Constitutional de novo deletion CNV encompassing REST predisposes to diffuse hyperplastic perilobar nephroblastomatosis (HPLN)

Abstract

Background:
Nephroblastomatosis is a recognised precursor for the development of Wilms tumour (WT), the most common childhood renal tumour. While the majority of WT is sporadic in origin, germline intragenic mutations of predisposition genes such as WT1, REST and TRIM28 have been described in apparently isolated (non-familial) WT.Despite constitutional CNVs being a well-studied cause of developmental disorders, their role in cancer predisposition is less well defined, so that the interpretation of cancer risks associated with specific CNVs can be complex.

Objective:
To highlight the role of a constitutional deletion CNV (delCNV) encompassing the REST tumour suppressor gene in diffuse hyperplastic perilobar nephroblastomatosis (HPLN).

Methods/results:
Array comparative genomic hybridisation in an infant presenting with apparently sporadic diffuse HPLN revealed a de novo germline CNV, arr[GRCh37] 4q12(57,385,330-57,947,405)x1. The REST tumour suppressor gene is located at GRCh37 chr4:57,774,042-57,802,010.

Conclusion:
This delCNV encompassing REST is associated with nephroblastomatosis. Deletion studies should be included in the molecular work-up of inherited predisposition to WT/nephroblastomatosis. Detection of delCNVs involving known cancer predisposition genes can yield insights into the relationship between underlying genomic architecture and associated tumour risk.

Full text

1
Hyder Z, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107087
SHORT REPORT
Constitutional de novo deletion CNV encompassing
REST predisposes to diffuse hyperplastic perilobar
nephroblastomatosis(HPLN)
Zerin Hyder,1 Adele Fairclough,1,2 Mike Groom,3 Joan Getty,3 Elizabeth Alexander,1
Elke M van Veen,1 Guy Makin,4,5 Chitra Sethuraman,6 Vivian Tang,7
D Gareth Evans ,1 Eamonn R Maher ,8,9 Emma R Woodward 1
Cancer genetics
To cite: Hyder Z,
Fairclough A, Groom M, etal.
J Med Genet Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
jmedgenet-2020-107087
For numbered affiliations see
end of article.
Correspondence to
Dr Emma R Woodward,
Manchester Centre for
Genomic Medicine, Manchester
University NHS Foundation Trust,
Manchester M13 9WL, Greater
Manchester, UK;
Emma. Woodward@ mft. nhs. uk
Received 17 April 2020
Revised 4 July 2020
Accepted 6 July 2020
© Author(s) (or their
employer(s)) 2020. No
commercial re- use. See rights
and permissions. Published
by BMJ.
ABSTRACT
Background Nephroblastomatosis is a recognised
precursor for the development of Wilms tumour (WT),
the most common childhood renal tumour. While the
majority of WT is sporadic in origin, germline intragenic
mutations of predisposition genes such as WT1, REST
and TRIM28 have been described in apparently isolated
(non- familial) WT.
Despite constitutional CNVs being a well- studied
cause of developmental disorders, their role in
cancer predisposition is less well defined, so that the
interpretation of cancer risks associated with specific
CNVs can be complex.
Objective To highlight the role of a constitutional
deletion CNV (delCNV) encompassing the REST tumour
suppressor gene in diffuse hyperplastic perilobar
nephroblastomatosis (HPLN).
Methods/results Array comparative genomic
hybridisation in an infant presenting with apparently
sporadic diffuse HPLN revealed a de novo germline
CNV, arr[GRCh37] 4q12(57,385,330–57,947,405)x1.
The REST tumour suppressor gene is located at GRCh37
chr4:57,774,042–57,802,010.
Conclusion This delCNV encompassing REST is
associated with nephroblastomatosis. Deletion studies
should be included in the molecular work- up of inherited
predisposition to WT/nephroblastomatosis. Detection
of delCNVs involving known cancer predisposition
genes can yield insights into the relationship between
underlying genomic architecture and associated tumour
risk.
INTRODUCTION
CNVs are an important source of human genetic
variation but can also be associated with disease.
