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A new case of microdeletion 5q11.2 with segmental 5q isodisomy and review of the literature.
Vanna Pecile(1), Raffaella Devescovi(2) , Stefania Capellani(1), Flavio Faletra(1), Elisabetta Lenzini(3)
1 Medical Genetics Division, Institute for Maternal and Child Health � IRCCS �Burlo Garofolo�, Trieste, Italy
2 Division of Child Neurology and Psychiatry-Institute for Maternal and Child Health � IRCCS �Burlo Garofolo�, Trieste, Italy
3  Molecular Cytogenetics Lab, Department of Child and Mother Health, University of Padova, Padova, Italy

Corresponding Author: Vanna Pecile Medical Genetics Division, Institute for Maternal and Child Health IRCCS �Burlo Garofolo�, via dell'Istria 65 Trieste, Italy Tel. +39403785583 Fax +39403785540   HYPERLINK "javascript:void(window.open('/groupware/imp/dynamic.php?page=compose&to=vanna.pecile%40burlo.trieste.it&popup=1','','width=820,height=610,status=1,scrollbars=yes,resizable=yes'))" vanna.pecile@burlo.trieste.it



Abstract
Deletions of the proximal regions of long arm of chromosome 5 are rare, reports describing patients with del 5q11.2 consist of 10 cases. Segmental UPD are stimated about 0.6% in the random/general population. Recently Snjiders Blok et al (2014) describe a new microdeletion 5q11.2  syndrome. We report a new case of microdeletion 5q11.1-q11.2 with associated segmental isodisomy 5q11.2-qter, patient presents psychomotor retardation, autism spectrum disorder, patent foramen ovale and speech delay and this case is compared with others reported cases from the literature to contribute to 5q11.2 microdeletion syndrome better definition.


Key words 
microdeletion 5q11.1-q11.2, segmental UPD, Autism Spectrum Disorder, SNP-array

Abbreviations: UPD: Uniparental disomy, ROHs: regions of homozygosity , SNPs: Single Nucleotide Polymorphisms, CGH: Comparative Genomic Hybridization, CNV: Copy Number Variation

Introduction
Uniparental disomy: is a genetic condition where offspring receives two chromosomal homologues from only one parent. If UPD is present on a segment, interstitial or telomere, of the homologues, the condition is referred to as a segmental UPD, and if the homologues are identical (copies of a single homologue from one of the parents), the condition is known as isodisomy. Segmental UPD is estimated to effect 0.6% of the population (2).
With the introduction of new technologies using oligo-SNP studies regions of homozygosity (ROHs) are being found more frequently and it affects all chromosomes (2). Deletions of the segment 5q11.q11.2 have rarely been reported and most reported cases of deletion affect the long arm of chromosome 5 involving different regions i.e. 5q11.2, 5q12.1, 5q12.3 e 5q13.2 .
Recently Snjiders Blok et al (2014) describe a new microdeletion 5q11.2 syndrome.
We report an additional case with microdeletion 5q11.1-q11.2 with associated segmental isodisomy 5q11.2-qter presenting psychomotor retardation, autism spectrum disorder, patent foramen ovale (PFO) and speech delay.
This case is compared with others reported cases from the literature the segment of which is only partially superimposed (because it is broader) than the one we found to contribute to 5q11.2 microdeletion syndrome better definition.
Patient
We present the case of a 2-year-old male patient, the first conceived by healthy non-  consanguineous parents who was born the 36� week of gestation as a result of artificially-induced childbirth due to reduced fetal growth, at 20week hemivertebra was detected. Birth weight was 2190g (3�-10� centile), length 45,3 cm ( 10-25 centile), head circumference 31cm. Apgar score was 7-9 and 10, during the neonatal period no distinguishing features were noted and reported. Height-weight development was 3-10 percentile. He was able to stay seated at 10 months, walk without assistance at 16 months and at about 1 year able to pronounce monosyllables. At 20 months the child was referred to our Pediatric Hospital for evaluation and diagnostic advice for cognitive retardation, autism, scoliosis, patent foramen ovale and speech delay Physical examination showed weight 10,2 kg (3-10 centile), height 81 cm (3-10 centile), occipito-frontal circumference 46,5 (3�). No seizures were reported even during episodes of high fever, dental growth was normal. There was no presence of cutaneous marks, abdominal ultrasound was normal. No dysmorphisms were observed. The ecocardiograph showed patent foramen ovale, EEG was normal. 
Patient received Autism Spectrum Disorder diagnosis based on clinical observation and according to DSM-V criteria (3). Diagnosis was confirmed by the Autism Diagnostic Observation Schedule � Second Edition (ADOS-2) (4) administered by experienced clinicians trained for research reliability. Furthermore, he was operated for unilateral inguinal hernia.

