SUFU: A potential novel gene responsible for Lhermitte Duclos disease

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SUFU: A potential novel gene responsible for Lhermitte Duclos disease

https://doi.org/10.21203/rs.3.rs-4271310/v1

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Lhermitte-Duclos disease (LDD) is a rare dysplastic gangliocytoma of the cerebellum, typically presenting as a hamartomatous lesion in the posterior fossa. PTEN and the PI3K/AKT/mTOR pathway are involved in the pathogenesis of LDD. We present a case of a patient who incidentally was detected with LDD. A novel, pathogenic, heterozygous, de novo, splice site variant c.183-2A > G (NM_016169.4) in the SUFU gene was identified with targeted next-generation sequencing from genomic DNA. SUFU, a tumor suppressor gene, negatively regulates the hedgehog (Hh) signaling pathway. SUFU also influences WNT and PTEN/AKT/mTOR signaling pathways. While SUFU pathogenic variants are associated with various central nervous system (CNS) tumors, this is the first reported link between SUFU and LDD. The study delves into the role of SUFU in LDD development, establishing the novel SUFU variant as a potential genetic marker for the disease. Sanger sequencing and gel electrophoresis were applied to RNA isolated from blood to show that the variant disrupts splicing. DNA extracted from tumor tissue underwent NGS with the TWIST Exome 2.0 Panel. Results unveiled the de novo pathogenic SUFU (c.183-2A > G) and PTEN (c.389G > A) variants. In conclusion, this study establishes the first reported association between LDD and a germline, de novo SUFU variant, and sheds light on the crucial role of SUFU in LDD pathogenesis. It contributes to the broader understanding of genetic factors influencing this rare cerebellar disorder.

Lhermitte Duclos

SUFU

PTEN

NGS

LDD, or dysplastic gangliocytoma of the cerebellum, is a rare benign mass primarily located in the posterior fossa [1, 2]. The pathological characteristics of LDD involve the displacement of the inner granular layer of the cerebellum by dysplastic ganglion cells of varying sizes, resulting in diffuse expansion [3]. Typically emerging in the third to fourth decades, LDD exhibits a broad age range without gender predominance. Symptoms vary in duration and may include cranial nerve palsies, gait instability, ataxia, and, in advanced stages, manifestations such as headaches, nausea, vomiting, and mental disorders [4–6].

LDD, typically sporadic, can also have a familial form linked to Phosphatase And Tensin Homolog (PTEN) gene variants. PTEN, a phosphatase, controls crucial cellular processes through the PI3K/PTEN/AKT pathway, impacting apoptosis, growth, migration, and differentiation. Inactivation of PTEN increases p-AKT levels and activates the mTOR pathway, promoting cell growth, proliferation, and survival. This pathway has a role in the pathogenesis of most LDD cases. [7]. Germline variants in the PTEN gene are responsible for developing Cowden syndrome, an autosomal-dominant disorder characterized by age-related penetrance. Cowden syndrome has multiple hamartomas, affecting tissues derived from all three germ layers [8].

SUFU, a tumor suppressor gene, negatively regulates the Hh signaling pathway [9]. Inactivation of SUFU unleashes control of GLI1 transcription factors, promoting tumorigenesis in various cancers [10, 11]. Homozygous SUFU variants cause Joubert syndrome, while heterozygous variants pose risks of medulloblastoma in childhood, gonadal tumors in adolescence, and BCC, Gorlin syndrome, and meningiomas in adulthood [12–14]. In recent studies, SUFU is a crucial link between the Hh and WNT pathways, interacting with SPOP and PTEN in clear renal cell carcinoma pathogenesis. Additionally, SUFU aids in the nuclear export of β-CATENIN, contributing to the negative regulation of WNT signaling [15]. Polydactyly/syndactyly and brain overgrowth, common in Hh pathway mutations, suggest a link between the Hh pathway and the PTEN/AKT/mTOR pathway, which is associated with overgrowth. In Steven D Klein's study, loss of SUFU triggers LAMTOR3-mediated AKT and mTOR activation. At the same time, GLI passes into the nucleus and inhibits mTOR negative regulators. These regulations lead to mTOR-mediated activation of p4EBP1 and p70S6K. (Fig. 1) [16].

