SNP Microarray in FISH Negative Clinically Suspected 22q11.2 Microdeletion Syndrome

The present study evaluated the role of SNP microarray in 101 cases of clinically suspected FISH negative (noninformative/normal) 22q11.2 microdeletion syndrome. SNP microarray was carried out using 300 K HumanCytoSNP-12 BeadChip array or CytoScan 750 K array. SNP microarray identified 8 cases of 22q11.2 microdeletions and/or microduplications in addition to cases of chromosomal abnormalities and other pathogenic/likely pathogenic CNVs. Clinically suspected specific deletions (22q11.2) were detectable in approximately 8% of cases by SNP microarray, mostly from FISH noninformative cases. This study also identified several LOH/AOH loci with known and well-defined UPD (uniparental disomy) disorders. In conclusion, this study suggests more strict clinical criteria for FISH analysis. However, if clinical criteria are few or doubtful, in particular newborn/neonate in intensive care, SNP microarray should be the first screening test to be ordered. FISH is ideal test for detecting mosaicism, screening family members, and prenatal diagnosis in proven families.


Introduction
The 22q11.2 microdeletion syndrome is the most common microdeletion syndrome and seen at a prevalence of 1 in 4000 to 6000 live births [1]. It is characterized by hemizygous microdeletion of ≤3 mb size of chromosome 22q11.2 locus in which several genes are lost. It is mostly spontaneous/de novo and in <10% cases are inherited [2][3][4]. It is frequently associated with multiple congenital anomalies, in particular cardiac anomaly (conotruncal cardiac anomaly such as Fallot's tetralogy, interrupted aortic arch, truncus arteriosus, or major aortopulmonary collateral), developmental delay, hypocalcaemia, immune deficiency, cleft palate or velopharyngeal insufficiency or swallowing difficulty, and dysmorphism (broad bulbous nose, square shaped tip of nose, short philtrum, telecanthus, slanting eyes, low set ears, etc.). FISH, until recent, was commonly used for precise genetic diagnosis of common microdeletion syndromes, including 22q11.2 microdeletion [5][6][7][8]. However, FISH provides information only on targeted locations and does not allow a comprehensive evaluation of the whole genome. In addition, atypical smaller deletions are difficult to identify by FISH due to failure in covering those locations by single FISH probe (outside the region of hybridisation by the FISH probe). Furthermore, it is difficult to detect 22q11.2 duplication by FISH due to variable size of signals as well as distraction with split signals of normal cells. Often 22q11.2 duplication displays features like 22q11.2 deletion syndrome [9]. Hence, FISH alone cannot provide reliable diagnosis for cases of 22q11.2 microdeletions/duplications syndrome. Further, if FISH is used for doubtful cases (if few numbers of clinical criteria are fulfilled by patient) then chances of detecting targeted deletion are less frequent [7,8]. High-resolution array CGH was used to investigate FISH negative for 22q11.2 deletions cases with conotruncal heart defects [10] and additional cases of 22q11.2 microdeletion/microduplication containing TBX1 gene were detected. In this study with SNP microarray we have assessed 101 cases of FISH negative/noninformative clinically suspected 22q11.2 microdeletion syndrome to determine whether any benefit is gained from using SNP microarray.

Result
A total of 101 DNA samples from FISH negative/noninformative clinically suspected 22q11.2 microdeletion syndrome were analyzed by SNP microarray successfully. Details of SNP microarray findings of all 101 FISH negative/noninformative samples are available as master table (Supplementary file 2). Out of 101 FISH negative for 22q11.2 microdeletion cases, SNP microarray detected several cases of chromosomal abnormalities (Table 1), 22q11.2 microdeletion/microduplication (Table 2), other microdeletion/microduplication cases (Table 3), and several cases of likely pathogenic CNVs (Table 4). SNP microarray also detected several cases of LOH/AOH of known UPD disorders (Table 5) and some more likely pathogenic UPD disorders (Table 6) in the study.
This SNP microarray study identified 6 cases of chromosomal abnormalities (Table 1). Clinical feature in brief is presented in Table 7 ( Figure 1). The study also identified eight cases of 22q11.2 microdeletions (7)/microduplication (1), mostly from FISH noninformative cases and 2 smaller CNVs (outside scope of FISH probe). Clinical feature in brief is presented in Table 8. This study detected 4 cases of other pathogenic CNVs (Table 3) and several cases of likely pathogenic CNVs (Table 4), which were outside the scope of FISH. Clinical features in brief of these cases are presented in Tables 9 and 10 ( Figure 2). Microarray analysis identified 9 cases of segmental LOH/AOH, those associated with known UPD disorders (Table 5). Clinical features in brief of these cases are presented in Table 11 ( Figure 3).