This is best studied for intellectual disability/
congenital abnormalities where a likely causative
CNV is detected in up to 15% of undiagnosed
cases.1 Although the role of constitutional CNVs
in cancer predisposition is less well understood,
particularly for large delCNVs involving a tumour
suppressor gene (TSG), causative CNVs have been
described in cancer predisposition mostly through
involvement of known cancer predisposition genes
(CPGs), for example, PMS2, NF1 and RB1.2 3
Nephroblastomatosis is defined as the presence
of multiple or diffuse nephrogenic rests persisting
beyond 36 weeks’ gestation. While the outcome of
nephroblastomatosis and nephrogenic rests is vari-
able, ranging from regression to neoplasia, nephro-
genic rests have been identified in 40% of unilateral
Wilms tumour (WT), and the presence of nephro-
blastomatosis increases the risk of developing a
subsequent WT.4
The underlying mechanisms predisposing to
nephroblastomatosis and WT are complex and not
fully understood. Germline variants predisposing to
WT may present as familial or sporadic cases in non-
syndromic and syndromic settings. Thus, a germline
mutation of the WT1 tumour suppressor gene may
present with a syndromic (Denys- Drash syndrome,
Frasier syndrome) or non- syndromic phenotype.
In addition, constitutional deletions of chr11p13
involving WT1 represent a classical contiguous
gene syndrome that predisposes to WT as part of
the WAGR syndrome. A further well- recognised
WT- CNV association is paternally derived duplica-
tions of chr11p15.5 causing Beckwith- Wiedemann
syndrome.5
Other constitutional CNVs involving genes more
typically undergoing somatic alteration in WT have
also been described in WT/nephroblastomatosis.
Constitutional gains of chr2p24.3 involving MYCN,
somatic gains of which are associated with poor
outcomes in WT, have been reported in both bilat-
eral WT and familial nephroblastomatosis/WT, and
a chr3p22.1 gain involving CTNNB1, commonly
somatically mutated in WT, reported in a child with
bilateral WT.6–8
Germline mutations in the RE1- silencing tran-
scription factor (REST) have been described in indi-
viduals with familial and sporadic non- syndromic
WT, but to our knowledge, neither WT nor
nephroblastomatosis- associated CNVs involving
REST have been described.9 Here, we report an
infant presenting with apparently isolated nephro-
blastomatosis and a de novo 0.5 Mb delCNV
encompassing REST.
METHODS
Array comparative genomic hybridisation (aCGH)
High- resolution whole- genome aCGH screen was
performed using DNA extracted from peripheral
blood using the Oxford Gene Technology (OGT)
CytoSure Constitutional v3 Array (8×60 k).
DNA digestion, labelling and hybridisation were
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2Hyder Z, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107087
Cancer genetics
performed according to the manufacturers’ instructions. Array
slides were scanned using the Agilent DNA G2505C Microarray
Scanner and images quantified using OGT CytoSure Feature
Extraction software. Data were then independently analysed
using OGT CytoSure Interpret software by two trained analysts.
CNV detection was based on a minimum four- probe inclusion
using Log2 thresholds of ≥0.35 (gain) and ≤−0.6 (loss). A
manual screen for mosaic aberrations was also performed.
Fluorescence in situ hybridisation
Cultures from parental bloods collected in lithium heparin were
harvested to create fixed cell suspensions and spread onto glass
slides. FISH was undertaken according to standard protocols
using probes (Empire Genomics; https://www. empiregenomics.
com/ fish- probes) RP11- 533F5 (orange) mapping to chr4q12
(within the deleted region) and RP11- 21L14 (green) mapping to
chr4p16.3 (control).
RESULTS
Clinical and pathological features
A female infant, the third child of distantly related parents, was
noted at age 2 months to have a large right- sided abdominal
mass. Pregnancy, birth and family history were unremarkable as
was clinical examination other than the abdominal mass. MRI
with contrast of the abdomen showed a large (10.8 cm×10.5
cm×14 cm) predominantly solid renal mass with cystic compo-
nents, replacing the entire right kidney (figure 1A). In view of
age at presentation and suspected diagnosis of a renal neoplasm,
she underwent immediate right nephrectomy.
Histological review demonstrated a lobular kidney with intact
capsule and multiple tumour nodules, the largest measuring 7.5
cm in diameter, and also a 4 cm haemorrhagic nodule (figure 1B).
Microscopy showed the presence of blastema and tubules,
without anaplasia (figure 1C). Following specialist histological
review, the diagnosis was considered to be diffuse HPLN rather
than WT. Imaging at the time of diagnosis and subsequently
(censored age 17 months) did not detect any evidence of
contralateral renal masses. Three- monthly surveillance imaging
is ongoing. There was no evidence of developmental delay or
clinical features suggestive of syndromic predisposition to WT/
nephroblastomatosis.
