Individuals with ring chromosome 13 may show characteristics observed in a deletion syndrome and could present a set of dismorphies along with intellectual disability, according to chromosomal segments involved in the genetic imbalance. Nevertheless, ring anomalies likewise is called “dynamic mosaicism”, phenomena triggered by the inner instability concerning the ring structure, thus leading to the establishment of different cell clones with secondary aberrations. Phenotypic features, such as growth failure and other anomalies in patients with this condition have been associated with an inherent ring chromosome mitotic instability, while recent studies offer evidence on a role played by the differential loss of genes implicated in development. Here, we observed similar mosaicism rates and specific gene loss profile among three individuals with ring chromosome 13 using GTW-banding karyotype analyses along with FISH and CGH-array approaches. Karyotypes results were: patient 1—r(13)(p13q32.3), patient 2—r(13)(p11q33.3), and patient 3—r(13)(p12q31.1). Array-CGH has revealed qualitative genetic differences among patients in this study and it was elusive in precise chromosomal loss statement, ranging from 13 Mb, 6.8 Mb, and 30 Mb in size. MIR17HG and ZIC2 loss was observed in a patient with digital anomalies, severe growth failure, microcephaly and corpus callosum agenesis while hemizygotic EFNB2 gene loss was identified in two patients, one of them with microphtalmia. According to these findings, it can be concluded that specific hemizygotic loss of genes related to development, more than dynamic mosaicism, may be causative of congenital anomalies shown in patients with ring 13 chromosome.
Ring chromosomes originate from the break and rejoining of both chromosome arms and consequently formation of a circular rearrangement, most often with a genetic loss of the extremities. Ring Chromosome 13 is observed in around 20% of ring cases in still-births and its prevalence is estimated in 1/58,000 births [
Some authors attribute the phenotypic features seen in ring 13 patients to a “ring chromosome syndrome” through a mechanism named “dynamic mosaicism” as postulated by Kosztolanyi [
Studies of genotype-phenotype correlation delineate the 13q- syndrome and thus provide an effective understanding of that clinical entity [
Here we present three new cases of individuals with ring chromosome 13 and their characterization using banding cytogenetics and fluorescence in situ hybridization (FISH) analysis along with array-comparative genomic hybridization (array-CGH) approach in order to evaluate the rate of cell mosaicism in each individual and likewise to ascertain differences in genetic profile on the genotype-phenotype correlation.
Patients previously referred to genetic counseling and diagnosed with ring chromosome 13, were reevaluated in the Genetics and Society Program–Biology Institute/Universidade Federal da Bahia (UFBA), Salvador, Brazil (Figure
Left to right, patients 1, 2, and 3 and their respective ring chromosome 13 (bottom).
The patient was born on October 04, 2012, female, healthy parents with normal karyotype. At birth, the mother was 24-years-old, G2/P1/A0, and reported infrequent fetal movements during pregnancy, beginning in the 4th month. Pregnancy was not accompanied by any medical care. The delivery was normal, with Apgar 8/9 at 1 and 5 minutes. The birth weight (1600 g) and height (42 cm) were below the 3rd percentile and microcephaly, floated nasal bridge, large ears and facial dysmorphysm were observed. In the 4th month a transfontanellar ultrasound showed mild ectasy of the right lateral ventricle corpus and a ventricular parenchymal increase. The patient showed global development delay and sat only with support in about the 8th month. Anterior fontanel closed between the 9th and 17th month. Serological tests were negative for Cytomegalovirus and Toxoplasma; inborn errors of metabolism tests were normal. After the first year, OFC was 35.5 cm (below 2nd percentile) and the child could not roll, crawl or walk without support.
The patient was born on March 26, 2008, male, healthy parents with normal karyotype. The birth weight (2620 g) and height (48 cm) were within the 3rd percentile. At 4½ years old, OFC was 47.5 cm (below the 2nd percentile). The mother reported that there were no complications during pregnancy, with cesarean delivery at term. The child had a weak cry, achieved cephalic equilibrium in a few months and lordosis was observed. The child walked after being two-years-old. Electroencephalogram was normal. Currently, the child is attentive and participatory, showing microcephaly, hypotonia, epicanthus, floated nasal bridge, high ogival palate, short neck, with small hands and feet.
