A Novel Variant of SMARCB1 in Rare Familial Schwannomatosis Identied by Whole-Exome Sequencing and Genotype-Phenotype Correlation Analysis.

Background: Variants in the tumor suppressor gene SMARCB1 could cause different conditions. In some cases, germline and somatic variants in SMARCB1 are implemented in schwannomatosis. But the genotype and phenotype correlation for variants in SMARCB1 has not been determined. Methods: A Chinese schwannomatosis family with an autosomal dominant inheritance pattern was recruited. Whole-exome sequencing (WES) was performed to discover the causative variant, followed by Sanger sequencing. We evaluated the Human Gene Mutation Database (HGMD) regarding SMARCB1 variants and validated associated phenotype records to assess phenotype-genotype relationships. Results: A novel deletion variant c.885_896delGAAGCTGTGCTC p.(295_299del(cid:0) in SMARCB1 was identied in the affected family members and cosegregated with phenotypes in the pedigree. About 51.1% of variants in SMARCB1 located in Snf5 subunit, 80.7% of variants were loss-of-function (LOF) variants, and more variants located in the Snf5 subunit of SMARCB1 in Rhabdoid tumour (67.8%) than that in schwannomatosis (25.7%). Conclusions: Our study expands the variant spectrum of SMARCB1 and the genetic background of schwannomatosis, conrms the clinical indications for genetic screening of the SMARCB1 gene, and has implications for genetic counseling in this disease.


Background
Schwannomatosis (MIM 162091) is a rare disorder characterized by the predisposition to develop benign multiple non-vesticular schwannomas and, in some cases, meningiomas and so on [1][2][3]. Examination and diagnosis of schwannomatosis has been hampered by the rarity of familial cases and the di culty in separating schwannomatosis from NF2 cases [4]. Schwannomatosis is very rare, having an incidence of 0.58 cases/1 million persons every year [5]. Familial and sporadic schwannomatosis accounts for about 15% and 85% of reported cases, separately [6]. SMARCB1, a tumor suppressor gene, is involved in very low proportion of schwannomatosis patients: about 50% of the familial cases in autosomal dominant fashion with incomplete penetrance, but no more than 10% of the sporadic cases [7].
Schwannomatosis shares clinical symptoms and it can be challenging to distinguish with neuro bromatosis type 2 (NF2), particularly in cases of mosaic conditions [8]. SMARCB1/INI1 is one of the core subunit of the ATP-dependent SWI/SNF chromatin remodeling complex, located at chromosomal position 22q11. 2 [9]. So far, only 135 SMARCB1 variants have been reported in the Human Gene Mutation Database (Professional 2021.2, http://www.hgmd.org). Variants in SMARCB1 can cause different phenotypes, and the exact genotype-phenotype correlation for the variants in SMARCB1 need to be further clari ed [10]. In this study, we revealed a novel variant c.885_896delGAAGCTGTGCTC (p.(296_299del)) (NM_003073.5) in SMARCB1 in a Chinese family with schwannomatosis.

Subjects and clinical evaluation
We observed a schwannomas family with autosomal dominant inheritance in our study. The diagnosis of schwannomas followed the consensus criteria of the National Institutes of Health, and the proband was con rmed with schwannomas by excisional biopsy and magnetic resonance imaging (MRI). Written informed consent was obtained from all participants, and this study was approved by the Medical Ethics

Clinical Manifestation
Three patients of the family were clinically diagnosed with schwannomas (Fig. 1A). The proband (III1), a 27-year-old woman, was born normally. Lumbar pain occurred during rope skipping at the age of 18 years. The symptoms were gradually aggravated after stopping activities, and the night pain was the most serious. The proband developed a mass the size of sour jujube in the right neck half a month before admission at the age of 20 years. Pain and numbness were manifested in the right upper limb after extrusion. The tumor in the superior trunk of the right brachial plexus was diagnosed after operation. The pathological results showed (right supraclavicular fossa) schwannoma. After a year (at the age of 21 years), because of the occupied spinal canal space, the proband was admitted to the hospital. Intraspinal schwannoma was diagnosed during the operation, and the pathological results showed the schwannoma in the thoracic 1, 2 spinal canal. At the age of 24 years, the mass was found on the posterior thigh, the occupied left sciatic nerve was diagnosed during the operation. and the pathological results suggested the schwannoma of the left lower limb. Two years later (at the age of 26 years), pains without obvious cause occurred in the left upper limb. She was admitted to hospital with multiple skin lesions with nodules ( The late proband ( 1), was dictated as schwannoma by clinical symptom descriptions without de nitive diagnosis, and died at the age of 69.

