Prenatal diagnosis and molecular cytogenetic characterization of de novo
partial trisomy 12q (12q24.21
qter) and partial monosomy 6q (6q27qter)
associated with coarctation of the aorta, ventriculomegaly and thickened
nuchal fold
Chih-Ping Chen a,b,c,d,e,f,g *, Yi-Yung Chen a, Schu-Rern Chern b, Peih-Shan Wu h,Jun-Wei Su a,i,
Yu-Ting Chen b, Li-Feng Chen a and Wayseen Wang b,j
a Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan b Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
c Department of Medicine, Mackay Medical College, New Taipei City, Taiwan d Department of Biotechnology, Asia University, Taichung, Taiwan
e School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan f Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwan g Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei,
Taiwan
h Gene Biodesign Co. Ltd, Taipei, Taiwan
i Department of Obstetrics and Gynecology, China Medical University Hospital, Taichung, Taiwan j Department of Bioengineering, Tatung University, Taipei, Taiwan
* Correspondence to: Chih-Ping Chen, MD
Department of Obstetrics and Gynecology, Mackay Memorial Hospital 92, Section 2, Chung-Shan North Road, Taipei, Taiwan
Tel: +886-2-25433535; Fax: +886-2-25433642, +886-2-25232448 E-mail: [email protected]
Highlights
We present concomitant del(6)(q27qter) and dup(12)(q24.21qter). The phenotype includes aortic coarctation and ventriculomegaly. We discuss the genotype-phenotype correlation.
Abstract
We present rapid aneuploidy diagnosis of de novo partial trisomy 12q (12q24.21qter) and partial monosomy 6q (6q27qter) by aCGH using uncultured amniocytes in a fetus with coarctation of the aorta, ventriculomegaly and thickened nuchal fold. We discuss the association of TBX3, TBX5 and MED13L gene duplication with coarctation of the aorta, and the association of RNASET2 gene haploinsufficiency with ventriculomegaly in this case.
Keywords: 6q deletion, 12q duplication, coarctation of the aorta, partial monosomy 6q, partial trisomy 12q, MED13L, RNASET2, TBX3, TBX5, ventriculomegaly
Abbreviations
MoM: multiples of the median; AFP: -fetoprotein; dup: duplication; del: deletion;
aCGH: array comparative genomic hybridization; FISH: fluorescence in situ hybridization; BAC: bacterial artificial chromosome; t: translocation; der: derivative;
The prevalence of unbalanced chromosome structural abnormalities detected in the newborn ranges from 0.052% to 0.061% (Jacobs et al., 1992). The prevalence of unbalanced reciprocal translocations at prenatal diagnosis has been a estimated to be 0.017% (Jacobs et al., 1992). Unbalanced reciprocal translocations detected at amniocentesis are frequently associated with ultrasound abnormalities (Chen et al., 2011). In a study of 40 cases of unbalanced reciprocal translocations detected at amniocentesis, Chen et al. (2011) found that 82.5% (33/40) of the cases presented fetal ultrasound abnormalities. Rapid aneuploidy diagnosis by molecular cytogenetic technology such as aCGH using uncultured amniocytes for rapid detection of unbalanced chromosome aberrations is currently available (Chen et al., 2012). Here, we present rapid aneuploidy diagnosis of de novo partial trisomy 12q (12q24.21qter) and partial monosomy 6q (6q27qter) associated with coarctation of the aorta, ventriculomegaly and thickened nuchal fold in a second-trimester fetus. Our presentation provides evidence for a correlation of chromosome and gene imbalances at chromosomes 12q and 6q with coarctation of the aorta and ventriculomegaly.
2. Methods and detection
2.1. Array-CGH
Whole-genome aCGH on uncultured amniocytes derived from 10 mL amniotic fluid was performed using NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA). The NimbleGen ISCA Plus Cytogenetic Array has 630,000 probes and a median resolution of 15-20 kb across the entire genome according to the manufacturer’s instruction.
2.2. Conventional cytogenetic analysis
Routine cytogenetic analysis by G-banding techniques at the 550 bands of resolution was performed. 16 mL amniotic fluid was collected, and the sample was subjected to in situ amniocyte culture according to the standard cytogenetic protocol. 5 mL cord blood and 5 mL parental blood were collected, and the samples were subjected to lymphocyte culture according to the standard blood cytogenetic protocol.
2.3. FISH
FISH analysis was performed on cultured amniocytes using a 12q24.33-specific BAC probe RP11-394D10 (133,524,016-133,698,086; spectrum green) and a 6q27-specific BAC probe RP11-417A5 (169,415,990-169,591,011; spectrum red) according to the standard FISH protocol.
