Genetic analysis of a novel TSC1 splice mutation causing tuberous sclerosis without neurological phenotypes

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by the development of benign tumors and lesions in multiple organ systems. The syndrome arises from heterozygous mutations in either TSC1 or TSC2. In this study, we identified a family with a TSC1 c.363 + 668G > C mutation exhibiting diverse clinical phenotypes. The proband and affected family members exhibited multifocal nodular pneumocyte hyperplasia (MMPH), renal hamartomas, bone marrow hyperplasia, and pulmonary lymphangioleiomyomatosis (LAM), with genetic co-segregation analysis confirming the association between the mutation and the clinical phenotype. Genetic co-segregation analysis demonstrated that the TSC1 c.363 + 668G > C mutation was consistently associated with the observed clinical features in this family. Using first-generation Sanger sequencing, we identified a heterozygous splicing variation located in intron 5 of TSC1 (NM_000368.5). In vitro cell and family Minigene results show that TSC1 c.363 + 668G > C mutation can lead to abnormal retention of 92 bp intron sequence in different positions, which may be related to the alternative splicing phenomenon that the same gene produces different splicing variants in different tissues or development stages. The 3D protein structure analysis using Chimera revealed that the mutation site was located at the 363rd base, within the intron between the 121st and 122nd amino acids. This mutation resulted in the insertion of a 92-base sequence, causing a frameshift that led to premature termination of the TSC1 protein after the translation of 26 amino acids. Additionally, the 121st amino acid was altered from lysine to asparagine, significantly shortening the mutated TSC1 protein. These findings provide critical experimental evidence supporting the potential pathogenic mechanism of the TSC1 c.363 + 668G > C mutation. Future research should focus on validating this splicing abnormality in patient-derived cells or tissues and investigating its impact on protein expression and functional activity to better understand its role in disease progression.Clinical trial number: K2024-09-144.

Fig. 1

(a) The family diagram of the TSC1 c.363 + 668G > C mutation reported in this study. Circles represent women, and squares represent men. Patients with the TSC1 gene mutation are marked in green. The proband is indicated by an arrow. Diagonal lines indicate death. In this family, four surviving patients show considerable clinical phenotypic heterogeneity. The proband’s condition is primarily characterized by pulmonary nodules (such as MMPH), while others exhibit lymphatic nodules (such as LAM). (b) The first-generation Sanger sequencing map of the TSC1 c.363 + 668G > C mutation. When the G→C mutation occurs, the originally unimodal signal becomes bimodal. (c) The first-generation Sanger sequencing map of the TSC1 wild type. In the absence of mutation, the signal corresponding to this base is unimodal.

Fig. 2

a. Shark leather spots on the back of the proband’s trunk. b, c. Brain MRI revealed slight hyperintensity on both T1 and T2 sequences in the left frontal lobe, along with a mildly elevated T2-FLAIR signal. These findings were suggestive of a demyelinating lesion or ischemic focus. d-g. Chest CT scan of the proband’s lung window showed multiple ground-glass nodules distributed across both lungs, ranging in size from 0.2 to 0.5 cm. Additionally, a mixed ground-glass density shadow was observed in the upper lobe of the left lung, with the corresponding bronchus appearing truncated. h, i. Chest CT scan of the proband’s father showed normal lung structures with no nodules or air sac lesions identified. j-l. Chest CT scan of the proband’s cousin revealed uniformly distributed air sacs of varying sizes throughout both lungs.

Fig. 3

(a) Nodular hyperplasia of MMPH multifocal alveolar epithelial cells at ×50 magnification. The lung shows scattered, multifocal, well-defined small nodules. (b) MMPH multifocal nodular hyperplasia of alveolar epithelial cells at ×100 magnification. The alveolar cavity narrows or collapses, with proliferation of alveolar epithelial cells. (c) MMPH multifocal nodular hyperplasia of alveolar epithelial cells at ×200 magnification. (d) MMPH multifocal nodular hyperplasia of alveolar epithelial cells at ×400 magnification. Alveolar epithelial cells proliferate, with most cells showing atypical features. The volume of both the cells and their nuclei increases, and pseudoinclusions or nucleoli are visible in some nuclei. (e) AAH at ×200 magnification. Small, localized lesions (usually ≤ 0.5 cm) show proliferated alveolar cells, which are round, cuboidal, or low columnar in shape, with round or oval nuclei. These cells display mild to moderate dysplasia and line the alveolar walls, occasionally extending into the respiratory bronchioles. Often, gaps between cells are present, and the cells do not form continuous clusters. (f) AIS adenocarcinoma in situ at ×200 magnification. It is composed of alveolar type II epithelium or Clara cells, with cancer cells growing strictly along the original alveolar wall. There is no evidence of other growth patterns, such as interstitial infiltration, vascular or pleural infiltration, or airway spread. (g) MIA at ×200 magnification. Part of the alveolar cavity is narrowed and collapsed, with homogeneous, gentle proliferation of alveolar epithelial cells along the alveolar wall. This forms a non-mucinous adenocarcinoma with an infiltration focus measuring less than 5 mm. (h) Bone marrow biopsy lesion map. Bone marrow biopsy confirmed active bone marrow hyperplasia, predominantly erythroid hyperplasia, with young red blood cells being the most common.

Fig. 4

PET-CT images showed multiple osteogenic lesions in cervical and thoracic vertebrae.

Fig. 5

a-d. Based on RT-qPCR analysis using four distinct primer sets targeting the TSC1 gene, the results reveal that patient expression levels are variably reduced compared with those of normal family members. e. T The figure illustrates the positions of the primers used in the RT qPCR experiments. f. After amplification with TSC1 PCR F/R primer in the family, the PCR amplification result is shown in the figure. g. The agarose gel electrophoresis results confirming the family minigene are presented.

Fig. 6

(a) The growth of wild-type and mutant cells for Minigene after transient transfection for 24 h. (b) After collecting the wild-type and mutant cells for precipitation, RNA was extracted and reverse-transcribed. The target gene was then amplified by PCR. (c) The amplified product was recovered and sequenced. After the c.363 + 668G > C mutation occurred, a 92 bp intron sequence was inserted between c.363 and c.364.

Fig. 7

Prediction and analysis of the 3D structure of the TSC1 protein. (a) The protein structure diagram of TSC1. (b) The local amino acid structure diagram of lysine 121 (121 LYS). The mutation occurs at the 363rd base, resulting in the insertion of a 92-base sequence into the intron between the 121 st and 122nd amino acids. (c) The predicted protein structure diagram of the TSC1 c.363 + 668G > C mutation. The red area in the diagram highlights the mutation site and the altered amino acid sequence structure. As a result, translation of the TSC1 protein continues for 26 additional amino acids before terminating prematurely. (d) The local amino acid structure of asparagine 121 (121 ASN) after the mutation. The 121 st amino acid is altered from lysine to asparagine. (e) Comparison of the structures of the TSC1 c.363 + 668G > C mutant protein and the wild-type TSC1 protein. The mutated TSC1 protein is significantly truncated.