Scientifica
Volume 2016 (2016), Article ID 5826431, 18 pages
http://dx.doi.org/10.1155/2016/5826431

Research Article

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

Ashutosh Halder, Manish Jain, and Amanpreet Kaur Kalsi

Reproductive Biology, AIIMS, New Delhi 110029, India

Received 28 December 2015; Revised 27 January 2016; Accepted 16 February 2016

Academic Editor: Albert Basson

Copyright © 2016 Ashutosh Halder et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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.

1. 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 [2–4]. 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 [5–8]. 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.

2. Material and Methods

This study was conducted in the Department of Reproduction Biology, All India Institute of Medical Sciences, New Delhi, India, from September 2011 to August 2014. A total of 101 FISH negative/noninformative (FISH was normal in 85 cases, was not attempted in 12 cases due to frozen/clotted samples, and failed in 4 cases due to few/lysed/clumped cells) clinically suspected 22q11.2 microdeletion syndrome cases were prospectively enrolled for the study using SNP microarray. FISH was carried out using noncommercial FISH probe (22q11.2; RP5-882J5; genomic coordinate/exact position of probe is unavailable; obtained from Uniba Biologia, University of Bari, Italy, by curtsy of Professor Mariano Rocchi, http://www.biologia.uniba.it/rmc/). SNP microarray was used to detect chromosomal abnormality (aneuploidy, triploidy, mosaicism, etc.) as well as microdeletion, microduplication, UPD/LOH, and so forth as described before [11]. All cases were referred from various hospitals of Delhi (northern India) for confirmation of clinical diagnosis. The Institutional Human Ethics Committee approved research protocol. Most patients (excluding very sick/intensive care patients) underwent clinical genetics evaluation (directly by our team or by referral physician) as per specific 22q11.2 microdeletion syndrome proforma (Supplementary file 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/5826431). About 2 mL EDTA blood sample was collected from the affected individual. SNP microarray study was carried out commercially either from Illumina (HumanCytoSNP-12 DNA Analysis Bead Chip Kit 300 K) or Affymetrix (GeneChip Human Mapping 750 K) platforms. Images were captured on iScan System or Cytoscan, respectively. Data were analyzed (primary) using Illumina’s KaryoStudio/GenomeStudio and Affymetrix ChAS software. Resolution was set as 0.1 mb for deletion, 0.5 mb for gain, and 3 mb for LOH/AOH. Secondary analysis was carried out using web data analysis resources like DECIPHER (GRCH37; version 9.4), OMIM, and UPD with cross reference to UCSC, NCBI, Ensembl, and DGV. Interpretation and reporting followed American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants [12].

3. 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.

Table 1: Details of chromosomal abnormalities in FISH negative suspected 22q11.2 microdeletion cases.

DBN
Locus/loci
CNV
Start
End
Size (mb)
Genes
Disease

93
18p11.32-p11.21
3
2842
15365878
15.3
89
Trisomy 18

18q11.1-q23
3
16786754
76115554
59.3
241

98
All chromosomes
3

Triploidy (mosaicism confirmed by FISH)

103
18q21.31-q23
1
54144903
78014582
23.8

Partial 18q monosomy

115
11q23.2-q25
3
113077541
134944006
21.8

Partial 11q trisomy (Jacobsen, Bartter 2, 11q)

116
All chromosomes
3

Triploidy (mosaicism confirmed by FISH)

205
21q11.2 q21.1
3
13609442
46923252
33.3
256
Trisomy 21

q21.2 q21.3

q22.11 q22.12

q22.13 q22.2

q22.3

FISH negative means normal FISH result or FISH that failed or FISH not carried out due to cell lysis (frozen sample) or clotted sample.

DBN: database number (CNV 1 = deletion; CNV 3 = duplication).

Table 2: Details of 22q11.2 microdeletion and/or microduplication in FISH negative suspected 22q11.2 microdeletion cases.

DBN
FISH
Locus
CNV
Start
End
Size (mb)
Genes (no)
Gene details

42
Not done due to frozen blood sample
22q11.21
1
18877787
21914652
3
58
DGCR6; PRODH; KIAA1647; DGCR9; DGCR10; DGCR2; DGCR2; DGCR14; TSSK2; GSC2; SLC25A1; CLTCL1; HIRA; MRPL40; C22orf39; C22orf39; UFD1L; CDC45L; CLDN5; LOC150185; SEPT5; GP1BB; TBX1; GNB1L; C22orf29; TXNRD2; COMT; COMT; COMT; COMT; ARVCF; C22orf25; MIR185; DGCR8; MIR1306; TRMT2A; RANBP1; ZDHHC8; Em:AC006547.7; RTN4R; MIR1286; DGCR6L; LOC375133; RIMBP3; ZNF74; SCARF2; KLHL22; MED15; POM121-like 1; DKFZp434N035; PI4KA; SERPIND1; SNAP29; CRKL; AIFM3; AIFM3; LZTR1; THAP7; FLJ39582; MGC16703; P2RX6; P2RX6; SLC7A4; P2RX6P; LOC400891

