A de novo atypical ring sSMC(22) characterized by array CGH in a boy with cat-eye syndrome
© Haltrich et al.; licensee BioMed Central Ltd. 2014
Received: 27 February 2014
Accepted: 21 May 2014
Published: 5 June 2014
Microduplications 22q11 have been characterized as a genomic duplication syndrome mediated by nonallelic homologous recombination between region-specific low-copy repeats. Here we report on a 19 years old boy with intellectual disability having an unexpected structurally complex ring small supernumerary marker chromosome (sSMC) originated from a larger trisomy and a smaller tetrasomy of proximal 22q11 harboring additional copies of cat eye syndrome critical regions genes.
Principal clinical features were: anorectal and urogenital malformations, total anomalous pulmonary venous return with secundum ASD, hearing defect, preauricular pits, seizure and eczema. The proband also presented some rare or so far not reported clinical findings such as hyperinsulinaemia, severe immunodeficiency and grave cognitive deficits.
Chromosome analysis revealed a mosaic karyotype with the presence of a small ring-like marker in 60% of cells. Array CGH detected approximately an 1,2 Mb single and a 0,2 Mb double copy gain of the proximal long arm of chromosome 22. The 1,3 Mb intervening region of chromosome 22 from centromere to the breakpoints showed no copy alteration. The karyotype of the patient was defined as 47,XY,+mar/46,XY.ish idic r(22)(q11.1.q11.21) × 4.arr 22q11(17,435, 645-18,656,678) × 3,(17,598,642-17,799,783) × 4 dn.
The present report is the first one with a detailed description of clinical presentation in a patient carrying an atypical size ring sSMC (22) analyzed by array CGH. The specialty of the finding is emphasized by the fact that although the patient had a mosaic sSMC and the amplified region was smaller than in typical cat eye syndrome cases, the clinical presentation was severe.
KeywordsSmall supernumerary marker chromosome sSMC ring(22) Cat eye syndrome Microarray
The sSMC 22q has been characterized as a genomic duplication syndrome mediated by nonallelic homologous recombination between region-specific low-copy repeats, designated LCR22s (eight different, ranging from LCR22-A to LCR22-H)  or by der(22)t(11;22)(q23.3;q11.2) originated from malsegregation of a parental balanced translocation between chromosomes 11 and 22 (known as Emanuel Syndrome, OMIM #609029) . A subset of SMCs is derived from proximal part of 22q11 and confers the tri- or tetrasomy for cat-eye syndrome (CES; OMIM #115470) chromosomal region. The original CESCR spans around 2Mb, from the centromere to the D22S57 marker, and includes two genes which are major candidates for relevant clinical features of CES: CECR1 between 17,659,680–17,702,744 Mb and CECR2 flanked by 17,956,630-18,033,845 Mb [3, 4]. Interstitial duplication of CESCR has been also reported in patients with clinical picture of CES, but without an sSMC .
There are two typical CES chromosomes: the smaller symmetrical type I CES chromosomes do not contain the DiGeorge critical region because both breakpoints are located in the proximal interval of the 22q11 deletion/duplication syndrome. The larger type II CES chromosomes harbor one (asymmetrical) or two (symmetrical) distal breakpoints and enclose one or two copies of DiGeorge critical region . Bartsch et al  described a third form (type III) bisatellited sSMC(22) with distal breakpoints outside of the LCRs22.
The more frequent attribute of CES phenotype [OMIM 115470] includes micrognathia, preauricular pits and/or tags, ocular coloboma, heart and kidney defects, genitourinary anomalies, anal atresia, normal to mild intellectual disability. The penetrance of these dysmorphic features is highly variable.
However over 210 cases of 22q11.2 microduplications have been reported so far, most of them overlap with the 3 Mb common typically deleted region (TDR) or 1,5 Mb DiGeorge critical region found in DGS/VCFS. The ring-like sSMCs generally occur with a lower frequency (0,14-0,72/100 in newborns)  and there are only a few details on ring sSMC(22) cases [9–11]. Here we characterize by array CGH an atypical ring sSMC with detailed description of clinical findings.
Chromosomes for cytogenetic analysis were obtained from 72-hour lymphocyte cultures. Analysis of the sSMC, present in G-banded karyotype was carried out using whole chromosome painting (WCP) 22 and acrocentric p-arm probes (Cytocell Technologies, Ltd., Cambridge, UK), satellite enumeration probe, SE 14/22, DiGeorge (N25, N85A3), subtelomeric 22q and 11q (Vysis/Abbott, Inc., Downers Grove, IL, USA) and a 22q11.1q11.21 specific oligonucleotide probe spanning 17,427-18,040 Mb close to the centromere (Agilent SureFISH, Santa Clara, CA USA).
