Research Letter
Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for
a small supernumerary marker chromosome derived from chromosome 15
Chih-Ping Chen
a,b,c,d,e,f *, Ming Chen
g,h,i, Yi-Ning Su
j, Schu-Rern Chern
b, Peih-Shan Wu
k,
Shun-Ping Chang
g,h, Yu-Ling Kuo
l, Wen-Lin Chen
aand Wayseen Wang
b,ma Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan b Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
c Department of Biotechnology, Asia University, Taichung, Taiwan
d School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan e Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwan f Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei,
Taiwan
g Department of Medical Research, Center for Medical Genetics, Changhua Christian Hospital, Changhua,
Taiwan
h Department of Genomic Medicine, Center for Medical Genetics, Changhua Christian Hospital,
Changhua, Taiwan
i Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan
j Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Taipei Medical
University, Taipei, Taiwan
k Gene Biodesign Co. Ltd, Taipei, Taiwan
l Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical
University, Kaohsiung, Taiwan
m 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]
A 34-year-old, primigravid woman underwent amniocentesis at 18 weeks of gestation because of
advanced maternal age. Cytogenetic analysis of the cultured amniocytes revealed mosaicism for a
small supernumerary marker chromosome (sSMC) and a karyotype of 47,XY,+mar[15]/46,XY[5].
Among 20 colonies of cultured amniocytes, 15 colonies had a karyotype of 47,XY,+mar, while the
rest five colonies had a karyotype of 46,XY. The parental karyotypes were normal. Level II
ultrasound findings were unremarkable. She underwent repeat amniocentesis at 29 weeks of
gestation. Array comparative genomic hybridization (aCGH) on the DNA extracted from the
uncultured amniocytes obtained from 10 mL of amniotic fluid was performed using NimbleGen
ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA). aCGH revealed no
genomic imbalance in the pericentromeric euchromatic regions of all 24 chromosomes.
Cytogenetic analysis of cultured amniocytes revealed a karyotype of 47,XY,+mar[12]/46,XY[10].
In 12 of 22 separated colonies of cultured amniocytes, a karyotype of 47,XY,+mar (Fig. 1) was
noted, while the other 10 colonies had a karyotype of 46,XY. Fluorescence in situ hybridization
(FISH) was performed on cultured amniocytes using the probes of Aquarius Satellite
Enumeration (Cytocell Inc.) and Vysis Prader-Willi/Angelman Region (Abbott Inc.). The probes
used included CEP15 (D15Z4, 15p11.1-q11.1; D15Z1, 15p11.2), CEP1/5/19, CEP6, CEP7,
CEP10, CEP18, CEP13/21 and CEP14/22 (Cytocell, Adderbury, Oxfordshire, UK); and LSI
SNRPN (15q11.2) and LSI PML (15q15) (Abbott Laboratories, Abbott Park, IL, USA). FISH
analysis revealed that the sSMC was positive for D15Z4 (Fig. 2) and negative for D15Z1, SNRPN
and PML (Fig. 3). FISH analysis also revealed a negative result for CEP1/5/19, CEP6, CEP7,
CEP10, CEP18, CEP13/21 and CEP14/22. The sSMC was derived from chromosome 15 without
involvement of the Prader-Willi/Angelman region. The karyotype at repeat amniocentesis was
47,XY,+mar .ish der(15)(D15Z4+, D15Z1-, SNRPN-, PML-)[12]/46,XY[10]. Methylation
analysis of the Prader-Willi/Angelman critical region (PWACR) by the methylation-specific
multiplex ligation-dependent probe amplification (MS-MLPA) kit of SALSA MS-MLPA
ME028-B1 Prader-Willi syndrome/Angelman syndrome (PWS/AS) probemix (MRC-Holland bv.
Amsterdam, The Netherlands) using the DNA extracted from the uncultured amniocytes excluded
uniparental disomy (UPD) 15 (Fig. 4). The parents decided to continue the pregnancy. At 40
weeks of gestation, a healthy male baby was delivered with a body weight of 3,004 g and a
karyotype of 47,XY,+mar[21]/46,XY[19] in cord blood.
sSMCs occurs in 0.075% of prenatal fetal cases and may or may not be associated with
phenotypic abnormalities depending on the origin of the chromosome and the presence of
euchromatic materials [1-4]. Prenatal diagnosis of sSMCs demands genetic counseling and
requires molecular cytogenetic techniques to identify the nature of the sSMC [5-15]. In the cases
with an sSMC, about 70% are de novo, 70% are derived from acrocentric chromosomes, and 70%
present no phenotypic effects [1-4,16]. In a study of 112 patients with constitutional SMCs
ascertained by FISH, Crolla et al [17] found that 68% (76/112) were from the acrocentric
chromosomes of 13/21, 14, 15 and 22, and among these acrocentric SMCs, 51% (39/76) were
derived from chromosome 15, indicating a high frequency of 35% (39/112) for SMC(15) in all
SMCs with known chromosomal origins.
