic hybridisation (array CGH) on a fetus showing increased nuchal translucency (NT).
Design Case study.
Setting Tertiary referral obstetrics unit.
Sample Pregnant woman attended the antenatal clinic.
Methods Conventional karyotyping and genetic test was carried out for the alpha-globin gene. High-resolution array CGH using the high-density 244K Agilent microarray was performed on fetal blood sample by cordocentesis to investigate the possibility of any genomic imbalance.
Main outcome measures Detection of chromosomal abnormality.
Results Karyotyping analysis showed 46,XY. Molecular genetic diagnosis confirms the fetus has Hb-H constant spring disease but cannot explain the increased NT to 3.2 mm. Array CGH analysis discovered a 1.32-Mb microdeletion on chromosome 16p13.11. Deletion at 16p13.11 has been implicated to predispose to autism and/or mental retardation. Baby was delivered at 40 weeks of gestation, and follow up was carried out at 3 months of age without sign of mental retardation/developmental delay.
Conclusions This case study demonstrated that array CGH can accurately calibrate the size and identify de novo interstitial chromosome imbalances. However, the presence of chromosome copy variants with unknown clinical significance currently limits its wider scale application in prenatal diagnosis and needs further investigations.
Increased nuchal translucency (NT) during the first trimester of pregnancy is a useful marker for the detection of chromosomal abnormalities.1 Conventional cytogenetic analysis by G-banding has been applied in prenatal diagnosis to identify numerical and structural chromosomal aberrations.2 However, conventional karyotyping yields lower band resolution (>4 Mb), and this makes detection of subtle abnormalities difficult, meaning that numerous common microdeletion syndromes are not detectable by karyotype. More recently, microarrays using large-insert clones, single nucleotide polymorphism array or oligo arrays can detect deletions or duplications (but not balanced structural rearrangements) identified by routine chromosomal analysis.3,4 Microarray-based comparative genomic hybridisation (array CGH) is based on the same principle as conventional metaphase CGH. In array CGH, the microarray contains probes that can be short (25- to 60-mer probe length) oligonucleotides or genomic fragments (up to 1 Mb). To identify copy number gain or loss from an individual patient, the whole genomic DNA from a patient’s DNA and a reference (normal) control DNA are differentially labelled with two different fluorophores. After the samples are co-hybridised to the same array, the fluorescence ratio generated from control versus patient for each probe represents the average copy number ratio between the patient’s and control’s DNA. A gain or loss of fluorescence signal intensity from the patient’s DNA indicates a retrospective gain or loss of patient’s DNA copy numbers. Therefore, the use of microarray probes with known human genome positions allows direct identification of the DNA copy number gains and losses within the genome.
In general, the resolution of array CGH is determined by two main factors: the density of probe coverage over the genome and the number and physical distance of neighbouring probes on the target chromosome. Hence, depending on the type and resolution of the microarrays, array CGH can detect copy number changes of any size from as small as few base pairs to submicroscopic deletions or duplications that are up to Mbs in size,3,4 which is well below the level of discrimination by G-banded karyotype analysis.5,6
Copy number variation (CNV) is a term collectively used to describe gains and losses of DNA sequences >1 kb in length.7 Such additional subtle changes are found in clinically significant syndromes, including subtelomeric chromosomal deletions, which account for approximately 6% of idiopathic mental retardation with or without congenital malformations.8 However, array CGH also offers rapid and high-throughput analysis on small amounts of DNA, which, apart from sensitivity and specificity, are the two most important prerequisites for any platform applied to prenatal diagnosis.
We anticipate that, given its ability to comprehensively detect genome-wide chromosomal aberrations at a resolution unattainable by other molecular cytogenetic techniques, array CGH has the potential to be used for prenatal diagnosis and may address many of the limitations of both conventional microscopic cytogenetic analyses and fluorescent in-situ hybridisation (FISH).9 However, there are several major questions that need to be addressed before the routine use of array CGH in prenatal medicine. First, the identification of small copy number changes may not be associated with an abnormal clinical phenotype, even if there are de novo. Second, we still know very little on (i) microdeletion/microduplication syndromes, (ii) CNVs and (iii) phenotype–genotype correlation, in other words what is a normal or abnormal CNV. Last but not least, the accuracy and reliability of array CGH in prenatal diagnosis has not yet been well established.
In this study, we applied the high-resolution genome-wide 244K array to identify chromosomal imbalances in fetus with increased NT to investigate the application of array CGH in prenatal diagnosis.
