P
RENATAL
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IAGNOSIS AND
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OLECULAR
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YTOGENETIC
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HARACTERIZATION OF A
S
MALL
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UPERNUMERARY
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ARKER
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HROMOSOME
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ERIVED
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ROM
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HROMOSOME
8
Chih-Ping Chen1,2,3,4,5,6*, Ming Chen7,8, Tsang-Ming Ko9, Gwo-Chin Ma7, Fuu-Jen Tsai4,10, Ming-Song Tsai11, Pei-Chen Wu1, Chen-Chi Lee1, Li-Feng Chen2, Wayseen Wang2,12
Departments of 1Obstetrics and Gynecology and 2Medical Research, Mackay Memorial Hospital, 5Institute of
Clinical and Community Health Nursing, 6Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, 9Genephile Geoscience Laboratory, Ko’s Obstetrics and Gynecology, 11Department of Obstetrics and Gynecology, Cathay General Hospital, and 12Department of Bioengineering,
Tatung University, Taipei; 3Department of Biotechnology, Asia University, 4School of Chinese Medicine,
College of Chinese Medicine, China Medical University, and 10Departments of Medical Research and Medical Genetics, China Medical University Hospital, Taichung; 7Departments of Medical Research and
Genomic Medicine, Center for Medical Genetic, 8Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan.
SUMMARY
Objective:To present prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 8 by multiplex ligation-dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), spectral karyotyping (SKY) and array comparative genomic hybridization (aCGH).
Case Report:A 42-year-old woman, gravida 6, para 3, underwent amniocentesis at 19 gestational weeks because of advanced maternal age. Amniocentesis revealed a de novo ring-shaped sSMC in all 13 colonies of the amniocytes. The karyotype was 47,XY,+mar. The MLPA showed duplications of 8p11.21-specific probes. At 24 gestational weeks, level II ultrasound revealed a left multicystic kidney in the fetus. Other internal organs were unremarkable. Repeat amniocentesis revealed a karyotype of 47,XY,+mar[25]/46,XY[2]. The sSMC was characterized by SKY and FISH, which showed a chromosome 8 origin of the sSMC. Oligonucleotide-based aCGH demonstrated a 4.4-Mb duplication of 8p11.21q11.1 [arr cgh 8p11.21q11.1 (42,637,263-47,062,180)×3]. The karyotype was 47,XY,+r(8) (p11.21q11.1)[25]/46,XY[2]. Polymorphic DNA marker analysis revealed no uniparental disomy for chromo-some 8. The woman elected to continue the pregnancy and at 34 gestational weeks, a 1,820 g male baby without craniofacial dysmorphism was delivered. At the age of 1 month, the infant was apparently normal except for left multicystic kidney disease and mild ventriculomegaly.
Conclusion:MLPA, SKY and aCGH are helpful in genetic counseling of prenatally detected sSMCs by providing the immediate information on the origin and the genetic contents of the sSMC. [Taiwan J Obstet Gynecol 2010; 49(4):500–505]
Key Words:aCGH, chromosome 8, MLPA, prenatal diagnosis, SKY, small supernumerary marker chromosome
*Correspondence to: Dr Chih-Ping Chen, Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan.
E-mail: [email protected] Accepted: July 22, 2010
Introduction
Small supernumerary marker chromosomes (sSMCs) are defined as structurally abnormal chromosomes that cannot be identified or characterized by conventional banding cytogenetics and are generally equal in size or smaller than a chromosome 20 [1–3]. The sSMCs are present in 0.044% of newborn infants and in 0.075% of prenatal cases [1,3,4]. Around 70% of sSMCs arise
de novo [5], and about 70% of sSMCs are derived from
acrocentric chromosomes [1,6], and 70% of de novo sSMCs have no phenotypic effects [4]. Prenatal diagnosis of sSMCs gives rise to difficulties in genetic counseling, and requires molecular cytogenetic technologies to iden-tify the nature of the aberrant chromosome [4,7–11]. Here we present our experience of prenatal diagnosis and molecular cytogenetic characterization of an sSMC derived from chromosome 8 by multiplex ligation-dependent probe amplification (MLPA), fluorescence
in situ hybridization (FISH), spectral karyotyping (SKY)
and array comparative genomic hybridization (aCGH).
