Low grade mosaic for a complex supernumerary ring chromosome 18 in an adult patient with multiple congenital anomalies
© van der Veken et al; licensee BioMed Central Ltd. 2010
Received: 7 April 2010
Accepted: 9 July 2010
Published: 9 July 2010
Several cases have been reported of patients with a ring chromosome 18 replacing one of the normal chromosomes 18. Less common are patients with a supernumerary ring chromosomes 18. High resolution whole genome examination in patients with multiple congenital abnormalities might reveal cytogenetic abnormalities of an unexpected complexity.
We report a 24 years old male patient with lower spinal anomalies, hypospadia, bifid scrotum, cryptorchism, anal atresia, kidney stones, urethra anomalies, radial dysplasia, and a hypoplastic thumb. Some of the anomalies overlap with the VACTERL association. Chromosome analysis of cultured peripheral blood lymphocytes revealed an additional ring chromosome in 13% of the metaphases. Both parents had a normal karyotype, demonstrating the de novo origin of this ring chromosome. FISH analysis using whole chromosome paints showed that the additional chromosomal material was derived from chromosome 18. Chromosome analysis of cultured fibroblasts revealed only one cell with the supernumerary ring chromosome in the 400 analyzed. To characterize the ring chromosome in more detail peripheral blood derived DNA was analyzed using SNP-arrays. The array results indicated a 5 Mb gain of the pericentromeric region of chromosome 18q10-q11.2. FISH analysis using BAC-probes located in the region indicated the presence of 6 signals on the r(18) chromosome. In addition, microsatellite analysis demonstrated that the unique supernumerary ring chromosome was paternally derived and both normal copies showed biparental disomy.
We report on an adult patient with multiple congenital abnormalities who had in 13% of his cells a unique supernumerary ring chromosome 18 that was composed of 6 copies of the 5 Mb gene rich region of 18q11.
Structural chromosomal abnormalities involving chromosome 18, including del(18q), del(18p) and r(18), are frequently occurring autosomal anomalies which are present in approximately 1/40,000 live births[1–4]. Ring chromosomes 18 have been reported in a significant number of cases. In most cases the ring chromosome 18 replaces a normal maternal or paternal chromosome 18. However, few cases have been described with supernumerary small ring chromosomes 18[3–13]. Timur et al. described a patient with a supernumerary r(18) chromosome that contained chromosomal material from 18p in 24% of his cells and presented with Klippel-Trenaunay Syndrome . Callen et al. described a patient with a short stature, normal intelligence and no dysmorphic features who had a supernumerary r(18) chromosome in 85% of the cells. Jenderny et al. described a phenotypically and mentally normal women who had a small ring chromosome 18 in 2% of her cells with breakpoints approximately at 18q11 and 18q23. In her daughter the ring chromosome 18 was not present supernumerary and replaced one of the normal chromosomes 18 in all her cells. In our patient a supernumerary ring chromosome 18 was detected in 13% of the metaphases of peripheral blood lymphocytes. The ring chromosome had a unique structural composition, which was different from the supernumerary ring chromosome 18 cases previously described.
The patient was born after an uncomplicated pregnancy of 37 weeks as the third son of healthy non-consanguineous parents with a weight of 2670 grams. There was a single umbilical artery. In the first week of life multiple congenital malformations were diagnosed, e.g. anal atresia, dysplastic lumbar and sacral vertebrae, penoscrotal hypospadia, cryptorchism on both sides, bifid scrotum, ureteral flow abnormality (dilatation of the higher urinary tract caused by vesico ureteral reflux, trabeculated bladder), radial dysplasia and thumb hypoplasia on the right upper extremity and preaxial polydactyly of the left hand. The diagnosis VACTERL-association was made, although there were no abnormalities of heart, trachea and esophagus. The urinary flow problems resulted in frequent infections and some episodes of urosepsis, finally causing chronic renal insufficiency. In his teens he underwent cholecystectomy because of an abnormal gallbladder full of stones. A pancreas fissum was seen. Neurologic investigation showed no signs of tethered cord syndrome. School performances were normal, there were no reasons for IQ testing. As an adult his height was on the 3rd centile and his headcircumference was normal.
Molecular and cytogenetic studies
In summary, we detected a unique supernumerary ring chromosome 18 in a male patient with various phenotypic aberrations. In 11-13% of peripheral blood lymphocytes the additional ring chromosome lead to an octasomy of ~5 Mb of the pericentromeric region of chromosome 18.
Small ring marker chromosomes have been shown to originate from the centromere and from the adjacent pericentric regions of a wide variety of chromosomes. Postzygotic formation of the ring chromosome or postzygotic instability resulting in loss during cell division my explain the mosaic state of the ring chromosome. The postzygotic instability can be caused by the instability and dysfunction of the centromere and/or instability of the ring chromosome. Cases with small ring chromosomes frequently show subclones lacking the ring chromosome resulting in mosaicism of ring containing cell lines and monosomic cell lines when the ring chromosome replaces one of the normal chromosomes [8, 9]. In the present case we found a paternally derived supernumerary ring chromosome in mosaic with a normal cell line. The mosaic supernumerary presence of the ring chromosome together with the observation of normal parental karyotypes may indicate that the ring was formed during sperm meiosis I, and that by subsequent non-disjunction and postzygotic instability of the ring a low grade mosaic for the r(18) was generated. Alternatively, the r(18) originated from a trisomic zygote generating normal diploid cell lines (trisomic rescue) through mitotic non-disjunction[11, 12]. Finally the presence of the supernumerary ring chromosome may be explained by the model proposed by Daniel et al. . According to this model, the ring chromosome may originate from an extra haploid pronucleus derived from a superfluous sperm that is usually degraded by DNAses or other means, but accidentally escaped degradation. After transfection into the zygote nucleus small ring chromosomes may occur. The extra haploid pronucleus may be generated by a multiple fertilization or a delayed incorporation into one blastomere of an extra sperm.