Prenatal diagnosis of an sSMC derived from chromosome 15 should be paid attention to the
involvement of the PWACR at 15q11-q13 and include a differential diagnosis of the inv dup(15)
or idic(15) syndrome (tetrasomy 15q), chromosome 15q11-q13 duplication syndrome and
maternal UPD 15. The inv dup(15) or idic(15) syndrome (tetrasomy 15q) is caused by an SMC
involving the inverted duplication of proximal chromosome 15 containing the PWACR and is
characterized by muscle hypotonia, developmental delay, intellectual disability and autistic
behavior [18]. Chromosome 15q11-q13 duplication syndrome (OMIM 608636) is characterized
by clinical features of autism, mental retardation, ataxia and epilepsy caused by 15q11q13
duplication [19-26]. Prenatal diagnosis of a sSMC(15) even without involvement of the PWACR
should also test UPD 15. Liehr et al [27] reported PWS with a karyotype of 47,XY,+min(15)
(pterq11.1:) and maternal heterodisomic UPD 15, and suggested the necessity to exclude the
presence of a UPD in prenatal diagnosis of a de novo sSMC.
Human chromosome 15q11-q14 region contains six clusters of chromosome 15 low-copy repeat
(LCR15) duplicons referred to as BP1~BP6 that mediate chromosomal rearrangements leading to
translocations, deletions, duplications, triplications and sSMCs [29-36]. The clinically relevant
sSMC(15) contains euchromatic 15q material especially the PWACR between BP2 and BP3. The
5.9-Mb 15q proximal region between BP2 and BP3 contains the PWACR and includes the genes
of MKRN3, MAGEL2, NDN, SNRPN, UBE3A, ATP10A, GABRB3, OCA2 and HERC2. In a
review of 32 cases with the sSMC(15), Eggermann et al [37] found that SMC(15) with
euchromatic content causes mental and psychomotor retardation, whereas SMC(15) without
euchromatic content has no influence on the carrier's phenotype but is associated with a high
incidence among infertile males. In a study of 20 patients with clinically relevant SMC(15) with
triplicated 15pter to BP3 or 15pterBP4::BP5pter, Kleefstra et al [36] found consistent
phenotypic abnormalities with a high prevalence of autistic behavior, attention problems,
aggressive behavior, anxiety and sleeping problems in theses patients. The present case belongs to
clinically irrelevant sSMC(15) that contains only heterochromatin and 15p material.
In conclusion, molecular genetic technologies such as FISH, aCGH and methylation analysis are
useful for rapid exclusion of the involvement of the PWACR and UPD 15 in prenatally detected
mosaic sSMC(15) at amniocentesis.
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-102-04 from Mackay Memorial Hospital, Taipei, Taiwan.
References
1. Liehr T, Claussen U, Starke H. Small supernumerary marker chromosomes (sSMC) in humans. Cytogenet Genome Res 2004; 107: 55-67.
2. Liehr T, Weise A. Frequency of small supernumerary marker chromosomes in prenatal, newborn, developmentally retarded and infertility diagnostics. Int J Mol Med 2007; 19: 719-31.
3. Liehr T, Ewers E, Kosyakova N, Klaschka V, Rietz F, Wagner R, et al. Handling small supernumerary marker chromosomes in prenatal diagnostics. Expert Rev Mol Diagn 2009; 9: 317-24.
4. Liehr T. Characterization of prenatally assessed de novo small supernumerary marker chromosomes by molecular cytogenetics. Methods Mol Biol 2008; 444: 27-38.
5. Chen C-P, Lin C-C, Li Y-C, Chern S-R, Lee C-C, Chen W-L, et al. Clinical, cytogenetic, and molecular analyses of prenatally diagnosed mosaic tetrasomy for distal chromosome 15q and review of the literature. Prenat Diagn 2004: 24; 767-73.
6. Chen C-P, Lin C-C, Su Y-N, Tsai F-J, Chen J-T, Chern S-R, et al. Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from chromosome 18 and associated with a reciprocal translocation involving chromosomes 17 and 18. Taiwan J Obstet Gynecol 2010a; 49: 188-91.