The patient was born on October 1st, 1989, female, healthy parents with normal karyotype. The birth weight was 1200 g (below 3rd percentile) while the height was not registered. The mother was 28 and the father was 21-years-old. Fetal movements were evident in the 4th month. Delivery was normal and premature (approximately 24 weeks), with amniotic fluid loss and suspicion of anoxya. The child remained in the ICU for 15 days and 4 days in the nursery. TORCH test was negative. Upon physical examination, microcephaly, hypertelorism, right thumb agenesis, heart murmur, dyspnea, and neonatal jaundice were observed. CT revealed corpus callosum agenesis. At 4 months, the patient weighed 4100 g and her height was 53 cm (both measurements below 3rd percentile) and OFC was 33 cm (below 2nd percentile). Currently, the proband presented cognitive disability, global development delay, hypotonia, absence of speech, and multiple dysmorphisms. Currently, she is completely dependent for daily life activities, and spends all the time in the sitting or lying position.
A total of 500 metaphases from each patient were attained by means of GTW-banding after 96 hours of peripheral blood cell cultive in RPMI 1640 medium and thymidine (both GibCo, USA) and synchronization following standard procedures [
FISH studies were performed according to manufacturer protocols (Cytocell, UK) using whole chromosome paint 13 probe (WCP13) and Rb1/Tel13q probe in order to characterize the origin of either ring or marker chromosomes observed in banding cytogenetics examinations. A total of 150 cells were addressed for each probe for all the patients in this study.
Whole-genome analysis was performed by means of the Agilent Human Genome CGH microarray 60-mer Oligonucleotide-based microarray (8 × 60 K, Agilent Technologies Inc., Santa Clara, CA) with a 40 kb resolution. Labeling and hybridization were performed according to the manufacturer protocols and analyses were made through the microarray scanner (G2600D) and the Feature Extraction software (v9.5.1) (Agilent Technologies). Image analyses were created by means of Agilent Genomic Workbench Lite Edition 6.5.0.18 along with the statistical algorithm ADM-2 and sensitivity threshold 6.0.
The patients described here showed a remarkable variation in phenotypes, with severity of clinical findings consistently being related to the extension of the deleted segments. Clinical data of them may be assessed in Table
Clinical and general findings of patients evaluated.
Patients | 1 | 2 | 3 |
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DOB (d/m/y) | 04/10/2012 | 26/03/2008 | 01/10/1989 |
Euchromatic deleted segment | q32.3q34 | q33.3q34 | q31.1q34 |
Segment size | 13.58 Mb | 6.87 Mb | 30 Mb |
Sex | F | M | F |
Weight at birth (g) | 1600 | 2620 | 1200 |
Height at birth (cm) | 42 | 48 | NK |
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Microcephaly | + | + | + |
Mental retardation | + | + | + |
Brain anomalies | − | − | + |
Development delay | + | + | + |
Hypotonia | + | + | + |
Facial dysmorphisms | + | − | + |
Prominent forehead | − | + | − |
Low-set hair | − | − | + |
Hirsutism | − | − | + |
Broad/flat nasal bridge | − | + | − |
Oblique eyelids | + | − | + |
Microphthalmia | − | − | + |
Nistagmus | − | − | + |
Strabismus | − | − | + |
Epicanthus | + | + | − |
Hypertelorism | + | − | − |
Micrognathism | − | − | + |
Microstomy | − | − | + |
Narrow and ogival palate | − | + | Nk |
Dysmorphic ears | + | − | + |
Low ears implantation | + | − | + |
Short neck | − | + | − |
Mammilary hypertelorism | − | − | + |
Congenital cardiopathy | − | + | + |
Skeletal anomalies | − | +mild lordosis | + |
Feet anomalies | − | − | + |
Hands anomalies | − | + | + |
Thumb hypoplasia/agenesis | − | − | + |
Parental karyotypes | Normal | Normal | Normal |
All individuals presented cognitive disability although with distinctive levels. Patient 2 (6.87 Mb deletion) showed milder clinical findings, while patient 3 (30 Mb deletion) was more affected and patient 1 (13.58 Mb deletion) apparently had an intermediate phenotype. Cases 1 and 3, which presented the largest deletions, additionally shared the following characteristics: low birth weight, microcephaly, facial dysmorphisms, oblique eyelids, and dysmorphic ears. The case 3 was the most clinically severe amongst the group, with brain anomalies, low-set hair, hirsutism, nistagmus, strabism, micrognathism. microstomy, mammilary hypertelorism, feet anomalies and thumb hypoplasia/agenesis. Interestingly, hands and skeletal anomalies along with congenital cardiopathy were observed only in patients 2 and 3 where the smaller and the larger deletions were detected.
GTG-banding analysis (adapted from [
Absolute and relative frequencies of clonal and nonclonal cytogenetic findings and distribution in probands (500 metaphases/individual).