47
Not done due to frozen blood sample
22q11.21
1
17257787
19792353
2.53
68
DGCR6; PRODH; KIAA1647; DGCR9; DGCR10; DGCR2; DGCR11; DGCR14; TSSK2; GSC2; SLC25A1; CLTCL1; HIRA; MRPL40; C22orf39; C22orf39; UFD1L; CDC45L; CLDN5; LOC150185; SEPT5; GP1BB; TBX1; GNB1L; C22orf29; TXNRD2; COMT; COMT; COMT; COMT; ARVCF; C22orf25; MIR185; DGCR8; MIR1306; TRMT2A; RANBP1; ZDHHC8; LOC150197; RTN4R; MIR1286; DGCR6L; LOC375133; RIMBP3; ZNF74; SCARF2; KLHL22; MED15; POM121L4P; TMEM191A; PI4KA; SERPIND1; SNAP29; CRKL; AIFM3; AIFM3; LZTR1; THAP7; FLJ39582; MGC16703; P2RX6; P2RX6; SLC7A4; P2RX6P; LOC400891

48
Not done due to frozen blood sample
22q11.21
1
19405537
19792353
0.38
16
PI4KA; SERPIND1; SNAP29; CRKL; AIFM3; AIFM3; LZTR1; THAP7; FLJ39582; MGC16703; P2RX6; P2RX6; SLC7A4; P2RX6P; LOC400891; TBX1

87
Not done due to sample clotting
22q11.21
1
18877787
21050552
2.1
47
DGCR6; PRODH; KIAA1647; DGCR9; DGCR10; DGCR2; DGCR2; DGCR14; TSSK2; GSC2; SLC25A1; CLTCL1; HIRA; MRPL40; C22orf39; C22orf39; UFD1L; CDC45L; CLDN5; LOC150185; SEPT5; GP1BB; TBX1; GNB1L; C22orf29; TXNRD2; COMT; COMT; COMT; COMT; ARVCF; C22orf25; MIR185; DGCR8; MIR1306; TRMT2A; RANBP1; ZDHHC8; Em:AC006547.7; RTN4R; MIR1286; DGCR6L; LOC375133; RIMBP3; ZNF74; SCARF2; KLHL22; MED15; POM121-like 1

182
Not done due to sample clotting
22q11.21
1
18877787
21811991
2.9
69
DGCR6; PRODH; KIAA1647; DGCR9; DGCR10; DGCR2; DGCR2; DGCR14; TSSK2; GSC2; SLC25A1; CLTCL1; HIRA; MRPL40; C22orf39; C22orf39; UFD1L; CDC45L; CLDN5; LOC150185; SEPT5; GP1BB; TBX1; GNB1L; C22orf29; TXNRD2; COMT; COMT; COMT; COMT; ARVCF; C22orf25; MIR185; DGCR8; MIR1306; TRMT2A; RANBP1; ZDHHC8; Em:AC006547.7; RTN4R; MIR1286; DGCR6L; LOC375133; RIMBP3; ZNF74; SCARF2; KLHL22; MED15; POM121-like 1; DKFZp434N035; PI4KA; SERPIND1; SNAP29; CRKL; AIFM3; AIFM3; LZTR1; THAP7; FLJ39582; MGC16703; P2RX6; P2RX6; SLC7A4; P2RX6P; LOC400891; POM121L8P; RIMBP3C; RIMBP3B; HIC2

188
Not done due to sample clotting
22q11.21
1
18877787
21798907
2.9
69
DGCR6; PRODH; KIAA1647; DGCR9; DGCR10; DGCR2; DGCR2; DGCR14; TSSK2; GSC2; SLC25A1; CLTCL1; HIRA; MRPL40; C22orf39; C22orf39; UFD1L; CDC45L; CLDN5; LOC150185; SEPT5; GP1BB; TBX1; GNB1L; C22orf29; TXNRD2; COMT; COMT; COMT; COMT; ARVCF; C22orf25; MIR185; DGCR8; MIR1306; TRMT2A; RANBP1; ZDHHC8; Em:AC006547.7; RTN4R; MIR1286; DGCR6L; LOC375133; RIMBP3; ZNF74; SCARF2; KLHL22; MED15; POM121-like 1; DKFZp434N035; PI4KA; SERPIND1; SNAP29; CRKL; AIFM3; AIFM3; LZTR1; THAP7; FLJ39582; MGC16703; P2RX6; P2RX6; SLC7A4; P2RX6P; LOC400891; POM121L8P; RIMBP3C; RIMBP3B; HIC2