7. Chen C-P, Lin C-C, Ko T-M, Tsai F-J, Chern S-R, Lee C-C, et al. Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from chromosome 21. Taiwan J Obstet Gynecol 2010b: 49; 377-80.
8. Chen C-P, Lin C-C, Su Y-N, Tsai F-J, Chern S-R, Lee C-C, et al. Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from chromosome 22. Taiwan J Obstet Gynecol 2010c; 49: 381-4.
9. Chen C-P, Chen M, Ko T-M, Ma G-C, Tsai F-J, Tsai M-S, et al. Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from chromosome 8. Taiwan J Obstet Gynecol 2010d; 49: 500-5.
10.Chen C-P, Chen M, Su Y-N, Tsai F-J, Chern S-R, Wu P-C, et al. Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 4. Taiwan J Obstet Gynecol 2011; 50: 188-95.
11.Chen C-P, Chang S-D, Su Y-N, Chen M, Chern S-R, Su J-W, et al. Rapid positive confirmation of mosaicism for a small supernumerary marker chromosome as r(8) by interphase fluorescence in situ hybridization, quantitative fluorescent polymerase chain reaction and array comparative genomic
hybridizationon uncultured amniocytes in a pregnancy with fetal pyelectasis. Taiwan J Obstet Gynecol
2012a; 51: 405-10.
12.Chen C-P, Chen M, Chern S-R, Wu P-S, Chang S-P, Lee D-J, et al. Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from ring chromosome 2. Taiwan J Obstet Gynecol 2012b: 51; 411-7.
13.Chen C-P, Ko T-M, Su Y-N, Chern S-R, Su J-W, Chen Y-T, et al. Prenatal diagnosis of mosaic tetrasomy 18p. Taiwan J Obstet Gynecol 2012c: 51; 625-9.
14.Chen C-P, Ko T-M, Chen Y-Y, Su J-W, Wang W. Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 22 associated with cat eye syndrome. Gene 2013a: 527; 384-8.
15.Chen C-P, Chen M, Su Y-N, Huang J-P, Chern S-R, Wu P-S, et al. Mosaic small supernumerary marker chromosome 1 at amniocentesis: prenatal diagnosis, molecular genetic analysis and literature review. Gene 2013b: 529; 169-75.
16.Huang B, Solomon S, Thangavelu M, Peters K, Bhatt S. Supernumerary marker chromosomes detected in 100,000 prenatal diagnoses: Molecular cytogenetic and clinical significance. Prenat Diagn 2006; 26: 1142-50.
17.Crolla JA, Youings SA, Ennis S, Jacobs PA. Supernumerary marker chromosomes in man: parental origin, mosacism and maternal age revisited. Eur J Hum Genet 2005; 13: 154-60.
18.Battaglia A. The inv dup(15) or idic(15) syndrome (Tetrasomy 15q). Orphanet J Rare Dis 2008; 3: 30. 19.Clayton-Smith J, Webb T, Cheng XJ, Pembrey ME, Malcolm S. Duplication of chromosome 15 in the
region 15q11-13 in a patient with developmental delay and ataxia with similarities to Angelman syndrome. J Med Genet 1993; 30: 529-31.
20.Baker P, Piven J, Schwartz S, Patil S. Duplication of chromosome 15q11-13 in two individuals with autistic disorder. J Autism Dev Disord 1994; 24: 529-35.
21.Bundey S, Hardy C, Vickers S, Kilpatrick MW, Corbett JA. Duplication of the 15q11-13 region in a patient with autism, epilepsy and ataxia. Dev Med Child Neuro 1994; l 36: 736-42.
22.Thomas JA, Johnson J, Peterson Kraai TL, Wilson R, Tartaglia N, LeRoux J, et al. Genetic and clinical characterization of patients with an interstitial duplication 15q11-q13, emphasizing behavioral phenotype and response to treatment. Am J Med Genet 2003; 119A: 111-20.
23.Bonati MT, Finelli P, Giardino D, Gottardi G, Roberts W, Larizza L. Trisomy 15q25.2-qter in an autistic child: genotype-phenotype correlations. Am J Med Genet 2005; 133A: 184-8.
24.Miller DT, Shen Y, Weiss LA, Korn J, Anselm I, Bridgemohan C, et al. Microdeletion/duplication at 15q13.2q13.3 among individuals with features of autism and other neuropsychiatric disorders. J Med Genet 2009; 46: 242-8.