The aim of this article is to present a rare case with both LDD and a germline, de novo SUFU variant, and to discuss the relationship between them.

2.1 Clinical Report

The case presented here is a 10-year-old female patient who was incidentally found to have a mass in the posterior fossa. This well-circumscribed mass in the cerebellum was detected by cranial magnetic resonance imaging (MRI), done due to the persistence of headaches after head trauma (Figure 2). Although she had no active complaints during the examination, the patient was taken into surgery with the preliminary diagnosis of medulloblastoma. However, The operation material examined by pathology was evaluated as dysplastic gangliocytoma (Figure 3A). The patient was referred to the medical genetics department, and there were no remarkable dysmorphic features on examination. During the 5-year follow-up, mild scoliosis and mild falx cerebri calcification were detected. The patient recently applied for plastic surgery with the complaint of a palpable mass on the skin of the mandible and thoracic spine. The pathology result was compatible with an epidermoid cyst. His family is healthy and has no history of tumors.

2.2 Germline Analysis

Genomic DNA was extracted from the proband's blood using the QIAamp DNA Mini QIA-cube Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Sanger sequencing of the PTEN gene was performed, but no clinically relevant variant was detected. Therefore, a targeted next-generation sequencing panel (TruSight Cancer Sequencing Panel, Illumina, San Diego, CA), which contains 94 high-risk genes associated with both common and rare cancers, was performed according to the manufacturer's instructions. The sequence data were analyzed using VariantStudio variant analysis software (Illumina) and Integrative Genomics Viewer (Broad Institute, Cambridge, MA, USA). Variants were filtered on mapping quality, call quality, minimum depth of coverage of 30 ×, and minor allele frequency < 1%. The detection threshold for constitutional analysis was 20% of reads. The technical sensitivity was greater than 95%.

Variants were assessed with in silico prediction programs (DANN, MutationTaster, BayesDel, GenoCanyon). Splice variants were interpreted using SpliceAI (v1.3) and SPIP (v1.0) software. Variants were annotated with the Genome Aggregation Database (gnomAD), Database of Short Genetic Variation (dbSNP), ClinVar, and Human Gene Mutation Database (HGMD). Variants were classified according to the recommendations of the American College of Medical Genetics (ACMG).

A novel, heterozygous, pathogenic splice site variant c.183-2A>G (NM_016169.4) in SUFU was detected. Segregation analysis was performed with NGS, and the variant was not found in the patient' s parents (Figure ). Sanger DNA sequencing confirmed the heterozygous variant in the patient and showed that his parents and healthy sister did not have this variant.

2.3 mRNA Analysis

Total RNA was extracted from blood using the hybrid-r blood RNA GeneAll® kit and then reverse-transcribed with the iScriptTM cDNA Synthesis kit (BIO-RAD, Waltham, MA, USA), following the manufacturer's instructions.

We analyzed the novel SUFU c.183-2G>A (NM_016169.4) variant by amplifying the complementary DNA of the SUFU mRNA via RealTime-PCR using the TaqTM Universal SYBR® Green Supermix kit (BIO-RAD, Waltham, MA, USA) and a specific primer set within the exons flanking exon 2 (forward in exon 1, GCCTTCGCTTCGCTCTTTC; reverse in exon 4, CTGCATCTGTGGGTCCTCTG). The PCR cycle included an initial denaturation step at 95 °C for 30 s, followed by 40 cycles consisting of denaturation at 95 °C for 15 s and an annealing step at 60 °C for 1 min. The PCR product, loaded onto a 4% agarose gel, revealed a second band of lower height (385 bp) in the patient compared to the control sample (520 bp), corresponding to the excision of exon 2 (long 135 bp) (Figure 3C). Sequencing of the PCR product confirmed the heterozygous excision of the entire exon 2 (Figure 3D).