191
Normal
22q11.21
3
18844632
19033532
0.18
6
DGCR6; PRODH; KIAA1647; DGCR9; DGCR10; DGCR2

244
Not done due to sample clotting
22q11.21
1
19004730
21800471
2.79
68
DGCR9, DGCR10, DGCR2, DGCR11, DGCR14, TSSK2, GSC2, SLC25A1, CLTCL1, HIRA, MRPL40, C22orf39, UFD1L, CDC45, CLDN5, LINC00895, SEPT5, SEPT5-GP1BB, GP1BB, TBX1, GNB1L, C22orf29, TXNRD2, COMT, MIR4761, ARVCF, TANGO2, MIR185, DGCR8, MIR3618, MIR1306, TRMT2A, RANBP1, ZDHHC8, LOC388849, LOC284865, LINC00896, RTN4R, MIR1286, DGCR6L, LOC729444, TMEM191B, PI4KAP1, RIMBP3, ZNF74, SCARF2, KLHL22, MED15, POM121L4P, TMEM191A, PI4KA, SERPIND1, SNAP29, CRKL, AIFM3, LZTR1, THAP7, THAP7-AS1, TUBA3FP, P2RX6, SLC7A4, P2RX6P, LOC400891, BCRP2, POM121L8P, RIMBP3C, RIMBP3B, HIC2

FISH negative means normal FISH result or FISH that failed or FISH not carried out due to cell lysis (frozen sample) or clotted sample.

DBN: database number (CNV 1 = deletion; CNV 3 = duplication).

Table 3: Details of other microdeletions and/or duplications in FISH negative suspected 22q11.2 microdeletion cases.

DBN
Locus/loci
CNV
Start
End
Size
Genes
Gene details/disease

88
15q11.2
3
22754322
23222284
0.46
6
Prader-Willi/Angelman syndrome and chromosome 15q11-q13 duplication syndrome (TUBGCP5; CYFIP1; CYFIP1; NIPA2; NIPA1; WHAMML1)

102
16p11.2
1
29661217
30347731
0.68
46
C16orf54; MAZ; PRRT2; C16orf53; MVP; MVP; CDIPT; LOC440356; LOC440356; SEZ6L2; ASPHD1; KCTD13; TMEM219; TAOK2; HIRIP3; INO80E; DOC2A; FLJ25404; FAM57B; ALDOA; ALDOA; ALDOA; ALDOA; PPP4C; TBX6; YPEL3; YPEL3; GDPD3; MAPK3; LOC100271831; CORO1A; LOC606724; BOLA2; GIYD1; SULT1A4; SULT1A4; LOC388242; LOC613037; IMAA; CD2BP2; TBC1D10B; MYLPF; SEPT1; ZNF48; ZNF771; DCTPP1

197
1q21.1
3
144943150
146916824
1.9
24
PDZK1; GPR89A; GPR89C; PDZK1; LOC200030; NBPF11; LOC728989; PRKAB2; PDIA3P; FMO5; FMO5; CHD1L; BCL9; ACP6; GJA5; GJA8; GPR89B; GPR89C; PDZK1; LOC200030; NBPF11; FLJ39739; PPIAL4B; PPIAL4A; NBPF14; PPIAL4F; NBPF15; NBPF15; NBPF16; PPIAL4E; NBPF16; PPIAL4F; LOC645166; LOC645166

229
15q11.1
3
20192086
22508324
2.3
13
GOLGA6L6; GOLGA8C; BCL8; POTEB; NF1P1; LOC646214; CXADR; POTEB; NF1P1; LOC727924; OR4M2; OR4N4; OR4N3P

q11.2

FISH negative means normal FISH result or FISH that failed or FISH not carried out due to cell lysis (frozen sample) or clotted sample.

DBN: database number (CNV 1 = deletion; CNV 3 = duplication).

Table 4: Details of likely pathogenic CNVs in FISH negative suspected 22q11.2 microdeletion cases.

DBN
Locus/loci
CNV
Start
End
Size
Genes
Gene details/disease

18
22q11.22
3
22901370
23307965
0.4
7
PRAME, LOC648691, POM121L1P, GGTLC2, MIR650, IGLL5; hsa-mir-650

7q11.21
1
64651296
65148399
0.49
3
INTS4L1, ZNF92, INTS4L2 (cutis marmorata, epicanthus, everted lower lip vermilion, intellectual disability, microcephaly, retinoblastoma, short nasal septum, and thick lower lip vermilion)

