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ABSTRACT Retinoblastoma (Rb) results from inactivation of both alleles of the _RB1_ gene located in 13q14.2. Whole-germline monoallelic deletions of the _RB1_ gene (6% of _RB1_ mutational

spectrum) sometimes cause a variable degree of psychomotor delay and several dysmorphic abnormalities. Breakpoints in 12 Rb patients with or without psychomotor delay were mapped to

specifically define the role of chromosomal regions adjacent to _RB1_ in psychomotor delay. A high-resolution CGH array focusing on _RB1_ and its flanking region was designed to precisely

map the deletion. Comparative analysis detected a 4-Mb critical interval, including a candidate gene protocadherin 8 (_PCDH8_). _PCDH8_ is thought to function in signalling pathways and cell

adhesion in a central nervous system-specific manner, making loss of _PCDH8_ one of the probable causes of psychomotor delay in _RB1_-deleted patients. Consequently, we propose to

systematically use high-resolution CGH in cases of partial or complete _RB1_ deletion encompassing the telomeric flanking region to characterize the putative loss of _PCDH8_ and to better

define genotype/phenotype correlations, eventually leading to optimized genetic counselling and psychomotor follow-up. SIMILAR CONTENT BEING VIEWED BY OTHERS A CRYPTIC MICRODELETION

DEL(12)(P11.21P11.23) WITHIN AN UNBALANCED TRANSLOCATION T(7;12)(Q21.13;Q23.1) IMPLICATES NEW CANDIDATE LOCI FOR INTELLECTUAL DISABILITY AND KALLMANN SYNDROME Article Open access 10 August

2023 _RBL2_ BI-ALLELIC TRUNCATING VARIANTS CAUSE SEVERE MOTOR AND COGNITIVE IMPAIRMENT WITHOUT EVIDENCE FOR ABNORMALITIES IN DNA METHYLATION OR TELOMERIC FUNCTION Article 13 May 2021 GENOMIC

BALANCING ACT: DECIPHERING DNA REARRANGEMENTS IN THE COMPLEX CHROMOSOMAL ABERRATION INVOLVING 5P15.2, 2Q31.1, AND 18Q21.32 Article Open access 10 September 2024 INTRODUCTION Retinoblastoma

(Rb) is a rare embryonic neoplasm of retinal origin resulting from inactivation of both alleles of the _RB1_ gene (MIM no.180200) located in chromosome band 13q14.2.1 Predisposition to Rb

must be suspected independently of family history and regardless of the clinical presentation, because 100 and 10% of bilateral and unilateral cases, respectively, carry an inherited or _de

novo_ germline mutation. Screening for the predisposing _RB1_ mutation should therefore be proposed to all Rb patients.2, 3 The pattern of mutations found in molecular studies revealed the

existence of 6% of complete deletions of the _RB1_ gene that are associated with variable phenotypes.4, 5 Interstitial 13q deletions involving _RB1_ and its flanking regions, initially

revealed by karyotype analyses in Rb patients, were found to be associated with dysmorphic, cranial and hand/foot abnormalities, psychomotor delay and hypotonia.6, 7 Correlations between the

size of the deletion and the phenotype were therefore investigated. Although a correlation between the size of the deletion and a specific pattern of malformations and dysmorphism was not

established, psychomotor delay was suspected to be restricted to patients harbouring a deletion that encompasses more than the 13q14 band.7 The size and location of the deletion may

therefore define the risk of psychomotor delay in a context of contiguous gene syndrome as previously demonstrated, for example, in neurofibromatosis type 1.8 The correlation between the

size of the deletion and psychomotor delay in Rb has not yet been determined because of the limited resolution of karyotype analysis. High-resolution analysis of deletions, for example, by

CGH, allows this issue to be properly addressed. This work is a nice follow-up of previous studies,9, 10 as it specifically tackles for the first time the issue of psychomotor delay in