Variant analysis of SMARCB1
Whole-exome sequencing and Sanger sequencing revealed the heterozygous variant of the affected family members c.885_896delGAAGCTGTGCTC (p.(296_299del)) in SMARCB1 (Fig. 1C). The variant in SMARCB1 was absent in the healthy family members, suggesting the variant cosegregated with the phenotype in this family (Fig. 1A). Multiple sequence alignment of 5 different species indicated the conservation around the 296_299 residues within the Snf 5 subunit of the SMARCB1 (Fig. S1). According to ACMG, the variant of SMARCB1 was predicted to be likely pathogenic (PM1+PM2+PP1+PP3). Moreover, the variant was novel because it did not present in dbSNP, HGMD database, nor had been previously reported.

Discussion
In this Chinese pedigree, a novel and rare deletion variant (c.885_896delGAAGCTGTGCTC p. (296_299del)) in exon 7 was identi ed, which results in a truncated protein of 4 amino acid residues instead of full-length INI protein.
SMARCB1 mutated familial schwannomatosis transmitted in autosomal dominant fashion with incomplete penetrance accounts for about 7-8% of reported cases. In our study, the variant (c.885_896delGAAGCTGTGCTC p.(296_299del)) in SMARCB1 has been shown to segregate with disease in the family members in 2 generations. Multiple sequence alignment of 5 different species reveals a high degree of conservation within the Snf5 subunit, implying its functional importance and the potential pathogenicity of the variant. The novel variant is a likely disease-causing loss of function variant according to the ACMG classi cation guidelines.
It is interesting that variants in SMARCB1 can cause two very different phenotypes: schwannomatosis or malignant rhabdoid tumours (MRTs), and it may depends on the the variant types and location within the domain. Nontruncating (missense or splicing) variants are the major inherited variants found in familial schwannomatosis. Moreover, a higher proportion of truncating variants were found in sporadic schwannomatosis patients. The most common alteration identi ed so far is found in exon 1, c.41C>A (p.Pro14His) and within the 3′UTR at c.*82C>T, indicating the variant hotspots at either end of the gene in both familial and sporadic forms of schwannomatosis [11,12]. In contrast, the variants in the central exons or which delete all, or large parts, of the coding sequence often occurred in rhabdoid tumours. However, no exact genotype-phenotype correlation between variants in SMARCB1 has been found so far. Intriguingly, the deletion variant (c.885_896delGAAGCTGTGCTC p.(296_299del)) in our study located in the central exon (exon 7), caused familial schwannomatosis. To assess the phenotype-genotype relationship of SMARCB1, We evaluated the Human Gene Mutation Database (HGMD) regarding SMARCB1 variants and validated associated phenotype records. Overall, we found that about 51.1% (69/135) of variants in SMARCB1 were located in Snf5 domain, 80.7% (109/135) of variants were loss-of-function (LOF) variants (Table 1). 44% (59/134) and 26.2%(35/134) of variants were related to rhabdoid tumour and schwannomatosis separately ( Table 2). We futher compared the variants in rhabdoid tumour and schwannomatosis, LOF variants are the main variant type in both diseases Rhabdoid tumour VS schwannomatosis: 79.7% (47/59) VS 82.9% (29/35)) ( Table 3), while more variants located in the Snf5 subunit of SMARCB1 in rhabdoid tumour (67.8%, 40/59) than that in schwannomatosis (25.7, 9/35) ( Table 2). These results indicated that LOF variants within the Snf5 subunit of SMARCB1 inclined to cause rhabdoid tumour.
Malignant peripheral nerve sheath tumors arising in schwannomatosis patients were reported in some cases [13]. But the prognosis on the schwannomatosis caused by SMARCB1 variant was uncertain. The oldest patient 1 in our study was up to 69 years old, and only showed multiple schwannomas with favorable outcome. Other patients (27 and 48 years-old, separately) in this pedigree has the similar symptoms, and need further follow-up.
Our study highlights the importance of genetic testing and counseling in patients with schwannomatosis, showed the consistent phenotype of the patients with the variant and expands the genetic background of schwannomatosis. Further and more studies of genotype and phenotype relationship are needed in order to better determine the cancer risk for carriers of SMARCB1 germline variants.

Declarations Acknowledgments
We'd like to express our gratitude to all the participants who participated in this investigation. The authors would like to thank the patient's family for participating in this study specially.
Authors' contributions CW contributed to the study conception and design. The manuscript was written by CW and YC. CW and XZ contributed to data retrieval and statistical analysis. Clinical data collection, genetic counseling and follow-up were performed by XK. WES analysis was performed by QL. CW performed Sanger sequencing. DNA was extracted by CC. CW, YC and CC modifed and proofread the paper. All authors read and approved the nal manuscript.

Availability of data and materials
The datasets used and analysed during the current study available from the corresponding author on reasonable request.