Implications of a Chr 7 q 21 . 11 Microdeletion and the Role of the PCLO Gene in Developmental Delay

We report here a 4-year-old boy with global developmental delay who was referred for karyotyping and fragile X studies. A small interstitial deletion on chromosome 7 at band 7q21 was detected in all cells examined. Subsequent molecular karyotype analysis gave the more detailed result of a 6.3 Mb heterozygous deletion involving the interstitial chromosome region 7q21.11. In this relatively gene-poor region, the presynaptic cytomatrix protein, Piccolo (PCLO) gene appears to be the most likely candidate for copy number loss leading to a clinical phenotype. G-banded chromosome analysis of the parents showed this deletion was inherited from the father. Molecular karyotype analysis of the father’s genome confirmed that it was the same deletion as that seen in the son; however, the father did not share the severity of his son’s phenotype. This cytogenetically-visible deletion may represent another example of a chromosomal rearrangement conferring a variable phenotype on different family members.

The proband's 7-year-old sister had no behavioural or developmental problems and was performing at an above-average level at school.The proband's father had had learning difficulties throughout his childhood which were referred to as dyslexia.He had particular difficulties in mathematics and English and required extra help and tuition.He was able to complete his high school studies and went on to obtain a training certificate and a diploma from a technical college.
Peripheral blood was taken for both routine G-banding and molecular karyotype analysis.In respect to the latter, genomic deoxyribonucleic acid (DNA) was isolated from the peripheral blood using the Gentra Puregene ® blood kit (QIAGEN Genomics, Bothell, Washington, USA) according to the manufacturer's instructions.Using the Affymetrix ® Cytogenetics Reagent Kit (Affymetrix, Santa Clara, California, USA) 0.1 micrograms of genomic DNA were labelled.The labelled DNA was applied to an Affymetrix ® Cytogenetics Array (2.7 million probes) according to the manufacturer's instructions, and the array was scanned.The data were analysed using the Affymetrix ® Chromosome Analysis Suite (ChAS), Version 1.0.1 and interpreted with the aid of the University of California Santa Cruz genome browser (hg18 assembly). 3outine G-banded chromosome analysis was undertaken in duplicate for the proband and his parents, which identified a subtle interstitial deletion in the long arm of one chromosome 7, both in the proband and his father [Figure 1] 46,XY,del(7) (q21.1q21.1).Molecular karyotype analysis of the proband confirmed a 6.3 Mb interstitial heterozygous deletion in the chromosome region 7q21.11(hg18 coordinates chr7: 79,573,975-85,833,865) [Figure 2], as well as a 458 kb terminal duplication in the chromosome region Xp22.33 (hg18 coordinates chrX:858,113-1,316,852).This terminal X chromosome region carries the CRFL2 gene (cytokine receptor-like factor 2), which is a receptor for thymic stromal lymphopoietin and so would not appear to play any role in the phenotype of the proband.Molecular karyotyping of the father   4 and cases reported in the DECIPHER database. 13The images were taken from the UCSC genome browser. 3howed the same 6.3 Mb heterozygous deletion in 7q21.11(hg18 coordinates chr7:79,573,967-85,835,041); the small X chromosome duplication event detected in the proband was not found in the father.The 6.3 Mb deletion was not detected in the paternal grandparents of the proband; hence, the deletion arose de novo in the father.Table 1 summarises the genes that are localised to the chromosome 7 region which were deleted in the proband.