Methods and Results

Conventional Cytogenetic Analysis
Peripheral blood lymphocytes of patient were cultured with phytohemagglutinin (PHA) and cells were harvested according standard cytogenetic techniques. Karyotype appeared normal from the analysis in metaphase chromosomes QFQ banded by standard methods (450 band stage ISCN, 2016).

SNP-array Analysis
Genomic DNA was purified from peripheral-blood samples. SNP array analysis was performed using the Human OmniExpress Exome-8 Bead Chip (Illumina Inc., San Diego, CA) containing 960,919 loci derived from phases I, II and III of the International HapMap project. The array includes over 274,000 functional exonic markers, delivering unparalleled coverage of putative functional exonic variant selected from 12,000 individual exome and whole-genome sequences.
A total of 200 ng of gDNA (50 ng/�l) was processed according to Illumina�s Infinium HD Assay Super protocol. Normalization of raw image intensity data, genotype clustering and individual sample genotype calls were performed using Illumina�s Genome Studio software v2011.1 (cnv partition 3.2.0). The CNVs were mapped to the human reference genome hg19 and annotated with UCSC RefGene. Allele detection and genotype calling were performed with Genome Studio software, B allele frequencies (BAFs) and log R ratios were exported as text files for PennCNV analysis.
Analysis conducted with SNP-array (Figure 1) detected a submicroscopic 5q11.1-q11.2 deletion (49,457,282-53,282,649) of approximately 3.8 Mb in size: 
arr[hg19] 5p11q11.2(46,399,093x2,49,457,282-53,282,649x1,53,282,979x2)
Moreover, an extensive homozygosity region of 127 Mb was detected (53,282,979-180,693,127) in chromosome 5q11.2-qter, leading to the suspicion of segmental UPD.
It was only possible to analyze and carry-out a comparison between the patient�s and the mother�s genotype, as the father was not present. This lead us to the hypothesis that the patient possesses a paternal UPD of the 5q11.2-qter segment as the deletion was present on the maternal chromosome. (Table 1 and Table 2).
In order to understand these results we presume that this rearrangement may be explained by a monosomy rescue mechanism of the long arm of a chromosome 5. Specifically, two different mechanisms are possible or delete the long arm of chromosome 5 in the maternal oocyte and subsequent rescue of the missed arm in the zygote using the paternal chromosome 5 as a mold, or instead deletion of the long arm of a maternal chromosome 5 in the early development zygotic stages and subsequently the rescue of the deleted arm using paternal chromosome 5 as a mold. In both hypotheses, a deletion of q11.12 segment also of the maternal chromosome 5 is observed because the chromosome rescue is not completely successful.