20
1q21.2
3
147828939
149723885
1.9
18
2-3-toe syndactyly, abnormality of the helix, aplasia cutis congenita over the scalp vertex, attention deficit hyperactivity disorder, clinodactyly of the 5th finger, facial asymmetry, flat occiput, gait ataxia, global developmental delay, hyperpigmentation of the skin, hypoplastic areola, long eyelashes, microcephaly, muscular hypotonia, pointed chin, short 3rd toe, short neck, short stature, skull asymmetry, and walking on tiptoes
FLJ39739; PPIAL4B; PPIAL4A; NBPF14; PPIAL4F; NBPF15; NBPF15; NBPF16; PPIAL4E; NBPF16; PPIAL4F; LOC645166; LOC645166; LOC388692; FCGR1C; HIST2H2BF; PPIAL4B; LOC728855

46
19p13.11
p12 p11
3
19863014
24556461
4.6
35
LOC284440; ZNF506; ZNF253; ZNF93; ZNF682; ZNF90; ZNF486; LOC284441; ZNF826; ZNF737; ZNF626; ZNF626; ZNF85; ZNF430; ZNF714; ZNF431; ZNF708; ZNF738; ZNF493; ZNF429; ZNF100; LOC641367; ZNF43; ZNF208; ZNF257; ZNF676; ZNF98; ZNF492; ZNF99; ZNF91; ZNF675; ZNF681; RPSAP58; ZNF254; LOC100101266

11p11.2
p11.12
3
48359268
51530241
3.1
10
OR4C45; OR4A47; FOLH1; LOC440040; OR4C13; OR4C12; LOC441601; LOC646813; OR4A5; OR4C46

49
1q31.3
4
193973521
197781198
3.8
13
KCNT2; CFH; CFHR3; CFHR1; CFHR4; CFHR2; CFHR5; F13B; ASPM; ZBTB41; CRB1; DENND1B; DENND1B

Xq21.1-q22.1
2
80368850
99441815
19
27
HMGN5; SH3BGRL; POU3F4; CYLC1; RPS6KA6; HDX; UBE2DNL; APOOL; SATL1; ZNF711; POF1B; CHM; CHM; DACH2; DACH2; KLHL4; CPXCR1; TGIF2LX; PABPC5; PCDH11X; PCDH11X; PCDH11X; NAP1L3; FAM133A; LOC643486; DIAPH2; RPA4; LOC442459

Xq23 q24
2
112244679
117105172
4.8
18
HTR2C; SNORA35; MIR764; MIR1912; MIR1264; MIR1298; MIR1911; MIR448; IL13RA2; LRCH2; RBMXL3; LUZP4; PLS3; PLS3; AGTR2; SLC6A14; CXorf61; KLHL13

Xq27.2-q28
2
141247401
148041505
6.7
36
MAGEC2; SPANXN4; SPANXN3; SLITRK4; SPANXN2; UBE2NL; SPANXN1; SLITRK2; SLITRK2; CXorf1; MIR890; MIR888; MIR892A; MIR892B; MIR891B; MIR891A; CXorf51; CXorf51; MIR506; MIR507; MIR508; MIR509-2; MIR509-3; MIR509-3; MIR509-1; MIR509-2; MIR509-3; MIR510; MIR514-1; MIR514-3; MIR514-3; ASFMR1; FMR1; FMR1NB; AFF2; AFF2

51
1q21.1
3
144007842
145384225
1.3
12
LOC728855; LOC728875; PPIAL4B; PPIAL4A; LOC728875; COAS3; NBPF20; PDE4DIP; PDE4DIP; PDE4DIP; SEC22B; NOTCH2NL; NBPF10

Xp22.33
2
449065
1705775
1.2
10
SHOX; CRLF2; CRLF2; CSF2RA; CSF2RA; IL3RA; SLC25A6; PP1164; ASMTL; P2RY8

71
15q11.2
1
20482360
22754322
2.2
19
CYFIP1; CYFIP1; p; WHAMML1; GOLGA9P; HERC2P2; GOLGA8E; MKRN3; MAGEL2; NDN; PWRN2; PWRN1; C15orf2; SNRPN; SNRPN; SNURF; SNURF

10q11.22
3
46167847
48338944
2.1
23
BMS1P1; FAM35B; SYT15; SYT15; GPRIN2; PPYR1; LOC728643; ANXA8; ANXA8L1; FAM25C; LOC642826; FAM35B2; ANTXRL; ANXA8L2; FAM21B; LOC642826; FAM25G; ANXA8; ANXA8L1; ZNF488; RBP3; GDF2; GDF10

197
1q21.1
3
144943150
146916824
1.9
24
PDZK1; GPR89A; GPR89C; PDZK1; LOC200030; NBPF11; LOC728989; PRKAB2; PDIA3P; FMO5; FMO5; CHD1L; BCL9; ACP6; GJA5; GJA8; GPR89B; GPR89C; PDZK1; LOC200030; NBPF11; FLJ39739; PPIAL4B; PPIAL4A; NBPF14; PPIAL4F; NBPF15; NBPF15; NBPF16; PPIAL4E; NBPF16; PPIAL4F; LOC645166; LOC645166

204
22q11.23
3
23980648
24240879
0.26
2
IGLL3; LRP5L (downslanted palpebral fissures, long palpebral fissure, moderate global developmental delay, short upturned nose, and wide nasal bridge)

FISH negative means normal FISH results or FISH that failed or FISH not carried out due to cell lysis (frozen sample) or clotted sample.