Molecular genetic and cytogenetic investigations
In view of the complexity and limitations of constitutional molec-
ular genetic investigations in nephroblastomatosis, aCGH was
undertaken as first- line investigation. This showed an approx-
imate 0.56 Mb interstitial deletion of chr4q, arr[GRCh37]
4q12(57,385,330–57,947,405)x1 (maximum size arr[GRCh37]
57,280,593–58,084,496) (figure 2A). This region contains the
eight protein coding genes: ARL9, HOPX, IGFBP7, NOA1,
POLR2B, REST, SPINK2 and THEGL. Literature review and
consideration of the three OMIM (https:// omim. org/ about)
morbid genes (IGFBP7, REST, SPINK2) revealed that while
homozygous mutations of IGFBP7 are associated with retinal
arterial macroaneurysm with supravalvular pulmonic stenosis10
and homozygous mutation of SPINK2 with failure of spermato-
genesis,11 constitutional intragenic mutation of REST has been
associated with predisposition to non- syndromic WT.9
FISH studies using the BAC probe, RP11- 533F5, mapping to
chr4q12 within the deleted region showed the presence of two
correctly located chr4 signals in each parental sample (minimum
five metaphase cells analysed), indicating likely de novo origin
of the deletion (figure 2B). Paternal aCGH targeted to chr4q12
showed no evidence of the deletion (data not shown). Neither a
maternal sample for targeted aCGH nor a proband sample for
FISH was available for analysis.
DECIPHER cases
We interrogated DECIPHER (DatabasE of genomiC varIa-
tion and Phenotype in Humans using Ensembl Resources)
(https:// decipher. sanger. ac. uk/ accessed 24 March 2020) using
the term ‘REST’ and noted four open access patients to have
delCNVs encompassing REST. The CNVs were larger than
the one presented (range 4.2 Mb to 29.2 Mb) and, for the two
with phenotypic data, neither was known to have nephroblas-
tomatosis/WT at the time of ascertainment (ages unknown)
(figure 2C).
DISCUSSION
This is the first description, to our knowledge, of a CNV
involving REST being associated with either nephroblastoma-
tosis or WT. Intragenic mutation of REST has been shown to
be involved in WT predisposition, although the precise mecha-
nisms by which intragenic mutation promotes tumourigenesis is
unclear, but likely results from disruption of its transcriptional
repressor function as indicated by the calculated lack of toler-
ance of REST to loss of function mutation (pLI=0.97).
On comparison with the open- access delCNV DECIPHER
cases encompassing REST, only this case was known to be asso-
ciated with nephroblastomatosis/WT. While this may reflect
the variable penetrance of REST, this delCNV is much smaller
than those reported in DECIPHER (0.5 Mb deletion vs 4.2 Mb
to 29.2 Mb; figure 2C). In our case, there were no syndromic
features, whereas for the two cases where clinical information
was available, this included intellectual disability, dysmorphic
features and congenital abnormalities but not renal anomalies or
neoplasia (ages unknown at data deposit).
Figure 1 MRI and histology images of renal lesion. (A) Contrast-
enhanced MRI showing right kidney replaced by multiple large solid
lesions of heterogeneous T2 signal. Upper panel, coronal section. Lower
panel, transverse section. Solid arrow, area of haemorrhage within lesion.
Open arrow, areas of hypo- enhancement within lesions. Arrowhead, rim
of enhancing normal renal tissue. (B) Nephrectomy specimen showing
multiple nodules of tumour replacing the entire kidney with one nodule
showing haemorrhage. (C) H&E stain at low power view (×10) showing
presence of blastema and tubules.