Discussion
Deletions of the proximal regions of long arm of chromosome 5 are rare. The regions that are mostly involved in these aberrations are: q11.2, q12.1, q12.3, q13.2 (1, 5-11).
Reports describing patients with del 5q11.2 consist of 10 cases (1,5,6,8,9,10) of which 5 have deletions of extended segments that also involve part of region q12.1 and a further (q12.3 Jalliard 3 case, q 13.2 a Jalliard 4 case), 6 cases involve part of region q11.1(of which Jalliard case 3 extends from q11 to q12.3) Three cases involve the region q12.3 (11, 7,8) (Figure 2).
In 2010 De Jong et al reported a case of del 5q11.2 (3.6Mb) where they tried to define the phenotype (cardiovascular anomalies and developmental delay) of the deletion by comparing it with the first case reported by Prescott (2005). Later Snjiders Blok et al (2014) defined microdeletion syndrome 5q11.2 by analysing four additional cases and defining the short overlap region (SRO) to 2.0 Mb and emphasizing that the phenotype of deletion 5q11.2 resembles that of deletion 22q11.2  and also to that of CHARGE syndrome. A recent report by Fontana et al (2016) reports a case of extended deletion (8.6 Mb) in band 5q11.2 and 5q12.1 associated with a developmental delay and dismorphysms.
The case we report with del 5q11.1-11.2 has cardiophaty, scoliosis and autistic spectrum disorders (Table 3), the deletion of 3.8Mb is among the smollest reported, comparable to that reported in the De Jong�case, but as reported by Fontana, no correlation was found between the size of deletion and the severity of the phenotype, presumably dependent on the genes involved. From the comparison with the other cases reported (Table 3), we see that our case present a joint segment in common with Snjiders Block case1, 2, 3, Prescott case, De Jong case, 3rd case of Jallard wich is much more extensive and the case of Peeters, also very extensive. By comparing the clinical signs of these cases it is noted that none of these presents seizures except that of Prescott having a much larger segment and the fourth case of Sniders Block. All  cases include developmental delay and three autistic behaviour (Snjiders 1 and 3 and the Peeters case) in addition to ours. It is difficult to correlate these features with the deletion.
Moreover, from the point of view of molecular analysis only partially overlapping is evident, we tried, in fact, to define the overlapping region of these case even if overlap is only partially in common with the region highlighted by Sniders Block et as shown in figure 2.
The comparison of all these cases was carried out in order to further define the critical region  of microdeletion 5q11.2 by evaluating the significance of the genes involved and their possible implication on autism.
Especially the cardinal gene DHX29, that encodes an ATP dependent RNA helicase involved in RNA translation and is associated with CHD7 that is related to Charge syndrome, is not involved in the deleted segment of our case.
In fact in common there is only NDUFS4 whose heterozygous mutations can lead to Leigh syndrome, but our case does not show homozygosity, but in the deleted area MIR851 can be found, which is involved in the post transcriptional gene expression regulation. It should also be considered that the array analysis on patient reported in the literature has been carried out with a-CGH technology that does not highlight the presence of ROH or IUPD that may be responsible for the different phenotypes and could alter the phenotype due to deletion alone. 
Certainly it is not possible to define the responsibility of isodisomy in the phenotype of our patient, in fact the presence of heterozygous mutations on the paternal chromosome may result in homozygous in our patient in the analysis of the presence of pathogenetic variations. 
Certainly, the collection of additional cases with SNP-array analysis methodology may contribute to the better definition of these phenotypes and also Exome analysis could lead to the identification of mutated genes on the 5q region responsible for the phenotype.















References
Snijders Blok C, Corsten-Janssen N, FitzPatrick DR, Romano C, Fichera M, et al (2014) Definition of 5q11,2 Microdeletion Syndrome Reveals Overlap with CHARGE Syndrome and 22q11 Deletion Syndrome Phenotypes. Am J Med Genet 164A:2843-2848 
Wang J-C, Ross L, Mahon LW, Owen R,  Hemmat M, et al. (2015) Regions of homozygosity identified by oligonucleotide SNP arrays: evaluating the incidence and clinical utility. European J.  Human Genetics 23: 663-671 
American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders. (2013) (5th ed) Arlington VA American Psychiatric Association
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Prescott K, Woodfine K, Stubbs P, Super M, Kerr B, et al (2005) A novel 5q11.2 deletion detected by microarray comparative genomic hybridisation in a child referred as a case of suspected 22q11 deletion syndrome. Hum Genet 116: 83-90 
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