Discussion
The deleted region of chromosome 7 encompasses 10 genes [Table 1].Of these, the Online Mendelian Inheritance in Man (OMIM) 4 database shows that 3 are disease-causing: OMIM 173510 (platelet glycoprotein IV deficiency), OMIM 142409 (hepatic growth factor), and OMIM 608166 (semaphorin 3E/ coloboma, heart defect, atresia choanae, retarded growth and development, genital abnormality, and ear abnormality [CHARGE] syndrome).In terms of the latter, disruption of, or mutations in, the SEMA3E gene are rare in patients with CHARGE syndrome, which is associated with mental retardation but usually occurs in combination with eye defects (coloboma) and heart anomalies that were not detected in the case reported here. 5Only one CHARGE patient has been described with a mutation in the SEMA3E gene, and this was a missense mutation resulting in a serine to leucine substitution at amino acid position 243. 5Of the remaining genes located in the deleted region, the presynaptic cytomatrix protein, Piccolo (PCLO), gene appears to be a likely candidate to be associated with developmental delay.
][8][9] Piccolo appears be involved in the cycling of synaptic vesicles at presynaptic nerve terminals of glutamatergic and gamma aminobutyric acid-ergic (GABAergic) central nervous system synapses.
Overexpression of the PCLO gene has been implicated in bipolar/mood disorders in humans. 10nterestingly, knockdown studies in mice show that Piccolo controls the extracellular levels of glutamate in the hippocampus when stimulated, and appears to play a pivotal role in synaptic plasticity in area CA1 and in hippocampus-dependent learning. 11t is tempting to speculate that the semaphorin genes that are localised to the deleted chromosome 7 region may also play a role, together with the PCLO gene, in the proband's phenotype.The semaphorins have roles in axonal guidance and haploinsufficiency for these proteins may affect normal brain development; however, animal modelling data are limited in terms of heterozygous knockdown studies of these genes.At least for semaphorin 3C, targeted disruption of the mouse orthologue leads to cardiovascular defects although only in the homozygous state; heterozygous mice are indistinguishable from their wild-type littermates. 12ignificantly, the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER) database 13 contains two patients' entries, cases 854 and 855 carrying deletions of 35.4 Mb and 17 Mb, respectively, that largely overlap the region identified in the case reported here; these patients exhibited a developmental delay/mental retardation phenotype.In addition, Courtens et al. reported a patient with moderate developmental delay and mild dysmorphic features with maternal monosomy in chromosome 7q21 (between 75.82 Mb and 92.59 Mb). 14 Also of relevance is the almost complete absence of large genomic variation among unaffected individuals within this region of chromosome 7 suggesting that copy-number variations (CNVs) in this region are not benign.
If Piccolo contributed to the phenotype observed in our case study, then a number of mechanisms might explain why the same deletion that was present in the proband's father resulted in a less severe clinical phenotype.Clinical variability may be due to a two hit model, or allelespecific expression. 15,16In respect of the former, an additional CNV, or a mutation below our detection threshold, may underlie differing clinical severity.In respect of the latter, it is unclear if there are parent-of-origin expression differences of the PCLO gene.8][19] This case represents another example of a chromosomal rearrangement conferring a variable phenotype on different family members. 20

Conclusion
This rare case is the first to report a small but detectable interstitial deletion in 7q11.23 that is paternally-inherited and appears to be implicated in developmental delay.Haploinsufficiency of the PCLO gene seems to be the most likely explanation for the clinical phenotype.Further cases of deletions that lie in this region of chromosome 7 should help confirm the role of Piccolo in developmental delay, and the mechanism underpinning clinical variability.

Figure 1
Figure 1 Panels A-C: Partial karyotype and ideograms of chromosomes 7 of proband and father.Panel A: The homologous chromosomes 7 from two metaphase spreads (M1 and M2) of the proband.Panel B: The homologous chromosomes 7 from two metaphase spreads (M1 and M2) of the proband's father.Panel C: An ideogram of the location of the deletion on chromosome 7 (left), and a normal chromosome 7 (right).

Figure 2
Figure 2 Panels A&B: Schematic of the chromosome 7 region that is deleted in the proband.Panel A: Ideogram of chromosome 7, together with the location and extent of the deletion detected in the proband (7q21.11;hg18 coordinates chr7: 79,573,975-85,833,865; boxed in red).Panel B: Shows the Refseq genes that are localised to the deleted region, those genes that are curated in the Online Mendelian Inheritance in Man (OMIM) database4 and cases reported in the DECIPHER database.13The images were taken from the UCSC genome browser.3