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Cancer genetics
Terminal exon truncating mutations of REST have been asso-
ciated with hereditary gingival fibromatosis (HGF) and splicing
defects with deafness12 13; neither clinical feature was known
to be present at censoring. While the reasons for these varying
phenotypes are not clear, they may reflect differing muta-
tional mechanisms, for example, a dominant negative effect
predisposing to HGF, alternative splicing products to deafness
and loss of function to tumourigenesis, in addition to protein
multi- functionality and differing downstream effects on cellular
pathways. This situation is not unprecedented with germline
disruption of CDH1 predisposes to both hereditary diffuse
gastric cancer and oro- facial clefting.14 15
The presence of nephroblastomatosis is a known precursor for
WT development with, in one study, 21/36 cases with HPLN
and not undergoing nephrectomy, subsequently developing WT.4
However, the somatic molecular changes promoting transition
from persistence of nephrogenic rests to nephroblastomatosis to
WT (or regression) are not fully understood, although somatic
alteration of WT1 is a recognised early step, being detected in
both nephrogenic rests and WT from the same individual.16
Given the natural ascertainment bias of individuals presenting
with WT rather than persistence of nephrogenic rests/nephro-
blastomatosis, the molecular basis of WT has been more exten-
sively studied. However, it is likely that, as with other hereditary
cancer predisposition syndromes, where there is underlying
germline disruption of a gene known to predispose to WT, this
predisposes to an early rate- limiting step in the tumourigenic
pathway, akin to germline mutation of APC predisposing to
adenomatous polyps, precursors of colorectal cancer.17
While germline mutations of REST have been associated with
WT, we are not aware that germline disruption of REST has been
investigated previously in cases of persistent nephrogenic rests/
nephroblastomatosis and our report would suggest that disrup-
tion of REST function is a critical step in tumourigenesis. Of
note, germline gain CNVs encompassing DDX1- MYCN have
been detected in a family with nephroblastomatosis and WT, and
separately in bilateral WT.6 7 When somatically acquired, MYCN
amplifications are associated with poor prognosis7; together,
these findings would suggest MYCN disruption also being critical
in WT development.
Although the majority of cases of WT are sporadic, where
syndromic features, bilateral, multifocal or very early onset
disease is present, consideration should be given to a possible
underlying genetic predisposition including clinical examina-
tion to identify subtle syndromic features. Our findings suggest
that where there is diffuse nephroblastomatosis, in the absence
of overt WT, molecular work- up ought be the same as for
WT. Molecular investigations of non- syndromic WT cases are
complex and, until recently, not always routinely available. The
new UK guidance for investigation of WT predisposition recom-
mends deletion/intragenic sequence analysis of WT1 and dele-
tion/methylation analyses of the chr11p15.5 imprinting region
(https://www. england. nhs. uk/ wp- content/ uploads/ 2018/ 08/ rare-
and- inherited- disease- eligibility- criteria- march- 19. pdf, accessed
29 June 2020). Considering this report and the published liter-
ature, we recommend that investigation (including deletion
analyses) of the more recently described WT- associated genes,
for example, REST, CTR9 and TRIM28, be included.9 18 These
Figure 2 Molecular, cytogenetic and database studies of constitutional CNV involving REST. (A) aCGH showing arr[GRCh37] 4q12(57,385,330–
57,947,405)x1. (B) FISH studies indicating no deletion, interchromosomal or large intrachromosomal rearrangement, in either parent, of the probe RP11-
533F5 (orange) which maps to 4q12 within the deleted region seen on microarray analysis in the proband. Image also shows the control probe RP11- 21L14
(green) mapping to 4p16.3 for positional information. Upper panel, paternal sample. Lower panel, maternal sample. (C) Schematic diagram of the four
delCNVs on DECIPHER encompassing REST, labelled by DECIPHER number, and the case presented relative to the midpoint of REST. Each bar represents
the CNV identified in the patient shown. The central axis represents the midpoint of REST, and the distance in base pairs from this midpoint is shown on the
horizontal axis.
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4Hyder Z, etal. J Med Genet 2020;0:1–5. doi:10.1136/jmedgenet-2020-107087
Cancer genetics
investigations should also be offered to very young onset appar-
ently isolated unilateral cases; recent publication has recom-
mended constitutional evaluation of such cases up to age 2
years.19 Where an underlying causative predisposition is present,
3- monthly screening ultrasound imaging to age 7 years has been
recommended for at- risk family members.20
Guidance for molecular investigation and subsequent manage-
ment of rare disease is challenging; therefore, we recommend
data pertaining to diagnostic genetic testing and screening should
be evaluated through prospective international patient cohorts
to assess yield and cost- effectiveness with a view to possible gene
specific guidance.