_RB1_-deleted patients. We used a dedicated _RB1_-customized CGH-array designed to define a critical interval and consequently identify candidate genes. This study also provides clues

concerning the role of CGH array in Rb molecular diagnosis and parent/patient information regarding genetic counselling. PATIENTS AND METHODS PATIENTS Diagnosis of Rb was established on the

basis of examinations by an ophthalmologist and a paediatrician, and by histopathological criteria when the tumour was available. Rb patients were offered genetic counselling, and individual

written consent was obtained from all sampled individuals or their legal guardians. In this series of 1160 consecutively ascertained cases, 320 mutations were found, and a total of 17

patients were diagnosed with a complete deletion of the _RB1_ gene by QMPSF or karyotype analyses. A sufficient amount of DNA was available for CGH analysis in 12 of these patients.

Psychomotor delay was reported either when a paediatrician, geneticist and/or psychometrician observed a delayed motor development or speech acquisition delay, or when the patient was taken

into care by a specialized educational structure (reported in Table 1 as a binary variable ‘yes’ or ‘no’). CYTOGENETIC ANALYSIS Karyotype analyses with RHG banding and FISH with an _RB1_

probe (Vysis, Downers Grove, IL, USA) were performed according to standard cytogenetic procedures. A customized CGH array centred on the _RB1_ locus was designed on a 1 × 385-K

oligonucleotide CGH microarray (Roche NimbleGen, Madison, WI, USA). The covered region corresponded to the genomic position Chr13:34000000–74000000 (Hg18), for example, a 100-bp resolution.

Data were analysed using VAMP software.11 CHARACTERIZATION AND SEQUENCING OF BREAKPOINTS First, MP/LC12, 13 was used to refine CGH analysis. MP/LC is a technique for the detection of

chromosomal rearrangements, which combines the advantages of semiquantitative multiplex PCR and quality of separation of DHPLC. Long-range PCRs were then performed using TripleMaster PCR

System (Eppendorf, Hamburg, Germany). Amplicons were sequenced using the BigDye Terminator V1.1 Cycle Sequencing Ready Reaction kit (Life Technologies, Carlsbad, CA, USA), followed by

electrophoresis in an ABI 3130xl (Life Technologies). RESULTS The 13q deletion in all 12 patients was characterized by our _RB1_-customized CGH array (Table 1 and Figure 1). The largest

deletion that was not detected by karyotype analysis measured 8.2 Mb and the smallest deletion detected by karyotype analysis measured 11 Mb. The karyotype resolution was therefore about 10 

Mb, which is consistent with routine diagnostic practice. The sequencing experiment (Figure 2) demonstrated good accuracy of the CGH array, as the mean difference of location between the

sequencing and CGH mapping results was equal to 1.08 kb (±1.7 kb, SD). Unfortunately, long-range PCRs and breakpoint sequencing failed in five cases due to low complexity and/or repeated

regions. Nevertheless, CGH resolution was sufficient to allow breakpoint location, for example, for _PCDH9_ (see below). Patients 1 to 6 presented a molecular microdeletion (not detected by

karyotype analysis), and patients 7 to 12 presented a cytogenetic deletion centred around the 13q14.2 band. One of the 6 patients with a molecular microdeletion and 5 of the 6 patients with

a cytogenetic deletion presented psychomotor delay (Table 1). Cytogenetic deletions in a context of Rb were therefore associated with psychomotor delay (_P_=0.03; Fisher’s exact test;

two-sided). To define the minimal deletion associated with psychomotor delay, patient 5 (with psychomotor delay) and patient 8 (without psychomotor delay) were excluded from the following

analysis because they presented documented fetal suffering or mosaicism documented by karyotype, respectively (Table 1; see Discussion). The largest deletion found without psychomotor delay

measured 8.2 Mb (Table 1, patient 6). Sequencing analysis (Figure 2) showed that the breakpoint were located inside the _EPSTI1_ and _FAM124_ genes. This interval (chr13:42449743_50715540)

of deletion was inherited from the patient’s father. In patients 2 and 4, who did not present psychomotor delay, sequencing analysis localized breakpoints inside the _DLEU2_ and _CDACC1_

genes. The deletion identified in patient 7 (chr13:13213373_54501464) was the smallest deletion (11.2 Mb) associated with psychomotor delay. The breakpoint was found inside the _ENOX1_ gene