Cytogenetic and array CGH analysis
G-banded chromosome analysis was performed using standard cytogenetic techniques. For array CGH analysis, the total genomic DNA (1 microgram) was extracted from the fetal blood from cordocentesis by Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). The patient’s consent had been obtained, and approval sought from the Ethics Committee on Human Research, The Chinese University of Hong Kong and the Prince of Wales Hospital (reference CRE 2007.059). Agilent human CGH microarray (Agilent Technologies, Santa Clara, CA, USA) containing unique oligonucleotides representing 244 000 probes (244K) with average probe spacing across the human genome of 6.4 Kb were used for the array CGH experiments. Labelling reactions were performed with 1 μg genomic DNA with Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Santa Clara, CA, USA) according to manufacturer’s protocol. The microarray chip was scanned by the Agilent Microarray Scanner. Data analysis was by the Agilent Feature Extraction 9.1 and CGH Analytics 3.4. In brief, a log2 expression ratios were computed and normalised for forward and reverse fluor (i.e. dye-swap) experiments using the CGH Analytics 3.4 software.
Putative chromosome copy number changes were defined by intervals of three or more adjacent probes with log2 ratios, suggestive of a deletion or duplication when compared with the log2 ratios of adjacent probes. The quality-weighted interval score algorithm (ADM2) was used to compute and assist the identification of aberrations for a given sample.
A 32-year-old Chinese nulliparous woman was first seen at 12 weeks of gestation for first-trimester combined screening for trisomy 21. She was known to be alpha-thalassaemia trait, but her partner’s red cell mean cell volume was normal (82 fl). The fetal NT measured 3.2 mm, and free-beta-human chorionic gonadotrophin (fb-hCG) and pregnancy-associated plasma protein A (PAPP-A) were 0.79 multiples of the median (MoM) and 0.76 MoM, respectively. As she was screened positive (risk of 1 in 200), chorionic villus sampling was performed. The fetal karyotype was 46,XY. A morphology scan at 21 weeks of gestation showed a normal nuchal fold and normal cardiac structures. However, there was placentomegaly with placental thickness of 3.52 cm (above 2 SD), and cardiomegaly was found with a cardiothoracic (CT) ratio of 0.52. The peak systolic velocity of the middle cerebral artery (PSVMCA) was 48.7 cm/second (>1.5 MoM).10 The liquor volume was normal. No other abnormality was detected. The fetus was suspected to have anaemia. Cordocentesis was thus performed at 23 weeks of gestation, which showed a fetal haemoglobin level of 6.7 g/dl with 40% Hb Barts and 60% Hb-F. It was therefore likely a case of haemoglobin H disease. Molecular studies showed the fetus to have Hb-H constant spring disease carrying the alpha 2 gene codon 142TAA→CAA constant spring mutation, which should not be transfusion dependent. The case was managed in conjunction with the haematology team, and their opinion was that the condition should not be transfusion dependent. Although the fetus had cardiomegaly, there were no other sign of hydrops fetalis and in utero blood transfusion was thus not performed. Instead, the woman was closely monitored with serial ultrasound scans, which showed normal fetal growth with mild cardiomegaly and a CT ratio of 0.59. The PSVMCA improved from 1.29 to 1.5 MoM. A 3.2-kg baby boy was delivered vaginally at 40 weeks of gestation. There were no dysmorphic features, and echocardiogram showed normal cardiac structure and function. The haemoglobin level was 13.4 g/dl at birth, and the baby was otherwise well. Regular follow up was conducted by the neonatologists, and no sign of developmental or growth delay was found during follow up at 3 months of age.
Cytogenetic and array CGH analysis
The cause of increased NT in this case was unknown initially. The fetal karyotyping was normal, 46,XY (Figure 1). Although fetal Hb-H may associate with increased NT, it has been shown that even in fetal alpha-thalassaemia major, the NT MoM is only increased by 19%.11 Therefore, the HB-H constant spring phenotype may not explain the significantly increased NT of 3.2 mm in this case. To investigate the presence of chromosomal imbalance, we performed array CGH analysis on the fetal and parental blood samples. As array CGH is not yet reliable on DNA extracted directly from amniotic fluid or CVS, it can only be carried out on fetal blood in our unit. Using the high-resolution 244K array platform from Agilent Technologies, a single copy number loss of chromosomal material, involving chromosome region 16:14876356 (within 16p13.11) to chromosome region16: 16199736, was detected in the fetal blood sample (Figure 1). Our array CGH results show that the deletion spans 1.32 Mb. To confirm our array CGH findings, to eliminate a false-positive finding and to identify the origin of this 16p13.11 microdeletion, a semi-quantitative polymerase chain reaction (PCR) technique was employed. Using six pairs of reported primers spanning 16p13.1,12 we identified the presence of a single copy deletion among the fetal DNA and there was no such deletion detected in the parental blood samples. In addition, we detected five chromosome CNVs ranging from 500 Kb to 1.26 Mb among the fetal DNA, and these were confirmed to be normal CNVs according to the Database of Genomic Variants.7
Our array CGH results indicate no presence of other chromosome aberrations detected by 244K array CGH across the fetal genomic DNA.