Cell lineage | Karyotype | Patient 1 | Patient 2 | Patient 3 |
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Normal | 46,X_ | — | — | 10 (2.0%) |
Monocentric ring | 46,X_,r(13) | 421 (84.2%) | 429 (85.8%) | 421 (84.2%) |
Monosomy 13 | 45,X_,‒13 | 59 (11.8%) | 40 (8.0%) | 46 (9.2%) |
Dicentric ring | 46,X_,dic r(13) | 3 (0.6%) | 21 (4.2%) | 5 (1.0%) |
Monosomy 13 + marker | 46,X_,‒13,+mar | 3 (0.6%) | 3 (0.6%) | 6 (1.2%) |
Deletion 13? | 46,X_,?del(13)(q31) | 1 (0.2%) | — | — |
Isochromosome 13q | 46,X_,i(13)(q10) | — | 1 (0.2%) | — |
Derivative13 | 46,X_,der(13) | 4 (0.8%) | — | — |
Monocentric ring × 2 | 47,X_,r(13) × 2 | 4 (0.8%) | 1 (0.2%) | 4 (0.8%) |
Dicentric ring × 2 | 47,X_,dic r(13) × 2 | 2 (0.4%) | 1 (0.2%) | 1 (0.2%) |
Monocentric ring + dicentric ring | 47,X_,r(13),+dic r(13) | 1 (0.2%) | 2 (0.4%) | 1 (0.2%) |
Monocentric ring + derivative 13 | 47,X_,r(13),+der(13) | 1 (0.2%) | — | — |
Monocentric ring + marker | 47,X_,r(13),+mar | 1 (0.2%) | 1 (0.2%) | 1 (0.2%) |
Dicentric ring + marker 1 + marker 2 | 49,X_,dic r(13),+mar1 × 2,+mar2 | — | 1 (0.2%) | — |
Pulverization | 46,X_,pvz(13) | — | — | 3 (0.6%) |
Monocentric ring + pulverization | 47,X_,r(13),+pvz(13) | — | — | 2 (0.4%) |
N= | 500 | 500 | 500 |
The FISH WCP13 probe allowed for the identification of the origin of both ring and marker chromosomes observed in GTW-banding cytogenetics examination. Micronuclei were likewise noted in all patients, and the use of WCP13 permitted the assessment of the chromosome 13 origin of such structures Figure
WCP13 probe (green): (a) patient 2, normal 13 and monocentric ring; (b) patient 1, two monocentric rings and normal 13; (c) patient 3, normal 13 and marker with positive hybridization (arrow); (d) patient 2, normal 13 and dicentric ring; (e) patient 1, single normal 13 in a monosomic cell; (f) patient 1, on the left, metaphase with monocentric ring and normal 13, near an interphasic nucleus bordered by a micronuclei, the latter also showing positive hybridization.
Rb1 (red)/Tel13q (green) probes: (a) patient 1, monocentric ring showing Rb1 signal and no Tel13q; (b) patient 2, single normal 13 in a monosomic cell; (c) patient 3, dicentric ring presenting double Rb1 signal; (d) Patient 2, marker chromosome without both Rb1 and Tel13q signals; (e) Patient 2, two dicentric rings with double Rb1 signal each; (f) Patient 3, normal cell.
All individuals showed a terminal deletion in 13q with genes losses Table
List of deleted genes
Gene | OMIM | Description | Patient 1 | Patient 2 | Patient 3 |
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605250 | ATP-binding cassette, subfamily C, member 4 |
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610620 | ADP-ribosylhydrolase 1 |
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605477 | Rho guanine nucleotide exchange factor 7 |
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605868 | ATPase, class VI, type 11A |
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137217 | ATPase, H+, K+ transporting, beta |
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612800 | Cysteinyl-tRNA synthetase 2 |
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609686 | Citrate lyase beta-like |
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120130 | Angiopathy, hereditary, with nephropathy, aneurysmal |
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120090 | Collagen IV, alpha-2 polypeptide |
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607408 | {Schizophrenia}, 181500 (2) |
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191275 | Dopachrome tautomerase |
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601184 | DNA J, E. coli, homolog of, subfamily C, member 3 |
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600527 | Eph-related receptor tyrosine kinase ligand 5 |
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133530 | Cerebrooculofacioskeletal syndrome 3 (3) |
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227600 | Factor X deficiency (3) |
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227500 | Factor VII deficiency (3) |
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601515 | Spinocerebellar ataxia-27, 609307(3) |
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600441 | Growth arrest- specific 6 |
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602446 | Glypican 5 |
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604404 | Glypican 6 |
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602042 | G protein-coupled receptor-18 |
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180381 | Oguchi disease-2, 258100 (3) |
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609401 | Heparan sulfate 6-O-sulfotransferase 3 |
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601566 | Squamous cell carcinoma, head and neck 275355 |
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600797 | {Diabetes mellitus, noninsulin-dependent} 125853 |
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604234 | Integrin, beta-like 1 |
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611613 | KDEL motif-containing 1 |
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153330 | Lysosome-associate membrane protein-1 |
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601837 | LIG4 syndrome, 606593 (3) |
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609416 | Micro RNA 17 |
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609417 | Micro RNA 18A |
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609418 | Micro RNA 19A |