2.4. QF-PCR
Polymorphic DNA marker analysis was performed on parental and fetal DNAs using small tandem repeat markers such as D6S969 (6q25.3; 159,282,599-159,282,708), D6S955 (6q26; 162,543,692-162,543,840), D6S1027 (6q27; 169,209,350-169,209,480), D12S1075 (12q23.3; 105,077,958-105,078,154), D12S2070 (12q24.22; 116,082,709-116,082,812), D12S378 (12q24.32; 124,662,996-124,663,160) and D12S1045 (12q24.33; 130,397,738-130,397,814).
2.5. Clinical description
A 27-year-old, primigravid woman was referred to the hospital at 19 weeks of gestation for ultrasound examination and genetic counseling because of an elevated maternal AFP level of 2.63 MoM examined at 17 weeks of gestation. Prenatal ultrasound at 19 weeks of gestation revealed ventriculomegaly in the fetus. Level II ultrasound examination at 21 weeks of gestation showed ventriculomegaly, thickened nuchal fold, a flat nasal bridge, micrognathia, hypertelorism, a single umbilical artery and coarctation of the aorta. The fetal biometry was equivalent to 19 weeks. Amniocentesis was performed. The amniotic fluid AFP level was 0.53 MoM. Whole-genome aCGH analysis on uncultured amniocytes detected a 4.4-Mb deletion at chromosome 6q27 and a 20.4-Mb duplication at chromosome 12q24.21-q24.33. Cytogenetic analysis of the cultured amniocytes revealed a derivative chromosome 6. The parental karyotypes were normal. Metaphase FISH analysis of cultured amniocytes using 6q and 12q subtelomeric probes showed a result consistent with the findings of aCGH and conventional karyotype. Polymorphic DNA marker analysis confirmed a paternal origin of the deletion. The parents elected to terminate the pregnancy, and a 434-g malformed female fetus was delivered. The cytogenetic result of cord blood lymphocytes was consistent with the prenatal diagnosis.
Whole-genome aCGH analysis on uncultured amniocytes detected a 4.4-Mb deletion at chromosome 6q27, or arr cgh 6q27 (166,693,141-171,115,067)1 and a 20.4-Mb duplication at chromosome 12q24.21-q24.33, or arr cgh 12q24.21q24.33 (113,469,298-133,851,895)3 (NCBI build 37) (Fig. 1). The deleted 6q27 region contains 52 genes including the OMIM genes of
FGFR1OP, RNASET2, THBS2 and TBP. The duplicated 12q24.21-212q24.33 region contains 259
genes including the OMIM genes of TBX3, TBX5, SDS, MED13L, HSPB8, ACADS, HNF1A,
ORAI1, HPD, BCL7A, EIF2B1, SCARB1, UBB, PUS1, C12orf65, ATP6V0A2, TCTN2 and DIABLO. The karyotype of cultured amniocytes was 46,XX,der(6)t(6;12)(q27;q24.21)dn (Fig. 2).
Metaphase FISH analysis on cultured amniocytes showed absence of the 6q subtelomeric probe signal and presence of the 12q subtelomeric probe signal on the derivative chromosome 6, indicating partial monosomy 6q and partial trisomy 12q (Fig. 3). QF-PCR analysis showed haploinsufficiency in D6S1027 and duplication in D12S2070, D12S378 and D12S1045. The result of D6S1027 was informative and showed a paternal origin of the deletion (Fig. 4). The karyotype of cord blood was 46,XX,der(6)t(6;12)(q27;q24.21)dn.
4. Discussion
Early prenatal features of fetuses with partial trisomy 12q or partial monosomy 6q have rarely been described. This is the first case report of prenatally detected de novo concomitant partial trisomy 12q (12q24.21qter) and partial monosomy 6q (6q27qter) following a sonographic diagnosis of ventriculomegaly, thickened nuchal fold and coarctation of the aorta.
The present case is associated with coarctation of the aorta and a 20.4-Mb duplication at chromosome 12q24.21-q24.33, encompassing the genes of TBX3 (OMIM 601621), TBX5 (OMIM 601620) and MED13L (OMIM 608771). Both TBX3 and TBX5 belong to members of the T-box factor family. Mutations of TBX3 cause ulnar-mammary syndrome (OMIM 181450) (Bamshad et al., 1997), and mutations of TBX5 are responsible for Holt-Oram syndrome (OMIM 142900) (Li et al., 1997). TBX3 has been found to control the sinoatrial node gene program and to impose pacemaker function on the atria (Hoogaars et al., 2007). TBX5 has been found to be associated with NKX2-5 and synergically promote cardiomyocyte differentiation (Hiroi et al., 2001).
and cerebellum (Muncke et al., 2003). Mutations of MED13L cause dextro-looped transposition of the great arteries (Muncke et al., 2003). Radio et al. (2010) first reported TBX2 gene duplication associated with coarctation of the aorta. We additionally report the association of
TBX3, TBX5 and MED13L gene duplication with coarctation of the aorta. We hypothesize that
coarctation of the aorta in this fetus could be related to over-expression of the genes of TBX3,
TBX5 and MED13L, suggesting parallel consequence of TBX3, TBX5 and MED13L gene dosage
imbalances in humans.