2.4 Tumour Analysis

According to the manufacturer's recommendation, DNA from the microdissected formalin-fixated paraffin-embedded (FFPE) tumor tissue was extracted with a DNeasy Blood Tissue Kit (Qiagen). The sequencing library was prepared with the TWIST Exome 2.0 Panel (Twist Bioscience), and next-generation sequencing (NGS) was performed on the NextSeq 550 platform (Illumina, San Diego, CA, USA). All procedures were performed according to the manufacturer's instructions. The sequence data were analyzed using A Variant Analysis Platform for Rare Disease, Cancer and Preventive Genomics (genomize-SEQ). Variant calling was performed according to a minor allele frequency threshold of 1% (gnomAD database); the detection threshold was 10% of reads, with coverage of 200 ×. The observed frequency of the variant in the patient's tumor site and histology is determined using databases such as the Catalog of Somatic Mutations in Cancer (COSMIC)18 and The Cancer Genome Atlas (TCGA). Variants were classified according to the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists (CAP) recommendations.

We present a rare case with LDD and germline SUFU variants and discuss their relationship. LDD can occur in isolation or with Cowden Syndrome, caused by loss-of-function (LOF) variants in the PTEN gene. [17, 18]. LDD has not been associated with variants in any other gene before. Therefore, PTEN gene Sanger sequencing was initially performed on this patient's genomic DNA, but no clinically relevant variant was detected. A new germline variant in the SUFU gene was subsequently identified with the targeted next-generation sequencing panel studied. In the segregation analysis, this variant was not detected in her family, so the variant was considered de novo. A novel de novo variant c.183-2A > G (NM_016169.4) affects an acceptor splice site in intron 1 of the SUFU gene. The variant disrupts RNA splicing and results in the absence or degradation of the protein product [19]. In-silico predictions (DANN, MutationTaster, SpliceAl, BayesDel, GenoCanyon) were aggregated deleterious for the replacement. The variant had not previously been reported in the public databases (gnomAD, HGMD, dbSNP). According to the ACMG criteria, the variant is pathogenic (PVS1-PM2-PS2). SUFU is a potential novel gene in LDD.

Variants in PTEN and SUFU were detected in the NGS analysis from FFPE tumor tissue. The PTEN variant c.389G > A (p.R130Q ) is enlisted in the Catalogue of Somatic Mutations in Cancer (COSMIC) database (COSV64288376), but The SUFU variant c.183-2A > G (NM_016169.4) is not included in the COSMIC database. A missense variant, p.Arg130Gln, is in exon 5 of PTEN and induces a conservative amino acid alteration within the Tyrosine-specific protein phosphatase domain (IPR000387) of the encoded protein sequence. This genetic alteration, demonstrated by published functional studies, manifests a detrimental impact, notably marked by a reduction in phosphatase activity and an elevation in pAKT levels [20, 21]. This variant is not found at a significant frequency in large population cohorts (gnomAD). This variant is in silico analysis, which is consistent with the idea that this missense variant exerts a deleterious effect on protein structure and function. Several clinical laboratories at ClinVar and an expert panel at ClinGen have classified it as pathogenic. [21, 22]. This variant is also interpreted as pathogenic according to the COSMIC database and considered oncogenic when mutated on the somatic level in PTEN syndrome-associated tumor forms. These forms include PTEN hamartoma tumor syndrome, breast cancer, endometrial cancer, prostate cancer, and gliomas [23]. The variant is also oncogenic for all solid tumors in the MSK's Precision Oncology Knowledge Base (OncoKB).

LDD is a rare benign tumor affecting the granular cell layer of the cerebellar parenchyma. It is primarily diagnosed in the middle age group. The onset of the symptoms may take up to 10 years. The symptoms of LDD are nonspecific and are caused by intracranial space-occupying formation. Our 10-year-old patient had no symptoms; incidentally, a mass was detected in the cerebellum. The patient was operated on with a preliminary diagnosis of medulloblastoma due to the location of the mass, age of the patient, and radiological images. The patient, who was evaluated as having dysplastic gangliocytoma in her pathology, has been followed for five years without progression.

Homozygous SUFU variants cause Joubert syndrome, while heterozygous variants pose risks of medulloblastoma in childhood, gonadal tumors in adolescence, and BCC, Gorlin syndrome, and meningiomas in adulthood [12–14]. A similar variant at the same position and splice site of the SUFU gene [c.183-2A > C (NM_016169.4)] has been previously reported in a case with Gorlin syndrome and medulloblastoma in ClinVar (RCV001990795.2) [12, 19]. Our patient had no history of medulloblastoma. However, during the 5-year follow-up of the patient, mild scoliosis, mild falx cerebri calcification, and two epidermoid cysts were detected as features of Gorlin syndrome. We informed the patient and referred her to a surveillance program as recommended by clinical guidelines.