DBN: database number (CNV 1 = deletion; CNV 3 = duplication).

Table 5: Details of LOH/AOH detected with known UPD (uniparental disomy) disorder in suspected 22q11.2 microdeletion.

DBN
Cytoband
Size (mb)
Gene number
UPD disorder
Genes
Remarks (associated CNVs and LOH/AOH)

17
11q13.4-q14.1
12.3
96
Dysmorphism and mental retardation
Unknown
One benign () CNV of 8 (p23.3-p23.2)
Two LOH/AOH of chromosome 11

45
14q32.2
3.0
11
Kagami-Ogata syndrome/Temple syndrome
DLK1, GTL2, RTL1, and so forth
No CNV
No other LOH/AOH

75
6p22.3-p22.1
7.4
112
Diabetes mellitus, transient neonatal, 1
ZFP57
No CNV
No other LOH/AOH

p21.33 p21.32

83
7p12.2-p11.2
7.3
29
Silver-Russell syndrome (7p11.2-p13)
GRB10
One benign () CNV (20q13.33)
Multiple LOH/AOH of other chromosomes

97
6q24.2-q25.1
8.2
49
Transient neonatal diabetes mellitus and isolated cleft lip and palate
PLAGL1
Two uncertain () CNVs (15q11.2)
One likely benign () CNV (10q22.3)
One LOH/AOH of 16p13.11-p12.3

110
7q31.32-q35
20
167
Silver-Russell syndrome (7q31-qter)
MEST
One likely benign () CNV (15q11.2)
Multiple LOH/AOH of chromosome 7 and other chromosomes

128
11p15.4
4.1
552
Beckwith-Wiedemann syndrome

No CNV
Multiple LOH/AOH of same/other chromosomes

220
11p15.5-p15.4
3.5

Beckwith-Wiedemann syndrome
CDKN1C
One benign () CNV 19p12-p11
Multiple LOH/AOH of other chromosomes

227a
15q11.2 q12
4.8
701
Prader-Willi/Angelman syndrome
SNRPN, SNORD116, UBE3A
No associated CNVs
Multiple LOH/AOH of other chromosomes

227b
11q24.2 q24.3
5.9
901
Dysmorphism and mental retardation
Unknown
No associated CNVs
Multiple LOH/AOH of other/same chromosomes

q25

DBN: database number.

Table 6: Large (>5 mb) UPD/LOH/AOH locus/loci detected in suspected 22q11.2 microdeletion.

DBN
Cytoband
Size (mb)
Gene number
Remarks (associated CNVs and LOH/AOH)

20
6p21.1 p12.3
5.7
38
One uncertain (3) CNV of chromosome 1 (q21.2)
Multiple LOH/AOH of chromosomes 6 and 1

43
4q21.23-q22.1
8.7
38
One benign (1) CNV of chromosome 9 (p24.3)
One LOH/AOH of chromosome 15

49
Xq21.1  q21.2 q21.31
19
28
Partial tetrasomy of chromosomes 1–8, 11–14, and 18 and partial disomy of chromosome X

q21.32 q21.33 q22.1

Xq27.2 q27.3 q28
6.7
25
Multiple LOH/AOH of chromosome X and one LOH/AOH of chromosome 1

73
2p25.2 p25.1
5.7
33
One likely benign (3) CNV of chromosome 4 (q22.3)
Multiple LOH/AOH of many chromosomes

2q21.1-q23.3
18
44

3p21.31-p14.2
13
161

3q21.1-3 q23
16
135

4q31.3-q35.1
32
112

6p21.1 p12.3
5.7
86

9p24.2-p23
8.3
29

10q25.1-q26.11
11.8
60

10q26.11-q26.3
12.8
68

11p15.4-p15.1
11.7
102

14q11.2 q12
8.8
63

14q12-q22.3
23.9
112

15q22.33-q25.2
14.4
155

18p11.31-p11.21
11.9
55

18q11.1-q12.3
22.8
85

76
17q12-q21.1
5.3
102
One benign (1) CNV of chromosome 14 (q11.2)
Multiple LOH/AOH of many chromosomes