Case Report
A 42-year-old woman, gravida 6, para 3, underwent amniocentesis at 19 gestational weeks because of advanced maternal age. Amniocentesis revealed a ring-shaped sSMC in all 13 colonies of the amniocytes. The karyotype was 47,XY,+mar (Figure 1). The parental karyotypes were normal. MLPA was applied to deter-mine the origin of the sSMC using the SALSA MLPA Kit P181-A2/P182-B1 Centromere (MRC-Holland bv, Amsterdam, the Netherlands; Figure 2). The result of MLPA indicated a duplication of the 8p11.21-specific
probe 05722-L05161 containing the FNTA gene and the 8p11.21-specific probe 06244-L06278 containing the HOOK3 gene. No duplication was found in the 8q11.21-specific probe 06239-L05745 (KIAA0146 gene) and the 8q11.21-specific probe 05757-L05195 (PRKDC gene). At 24 gestational weeks, level II ultrasound revealed a left multicystic kidney in the fetus. Other inter-nal organs were unremarkable. Repeat amniocentesis revealed a karyotype of 47,XY,+mar[25]/46,XY[2]. The sSMC was characterized by SKY using 24-color SKY probes (Applied Spectral Imaging, Carlsbad, CA, USA) and FISH using a WCP8-specific gene probe (Cytocell, Adderbury, Oxfordshire, UK; Figures 3 and 4). SKY and FISH showed a chromosome 8 origin of the sSMC. Oligonucleotide-based aCGH demonstrated a 4.4-Mb duplication of 8p11.21q11.1 [arr cgh 8p11.21q11.1 (42, 637,263-47,062,180)×3] (Figure 5). The aCGH results indicated a duplication of 8p from the 8p11.21-specific probe A_16_P18291023 (42,637,263 bp) (NCBI Build 36) to the 8p11.1-specific probe A_16_P18292593 (43,647,122 bp) and a duplication of 8q at the 8q11.1-specific probe A_14_P133856 (47,062,180 bp) [Cyto Scan gene chip (Agilent customer array, Changhua Christian Hospital, Changhua, Taiwan)]. The karyo-type was 47,XY,+r(8)(p11.21q11.1)[25]/46,XY[2]. Polymorphic DNA marker analysis of the cultured amniocytes revealed no uniparental disomy for chromo-some 8. The woman elected to continue the pregnancy and at 34 gestational weeks, a 1,820-g male baby with-out craniofacial dysmorphism was delivered by cesarean section because of maternal preeclampsia. At the age of 1 month, the infant had a weight of 2,090 g (<3rdcentile) and a height of 43 cm (< 3rdcentile). He was apparently normal except for left multicystic kidney disease and mild ventriculomegaly.
Figure 1.The G-banded karyotype of 47,XY,+r(8)(p11.21q11.1). The arrow indicates a supernumerary marker chromosome (SMC) derived from chromosome 8.
Discussion
Our presentation shows the usefulness of the MLPA Centromere kit, SKY and aCGH in the identification of an sSMC derived from chromosome 8. MLPA, first described by Schouten et al [12], is a molecular method to detect gene dosage abnormalities in a wide range of conditions by relative quantification of multiple DNA target sequences in one polymerase chain reaction with the input of 20 ng or more DNA but without the
requirement of living cells or cell cultures. It can be easily automated and results can be obtained within 30 hours [13]. In MLPA, the probes added to the sam-ples are amplified and quantified [14]. The MLPA kit for rapid aneuploidy diagnosis is commercially avail-able. Both the SALSA MLPA P181-A2 and P182-B1 Centromere kits contain one probe for the short arm and one probe for the long arm of chromosomes other than acrocentric chromosomes, and two probes for the long arm of chromosomes 13, 14, 15, 21 and 22.