To ascertain the segmental composition of the sSMC, a high resolution genomic scan using ISCA plus design array of Nimblegen-Roche containing 1.4 M probes per subarray (assembly GRCh37/hg19) was performed in the DNA samples of the patient. This CGX microarray provides a mean average resolution of approximately 15-20 Kb. The subarrays were scanned on NimbleGen MS 200 microarray scanner and data were extracted and analysed using NimbleScan, SignalMap and Deva 1.1 softwares (Roche NimbleGen Inc.). DNA CNVs were mentioned as gain or loss in a linear ratio and the length of the variation was given in megabase (Mb).
Classical CES is usually associated with a supernumerary isodicentric bisatellited chromosome 22 symmetrical or asymmetrical, depending on the proximal or distal localization of the two recurrent deletion/duplication breakpoints .
We identified an asymmetric dicentric sSMC(22) originated from a larger trisomy and a smaller tetrasomy of 22q11 harboring two additional copies of CECR1 and one of CECR2, and no extra copies of DiGeorge critical regions and chromosome 22 satellites. The ring shape was suggested by G-band and DAPI stained aspect of sSMC. The symmetric localization of the two 22q11 control oligonucleotid segments on the FISH images led us to hypothesize that the starting point of this sSMC(22) was a classical inv dup(22)(q11.21) dicentric chromosome and its breakages would have led to an “inv dup del” that stabilizes itself through circularization. All of the proximal and distal duplication breakpoints of the ring sSMC stretched into the largest, LCR22-A region which is considered to be the recurrent proximal deletion endpoint of TDR seen in 85% of 22q11.2 deletion syndrome cases (Figure 5a, b) . However, similar breakpoints and even the same gene content have been published [12, 13], were not specifically in ring shape. The reported ring 22 chromosomes were accompanied by the absence of normal chromosome and/or were the results of centric fission 22 [11, 14–16]. The majority of ring sSMC(22) cases was prenatally detected, with only some poor clinical details, lacking a precise delineation by sensible techniques such as microarray [9, 10]. The present report is the first one with a detailed description of clinical presentation in a patient carrying a ring sSMC(22) atypical in size which was analyzed by array CGH. Eventhough, the presented patient’s CES chromosome was in 60% mosaic and smaller than typical type I CES, he carried almost all major (cardiovascular, urogenital and gastro-intestinal) and minor (micrognathia, preauricular pits, kypho-scoliosis, pes planus) as well neurological (speech delay, intellectual disability) abnormalities characteristic for CES. He suffered from some medical issues commonly reported in individuals with Emanuel syndrome such as recurrent urinary tract and ear infections, sinusitis, low immunoglobulin levels, constipation, gastroesophageal reflux, hearing loss . The proband additionally presented some rare or previously not reported clinical findings such as hypoglycaemia, hyperinsulinaemia and psychiatric troubles.
The gene content of the present sSMC(22) (Figures 5c,d, 6c) included two extra copies of CECR1(OMIM 607575), CECR3 and CECR6 and a single additional copy of CECR2 (OMIM 607575), CECR7 CESCR candidate genes. Homo sapiens CECR1 gene encodes two distinct adenosine deaminases, ADA1 and ADA2. The ADA1 deficiency causes severe combined immunodeficiency syndrome and ADA2 protein may act as a growth factor and has adenosine deaminase activity converting adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. Extra-cellular adenosine is an important regulatory molecule with a low physiological concentration that can rapidly increase during tissue damage and inflammation. ADA2 is specifically secreted by antigen-presenting cells and may navigate to sites with a high concentration of adenosine by binding to specific cell surface receptors . The role of ADA2 is to induce differentiation of monocytes into macrophages as well as to stimulate the proliferation of macrophages and CD4+ T cells. The presented patient suffered from severe immunodeficiency, recurrent episodes of bacterial infection with sepsis, almost permanent herpes and/or stomatitis. We can speculate that ADA2 overhead production due to the tetrasomy of CECR1 could perturb the role of extra-cellular adenosine in adaptive immunity . Current discovered high resolution protein structure of ADA2 revealed two specific protein domains of novel folds that mediate the protein dimerization and are responsible for the receptor-mediated growth factor activity of ADGF/ADA2 proteins .