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609419 | Micro RNA 19B1 |
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609420 | Micro RNA 20A |
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611549 | Sodium leak channel, nonselective |
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232000 | Propionicacidemia, 606054 (3) |
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179540 | RAP2, member of RAS oncogene superfamily (K-rev) |
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605182 | Ras p21 protein activator 3 |
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601295 | Bile acid malabsorption, primary (3) |
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600544 | Solute carrier family 15 (oligopeptide transporter) |
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609680 | SLIT- and NTRK-like family, member 5 |
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609681 | SLIT- and NTRK-like family, member 6 |
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602148 | SRY (Sex determining region Y) box-1 |
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604974 | SRY (Sex determining region Y) box-21 |
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604984 | Serine/Threonine protein kinase 24 |
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189902 | Transcription factor Dp-1 |
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603969 | Tumor necrosis factor ligand superfamily, member 13B |
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190470 | Tripeptidyl peptidase II |
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605898 | UDP-glucose glycoprotein glucosyltransferase 2 |
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605530 | UPF3 regulator of nonsense transcripts |
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603073 | Holoprosencephaly-5, 609637 (3) |
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Schematic distribution of deleted genes and sizes of deletions of probands (red bars). Order of appearance of genes in the bands does not necessarily reflect the actual sequence in the chromosome (ideogram from above extracted from UCSC Genome Browser Gateway).
aCGH profile indicating the segment deletions on chromosome 13q.
In this study, classical cytogenetic tools were combined with biomolecular approaches in order to assess the cell mosaicism in individuals with ring chromosome 13 and to perform genotype-phenotype association to the 13q- syndrome. Brown et al. [
FISH analysis with WCP13 confirmed the chromosome 13 origin of small markers, large dicentric rings, and even pulverized material. Interestingly, micronuclei eventually observed in cytogenetics examination showed positive WCP13 hybridization, indicating the loss of ring chromosomes. In fact, Ford et al. [
The low-rate mosaicisms observed in cytogenetic examination were not detected by a-CGH, as described elsewhere [
GTW-banding such as FISH analysis in a comprehensive number of cells for each patient did not reveal significant differences in the frequencies of cell clones among individuals, despite the unequal size of deletions observed in the group, as referred to by Sodre et al. [
Kosztolanyi [
Further, Rossi et al. [
More recents evaluations by the means of aCGH have allowed the identification of a number of genes in deleted chromosome regions. However, only few reports postulate an improvement of a genotype-phenotype correlation with this new approach [
Concerning 13q- haploinsufficiency, Amor et al. [
The
An-/microphthalmia, a condition which has been linked to both the EFNB2 gene [
Although a gene could apparently lack a causative link to a phenotypic trait, one cannot exclude its involvement in that feature. Another mechanism proposed to explain the clinical anomalies in deletions would be the “unmasking” of recessive genes lying in the normal homologue chromosome through the loss of chromosomal segments in the rearrangement [
In this study we observed the presence, though in very low frequency, of marker chromosomes with deletion of Rb1. One knows that the occurrence of constitutive mutation at one of the Rb1 alleles is implicated in an estimated risk of 400x in retinoblastoma and other malignancies [
This study agree with the point of view of Rossi et al. [
We believe the combination of multiple cytogenetics and cytomolecular approaches to be critical for the genetic evaluation of syndromic and nonsyndromic individuals with ring chromosome 13 syndrome.
The Array-CGH approach is currently an indispensable tool for the evaluation of cryptical aberrations as well as for performing genotype-phenotype studies. However, classical cytogenetics strategies are still the methods of choice in the investigation of either cell mosaicism or balanced rearrangements.
Additional genotype-phenotype studies are needed in order to associate genes to their theoretical functions, thus providing a guide for further genomic research.
Our data indicates that the deletion of development genes may be the main cause of phenotypic variation, at least in cases of ring chromosome 13.
This study was approved by the Ethics Committee of ASSOCIACAO DAS PIONEIRAS SOCIAIS–Brasília/Distrito Federal–Brazil, under registration CAAE: 25847314.5.1001.0022. Written informed consent was obtained from the patients’ parents.
The authors declare that they have no conflicts of interest.
The authors thank Dr. Renata Lúcia Ferreira de Lima and Dr. Rafaela Mergener for the valious suggestions. The work has the partial support of RedeBRIM project.