(Figure 2). Combining these results with those from patient 6, the chr13:50715540_54501464 interval defined an _RB1-_flanking telomeric region where candidate genes for psychomotor delay may

be found (Figure 1). Twenty-five Refseq genes have been reported inside this region, including _PCDH8_ (Supplementary Table 1). DISCUSSION High-resolution CGH reliably defined a deleted

interval not associated with psychomotor delay (chr13:42449743_50715540; Table 1 and Figure 1). This deletion was inherited (Table 1), conferring a high degree of confidence to our results,

by excluding the mosaicism bias. On the other hand, mosaicism led to exclusion of patient 8, as a mosaic status can be associated with an attenuated phenotype and should not be considered in

terms of the genotype/phenotype relationship.14 As Rb is a disease with a high rate of _de novo_ mutations, an attenuated phenotype (ie, an absence of psychomotor delay in a patient

harbouring a cytogenetic deletion) in first-generation mutation carriers is not unexpected.15, 16 Patient 5 was also excluded from the analysis because fetal suffering can be responsible for

psychomotor delay, thereby introducing another analysis bias. The breakpoints of the deletion located in patients with normal development demonstrated that _DLEU2_, _CDACC1_, _EPST1_ and

_FAM124A_ genes, disrupted by the deletion, therefore cannot be associated with psychomotor delay. A breakpoint in the _ENOX1_ gene was also found in a patient with psychomotor delay.

Inhibition of _ENOX1_ has been reported to decrease angiogenesis in tumour growth.17 _ENOX1_ was found with a high but not exclusive expression level in fetal brain (see http://biogps.org),

but no other data are available in the literature to incriminate this gene in the context of psychomotor delay. Comparative analysis of deleted intervals in delayed and non-delayed patients

identified _PCDH8_ as a candidate gene for psychomotor delay (Figure 1). _PCDH8_ (MIM no. 603580), for protocadherin 8, is located in 13q14.3, and belongs to a subclass of cadherins.18

_PCDH8_ has a brain-specific expression making this gene a good candidate gene for psychomotor delay. Furthermore, previous linkage data suggested _PCDH8_ as a candidate gene for

schizophrenia.19 Also of interest is that protocadherin _PCDH19_ has been previously involved in the female-restricted epilepsy and mental retardation syndrome.20 Interestingly, 2 patients

(Figure 1; patient 7 and 11) with psychomotor impairment and loss of a _PCDH8_ copy suffered from epilepsy. Also, our data clearly point to _PCDH8_ as one of the putative genes responsible

for psychomotor delay in the context of Rb, acting either directly or indirectly via regulatory mechanisms. An autosomic dominant model linked to _PCDH8_ loss-of-function could be suspected,

but a recessive model driven by epigenetic inactivation of the second allele of _PCDH8_ cannot be excluded, as the _PCDH8_ promoter has been found to be methylated.21 Two previous studies

also suggested the role of _PCDH8_, but did not formally map this gene, as confounding factors such as mosaicism and alternative causes of psychomotor delay were not evaluated.9, 10

Nevertheless, one study described a few deleted patients without _PCDH8_ involvement, who did present psychomotor delay, leading authors to designate _NUFIP1_ as another possible candidate

gene.10 Overall data show that loss of neither _PCDH8_ nor _NUFIP1_ can explain all delayed cases, but _PCDH8_ loss is always associated with psychomotor delay, as opposed to _NUFIP1_ (see

patient 6, Figure 1). Another interesting finding was that the deletion breakpoints in patient 9, who presented psychomotor delay, were located inside the _PCDH9_ gene (Figure 1). On the

basis of the role of protocadherins in neuronal development and neuronal plasticity,22 a _PCDH_ dose/effect in the expression of psychomotor delay, implying genetic heterogeneity, could be