84
3p21.31-p21.1
7
34
No CNVs; multiple LOH/AOH of many chromosomes

85
8q23.1 q23.2 q23.3
6
14
No CNVs
No other LOH/AOH

87
6q21 q22.1 q22.2 q22.31
12
60
No CNVs
One LOH/AOH of chromosome 11

101
6q14.1-q15
10.5
51
One benign CNV

105
1q23.1-q31.1
29.5
232
Multiple benign/likely benign CNVs
Multiple LOH/AOH of many chromosomes

3q27.1-q29
13.6
108

5q33.3-q35.3
24.7
195

7p14.2 p14.1
5.3
22

10p15.1 p14
5.4
34

16q22.3-q23.3
10
44

17p13.1-p11.2
11.7
124

18q12.1-q12.3
13
46

112
5p15.2 p15.1
7
18
One benign CNV
LOH/AOH of chromosome 11 (p11.2 p11.12)

9q31.1-q32
8.3
71

169
1p13.3-p12
9.9
101
2 likely benign CNVs of chromosomes 8 and X
Multiple LOH/AOH of many chromosomes

5q35.1-q35.3
8
107

10q11.21 q11.22
5.4
49

10q21.1-q22.1
19
81

10q24.1-q25.2
14
133

12q22-q24.32
34.3
281

14q23.3-q32.2
30.3
231

19q13.32-q13.42
7.2
272

20q13.13-q13.32
8
43

22q11.1-q12.1
11.4
173

Xp22.32-p21.1
27
123

Xq23 q24
9.8
51

178
5p13.3 p13.2
5.8
37
No CNV

17p11.2-q11.2
9.9
125
No other LOH/AOH

182
13q14.2-q21.32
18.6
75
No CNV
No other LOH/AOH

6p22.3-p21.33
10.3
269

2q32.3 q33.1
5.2
717

2q34-q36.1
9.6
765

2q36.1-q37.1
7.4
622

7p22.2-p21.3
5.7
49

189
17q21.32-q23.2
13.3
175
No CNV

200
4q32.3-q34.3
9.7
616
No CNV
2 other LOH/AOH

2q11.1-q12.1
8.8
996

2p12-p11.2
8.2
844

2p22.3 p22.2 p22.1
6.1
719

3p26.1 p25.3 p25.2 p25.1
7.8
830

15q23-q24.3
8
1205

204
3q11.2 q12.1 q12.2 q12.3
6
49
One likely benign CNV
No other LOH/AOH

16p12.1 p11.2 p11.1
8
107

16q11.2 q12.1 q12.2
7
40

211
5q21.3-q22.3
7.3
30
No CNV

14q22.1-q23.1
7.3
51
One LOH/AOH of chromosome 7

214
18q11.2-q12.1
5.6
29
One likely benign CNV

6p25.2-p24.3
5.5
42
Two LOH/AOH of chromosomes 12 and 19

228
8q24.21 q24.22
5.5
20
No CNV or no other LOH/AOH

To investigate its clinical importance/significance/associations/and so forth there is a need for further study using SNP microarray of parents.

DBN: database number.

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).

Table 7: Clinical details of chromosomal abnormalities detected by SNP microarray.

Locus/loci (CNV)
Microarray diagnosis
Clinical details
Referral reason

18p11.32-p11.21
18q11.1-q23 ()
Trisomy 18
Seven-month-old male with acyanotic congenital heart defect (CHD)
CHD
To exclude 22q11.2 microdeletion

All chromosomes ()
Triploidy (mosaicism confirmed by FISH; 33% triploid cells)
Five-year-old female (at first visit) and now 13 years old without any secondary sex character (Figure 1), less fetal movement during pregnancy, cesarean section delivery due to nonprogress of labor, feeding problem, DTGV (dextrotransposition of great vessels) operated and ASD (atrial septal defect), global developmental delay (GDD), broad thumb and toe, convulsion (controlled), hypertelorism, bulbus nose, rocker bottom feet, and periventricular leukomalacia on MRI
CHD
To exclude 22q11.2 microdeletion

18q21.31-q23 ()
Partial 18q monosomy
One-month-old male in intensive care unit (ICU), acyanotic CHD with congestive cardiac failure (CCF), and Noonan syndrome like face, plagiocephaly, low set ears, upslanted eyes, sandal gap, and so forth
?Noonan syndrome

11q23.2-q25 ()
Partial 11q trisomy (Jacobsen, Bartter 2, 11q)
Seven-day-old female neonate in ICU, asymmetric IUGR (intrauterine growth restriction), preterm, multiple malformations, respiratory infection, VSD (ventricular septal defect), hypoplastic nail and alae nasi, blepharophimosis, hypotonia, and so forth
CHD
To exclude 22q11.2 microdeletion

All chromosomes ()
Triploidy (mosaicism confirmed by FISH; 24% triploid cells)
Eight-year-old female with dysmorphism (broad nose, cleft palate, thin lip, and long philtrum), long slender fingers, GDD, recurrent respiratory infection, behavioral problem, and so forth
Broad nose, behavioral problem
To exclude 22q11.2 microdeletion

21q11.2 q22.3 ()
Trisomy 21
Three-month-old male in Pediatric ICU with CCF, very sick
?CHD
To exclude 22q11.2 microdeletion

Table 8: Clinical details of 22q11.2 CNVs detected by SNP microarray.

