De Novo Duplication of 7 p 21 . 1 p 22 . 2 in a Child with Autism Spectrum Disorder and Craniofacial Dysmorphism

The duplication of the short arm of chromosome 7 as de novo is extremely rare. The phenotype spectrum varies depending on the region of duplication. We report a case of de novo duplication of chromosomal region 7p21.1p22.2 in a three-year-old male child with autism who presented to the Sultan Qaboos University Hospital in Muscat, Oman, in January 2012. The patient was diagnosed with craniofacial dysmorphism, global developmental delay, hypotonia and bilateral cryptorchidism. The duplication was detected by conventional G-banded karyotype analysis/fluorescence in situ hybridisation and confirmed by array comparative genomic hybridisation. To the best of the authors' knowledge, this is the first report of chromosomal region 7p21.1 involvement in an autistic patient showing features of a 7p duplication phenotype. Identifying genes in the duplicated region using molecular techniques is recommended to promote characterisation of the phenotype and associated condition. It may also reveal the possible role of these genes in autism spectrum disorder.

Upon examination, the patient would not speak spontaneously and rarely vocalised directly to either the examiners or his parents.Non-verbal communications (including gestures, smiling, pointing or using other body parts) and the range of distinct facial expressions were very limited.Methods of communication were not coordinated with eye contact and the child showed limited enjoyment during interactions.The patient did not request objects and was not interested in showing objects to others.He showed no interest in symbolic/pretend play but demonstrated unusual sensory interest in materials (e.g.licking objects).The patient showed stereotypical motor repetitive behaviour (repetitive hand flicking) and persistent odd hand positioning in the form of bilateral wrist flexion.No disruptive, aggressive or self-injurious behaviours were noted.His communication and reciprocal social interaction scores fell within the range of ASD.Based on the above clinical and multidisciplinary evaluations, the patient was diagnosed with ASD.
The patient had delayed developmental milestones, demonstrating head control at the age of 12 months old, sitting with support by 14 months old, standing with support by two years old and walking by the time he was three years old.His hearing test results were normal, although an eye examination showed hypermetropic astigmatism.During a follow-up ophthalmological appointment, a slightly enlarged cup-to-disc ratio was noticed, indicating glaucoma.
Features of craniofacial dysmorphism were observed, including a high forehead with a prominent metopic ridge; brachycephalic and plagiocephalic closed anterior fontanelle; a low v-shaped posterior hairline; well-arched thin eyebrows; hypoplastic alae naris; narrow anteverted nares; a bulbous nasal tip; ears with prominent crus and overfolded helices; a short philtrum; a prominent lower lip; micrognathia; and bilateral cryptorchidism.The patient had undergone an operation for the cryptorchidism.The coronal sutures were open but appeared narrow.The height and weight of the patient were both at the 50-75 th centile, with head circumference at the 25 th centile.Magnetic resonance imaging of the brain, echocardiography and abdominal ultrasonography findings were unremarkable.The parents refused permission for photographs of the child to be published.
Peripheral blood chromosomal G-band karyotyping analysis was performed with 400-550 band resolution, which showed a 46, XY, dir dup(7) (p21p22) karyotype [Figure 1A].Fluorescence in situ hybridisation using a subtelomeric probe (Cytocell Ltd., Cambridge, UK) showed the presence of telomeres on both short arms of chromosome 7 [Figure 1B].The parental karyotypes were normal, indicating the 7q duplication in the child to be a de novo occurrence.
Array-based comparative genomic hybridisation (aCGH) using 8x60k oligoarray platforms (Oxford Gene Technology, Begbroke, Oxfordshire, UK) confirmed the presence of a cytogenetically visible duplication within the short arm of one chromosome 7. Analysis using CytoSure ™ Interpret software, Version 3.4.8(Oxford Gene Technology), showed that this duplication was ~16.5 million bases (Mb) in size.The breakpoints were also refined.The duplication involving the region 7p22.  of 7p in a few cases or smaller terminal 7p segments in others. 2 Arens et al. reported complete 7p trisomy (without the involvement of any other chromosomes) in two patients. 6Similar diagnoses have been made in five other patients. 4,7Many phenotypic features common to 7p duplication syndrome were present in the patient in the current report [Table1]; 1,4,10,11 these have also been described in earlier case reports. 3,6,8otably, the patient described in the current report did not have any cardiovascular abnormalities and thus had a better prognosis compared to patients in previous reports who died early as a result of these abnormalities. 3,6,8idence suggests that most 7p duplications occur due to malsegregation of parental balanced translocations or abnormal recombination of parental chromosome inversions and that these duplications rarely result from de novo partial 7p direct [Figure 2].This analysis was done commercially.No other imbalances were detected.The duplication interval contained 67 annotated genes, of which six had morbid entries in the Online Mendelian Inheritance in Man ® (OMIM) catalogue.However, the genes in this region were not directly disrupted by the breakpoints.

