Identification of a de novo TSC2 variant in a Han-Chinese family with tuberous sclerosis complex

Abstract

Background:

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with a variety of clinical findings. Variants in the TSC complex subunit 1 gene (TSC1) or the TSC complex subunit 2 gene (TSC2) are responsible for TSC.

Methods:

Physical examinations, computed tomography scans, and light microscopy analyses were performed on the TSC patient from a Han-Chinese pedigree. Whole-exome sequencing combined with Sanger sequencing were performed on the family members.

Results:

The TSC patient showed typical clinical features, including facial angiofibromas, gingival fibromas, a shagreen patch, hypomelanotic macules, ungual fibromas, subependymal nodules, multiple pulmonary cysts, and renal hamartomas. A de novo heterozygous c.5146delG (p.Ala1716Profs*110) variant in the TSC2 gene was identified in the TSC patient of the Han-Chinese family. To our knowledge, this is the first report of the TSC2 c.5146delG variant associated with TSC.

Conclusion:

The study expanding the disease-causing variant spectrum, suggests that whole-exome sequencing combined with Sanger sequencing may be a method for TSC diagnosis and differential diagnosis, and may facilitate the development of genetic counseling and targeted gene therapy for this disease.

1. INTRODUCTION

Tuberous sclerosis complex (TSC) is a dominantly inherited disorder with a variety of clinical findings.^1,2 The frequency of TSC is estimated at 0.10‰–0.17‰ of live births.³ TSC manifests with the formation of hamartomas in the skin, central nervous system, lungs, kidneys, and heart, which results in multiple organ dysfunction as various types of cells replace the normal parenchymal cells.^2,4 The clinical presentation spectrum of TSC varies widely in different individuals, and testing for variants in the TSC causative genes may better define patient cohorts.⁵ The TSC complex subunit 1 gene (TSC1, encoding hamartin) and the TSC complex subunit 2 gene (TSC2, encoding tuberin) are two genes that are responsible for TSC.¹ This study performed a combination of whole-exome sequencing (WES) and Sanger sequencing on a Han-Chinese family with TSC, and a de novo heterozygous TSC2 c.5146delG (p.Ala1716Profs*110) variant was identified as the disease-causing variant.

2. METHODS

2.1. Subjects and clinical evaluation

A Han-Chinese pedigree with TSC was obtained from Hunan Province, China (Fig. 1A). The TSC diagnosis was established according to the diagnostic criteria recommended by the 2012 International Tuberous Sclerosis Complex Consensus Group.² Peripheral blood samples were obtained from the TSC patient (II:2; Fig. 1A) and three unaffected family members (I:1, I:2, and II:1; Fig. 1A). The study was carried out in accordance with the World Medical Association Declaration of Helsinki, and was ratified by the Institutional Review Board of the Third Xiangya Hospital, Central South University. Voluntary signed informed consents were got from all participants. Physical examinations, computed tomography (CT) scans and light microscopy analyses were performed on the family members.

Fig. 1.

Pedigree and genetic analysis of the family with TSC. A, Pedigree of the TSC family. Filled symbol represents the affected member, and empty symbols represent unaffected members. A square represents a male, and a circle represents a female. The patient is indicated by an arrow. B, Sequence with a TSC2 c.5146delG allele and a wild-type allele of the patient (II:2). C, Sequence with wild-type alleles of the patient’s father (I:1). N = normal allele; TSC = tuberous sclerosis complex; TSC2 = the TSC complex subunit 2 gene; V = allele with TSC2 c.5146delG variant.

2.2. DNA extraction and exome capture

Phenol-chloroform procedure was used for genomic DNA extraction.⁶ The Covaris Ultrasonic Sample Processor (Covaris, Woburn, MA) was used to randomly fragment genomic DNA into 180–280 bp.⁷ These size-selected fragments were end-repaired, dA-tailed, and adapter-ligated. After enrichment, the exome capture of the DNA library was performed by xGen Exome Research Panel (Integrated DNA Technologies, Inc., Coralville, IA). To comprehensively search for the disease-causing variants, exclude variants related to diseases with similar features, and make predictions for potential disease risk, WES for the TSC patient and her parents was performed by the Cipher Gene LLC (Beijing, China) using an Illumina NovaSeq6000 platform.

2.3. Variant analysis

After raw data filtration, the generated clean data were mapped to the human reference genome (GRCh38/hg38). Genome Analysis Toolkit was used to sort sequencing alignments and remove duplicate reads, and single nucleotide polymorphisms and insertions and deletions were then detected.⁸ Annotate Variation software was used for high-confident variant annotation. Nonsynonymous variants were filtered by public databases to remove common variants.⁹ After variant evaluation by Online Mendelian Inheritance in Man, Single Nucleotide Polymorphism database, Human Gene Mutation Database, and ClinVar, in silico programs were used for protein function prediction. American College of Medical Genetics and Genomics guidelines for the sequence variant interpretation were used to classify variants as “pathogenic,” “likely pathogenic,” “uncertain significance,” “likely benign,” and “benign” variants. Sanger sequencing using the ABI3500 genetic analyzer (Life Technologies, Foster City, CA) was performed on the TSC patients and other three family members to verify the candidate disease-causing variants.¹⁰ The primers were designed as previously described,¹¹ and the sequences were 5′-AAGTCTCCCCAGACATGGAG-3′ and 5′-TAGATATCGGTGGGGTTGGA-3′. The candidate disease-causing variants were filtered against our Chinese in-house exome databases (including 652 Chinese independent normal controls without TSC).