The ring chromosome generated in such a complex chain of events must contain 4 centromeres. We tried to confirm this using FISH probes (CEP18) for centromere 18. However, while normal signals were observed on the normal paternal and maternal chromosomes 18, no centromere 18 signals were found on the ring chromosome 18 (data not shown). Since cell division is likely to be hampered by multiple functional centromeres, most of the centromeres on the supernumerary ring were likely to be inactivated. However, since FISH did not reveal the presence of centromere 18 signals on the ring chromosome it is possible that the centromeres were actually lost by an unknown other mechanism of ringformation and that neocentromeres might have been generated .
Clinical characterstics of the patient compared with other cases with supernumerary ring chromosomes 18 and other abnormal chromosomes 18
 case 9
 case 18-2
 child 3
patients with supernumerary ring chromosomes 18
patients with abnormal chromosomes 18
TOP 22wks 520 g
Developmental delay/mental retardation
Atrial septal defect
Single umbilical artery
Dysplastic lumbar and sacral vertebrae
Vesico ureteral reflux
Hypertrophy of the lower limb
Long tapering fingers
Elongated, thin feet
Size discrepancy kidneys
Renal arteriovenous malformations
Unilateral cleft lip and palate
Anotia with atresia
Low set ear
Left index finger crossed over 3rd and 4th fingers
2nd toe crossed over 3rd toe
Open ductus botalli
To explain the phenotype of the patient by the aberrant expression of genes present on the ring chromosome is difficult. The large number of genes (~25 RefSeq genes) present in the chromosomal region of 18q10 - 18q11.2, including the OMIM disease genes RBBP8, NPC1, LAMA3, makes it complex to pinpoint the relative contribution of each gene to the phenotype. Furthermore, disease phenotypes are in most cases linked to deletions, while our patient presented with an octasomy of the particular genomic region.
We described the de novo occurrence of a unique supernumerary ring chromosome leading to a partial octasomy of chromosome 18 in 11-13% of the peripheral blood lymphocytes of an adult male patient with phenotypic aberrations. The present case clearly illustrates the potential complexity of chromosomal aberrations in patients with phenotypic abnormalities.
All studies were conducted with approval of the institutional review board at Erasmus MC Rotterdam. Informed consent was given and the study was performed according to the tenets of the Declaration of Helsinki. GTG-banded metaphases obtained from peripheral blood and fibroblast cultures were karyotyped using standard procedures. Karyotypes were obtained from the patient and both parents. Results were described in accordance with the ISCN 2009.
Fluorescence in situ hybridization (FISH)
For FISH analysis of the ring chromosome the BAC clones RP11-79F3 (18q11.2) and RP11-411B10 (18p11.21) were selected from the University of California Santa Cruz (UCSC) http://genome.ucsc.edu/[26, 27] genome browser (University of California, Santa Cruz, CA). Ten micrograms of DNA was isolated using an AutoGenPrep 3000 robot (Autogen, Holliston, MA) and after whole genome amplification (WGA, Repli-G) (Qiagen, Venlo, The Netherlands), the DNA was digested and labeled (Random Prime labeling system) (Invitrogen/Life Technologies, Carlsbad, CA) with Bio-16-dUTP or Dig-11-dUTP (Roche, Almere, The Netherlands). The FISH experiments were performed in duplicate according to standard protocols with minor modifications. The FISH probes were validated on control metaphase spreads. The whole chromosome 18 paint probe (wcp18) (Euro-Diagnostica, Malmö, Sweden) was used according to the same FISH protocol. FISH slides were analyzed with an Axioplan 2 Imaging microscope (Carl Zeiss, Sliedrecht, The Netherlands) and images were captured using Isis software (MetaSystems, Altlussheim, Germany).
Array whole genome analysis
Genomic DNA was extracted from peripheral blood (Qiagen). DNA quantity (20-80 μg in 20 μL) was measured with a spectrophotometer (model ND-1000) (NanoDrop Technologies, Wilmington, DE), and quality was assayed on a bioanalyzer (model 2100) (Agilent, Palo Alto, CA). WGA was performed according to the manufacturer's instructions. The array platforms used were the single nucleotide polymorphism (SNP) microarray Affymetrix 250 K Nsp1 (Affymetrix, Santa Clara, CA) and the Illumina 610 quad arrays (Illumina, San Diego, CA). Copy number data was analyzed using Beadstudio software (Illumina) and Nexus software (Nexus BioDiscovery, El Segundo, CA).
DNA-analysis by MLPA and direct sequencing of the coding regions of the genes SALL1, SALL4 en TBX5 of the patient showed no pathogenic abnormalities.
GeneScan microsatellite markers
DNA was acquired from peripheral blood samples of the patient and both parents. Eight microsatellite markers were selected from the UCSC genome browser for identification of the parental origin of the ring chromosome: D18S1149, D18S1104, D18S869, D18S480, D18S1108, D18S1107, D18S66, and D18S819. For all microsatellites the same PCR program was used. After the initial denaturation (10 min, 95°C), 30 cycles were performed for denaturation (45 sec, 95°C), annealing (45 sec, 56°C), and elongation (1 min 30 sec, 72°C), followed by an extra elongation period of 10 min (72°C). PCR product sizes were determined using an ABI 3730xl capillary sequencer (Applied Biosystems/Life Technologies, Carlsbad, CA). Genotyping of the microsatellite markers was performed using GeneMarker software (SoftGenetics, State College, PA).
Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
We would like to thank Ruud Koppenol for his contribution to the preparation of the figures.
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