Towards New Approaches to Evaluate Dynamic Mosaicism in Ring Chromosome 13 Syndrome

1. Introduction

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 [1, 2]. Clinical findings seem associated with the size of the rings, concerning the extent of deleted segments, but also on the ring instability, which results in more severe features [3–9]. Phenotype usually consists of anencephaly, aprosencephaly or encephalocele along multiple and more severe congenital anomalies in minor rings (larger deletions) involving bands 13q14 and/or q22. Larger ring 13 cases (smaller deletions) show milder phenotypic implications including ophthalmic and extremities malformations and a prolonged life expectancy. In cases with even larger rings, individuals may not present any dismorphic traits, although a mental deficiency would be detected [10]. Clinical characteristics observed in ring 13 individuals usually overlap with linear 13q deletions, although more severe traits are commonly diagnosed because of the mitotic instability of the ring.

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 [11], while recent reports point to a loss of development genes during the ring formation as a main role in the process.

Studies of genotype-phenotype correlation delineate the 13q- syndrome and thus provide an effective understanding of that clinical entity [2, 12–17]. Identification of specific genes and their respective association to clinical traits has led, for instance, to the establishment of ZIC2 as a hallmark in cases of 13q-, due to the fact that this gene loss is causative of holoprosencephaly [18]. Additional studies have been trying to associate a number of genes to the sort of clinical characteristics found in this syndrome, for instance, EFNB2 to microphtalmia [13, 15] and ARHGEF7 to mental retardation/microcephaly [19, 20].

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.

2. Materials and Methods

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 1). Peripheral blood samples for GTW-banding and FISH karyotype, and aCGH were obtained from probands after informed consent.

3. Casuistics

4. Results

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 1.

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.

4.2. Fluorescence In Situ Hybridization (FISH)

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 2(f). The Rb1/Tel13q probe indicated deletion of subtelomere 13q in the whole ring chromosomes of probands in this study. Otherwise, Rb1 structural deletion was observed in very small ring chromosomes (markers). Nevertheless, the presence of a normal karyotype cell clone in patient 3 was also confirmed Figure 3(f).

4.3. Array-Comparative Genomic Hybridization

All individuals showed a terminal deletion in 13q with genes losses Table 3. Patient 1 revealed a 13.58 Mb loss affecting bands q32.3–q34 between genomic positions 101475155–115059020. Patient 2 presented a 6.87 Mb deletion affecting bands q33.3–q34 between genomic positions 108181836–115059020. Patient 3 showed a deletion of 30 Mb affecting bands q31.1–q34 between genomic positions 85.075.294–115.059.020 (UCSC Genome Browser on Human Feb. 2009 (grhH37/hg19) (Figures 4 and 5).

Table 3. List of deleted genes ^∗ in probands.