Microarray details
Genes
TBX1/DGCR2/DGCR6/DGCR14/DGCR8
Clinical details
Other CNVs

3 mb deletion
58
Y/Y/Y/Y/Y
Male of 3+ years with tetralogy of Fallot (TOF; operated) and facial dysmorphism, broad nose, feeding difficulty, and so forth
Nil

2.5 mb deletion
65
Y/Y/Y/Y/Y
7-year-old female with TOF and dysmorphism
7q11.21 ()

0.38 mb deletion
15
Y/N/N/N/N
1.5-year-old male with TOF
Nil

2.17 mb deletion
49
Y/Y/Y/Y/Y
34-year-old male referred from anesthesia ICU for hypoparathyroidism, hypocalcemia, recurrent fungal infection, seizure, and respiratory failure (since last 45 days)
Nil

2.9 mb deletion
69
Y/Y/Y/Y/Y
1.5-year-old male with TOF, facial dysmorphism, GDD, and speech delay
Nil

2.9 mb deletion
69
Y/Y/Y/Y/Y
45-day-old female with TOF, recurrent intractable seizure, recurrent infection, suckling difficulties, low calcium, absent thymic shadow on X-ray, and history of polyhydramnios during pregnancy
Nil

0.18 mb duplication
6
N/Y/Y/N/N
10-month-old female with CHD, frontal bossing, prominent metopic suture, hypertelorism, V shaped lip, dysplastic ear, wide spaced nipple, pectus carinatum, mid gut volvulus, and so forth
2p22.3 ()
11p11.12 ()
14q11.2 ()

2.79 mb deletion
68
Y/Y/Y/Y/Y
3-year-old male with seizure, GDD, dysmorphism, high arched palate, long slender fingers, low parathyroid hormone (PTH), low calcium, recurrent infection, and so forth
Nil

Table 9: Clinical details of other pathogenic CNVs detected by SNP microarray.

Microarray details
Genes
Diagnosis
Clinical details
Other CNVs

15q11.2
0.46 mb duplication
6
15q11-q13 duplication
Three-month-old male with TOF, feeding difficulty, hypocalcemia, dysmorphism, poly/syndactyle, hypoplastic mandible, IUGR, and so forth
10q11.22 ()

16p11.2
0.68 mb deletion
46
16p11.2 deletion
4-month-male with CHD (DORV, PS/pulmonary stenosis), blepharophimosis, ptosis, and so forth
15q12 ()
14q11.2 ()

1q21.1
1.9 mb duplication
24
1q21.1 duplication
Male of 2+ years with TOF, broad nose, thin upper lip, absent philtrum, small and low set ears, antimongoloid slant, telecanthus, long slender fingers, widow peak, and so forth
11p11.12 ()

15q11.1 q11.2
2.3 mb duplication
13
15q11.1 q11.2 duplication
1-year-old male with CHD (VSD), dysmorphism, GDD, and so forth
14q11.2 ()

Table 10: Clinical details of likely pathogenic CNVs detected by SNP microarray.

Locus/loci
Microarray details
Genes
Clinical details

22q11.22
7q11.21
0.4 mb duplication
0.49 mb deletion
7
3
2-year-old male with TOF

1q21.2
1.9 mb duplication
18
9-year-old male with TOF (operated)

19p13.11-12
11p11.2
4.6 mb duplication
3.1 mb duplication
35
10
17-year-old female with TOF (operated)

1q31.3
Xq21.1-24
Xq27.2-28
3.8 mb triplication
23.8 mb duplication
6.7 mb duplication
13
27
18
13-year-old male with TOF (operated)

1q21.1
Xp22.33
1.3 mb duplication
1.2 mb duplication
12
10
4-year-old male with TOF (operated)

15q11.2
10q11.22
2.2 mb deletion
2.1 mb duplication
19
23
One-month-old male with hypocalcaemia (Ca 5.6; PTH-46), convulsion, osteopenia, squint, small toe, deep furrow feet, and so forth (Figure 2)

22q11.23
0.26 mb duplication
2
6-year-old female with dysmorphism, square nose tip, proportionate short stature, cyanotic CHD (tricuspid atresia, ostium secundum ASD, etc.), clubbing, tracheal shift, right pneumothorax with lung collapse, right anotia, synophrys, pear shaped nose, webbed neck, and so forth

Table 11: Clinical details of LOH/AOH detected by SNP microarray with known UPD disorders.