Gliomas and meningiomas from CNS tumors have been associated with the deletion of the q arm of chromosome 10. LOF variants in the PTEN located at 10q23 are observed in gliomas, while LOF variants in the SUFU located at 10q24 are detected in meningiomas [8]. Our patient has germline SUFU and somatic PTEN pathogenic variants. Somatic PTEN pathogenic variants were identified in sporadic LDD patients during adulthood. However, these studies did not observe somatic PTEN pathogenic variants in young individuals and children with LDD. This situation suggests a potential age-related pattern. [8].

In the study by Ty W. Abel et al., immunohistochemical results indicate that the PTEN/AKT/mTOR pathway is involved in the pathogenesis of most LDD cases. Most lesions were negative for PTEN, suggesting that the inhibitory effect of this protein on the pathway was lost. Consistent with this interpretation, nearly all tumors showed immunoreactivity to p-AKT in abnormal ganglion cells, while one of the patients studied, a 13-year-old male, was positive for PTEN negative for p-AKT. This result suggests that other pathways play a role, especially in young patients.[7]. In Xiao-Ping Zhou's study, the PTEN/AKT/mTOR pathway was responsible for the pathogenesis in most cases. However, in three cases aged 1, 3, and 11 years, there was no PTEN-related variant, and the tissues were PTEN positive, although p-AKT was also positive [24]. In studies, while the PTEN/AKT/mTOR pathway was active in the pathogenesis of adult LDD patients, p-AKT was positive in child and adolescent cases even when there was no LOF variant in PTEN. This result suggests that dysfunction in genes other than PTEN causes p-AKT positivity in early LDD cases during childhood and adolescence. Additionally, the intricate genomic architecture of these tumors contributes to a significant diversity, thereby predisposing individuals harboring multiple genetic variants to an earlier onset of LDD manifestation.

Although SUFU is primarily a negative regulator of the Hh pathway, it has also been shown to play a role in the PI3K/AKT/mTOR pathway in macrocephaly-associated conditions [16]. It also regulated the ß-catenin pathway via PTEN in the pathogenesis of ccRCC [15]. This result suggests that there is a connection between PTEN and SUFU. In conclusion, we present a 10-year-old case with LDD and the germline pathogenic SUFU variant and It shows that LOF of SUFU is associated with the pathogenesis of LDD.

The emergence of NGS technology and the widespread use of multi-gene testing in clinical practice has accelerated the detection of hereditary cancer syndromes and reduced costs [25]. Detection of variants that cause cancer is critical in the clinical management of the patient, both in determining the cancer risk and, if necessary, in reducing the risk of cancer that may develop in the patient. They may include screening programs or conservative surgeries. In addition, detecting these variants is necessary for family screening, risk assessment, and family planning.

Although germline and pathogenic variants in the SUFU gene are associated with various cancers in the central nervous system, their association with LDD has not yet been demonstrated. The patient is the first case in the literature to show an association between LDD and a germline, novel de novo SUFU variant.

AUTHOR CONTRIBUTIONS

Conception and design: Özge Güngör, Aslı Ece Solmaz

Development of methodology: Özge Güngör, Aslı Ece Solmaz Data collection (including animal provisioning, patient management, facility arrangement, etc.): Özge Güngör, Aslı Ece Solmaz, Haluk Akın, Taner Akalın

Writing, review, and/or revision of the manuscript: Özge Güngör, Aslı Ece Solmaz

Administrative, technical, or material support: Özge Güngör, Aslı Ece Solmaz, Taner Akalın

Study supervision: Aslı Ece Solmaz, Taner Akalın, Emin Karaca, Haluk Akın

ACKNOWLEDGMENTS

The authors express their gratitude to the patient for granting consent for publication.

CONFLICT OF INTEREST STATEMENT

The authors assert that they do not possess any conflicting interests.

ETHICS STATEMENT

The patient has given their written informed consent for genetic research and publication.

FUNDING DECLARATİON

There was no Funding

DATA AVAILABILITY

All data are available upon request.

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No competing interests reported.

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SUFU: A potential novel gene responsible for Lhermitte Duclos disease

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