Discussion and conclusions
First-trimester screening using a combination of age and NT, PAPP-A and fb-hCG has been shown to be an effective way to screen for aneuploidy, with a detection rate of 90% at a false-positive rate of 5%.13 However, in fetuses with increased NT but normal karyotyping, many associations with other abnormalities such as syndromal disorders or cardiac abnormalities have been described.14 It is still unknown whether they are secondary to chromosomal abnormalities, which are not detectable with conventional karyotyping methods. In this single case study, we demonstrate the application of array CGH in prenatal diagnosis, which permits the rapid identification of a microdeletion at 16p13.11 in a fetus with increased NT.
Array CGH enables comprehensive high-resolution genome screening at approximately 19.2 Kb (because genomic changes were defined by intervals of three or more adjacent probes) across the genome, and mapping of DNA sequences not reachable by conventional karyotyping or FISH. More importantly, the reporting time for this comprehensive screening across the human genome only takes only 3 days, requires as little as 1 microgram DNA and could precisely identify that the deleted region at 16p13.11 included the critical region predisposing to autism and/or mental retardation.12 Currently, for array CGH, any copy number changes will require further verification by quantitative PCR or FISH that may take an additional 7–60 days.
For array CGH studies, parental DNA is always needed for analysis to determine if the finding is de novo, and parental bloods should be taken with the fetal sample. The presence of de novo chromosome CNVs can be difficult to interpret as a ‘positive’ result, especially when involving chromosomal segments, of which the clinical significance is still unclear. This means that further investigation and counselling of such cases can be difficult and time consuming. For example, in this case, an obstetrician as well as a clinical geneticist were involved in counselling the woman. We first search through the Database of Genomic Variants for all the five CNVs we identified and excluded all of them except the microdeletion at 16p13.11 to be pathogenic CNVs. For the 16p13.11 deletion, we further searched the DECIPHER database, and it revealed another two cases: DECIPHER15 patient number 00000605 and 00001497; both had del(16)(p12.3;p13.11) spanning a 2.6-Mb deletion containing the 16p13.11 region identified in our case.11 Although there was no consistent morphological abnormality noted in these two cases, or any information regarding the antenatal NT, both have mental and cognitive function abnormalities such as mental retardation/developmental delay. Additional literature search from PubMed show that 16p13.1 deletion predisposes to autism and/or mental retardation.12 This further supported that the novel 16p13.11 deletion we detected may predispose to developmental delay. The parents, who were both university graduates in the medical profession, decided to continue the pregnancy as this microdeletion does not cause any severe syndromic malformations.
This case raises a potential shortcoming of array CGH in prenatal diagnosis; this is the predictive value of a positive finding. In this case, the only abnormality was the microdeletion of chromosome 16p13.11 but no deletion over 16p13.3 as described in the ATR-16 syndrome (Figure 1B). There is as yet no antenatal detail from these two DECIPHER cases or from other literature to support the association between the increased NT and this molecular defect. We think that even in cases with Hb Barts disease, the NT could still be normal. Thus, there may be other reasons—like the microdeletion—which may have led to the increase in NT. However, more cases will be required to look for the relationship, and we have continuing study to look into this.
In summary, this study marks the first time that high-resolution 244K array CGH has been used for identification and precise mapping of the copy number changes associated in a fetus with increased NT. Comparing with conventional karyotyping, CGH is more costly, but it requires only 1 microgram of extracted DNA, and the reporting time is 3 days, making it superior to conventional karyotyping. However, array CGH will also pick up a large number of CNVs. The high frequency of CNVs in the human genome could generate a considerable number of ‘false-positive’ results that create great challenges in application of array CGH for prenatal diagnosis. Therefore, our study illustrates the importance of launching comprehensive studies on array CGH prenatal application to increase our knowledge on these chromosomal variations including CNVs before high-resolution array CGH is used in routine clinical practice. We believe that our results provide proof of principle that array CGH has great potential for first-trimester prenatal diagnosis in the future, especially for detecting microdeletions that cannot be detected by microscopic karyotype analysis.
Disclosure of interests
None of the authors has conflict of interest.
Contribution to authorship
K.W.C. and L.W.L. designed the research; T.K.L. and T.Y.L. performed the research; T.Y.F. and C.C.W. contributed new reagents/analytic tools; K.W.C., L.W.L. and T.K.L. analysed the data; L.W.L. and K.W.C. wrote the paper.
Details of ethics approval
Ethic approval had been sought from the Ethics Committee on Human Research, The Chinese University of Hong Kong and the Prince of Wales Hospital (CRE 2007.059) on 13 June 2007.
Research funding by Agilent university research grant from Agilent Technologies. (Project ID: 6902238). The Hong Kong Research Grants Council (RGC) earmarked grant and the Direct grant for research at Chinese University of Hong Kong. Choy KW was supported by the Global Scholarship Programme for Research Excellence-CNOOC Grant.
The authors are thankful to Loreta Lee and Wandy Liu for excellent technical assistance.