CECR1 gene expression in the outflow tract and atrium of the heart as well as in the facial/auditory-vestibular cranial nerve ganglion suggests its involvement in heart malformation and facial defects. Recent studies have been demonstrated that extra-cellular adenosine can act as an anti-insulin hormone, stimulating a release of glucose from stores in the Drosophila model . In this model increased extracellular adenosine is associated with hyperglycemia and impairment in energy storage. A similar role for adenosine signaling through adenosine receptors stimulating, the glucose release was described for mammalian liver cells , suggesting that this role of extracellular adenosine and the mechanism of action are evolutionary conserved from flies to mammals.
The GAB4 is a member of docking proteins family closely related to the insulin growth factor, cytokine and antigen receptors as well as cell adhesion molecules contributing to signal diversification by channeling the information from activated receptors into signaling pathways. These scaffold proteins play critical roles in protein-protein interactions in a variety of physiological processes as well as in disorders including cancer and inflammation, Alzheimer and cardiovascular diseases [21, 22]. We can hypothesize that gene dosage alterations of GAB4 and ADA2 and their possible functional interactions could be subject of changes in carbohydrate metabolism and these are likely the cause of hypoglycaemia susceptibility of the presented patient. It is noteworthy that transient hypoglicaemia similar to those seen in our patient have also been reported in a previous CES phenotype r(22) case .
The CECR2 is known as a chromatin remodeling gene, its candidate downstream genes are mesenchymal and ectodermal transcription factors involved in neural tube closure and inner ear development [23, 24]. Overexpression influence on the development of brain, eye and ear might be responsible for frequent abnormalities of these organs in CES patients. In our patient, the most frequent eye abnormality, coloboma was absent; instead he had Duane anomaly described only in a smaller number of CES patients .
MICAL3 (OMIM 608882) and TUBA8 (OMIM 605742) are responsible for cytoskeletal structures essential for neuronal migration and function , BID (OMIM 601997) regulates the cross-talk of cell cycle arrest and apoptosis . The overdosage of these three distal genes could be related to the neurologic impairment in our as well as in other CES patients.
The protein encoded by IL17RA (interleukin 17A receptor, OMIM 605461) gene is a ubiquitous type I membrane glycoprotein that binds with low affinity to interleukin 17A. Interleukin 17A and its receptor play a pathogenic role in many inflammatory and autoimmune diseases. Increased gene dosage could negatively impact the CES patient’s infectious conditions. The altered immune response function of this gene is not mentioned in other CES reports .
Out of the thirteen patients with ring sSMC(22) listed in the sSMC database  not a single one was identical with the case presented here and only two cases (reported by Ballif et al  and SW Cheung with scarce clinical description) were analyzed by array CGH. A similar but smaller double ring sSMC(22) was described by Mears et al in 1995 . The fact that both patients (published by Mears and presented here) were carriers of a smaller than typical type I CES with four copies of CECR1 and possessed all main CES abnormalities suggests that CECR1 gene dosage and functional interactions among amplified genes  are the predictable determining factor to express the CES phenotype and the size of sSMC(22) is rather a contributing factor to phenotypic variability.
Here we fully characterized a highly complex ring sSMC(22) containing extra copies of CES critical regions by high resolution array CGH. On basis of the sSMC database our ring is a unique finding being atypical in size and construction. The proband presented so far rare or not reported clinical findings such as hypoglycaemia, hyperinsulinaemia, severe immunodeficiency and grave cognitive deficits. The array CGH provided to be an optimal technique to determine the duplication breakpoints while chromosome banding provided information on mosaic feature and ring morphology of the sSMC. The FISH examination with whole chromosome painting probes is not a sufficiently sensitive technique to identify the origin of sSMCs but the use of adequate oligonucleotide probes is an efficient tool for confirmation of the array finding and level of mosaicism. This case illustrates the importance of reporting rare and unusual CES chromosome carrier patients to delineate more precisely the genotype-phenotype correlations and to evaluate the incidence of this type of ring sSMC(22) in the general population.
Written informed consent was obtained from the patient’s parents for publication of this case report, portrait and accompanying images. A copy of the written consent is available by the Editor-in –chief of this journal.
- ADA1 and ADA2:
Atrial septal defect
CES critical region
Comparative genomic hybridisation
GRB2-Associated binding protein family, member 4
(interleukin 17A receptor) gene
Low-copy repeats of chromosome 22
Microtubule associated onoxygenase, calponin and LIM domain containing 3 gene
Satellite enumeration probe
Small supernumerary marker chromosome
Typically deleted region
Tubulin, alpha 8 gene
- BID BH3:
Interacting domain death agonist isoform 1
Whole chromosome painting probe.
The authors wish to thank the patient and his parents who participated in this study.
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