Clinico-radiological and molecular characterization of a child with ring chromosome 2 presenting growth failure, microcephaly, kidney and brain malformations

Background Ring chromosome 2 is a rare constitutional abnormality that generally occurs de novo. About 14 cases have been described to date, but the vast majority of papers report exclusively conventional cytogenetic investigations and only two have been characterized by array-CGH. Results Here we describe the clinical, neuroradiological, and molecular features of a 5-year-old boy harbouring a ring chromosome 2 presenting with severe growth failure, facial and bone dysmorphisms, microcephaly, and renal malformation. Brain MR with diffusion tensor imaging revealed simplified cortical gyration, pontine hypoplasia, and abnormally thick posterior corpus callosum, suggesting an underlying axonal guidance defect. Cytogenetic investigations showed a karyotype with a ring chromosome 2 and FISH analysis with subtelomeric probes revealed the absence of signals on both arms. These results were confirmed by array-CGH showing terminal deletions on 2p25.3 (~439 kb) and 2q37.3 (~3.4 Mb). Conclusions Our report describes a new patient with a ring chromosome 2 completely characterised by array-CGH providing additional information useful not only to study genotype-phenotype correlation but also to validate the role of already reported candidate genes and to suggest novel ones which could improve our understanding of the clinical features associated with ring chromosome 2. Electronic supplementary material The online version of this article (doi:10.1186/s13039-015-0121-z) contains supplementary material, which is available to authorized users.


Background
Ring chromosomes result from one or two distal breaks followed by fusion of the broken ends and loss of the acentric fragments, but they can also be formed by fusion of subtelomeric sequences without apparent loss of genetic material. The ring chromosome phenotype is characterized by growth failure and intrauterine growth retardation (IUGR). According to Cote et al. [1] and Kosztolanyi et al. [2], which defined the "Ring syndrome", suggested that the common growth deficiency, observed across many patients with diverse ring chromosomes, was due to mitotic instability and tissue-specific mosaicism.
Recently, FISH and CGH studies demonstrated that, in most cases, a cryptic deletion is the cause of the phenotypic abnormalities in apparently intact rings.
Here, we report on a boy with a ring chromosome 2 presenting growth failure, microcephaly, dysmorphic features, L-shaped kidney, and brain malformations studied with brain MRI and diffusion tensor imaging.

Case presentation
The patient is a 5-year-old boy, only child of healthy non-consanguineous parents. He was born to a 28-yearold mother whose pregnancy was complicated by IUGR and oligohydramnios, requiring caesarean section at 35 weeks of gestation. The Apgar score was 9 and 10 at 1 and 5 minutes, respectively. Physical examination at birth revealed left clubfoot (congenital talipes equinovarus) that was treated with a plaster cast. Birth weight was 1740 g (<3 rd centile), length 40.5 cm (<3 rd centile) and head circumference 29.5 cm (<3 rd centile). He developed mild respiratory distress requiring continuous positive airway pressure (nCPAP) for 72 hours. Echocardiogram depicted patent ductus arteriosus that disappeared spontaneously at follow-up. Abdominal ultrasound revealed crossed renal ectopia resulting in an L-shaped kidney (the left crossed kidney assumed a transverse position in the pelvis), while brain ultrasound showed incomplete opercularization.
At the age of 2 years, weight and length were well below the 3 rd centile and head circumference was 4 SD below the mean. Dysmorphic features ( Figure 1A,B) included round face, short forehead, medial eyebrow flare, wide nasal bridge, hypertelorism, epicanthal folds, broad nasal tip with prominent columella, long philtrum, thin upper lip, mild protruding ears, short neck, small hands with brachydactyly and clinodactyly of II left digit and V digit bilaterally, feet with broad big toes, brachydactyly, and left clubfoot ( Figure 1C,D). Neurological evaluation revealed mild developmental delay (he had a developmental age of 15 months at the age of 2 years in according to Griffith scale) and global hypotonia. He walked with support at 14 months and emitted vocalizations and babbling at the age of 19 months. The ophthalmologic evaluation was negative.
At the age of 2 years, brain MRI revealed microcephaly with simplified cortical gyration, bilateral hippocampal malrotation, and pontine hypoplasia. The corpus callosum appeared abnormally thickened at the level of the splenium and the lateral ventricles were dysmorphic (Figure 2A,B). Diffusion tensor imaging (DTI) and tractography (DTT) confirmed the disproportion between the anterior and posterior segments of the corpus callosum with an abnormally thick splenium. No ectopic callosal bundles were found. Interestingly, the tapetum was hyperplasic and caused a peculiar inward deformation of the lateral walls of the ventricular trigones ( Figure 2C,D).