Discussion
We have been investigating microdeletion syndrome using FISH since 2005. Our experience with FISH in microdeletion syndrome including 22q11.2 microdeletion syndrome (most common microdeletion syndrome) is unsatisfactory as FISH detects approximately 8% of cases of 22q11.2 microdeletions [7,8]. The reasons for low positive rate of 22q11.2 microdeletion syndrome using RP5-882J5 PAC clone (FISH probe for 22q11.2 locus) were poor clinical inclusion criteria (often single criteria) and poor quality of sample referral (clotted/frozen/clumped blood samples/cells leading to FISH failure or omission of FISH tests). In our experience (8,(11)(12)(13), FISH probe derived from RP5-882J5 PAC clone identifies all typical deletions (≥2 mb); however, it is unable to detect atypical deletions of ≤0.5 mb sizes (at proximal or distal LCRs). Number of diagnostic criteria fulfilled by our patients ranged from one to eight and most consistent referral criteria were broad nose, TOF, swallowing difficulty, and hypocalcaemia/convulsion. Patient often was referred to our laboratory for 22q11.2 FISH testing based on a single clinical feature, namely, CHD (TOF) or convulsion. These lead to lower deletion detection rate. Poirsier et al. [13] also observed poor clinical criteria for FISH test referral.
In this prospective study, SNP microarray was carried out in 101 FISH negative/noninformative clinically suspected 22q11.2 microdeletion syndrome cases to assess the role of SNP microarray in the evaluation of clinically suspected 22q11.2 microdeletion syndrome. There were 12 samples that were not processed for FISH due to freezing or clotting. Microarray was successfully carried out in all 101 cases. Chen et al. [10], like this study, investigated FISH negative 22q11.2 deletion cases using high-resolution array CGH. They have found better diagnostic sensitivity of array CGH over FISH in fetuses with cardiac abnormalities associated with deletion 22q11.2 and duplication 22q11.2 syndromes. In the present study we have also detected several cases of chromosomal abnormalities (trisomy, triploidy, partial monosomy, or partial trisomy) and other pathogenic/likely pathogenic CNVs (Tables 1, 3, and 4). We have also picked up some smaller specific deletions/duplications outside the region of hybridisation of the FISH probe. Clinically suspected specific CNV (22q11.2) was detectable in approximately 8% of cases by SNP microarray over and above                 known and well-defined UPD disorders. Several cases of suspected LOH/AOH were also identified; however, confirmation of association needs additional investigation using SNP microarray/QF PCR of their parents before claiming causative.
One of the major referral criteria for our patients was congenital heart disease, mainly conotruncal heart defect [7]. Several studies have established the importance of 22q11.2 CNVs in the etiology of congenital heart disease (CHD) with or without other associated malformations [14,15]. Patients with 22q11.2 duplication also present clinical features like 22q11.2 deletion syndrome, including heart defect [16]. In 22q11.2 duplication syndrome, most prevalent heart defect was conotruncal heart defect [17]. This is also true in animal experiment where mice carrying extra copies of TBX1 gene   [13,19]. The role of TBX1 in heart development has been already demonstrated in mice [20] and TBX1 mutations have also been identified in individuals with TOF [21]. We have also observed TBX1 deletion with TOF, in our series. This study detected several cases of LOH/AOH of known UPD disorders (Table 5) and some more likely pathogenic UPDs (Table 6); however, further study using SNP microarray on parental DNA is required before reporting associations with specific developmental disorders.
Our study indicates that microarray should be first tier of test when samples are scant, lysed, or clumped/clotted/frozen as FISH or conventional cytogenetics are bound to fail/noninformative. This study suggests that microarray is a superior  technique in clinically doubtful cases as well as ICU admissions on life support and 22q11.2 microdeletion is suspected on the basis of convulsion and/or cardiac defect/failure. Furthermore, in early weeks of life dysmorphology and malformation detection is difficult, in particular sick neonate on life support. We have observed in this study that clinically suspected microdeletion syndrome cases are frequently associated with second/more hits (deletion or duplication) elsewhere in the genome (tables/associated CNVs). Microarray also detected several cases of chromosomal aneuploidy, partial aneuploidy, triploidy, and so forth. This approach of DNA microarray will provide the highest chance of making a diagnosis and sparing the patient unnecessary diagnostic testing from many places, in addition to saving crucial times. Our conclusion is in agreement with the consensus statement [22]. We conclude that more strict clinical criteria should be followed for FISH test. If clinical diagnosis is uncertain or doubtful then microarray should be the first screening test. This is most important with newborn/neonate in intensive care unit as clinical criteria are few and difficult to elicit. Microarray is applicable in all samples irrespective of frozen, lysed, clotted, clumped, and other samples. Furthermore, SNP microarray provides information on aneuploidy, triploidy, partial aneuploidy, and associated small CNVs (often many) besides information on LOH/AOH (indicating UPD disorders/in case of consanguinity homozygosity of recessive disorders). FISH may be used for detecting mosaicism, screening family members, prenatal diagnosis, and preimplantation diagnosis of specific deletions in proven family.