25.Piard J, Philippe C, Marvier M, Beneteau C, Roth V, Valduga M, et al. Clinical and molecular characterization of a large family with an interstitial 15q11q13 duplication. Am J Med Genet 2010; 152A: 1933-41.
26.Burnside RD, Pasion R, Mikhail FM, Carroll AJ, Robin NH, Youngs EL, et al. Microdeletion/ microduplication of proximal 15q11.2 between BP1 and BP2: a susceptibility region for neurological dysfunction including developmental and language delay. Hum Genet 2011 130: 517-28.
27.Liehr T, Brude E, Gillessen-Kaesbach G, König R, Mrasek K, von Eggeling F, et al. Prader-Willi syndrome with a karyotype 47,XY,+min(15)(pter→q11.1:) and maternal UPD 15 – case report plus review of similar cases. Eur J Med Genet 2005; 48: 175-81.
28.Amos-Landgraf JM, Ji Y, Gottlieb W, Depinet T, Wandstrat AE, Cassidy SB, et al. Chromosome breakage in the Prader-Willi and Angelman syndromes involves recombination between large, transcribed repeats at proximal and distal breakpoints. Am J Hum Genet 1999; 65: 370-86.
29.Christian SL, Fantes JA, Mewborn SK, Huang B, Ledbetter DH. Large genomic duplicons map to sites of instability in the Prader-Willi/Angelman syndrome chromosome region (15q11–q13). Hum Mol Genet 1999; 8: 1025-37.
30.Pujana MA, Nadal M, Guitart M, Armengol L, Gratacòs M, Estivill X. Human chromosome 15q11-q14 regions of rearrangements contain clusters of LCR15 duplicons. Eur J Hum Genet 2002; 10: 26-35. 31.Roberts SE, Maggouta F, Thomas NS, Jacobs PA, Crolla JA. Molecular and fluorescence in situ
hybridization characterization of the breakpoints in 46 large supernumerary marker 15 chromosomes reveals an unexpected level of complexity. Am J Hum Genet 2003; 73: 1061-72.
32.Sahoo T, Shaw CA, Young AS, Whitehouse NL, Schroer RJ, Stevenson RE, et al. Array-based comparative genomic hybridization analysis of recurrent chromosome 15q rearrangements. Am J Med Genet 2005; 139A: 106-13.
33.Makoff AJ, Flomen RH. Detailed analysis of 15q11-q14 sequence corrects errors and gaps in the public access sequence to fully reveal large segmental duplications at breakpoints for Prader-Willi, Angelman and inv dup(15) syndromes. Genome Biol 2007; 8: R114.
34.Makoff AJ, Flomen RH. Common inversion polymorphisms and rare microdeletions at 15q13.3. Eur J Hum Genet 2009; 17: 149-50.
35.Mignon-Ravix C, Depetris D, Luciani JJ, Cuoco C, Krajewska-Walasek M, Missirian C, et al. Recurrent rearrangements in the proximal 15q11-q14 region: A new breakpoint cluster specific to unbalanced translocations. Eur J Hum Genet 2007; 15: 432-40.
36.Kleefstra T, de Leeuw N, Wolf R, Nillesen WM, Schobers G, Mieloo H, et al. Phenotypic spectrum of 20 novel patients with molecularly defined supernumerary marker chromosomes 15 and a review of the literature. Am J Med Genet 2010; 152A: 2221-9.
37.Eggermann K, Mau UA, Bujdosó G, Koltai E, Engels H, Schubert R, et al. Supernumerary marker chromosomes derived from chromosome 15: Analysis of 32 new cases. Clin Genet 2002; 62: 89-93.
Figure Legends
Fig. 1. The G-banded karyotype of 47,XY,+mar. mar = marker chromosome.
Fig. 2. Fluorescence in situ hybridization (FISH) using the probe of D15Z4 (15p11.1-q11.1; spectrum green) shows a positive hybridization signal on the marker chromosome (mar).
Fig. 3. FISH using the probes of D15Z1 (15p11.2; spectrum green), LSI SNRPN (15q11.2; spectrum red) and LSI PML (15q15; spectrum green) shows absence of the green and red signals on the marker chromosome (mar).
Fig. 4. Methylation analysis using SALSA MS-MLPA ME028-B1 PWS/AS probemix shows no evidence of UPD 15. (A) Fetus (uncultured amniocytes). (B) Negative control (wild type). (C) Positive control (PWS UPD type). (D) Positive control (AS UPD type). UPD = uniparental disomy, PWS = Prader-Willi syndrome, AS = Angelman syndrome.