Gene	OMIM	Description	Patient 1	Patient 2	Patient 3
ABCC4	605250	ATP-binding cassette, subfamily C, member 4			∗
ADPRHL1	610620	ADP-ribosylhydrolase 1	∗	∗	∗
ARHGEF7	605477	Rho guanine nucleotide exchange factor 7	∗	∗	∗
ATP11A	605868	ATPase, class VI, type 11A	∗	∗	∗
ATP4B	137217	ATPase, H+, K+ transporting, beta	∗	∗	∗
CARS2	612800	Cysteinyl-tRNA synthetase 2	∗	∗	∗
CLYBL	609686	Citrate lyase beta-like			∗
COL4A1	120130	Angiopathy, hereditary, with nephropathy, aneurysmal	∗	∗	∗
COL4A2	120090	Collagen IV, alpha-2 polypeptide	∗	∗	∗
DAOA	607408	{Schizophrenia}, 181500 (2)	∗		∗
DCT	191275	Dopachrome tautomerase			∗
DNAJC3	601184	DNA J, E. coli, homolog of, subfamily C, member 3			∗
EFNB2	600527	Eph-related receptor tyrosine kinase ligand 5	∗		∗
ERCC5	133530	Cerebrooculofacioskeletal syndrome 3 (3)	∗		∗
F10	227600	Factor X deficiency (3)	∗	∗	∗
F7	227500	Factor VII deficiency (3)	∗	∗	∗
FGF14M	601515	Spinocerebellar ataxia-27, 609307(3)	∗		∗
GAS6	600441	Growth arrest- specific 6	∗	∗	∗
GPC5	602446	Glypican 5			∗
GPC6	604404	Glypican 6			∗
GPR18	602042	G protein-coupled receptor-18			∗
GRK1	180381	Oguchi disease-2, 258100 (3)	∗	∗	∗
HS6ST3	609401	Heparan sulfate 6-O-sulfotransferase 3			∗
ING1	601566	Squamous cell carcinoma, head and neck 275355	∗	∗	∗
IRS2	600797	{Diabetes mellitus, noninsulin-dependent} 125853	∗	∗	∗
ITGBL1	604234	Integrin, beta-like 1	∗		∗
KDELC1	611613	KDEL motif-containing 1	∗		∗
LAMP1	153330	Lysosome-associate membrane protein-1	∗	∗	∗
LIG4	601837	LIG4 syndrome, 606593 (3)	∗	∗	∗
MIR17	609416	Micro RNA 17			∗
MIR18A	609417	Micro RNA 18A			∗
MIR19A	609418	Micro RNA 19A			∗
MIR19B1	609419	Micro RNA 19B1			∗
MIR20A	609420	Micro RNA 20A			∗
NALCN	611549	Sodium leak channel, nonselective	∗		∗
PCCA	232000	Propionicacidemia, 606054 (3)			∗
RAP2A	179540	RAP2, member of RAS oncogene superfamily (K-rev)			∗
RASA3	605182	Ras p21 protein activator 3	∗	∗	∗
SLC10A2	601295	Bile acid malabsorption, primary (3)	∗		∗
SLC15A1	600544	Solute carrier family 15 (oligopeptide transporter)			∗
SLTRK5	609680	SLIT- and NTRK-like family, member 5			∗
SLTRK6	609681	SLIT- and NTRK-like family, member 6			∗
SOX1	602148	SRY (Sex determining region Y) box-1	∗	∗	∗
SOX21	604974	SRY (Sex determining region Y) box-21			∗
STK24	604984	Serine/Threonine protein kinase 24			∗
TFDP1	189902	Transcription factor Dp-1	∗	∗	∗
TNFSF13B	603969	Tumor necrosis factor ligand superfamily, member 13B	∗	∗	∗
TPP2	190470	Tripeptidyl peptidase II	∗		∗
UGGT2	605898	UDP-glucose glycoprotein glucosyltransferase 2			∗
UPF3A	605530	UPF3 regulator of nonsense transcripts	∗	∗	∗
ZIC2	603073	Holoprosencephaly-5, 609637 (3)			∗

5. Discussion

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. [24] describe three deletion groups in 13q: group 1, deletions proximal to q32, group 2, breakpoints within q32, and group 3, deletions of q33 and q34. Patients 1—r(13)(p13q32.3)—and 3—r(13)(p12q31.1) were classified as deletion group 2, while patient 2—r(13)(p11q33.3) was compatible with group 3.

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. [25] inferred that the majority or even the totality of micronuclei would correspond to either whole chromosomes or chromatids due to mitotic arrestment. On the other hand, Rb1/Tel13q- indicated deletion of subtelomeric 13q sequences in all ring chromosomes as well as in their derivatives. Hemizygotic Rb1 deletion was revealed in very small marker chromosomes at a low incidence.

The low-rate mosaicisms observed in cytogenetic examination were not detected by a-CGH, as described elsewhere [2, 8, 26–28]. According to Liehr et al. [29], cytogenetics is still considered as the gold-standard test for mosaicism identification while the refinement of aberrations should be performed by cytomolecular approaches. In fact, a-CGH was a determinant to accurately address the size of deletions in probands. Data obtained directly correlated to the severity of patient’s clinical findings, as reported elsewhere [2, 13, 15–17].

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. [7]. This is in disagreement with Kosztolanyi [11] postulated, who pointed to a correlation between ring size and ring instability. In each proband, the clonal distributions were very close and showed the same proportion reported elsewhere in cases of ring chromosomes [1, 2, 7, 28, 30–34].