3. RESULTS

3.1. Clinical features

The TSC patient is a 30-year-old female. Physical examinations revealed specific skin and intraoral lesions, including facial angiofibromas, gingival fibromas, a shagreen patch, hypomelanotic macules, and ungual fibromas (Fig. 2). An axial CT scan of the brain showed subependymal nodules along the wall of the lateral ventricles (Fig. 3A, B). An axial CT scan of the chest showed multiple, bilateral pulmonary cysts which supported a diagnosis of lymphangioleiomyomatosis (Fig. 3C). An axial abdominal CT scan showed renal hamartomas (Fig. 3D). Light microscopy analysis of the facial lesions supported the clinical diagnosis of facial angiofibromas (Fig. 3E).

Fig. 2.

Skin and intraoral lesions of the patient with TSC. A, Multiple facial angiofibromas. B, Gingival fibromas (black arrows). C, A shagreen patch. D, Hypomelanotic macules. E, Ungual fibromas. TSC = tuberous sclerosis complex.

Fig. 3.

CT imaging of the brain, chest, and abdomen of the affected individual with TSC. A and B, Axial cranial CT, demonstrating subependymal nodules. C, Axial chest CT, demonstrating multiple, bilateral pulmonary cysts. D, Axial abdominal CT, demonstrating renal hamartomas. E, Pathological imaging of the affected individual’s facial lesions skin tissue at 10× by HE stain. Fibers in the superficial dermis revealed hyperplasia, and small vessels appeared proliferation. CT = computed tomography; HE = hematoxylin-eosin; TSC = tuberous sclerosis complex.

3.2. Genetic findings

On average, 69.51 million reads were generated by WES and there were 69.49 million reads mapped to the reference genome. The sequencing depth on the target region was 125.05 on average, and the target sequence covered 99.50% of the region at 10× or greater. The coverages of TSC1 and TSC2, as well as the folliculin gene (FLCN, the disease gene of Birt-Hogg-Dubé syndrome), were all 100% in the patient and her parents. Variants with minor allele frequency <5% or which were not recorded in databases including Exome Aggregation Consortium_East Asian (ExAC_EAS), ExAC_All, 1000 Genomes Project, and Genome Aggregation Database were obtained. Variants with minor allele frequency ≥5% in only one database or not in databases were retained to reduce false-negative rate. Variants predicted to affect splicing were also retained. After variant evaluation, MutationTaster (prediction result = disease-causing or disease-causing automatic), Polymorphism Phenotyping v2 (PolyPhen-2, output value ≥0.909), Functional Analysis through Hidden Markov Models (FATHMM, prediction result = damaging) and Likelihood Ratio Test (LRT, prediction result = deleterious) were further used to screening for candidate variants. A total of 1845 variants which passed the selection criteria were filtered from the patient, and 3109 variants were filtered from the patient and her parents. According to the American College of Medical Genetics and Genomics guidelines for the sequence variant interpretation and the clinical phenotype, a heterozygous TSC2 c.5146delG (p.Ala1716Profs*110) variant was identified in the patient as a candidate disease-causing variant. No other potential disease-causing variants associated with TSC were identified. The variant was not detected in her unaffected parents by WES. By Sanger sequencing, the TSC2 c.5146delG variant was further verified in the TSC patient (Fig. 1B), and this variant was absent in all other family members (Fig. 1C), suggesting that the variant was a de novo variant. The TSC2 c.5146delG variant was not found in our Chinese in-house exome databases.

4. DISCUSSION

The TSC1 and TSC2 genes were reported as the causative genes of TSC in the 1990s, however, no disease-causing variants were found in 10%–15% of individuals having a clinical TSC diagnosis.^12–14 The possible explanations may be a few disease-causing variants escaping detection, clinical misdiagnosis, or interference by uncertain factors, such as genetic, epigenetic, or environmental factor modification.

The TSC2 gene, which encodes a protein named tuberin, is located on chromosome 16p13.3.¹⁵ Approximately 60% of all TSC cases are due to the TSC2 disease-causing variants.¹⁶ In this study, a TSC2 c.5146delG variant was identified in a TSC patient. To our knowledge, this is the first report of the TSC2 c.5146delG variant associated with TSC, expanding the disease-causing variant spectrum. The TSC2 c.5146delG variant may lead to the reading frameshift and the C-terminal region defect, which may impact the functional “asparagine-thumb” GTPase activating protein domain and result in function-obliterated proteins, destroying the complex formed by hamartin (encoded by TSC1) and tuberin.¹⁷ This deficiency results in overactivation of the distal mammalian target of rapamycin pathway, and reduces the dephosphorylation of downstream components such as ribosomal protein S6 kinase 1.^1,16 Abnormal S6 kinase 1 protein can reduce or even eliminate the inhibition of cell proliferation, angiogenesis, and metabolism. It may result in uncontrolled cell proliferation and tissue type disorganized arrangement forming angiofibromas, hypomelanotic macules, and subependymal nodules.^16,18

In conclusion, a de novo heterozygous TSC2 c.5146delG (p.Ala1716Profs*110) variant was identified as the disease-causing variant in a Han-Chinese TSC family. WES combined with Sanger sequencing may be a method to confirm TSC diagnosis and exclude TSC with phenotypic related diseases such as Birt-Hogg-Dubé syndrome. This report may have significant implications on genetic counseling and targeted gene therapy for this disease.

ACKNOWLEDGMENTS

This study was supported by the National Key Research and Development Program of China (grant number 2016YFC1306604), National Natural Science Foundation of China (grant numbers 81800219 and 81873686), Natural Science Foundation of Hunan Province (grant numbers 2018JJ2556, 2019JJ50927, 2020JJ3057, and 2020JJ4830), Scientific Research Project of Health and Family Planning Commission of Hunan Province, China (grant numbers B20180729, B20180760, and C20180741), and the Fundamental Research Funds for the Central Universities of Central South University (grant number 2019zzts831).

We thank the patient, her family, and referral physicians for their cooperation and contribution.