In addition to CNVs detected in children and adults presenting
with a neoplasm, CNVs involving WT predisposition genes and
other CPGs may be detected as part of investigation of devel-
opmental delay where there is no personal or family history of
neoplasia. In particular, delCNVs involving tumour suppressor
CPGs are found in 0.3%–0.4% of aCGH investigations under-
taken for investigation of developmental delay/congenital abnor-
malities.2 However, accurate prediction of tumour risks in such
cases is difficult because the relevant CNVs are usually heteroge-
neous, affecting a wide variety of CPGs, and for any specific CPG
the size and location of the delCNV also varies. Although whole
gene tumour suppressor CPG deletions are generally associated
with a similar phenotype to loss- of- function intragenic muta-
tions, the consequences can be difficult to interpret where a large
delCNV is detected because of the potential modifying influence
of genes within the deletion.2 3 21 22 Tumour risks may also be
modified by non- coding region effects on long- range regulatory
elements and disruption of topology- associated domains. For
those TSGs that follow a two- hit model of tumourigenesis in
which the somatic ‘second hit’ is frequently total or segmental
loss of a chromosome, this could result in homozygous loss
of many genes in the cancer cell and produce a non- viable or
synthetic lethal state. Such effects might be predicted to be
more likely for large delCNVs but will be specific for individual
TSGs, for example, for RB1 a ~50 Mb deletion was associated
with bilateral retinoblastoma in addition to multiple congenital
abnormalities, whereas for the VHL TSG deletion that includes
the BRK1 gene, the risk of renal cell carcinoma is drastically
reduced.2 21 In the present study, we note that neoplasia has
only been associated with the smallest REST- associated deletion
(limited clinical information available for the larger deletions).
We strongly suggest that, whenever possible, clinical and aCGH
data should be deposited in public repositories (eg, DECIPHER)
so that CNV genotype–phenotype relationships can be char-
acterised and clinical management of individuals with CNVs
affecting CPGs is improved.
Large CNVs and whole exon deletions of WT/nephroblas-
tomatosis predisposition genes may not always be detected by
targeted resequencing techniques. Specific molecular studies to
detect such pathogenic variants should be included when inves-
tigation of the inherited predisposition to WT/nephroblastoma-
tosis is undertaken.
With on- going advances in bioinformatics and genomic
sequencing technologies (eg, long- range sequencing), structural
variant and CNV data will be increasingly generated, necessi-
tating the role of constitutional structural variants in cancer
predisposition to become an increasingly important focus for
clinical genetics practice.
Author affiliations
1Manchester Centre for Genomic Medicine, Manchester University NHS Foundation
Trust, Manchester, UK
2NW Genomic Laboratory Hub, Manchester University NHS Foundation Trust,
Manchester, UK
3NW Genomic Laboratory Hub, Liverpool Women’s Hospital, Liverpool, UK
4Department of Paediatric Oncology, Royal Manchester Children’s Hospital,
Manchester, UK
5Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine
and Health, The University of Manchester, Manchester, UK
6Department of Paediatric Histopathology, Manchester University NHS Foundation
Trust, Manchester, UK
7Department of Radiology, Royal Manchester Children’s Hospital, Manchester
University NHS Foundation Trust, Manchester, UK
8Department of Medical Genetics, University of Cambridge, Cambridge, UK
9Department of Clinical Genetics, Cambridge University Hospitals NHS Foundation
Trust, Cambridge, UK
Twitter Emma R Woodward @ER_Woodward
Acknowledgements We thank Kidscan (Charity #109406) for financial support.
This study makes use of data generated by the DECIPHER community. A full list of
centres who contributed to the generation of the data is available online (http://
decipher. sanger. ac. uk) and via email ( decipher@ sanger. ac. uk).
Contributors ERW and ERM planned the study. AF, MG, JG, EA, EMvV, GM, CS, VT
and ERW contributed to the acquisition, analysis and interpretation of the data. The
manuscript was critically appraised and approved by all authors.
Funding ERW and DGE are supported by the Manchester NIHR Biomedical
Research Centre (IS- BRC-1215-20007). ERM is funded by a European Research
Council (Advanced Researcher Award), NIHR (Senior Investigator Award and
Cambridge NIHR Biomedical Research Centre) and the Cancer Research UK
Cambridge Cancer Centre. The University of Cambridge has received salary support
in respect of ERM from the NHS in the East of England through the Clinical Academic
Reserve. Funding for the DECIPHER project was provided by the Wellcome Trust.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
ORCID iDs
D GarethEvans http:// orcid. org/ 0000- 0002- 8482- 5784
Eamonn RMaher http:// orcid. org/ 0000- 0002- 6226- 6918
Emma RWoodward http:// orcid. org/ 0000- 0002- 6297- 2855
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