proposed in the context of complete _RB1_ deletion. In summary, we demonstrated that loss of _PCDH8_ in the context of complete deletion of _RB1_ should alert geneticists to the risk of

psychomotor delay. Fine mapping of deletion breakpoints is therefore mandatory in Rb patients in case of the following: (i) complete or partial _RB1_ deletion encompassing a flanking region

and (ii) mental delay either isolated or associated with dysmorphic features. This could be performed by CGH array of the chromosome 13 or, in the near future, by a global approach such as

massively parallel sequencing. This second line of investigation will precisely define the deleted genes flanking _RB1_, and thereby improve genetic counselling/information and define the

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connecting brain regions. _Neuroscience_ 2010; 170: 189–199. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by grants from the ‘Programme

Incitatif et Coopératif sur le Rétinoblastome’ (Institut Curie) and RETINOSTOP. We thank Laurence Desjardins, Virginie Moncoutier, Carole Tirapo, Catherine Dubois d’Enghien, Anthony Laugé,

Isabelle Eugène, Sandrine Miglierina, Catherine Rougeron and Catherine Gilbon for their helpful support during the study. We also thank clinicians from the GGC (Groupe Génétique et Cancer)

for referring patients. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Département de Biologie des Tumeurs, Institut Curie, Paris, France Laurent Castéra, Catherine Dehainault, Dorothée

Michaux, Jérôme Couturier, Dominique Stoppa-Lyonnet, Marion Gauthier-Villars & Claude Houdayer * Service d’Ophtalmologie, Institut Curie, Paris, France Livia Lumbroso-Le Rouic * Service

de Pédiatrie, Institut Curie, Paris, France Isabelle Aerts & Francois Doz * Université Paris Descartes, Sorbonne Paris Cité, Paris, France Francois Doz, Dominique Stoppa-Lyonnet & 

Claude Houdayer * Hôpital Necker Enfants Malades, INSERM U781, Paris, France Anna Pelet * Institut Curie, INSERM U830, Paris, France Jérôme Couturier, Dominique Stoppa-Lyonnet & Claude

Houdayer Authors * Laurent Castéra View author publications You can also search for this author inPubMed Google Scholar * Catherine Dehainault View author publications You can also search

for this author inPubMed Google Scholar * Dorothée Michaux View author publications You can also search for this author inPubMed Google Scholar * Livia Lumbroso-Le Rouic View author

publications You can also search for this author inPubMed Google Scholar * Isabelle Aerts View author publications You can also search for this author inPubMed Google Scholar * Francois Doz

View author publications You can also search for this author inPubMed Google Scholar * Anna Pelet View author publications You can also search for this author inPubMed Google Scholar *

Jérôme Couturier View author publications You can also search for this author inPubMed Google Scholar * Dominique Stoppa-Lyonnet View author publications You can also search for this author

inPubMed Google Scholar * Marion Gauthier-Villars View author publications You can also search for this author inPubMed Google Scholar * Claude Houdayer View author publications You can also

search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Claude Houdayer. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest.

ADDITIONAL INFORMATION Supplementary Information accompanies the paper on European Journal of Human Genetics website SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE 1 (DOC 50 KB) RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Castéra, L., Dehainault, C., Michaux, D. _et al._ Fine mapping of whole _RB1_ gene deletions in retinoblastoma

patients confirms _PCDH8_ as a candidate gene for psychomotor delay. _Eur J Hum Genet_ 21, 460–464 (2013). https://doi.org/10.1038/ejhg.2012.186 Download citation * Received: 03 April 2012 *

Revised: 20 June 2012 * Accepted: 17 July 2012 * Published: 22 August 2012 * Issue Date: April 2013 * DOI: https://doi.org/10.1038/ejhg.2012.186 SHARE THIS ARTICLE Anyone you share the

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Nature SharedIt content-sharing initiative KEYWORDS * retinoblastoma * _RB1_ * _PCDH8_ * CGH array * psychomotor delay * large-scale rearrangement