1q12 2q11 .2 3q11 .2 4q12 5q11 .2 6q12 7q21 .11 8q11 .21 9q13 10q11 .2 11q12 12q12 13q12 14q11 .2 15q11 .2 16q12 17q11 .1 18q11 .1 19q12 20q11 .21 21q11 22q11 .2 Xq11 .2 1p11 .2 2p11 .2 3p11 .2 4p11 5p13 6p11 7p11 .2 8p11 9p13.2 10p11 11p11 .2 12p11 .21 16p11 .2 17p11 .2 18p11 .22 19p11 20p11 .2 Xp11 .23 13q11 14q12 15q11 .2 21q11 22q11 .2 1p11 .2 2p12 3p11 .2 4p11 5p12 6p12 7p11 .2 8p11 9p11 10p11 11p11 .2 12p11 .21 13q11 14q11 .2 15q11 .2 16q11 .2 17p11 .2 18p11 .22 19p11 20p11 .21 21q1 22q11 .2 Xp11 .22 1q21 .1 2q11 .2 3q11 .2 4q12 5q11 .2 6q11 7q11 .21 8q11 9q13 10q11 .2 11q12 12q12 13q11 14q11 .2 15q11 .2 16q12 17q11 .1 18q11 .2 19q12 20q11 .21 21q11 22q11 .2 Xq11 .2 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 P181 ratio P182 ratio
Figure 2.Multiplex ligation-dependent probe amplification shows three copies of two targets on chromosome 8p11 consis-tent with the diagnosis of marker chromosome 8 and a duplication of 8p11. Arrows indicate the chromosome 8p11 targets.
2 1 6 13 19 20 21 22 X Y 14 15 Marker 16 17 18 7 8 9 10 11 12 3 4 5
Each probe is located close to the centromere of a specific chromosome. The SALSA MLPA Kit P181-A2/P182-B1 Centromere is designed to detect deletions/ duplications of the designated pericentromeric regions
of each chromosome. For chromosome 8, the desig-nated regions are FNTA and HOOK3 at 8p11.21, and
KIAA0146 and PRKDC at 8q11.21. This MLPA
Centro-mere kit is especially useful for rapid identification of the chromosome origin of an sSMC. In addition to MLPA and SKY, aCGH has the ability to detect DNA dosage imbalance including deletions and duplications in the pericentromeric euchromatic regions and is use-ful for the characterization of the genomic imbalance in the sSMC.
To date, at least 11 patients with an sSMC(8) derived from r(8)(::p23.1q11::), min(8)(:p11.22∼11.21 q11.1:), r(8)(::p11.21q11.1::), min(8)(:p11.21 q11.1:), r(8)(::p11.21q11.21::), min(8) (:p11q11:), min(8)(:p11.1q11.21:), r(8)(::p11.1q12.1::), min(8) (:p11q11.2:) or r(8)(::p10q12::) have been docu-mented without clinical findings [2,15–22]. However, at least 10 patients with an sSMC(8) derived from r(8)(::p11.22∼11.21q11.1::), min(8)(:p11.21 q11.1:),min(8)(:p11.22∼11.21q11.1:),min(8) (:p11.21∼11.22q10:), dic(8;8)(::p11.21q11.1:: p11.21q11.1::), r(8)(::q10 p11.2::p11.2 q10::) or r(8)(::p11q11::) have been documented with clini-cal findings [2,22–27]. mar 8 8 Marker (WCP8)
Figure 4.Fluorescence in situ hybridization using chromosome 8 whole chromosome painting probe shows a chromosome 8 origin of the small supernumerary marker chromosome. mar= marker chromosome; WCP= whole chromosome pointing probe. p23.2 p22 p21.2 41 .0 Mb 43.8 Mb 46.6 Mb 49.4 Mb p12 p11.22 q11.22 q12.1 q12.3 q13.2 q21.11 q21.13 q21.3 q22.2 q23.1 q23.3 q24.12 q24.21 q24.23 42,637,263 (A_16_P18291023) 43,647,122 (A_16_P18292593) 47,062,180 (A_14_P133856)
Figure 5.Oligonucleotide-based array comparative genomic hybridization (aCGH) demonstrates a 4.4 Mb duplication in 8p11.21q11.1 [arr cgh 8p11.21q11.1 (42,637,263-47,062,180)×3]. The aCGH shows a duplication of 8p from the 8p11.21-specific probe A_16_P18291023 (42,637,263 bp) (NCBI Build 36) to the 8p11.1-specific probe A_16_P18292593 (43,647,122 bp) and a duplication of 8q at 8q11.1-specific probe A_14_P133856 (47,062,180 bp).