Locus/loci
Genes
Clinical details
UPD disorders

11q13.4-q14.1
96
9-year-old male with TOF (operated)
Dysmorphism and mental retardation

14q32.2
11
5-year-old male with TOF (operated)
Kagami-Ogata syndrome/Temple syndrome

6p22.3-p21.32
112
4-year-old male with TOF (operated), cleft lip and palate (repaired), broad and bifid nose, small left cornea and eye, GDD, hearing problem, being still unable to suck/drink, long fingers, small philtrum, mental retardation, and so forth (Figure 3)
Diabetes mellitus, transient neonatal, 1

7p12.2-p11.2
29
3.5-year-old male with TOF (operated), GDD, and previous 2 siblings with CHD
Silver-Russell syndrome (7p11.2-p13)

6q24.2-q25.1
49
10-month-old male with cyanotic CHD, GDD, obesity, hypospadias, no cryptorchidism, dysmorphism, and one elder sister who has CHD
Transient neonatal diabetes mellitus and isolated cleft lip and palate

7q31.32-q35
167
14-month-old female with TOF, small nose, wide philtrum, narrow forehead, low frontal hairline, GDD, broad nose, short stature, bilateral cataract, and previous 3 siblings with malformations (including CHD in one)
Silver-Russell syndrome (7q31-qter)

11p15.4
552
1.5-year-old male with cyanotic CHD, dysmorphism, clubbing, dysplastic small low set ears, weight of 8 kg (overweight), and so forth
Beckwith-Wiedemann syndrome

11p15.5-p15.4

6-day-old very sick (in ICU) male with dysmorphism, CCF, and so forth
Beckwith-Wiedemann

15q11.2-q12
701
9-year-old male with TOF (operated)
Prader-Willi/Angelman

11q24.2-q25
901
5-year-old male with TOF (operated)
Dysmorphism and mental retardation

11q13.4-q14.1
96
4-year-old male with TOF (operated), cleft lip and palate (repaired), broad and bifid nose, small left cornea and eye, GDD, hearing problem, being still unable to suck/drink, long fingers, small philtrum, mental retardation, and so forth
Dysmorphism and mental retardation

(a)

(b)

(c)

(d)

Figure 1: Mosaic triploidy case showing facial profile (a), flat rocker bottom foot ((b) clinical and (c) X-ray), and triploid metaphase cell (d).

(a)

(b)

(c)

Figure 2: Male child with 15q11.2 deletion and 10q11.22 duplication showing squint, small toe and deep furrow feet.

(a)

(b)

Figure 3: Male child with LOH/AOH of 6p22.3-p21.32 showing repaired cleft lip, broad and bifid nose, small left cornea and eye, long fingers, and rough creased skin over hands.

4. 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, 11–13), 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 FISH, mostly from FISH failure samples (frozen/clotted samples; FISH not tried). In fact, it was unexpected from FISH to identify small atypical deletions outside the region of hybridization in two cases (database numbers 48 and 191), which were identified by microarray. Thus, microarray contributed actual improvement by ~2% above FISH for specific 22q11.2 deletions. However, SNP microarray provided many other pathogenic/likely pathogenic microdeletions/duplications besides aneuploidy/partial aneuploidy, triploidy, UPD, and other disorders. We have also observed poor clinical criteria as the leading contributor of failure to detect specific 22q11.2 deletion. Variations in deletion size and/or break point difference (with genes involvement) as well as other CNVs with or without LOH were evident. This study also identified several cases of LOH/AOH loci with 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 display full clinical picture of 22q11.2 deletion syndrome [18]. In this study we have found 2 cases of atypical small CNVs of 22q11.2 (duplication of 0.18 mb spanning 18844632–19033532 and containing genes like DGCR6, PRODH, KIAA1647, DGCR9, DGCR10, and DGCR2 and deletion of 0.38 mb spanning 19405537–19792353 and containing genes like UFD1L, PI4KA, SERPIND1, SNAP29, CRKL, AIFM3, AIFM3, LZTR1, THAP7, FLJ39582, MGC16703, P2RX6, P2RX6, SLC7A4, P2RX6P, LOC400891, and TBX1) with multiple malformations including cardiac malformations. These atypical small deletions are rarely reported [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.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

The study was supported by grant from Indian Council of Medical Research, New Delhi, India. Authors thank Departments of Pediatrics, Pediatric Surgery, Cardiovascular Surgery, and Cardiology of All India Institute of Medical Sciences and various hospitals of Delhi, India, for referring the cases for the research/service, and also they thank the family members of patients for their cooperation during the study. Authors acknowledge Professor Mariano Rocchi (University of Bari, Italy) for providing molecular probes (PAC/BAC clones) for FISH study.

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