Discussion
5][6][7][8]10,11,13 In their review of the literature, Cai et al. found that 50% of 7p duplications were the result of balanced reciprocal translocation carriers. 3Reish et al. suggested that these could be an entire duplication duplication. 2,3The critical region for physical and mental abnormalities is 7p15-pter; 2 for craniofacial dysmorphism, it is 7p21. 2,3,9Both these patient groups, viz.those with physical and mental abnormalities, have many specific features in common.The range of severity may depend on the size and genes involved.
The segmental size of the duplication in the current patient was larger than those reported elsewhere. 10,11Furthermore, there was an association with ASD.Notably, two reports have associated 7p duplication with this disorder. 4,12Wolpert et al. described an inverted duplication of 7p14.1p11.2 in an autistic adult, who lacked many of the characteristic features found in 7p duplication syndrome. 4This may indicate that the critical region was distal.The patient was normocephalic, had normal developmental milestones, meatal stenosis, bilateral esotropia and mild scoliosis. 4Cukier et al. reported a pair of autistic first cousins carrying two microduplications, one of whom had a tandem duplication on 7p21 replicating part of the neurexophilin 1 (NXPH1; OMIM entry *604639) and islet cell autoantigen 1 (ICA1; OMIM entry *147625) genes. 12The patient in the current report was a child with direct duplication, clinical features of microcephaly and delayed milestones.None of the other features reported by Cukier et al. were present in the current patient. 12However, both patients had common autistic phenotypes and behavioural features. 12The duplication was more proximal in Cukier et al. 's case, 12 whereas it was distal in the current patient.
It has been suggested that the region within the duplication interval (7p21.1 to 7p22.2) observed in the current patient is the critical region for manifestations of the 7p duplication phenotype. 13This region of 7p contains the OMIM morbid gene TWIST1, duplications of which are thought to be the cause of the large fontanelles in these patients; 13 it is hence likely to be the cause of the current patient's clinical phenotype.The duplication region of this patient encompassed the whole of the TWIST1, ICA1 and NXPH1 genes.These genes, however, were not directly disrupted by the breakpoints of the duplication.The aCGH analysis could not determine whether this duplication might have a position effect on the regulation of these genes.
Parental chromosomal studies confirmed that the duplication in the presently reported patient was de novo and did not occur from a balanced chromosomal rearrangement, which may sometimes occur as insertional translocations. 14These translocations underlie 2.1% of apparently observed de novo interstitial copy number changes detected by aCGH. 14urther molecular analysis is worth considering for genes of the duplicated region, particularly when they are associated with ASD phenotypes, for further delineation of genotype-phenotype correlations.

Conclusion
A de novo duplication of chromosomal region 7p21.1p-22.2 was identified in a three-year-old autistic child with craniofacial dysmorphism, global developmental delay, hypotonia and bilateral cryptorchidism.Characterising the genes involved in duplication regions may help in understanding the genotype-phenotype correlation in 7p duplication patients.It may also reveal the possible role of these genes in ASD.aCGH analysis is an important tool in revealing genetic abnormalities among children with intellectual disabilities and dysmorphic features, as these are usually not detected by conventional cytogenetics.

Figure 2 :
Figure 2: Array comparative genomic hybridisation of an autistic child with craniofacial dysmorphism showing a duplication interval of ~16.5 million bases within the breakpoints at chromosome 7p22.2and 7p21.

Table 1 :
Comparative analysis of cases of de novo 7p duplication in the literature