Discussion
Ring chromosomes are rare and present a classical phenotype characterized by minor dysmorphic facial features and mental retardation. According to Cote et al. [1] and Kosztolanyi, [2], the "Ring syndrome" is essentially characterised by intrauterine growth retardation (IUGR), and failure to thrive, which can be the sole major physical abnormalities. The common growth deficiency observed in many patients with different ring chromosomes suggested that this phenomenon is due to mitotic instability of the ring chromosome and tissue-specific mosaicism [2].
The application of FISH and, more recently, of array-CGH demonstrated that, in most cases, a cryptic deletion could determine phenotypic abnormalities in apparently intact rings. Our patient carried a ring chromosome 2 with a 2pter-p25.3 deleted region of~470 kb and a concomitant deleted region of~3.405 Mb at bands 2q37.3qter. This ring chromosome appeared to be a mosaic with a proven de novo origin.
The ring chromosome described by López-Uriarte et al. [12] presented a deleted region of 139 kb at 2p25.3 including only FAM110C gene, while the deleted region  In our opinion, the patient described by Lopez-Uriarte et al. [12] showed the features of the so-called "Ring syndrome", characterised by growth failure, microcephaly, and minor dysmorphic features. Some features of the "Ring syndrome", as IUGR and failure to thrive, were present also in our patient, who however showed other  manifestations that could be the result of the deleted regions of ring chromosome 2. In fact, our case has a 3.4 Mb microdeletion in 2q37.3. It is known that 2q37 deletions have been associated to the Brachydactyly-Mental Retardation syndrome (MIM 600430), a contiguous gene syndrome characterized by haploinsufficiency or heterozygous mutations in the HDAC4 gene. The human HDAC4 gene encodes a chromatin-remodelling factor, histone deacetylase 4, which cooperatively regulates gene expressions with other transcription factors in the physiological process of development and differentiation of various tissues [13,14].
The deleted region in our patient contained also KIF1A gene. KIF1A belongs to the kinesin 3 family, a large superfamily of molecular motors using microtubules as a "rail" to transport cargo along and chemical energy of ATP to drive conformational changes that generate motile force. Active transport of proteins along directional cytoskeletal filaments is fundamental for neuronal function and survival, because most of the proteins required in the axon and nerve terminals need to be transported from the cell body [18]. Homozygous or compound heterozygous mutation in the KIF1A is implicated in mental retardation, autosomal dominant 9 (MIM 614255), neuropathy, hereditary sensory, type IIC (MIM 614213) and spastic paraplegia 30, autosomal recessive (MIM 610357).
Interestingly, our patient presented brain anomalies such as microcephaly with simplified cortical gyration associated with pontine hypoplasia, hippocampal malrotation and callosal abnormalities. In particular, the corpus callosum was dysmorphic with an abnormally thick splenium. DTI revealed the presence of a hypertrophic splenium suggesting an underlying axonal guidance defect altering the normal corpus callosum development. Intriguingly, another gene included in the deleted region of the present case, FARP2, encodes for a GTPase involved in neurite growth and axonal guidance. In particular, FARP2 is an immediate downstream signalling molecule of the Sema3A receptor complex that mediates repulsion of outgrowing axons and suppression of neuronal adhesion [19].
This peculiar combination of brain malformations has never been described in patients with ring chromosome 2 or 2q37.3 syndrome. This suggests that other pathogenic genes like TWIST2, NDUFA10, SEPT2, GAL3ST2, and NEU4, present in the deleted region, could have contributed to the overall phenotype of our patient.
Actually, the genes included in the deleted region 2pter-p25.3 (FAM110C, SH3YL1, ACP1, FAM150B) did not appear to be causative of the phenotype of our patient, but we cannot exclude this hypothesis.

Conclusions
In conclusion, our report describes a new patient with a ring chromosome 2 completely characterised by array-CGH proving additional information useful not only to study genotype-phenotype correlation but also to validate the role of already reported candidate genes and to suggest novel ones which could improve our understanding of the clinical features associated with ring chromosome 2.

Cytogenetic and CGH analyses
Standard GTG banding was performed at a resolution of 400-550 bands on metaphase chromosomes from peripheral blood lymphocytes of the patient and his parents. FISH analysis was performed using Aquarius Subtelomere Specific Probe 2p (D2S2983) (Spectrum Green)/2q (D2S2986) (Spectrum Orange) (Cytocell; http://www.cytocell.com). To better characterize the rearrangement, array-CGH analysis was performed using a 180 K platform with~13 kb overall median probe spacing (Agilent Technologies, Santa Clara, CA). Labelling and hybridization were performed following the protocols provided by the manufacturers. A graphical overview was obtained using the Agilent Genomic Workbench Lite Edition Software 6.5.0.18.

Brain MRI
Brain MRI was performed with a 1.5 T scanner (Achieva 2.6, Philips, Best, the Netherlands). DTI data were acquired  [12] and Chen et al. [11].