The present case is associated with ventriculomegaly and elevated maternal serum AFP, and a 4.4-Mb deletion at chromosome 6q27, encompassing the genes of RNASET2 (OMIM 612944). Li et al. (2011) recently reported prenatal diagnosis of a 4.9-Mb deletion at 6q27 (Chr. 6: 165,927,396-170,809,934 bp) in a pregnancy with elevated maternal serum AFP and fetal ventriculomegaly. RNASET2 has been found to be strongly expressed in temporal lobe, capsula interna, cerebral cortex, hippocampus and corpus callosum of fetal brain, and mutations of
RNASET2 has been associated with cystic leukoencephalopathy without megalencephaly (OMIM
612950) (Henneke et al., 2009). We speculate that haploinsufficiency of RNASET2 may be responsible for ventriculomegaly in this case.
In summary, we present rapid aneuploidy diagnosis of de novo partial trisomy 12q (12q24.21qter) and partial monosomy 6q (6q27qter) by aCGH using uncultured amniocytes in a fetus with coarctation of the aorta, ventriculomegaly and thickened nuchal fold. We discuss the genotype-phenotype correlation in this case.
Acknowledgements
This work was supported by research grants NSC-99-2628-B-195-001-MY3 and NSC-101-2314-B-195-011-MY3 from the National Science Council and MMH-E-101-04 from Mackay Memorial Hospital, Taipei, Taiwan.
Appendix A. Supplementary data
Bamshad, M., et al. 1997. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16, 311-315.
Chen, C.-P., et al. 2011. Unbalanced reciprocal translocations at amniocentesis. Taiwan J Obstet Gynecol 50: 48-57.
Chen, C.-P., et al. 2012. Rapid aneuploidy diagnosis of partial trisomy 7q (7q34qter) and partial monosomy 10q (10q26.12qter) by array comparative genomic hybridization using uncultured amniocytes. Taiwan J Obstet Gynecol 51: 93-99.
Henneke, M., et al. 2009. RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection. Nat. Genet. 41, 773-775.
Hiroi, Y., et al. 2001. Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat. Genet. 28, 276-280.
Hoogaars, W.M.H., et al. 2007. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 21, 1098-1112.
Jacobs, P.A., Browne, C., Gregson, N., Joyce, C., White, H. 1992. Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding. J. Med. Genet. 29, 103-108.
Li, P., et al. 2011. Genomic characterization of prenatally detected chromosomal structural abnormalities using oligonucleotide array comparative genomic hybridization. Am. J. Med. Genet. 155A, 1605-1615. Li, Q.Y., et al. 1997. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T)
gene family. Nat. Genet. 15, 21-29.
Muncke, N., et al. 2003. Missense mutations and gene interruption in PROSIT240, a novel TRAP240-like gene, in patients with congenital heart defect (transposition of the great arteries). Circulation 108, 2843-2850.
Radio, F.C., et al. 2010. TBX2 gene duplication associated with complex heart defect and skeletal malformations. Am. J. Med. Genet. 152A, 2061-2066.
Figure Captions
Fig. 1. Array comparative genomic hybridization on uncultured amniocytes shows a 4.4-Mb deletion in chromosome 6q27 [arr cgh 6q27 (166,693,141-171,115,067)1] and a 20.4-Mb duplication in chromosome 12q24.21-q24.33 [arr cgh 12q24.21q24.33 (113,469,298-133,851,895)3] (NCBI build 37). (A) Whole genome view, (B) chromosomal view and (C) zoom in view.
Fig. 2. The G-banded karyotype of the cultured amniocyte shows a derivative chromosome 6, or der(6). The arrows indicate the breakpoints.
Fig. 3. Metaphase fluorescence in situ hybridization (FISH) analysis shows absence of the 6q subtelomeric probe signal (spectrum red) and presence of the 12q subtelomeric probe signal (spectrum green) on the der(6) in a cultured amniocyte. Interphase FISH analysis shows three green signals and one red signal in a cultured amniocyte.
Fig. 4. Representative electrophoretograms of quantitative fluorescent polymerase chain reaction assays using polymorphic markers of D6S955 (6q26; 162,543,692-162,543,840) and D6S1027 (6q27; 169,209,350-169,209,480). The marker D6S955 is outside the deleted region and shows two peaks of equal fluorescent activity from two different parental alleles in the fetus. The marker D6S1027 is within the deleted region and shows only one peak of fluorescent activity from the maternal allele in the fetus, indicating a paternal origin of the deletion.
Appendix A. Supplementary data
Fig. S1.Level II ultrasound examination at 21 weeks of gestation shows (A) a flat nasal bridge (arrow), (B) ventriculomegaly, (C) coarctation of the aorta (arrow) and (D) thickened nuchal fold.