Kosztolanyi [31] attributes the mitotic ring instability to a unique entity called “ring syndrome”, irrespective of the chromosomal origin, whose characteristics would include extreme growth failure, few or no minor anomalies and moderate to high mental retardation. The phenotypic characteristics would be due to the apoptosis of nonviable cells with secondary aberrations and thus lead to growth failure. However, Sodre et al. [7] pointed out that cells derived from secondary ring aberrations could multiply and survive in vivo, and so contribute to the phenotypic variations observed in patients with ring chromosomes, in opposition to the apoptotic mechanism of Kosztolanyi [31].

Further, Rossi et al. [5, 6] pointed out evidence of specific losses of genetic elements to be the real cause of short stature in ring patients. The authors refer to a group of ring 15 patients whose probands with short stature presented deletion of IGF1R (insulin-like growth factor 1 receptor precursor). Additionally, Glass et al. [35] reported the same results in another cohort of ring chromosome 15 individuals. Strong evidence in the latter study against the theory of a ring chromosome syndrome was the fact that, in the majority of cases, a cryptic deletion was the current cause of the phenotypic anomalies.

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 [2, 9, 12–16, 27, 28, 36–39].

Concerning 13q- haploinsufficiency, Amor et al. [40] described a boy with growth hormone deficiency and hand anomalies which presented a neocentric ring 13q31–32. The authors suggested three candidate genes that might be contributing to the boy’s phenotype, GPC5 and FARP1 for the hand anomalies and SOX21 for the short stature. Other authors also suggested the association of GPC5 with those anomalies [14, 15, 36]. However, recent evidence shown by DePontual et al. [39] attributes this to the miRNA cluster MIR17HG, and not to GPC5, a causative factor in the syndrome of growth failure, hand anomalies, and microcephaly. According to the authors, this syndrome would be the first example of a miRNA gene responsible for a syndromic development defect in humans. Our patient 3 showed short stature, microcephaly, and hand anomalies with thumbs aplasia/hypoplasia and the patient lacks the MIR17HG cluster. Nevertheless, one of the DePontual’s patients also presented hypoplasic thumb. Kirchhoff et al. [14] referred to two patients with thumb a-/hypoplasia and indicated a separate entity within the 13q31.3q33.1 segment. It seems essential to carry out new investigations aimed at the miRNAs as possible causes of novel human syndromes, as according to DePontual et al. [39], new evidence has been emerging about the miRNAs in development modulation.

The ZIC2 gene is considered a hallmark in 13q- syndrome as it is implicated with brain anomalies [13–15, 18, 41]. Patient 3 showed hemizygotic loss of this gene and presented brain anomalies. Patient 1, despite belonging to deletion group 2, involving band 13q32.3 where ZIC2 is located, did not show brain anomalies and presented both alleles of this gene.

An-/microphthalmia, a condition which has been linked to both the EFNB2 gene [13, 15] and the SOX1 gene [14] was noted only in individual 3, although both patients 1 and 3 showed deletion of EFNB2 and the three cases revealed haploinsufficiency of SOX1. Thus, our data weakens an association of both genes to eye malformations. On the other hand, SOX1 has also been implicated in microcephaly and cortical malformations [14, 19] together with ARHGEF7, deleted in our group [14, 19, 20]. Microcephaly was a condition observed in all probands in this study and is a common finding in patients with 13q deletions [13, 14, 24]. A minimal critical deletion for this trait was determined for 13q34 [2, 17].

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 [5, 6, 42]. The emergence of a trait, in this case, would depend on the alleles lying in the normal homologue.

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 [43]. Despite the fact that a cytogenetic loss of Rb1 would not necessarily lead to the development of neoplasies [44], rare cases of ring chromosome 13 related to retinoblastoma were reported [45].

This study agree with the point of view of Rossi et al. [5, 6] that phenotypic features in ring cases depend mainly on the size of deleted segments and respective loss of development genes, although the effect of the ring instability could not be excluded. However additional investigations to clarify this phenomenon are yet recommended.

6. Conclusions

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.

Consent

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.

Conflicts of Interest

The authors declare that they have no conflicts of interest.