An SMC(8) with a duplication of p11.21q11.1 can be associated with clinical findings. Batanian et al [23] reported a 5-year-old boy with 72% mosaicism for min(8)(:p11.21q11.1:), attention deficit disor-der, seizures and developmental delay. Batanian et al [23] also reported an 11-year-old girl with an SMC(8) or min(8)(:p11.22∼p11.21q11.1:), anomalous pul-monary venous return, idiopathic thrombocytopenia, precocious puberty and obesity. Anderlid et al [24] reported a 3-year-old boy with 27% mosaicism for r(8)(p10) de novo or r(8)(::p11q11::), mental retarda-tion and behavior problems. Herry et al [25] reported a 29-year-old man with 76% mosaicism for r(8)(::p11 q11::) and mild intellectual delay but no dysmorphic features. Brecevic et al [26] reported a female infant with 60% mosaicism for r(8)(::p11.21q11.1::) and global developmental delay. Liehr et al [2] reported a 43-year-old man with 60% mosaicism for dic(8;8) (::p11.21q11.1::p11.21q11.1::), developmental delay, severe mental retardation and autistic behavior. Ballif et al [27] reported a patient with an SMC(8) or min(8)(:p11.22∼p11.21q10:) and developmental delay. Ballif et al [27] also reported a patient with 47% mosaicism for r(8)(::q10p11.2::p11.1q10:) and dev-elopmental delay. Liehr [22] reported a 15-year-old boy with SMC(8) or r(8)(::p11.22∼11.21q11.1::) and atyp-ical autism. Liehr [22] also reported a 3-year-old girl with an SMC(8) or min(8)(:p11.21q11.1:), developmental and mental retardation, and psychomotor deficiencies. The present case had a 1-Mb 8p duplication encompassing 8p11.21-p11.1. The duplicated seg-ment contained eight functional genes: CHRNB3,
CHRNA6, THAP1, RNF170, HOOK3, FNTA, HGSNAT
and A26A1, and seven hypothetical genes: FLJ23356,
LOC100131789, LOC643654, LOC347028, LOC1001 28173, LOC100130474 and LOC100130767. The genes CHRNB3 and CHRNA6 are associated with nicotinic
acetylcholine receptors. Mutations in the THAP1 gene are related to autosomal dominant torsion dystonia-6. RNF170 is a putative LAG1-interacting ring finger protein. The Golgi-associated HOOK3 protein is a member of the microtubule-binding proteins. The gene FNTA encodes the α subunit of CAAX farnesyltransferase and HGSNAT encodes heparan-α-glucosaminide N-acetyltransferase, with mutations of HGSNAT associated with an autoso-mal recessive disorder of mucopolysaccharidosis type IIIC. A26A1 or POTE8 is a prostate-, ovary-, testis-expressed protein on chromosome 8. The present case clinically manifested as multicystic kidney disease and ventriculomegaly but no dysmorphic features. An increase of the genes within the duplicated segment in this case has not previously been reported to be associated with multicystic kidney disease or ventriculomegaly.
With the advent of MLPA, SKY and aCGH, de novo non-acrocentric sSMCs can be well characterized by molecular cytogenetic technologies. We conclude that MLPA, SKY and aCGH are helpful in genetic counseling of prenatally detected sSMCs by providing immediate information on the origin and genetic contents of the sSMC.
Acknowledgments
This work was supported by research grants NSC-96-2314-B-195-008-MY3 and NSC-97-2314-B-195-006-MY3 from the National Science Council, and MMH-E-99004 from Mackay Memorial Hospital, Taipei, Taiwan.
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![Figure 5. Oligonucleotide-based array comparative genomic hybridization (aCGH) demonstrates a 4.4 Mb duplication in 8p11.21 q11.1 [arr cgh 8p11.21q11.1 (42,637,263-47,062,180) ×3]](https://thumb-ap.123doks.com/thumbv2/9libinfo/8985102.282522/4.892.194.731.585.1030/figure-oligonucleotide-based-comparative-genomic-hybridization-demonstrates-duplication.webp)
