TSC1 intragenic deletion transmitted from a mosaic father to two siblings with cardiac rhabdomyomas: Identification of two aberrant transcripts

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Abstract

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by non-cancerous tumors in multiple organs including the brain, kidney, lung, heart, and skin. We encountered a Japanese family consisting of two siblings (a four-year-old boy and a one-year-old girl) with multiple cardiac rhabdomyomas conveying a high risk of TSC and apparently unaffected sibling (a two-year-old girl) and parents. Whole exome sequencing and application of Integrative Genomic Viewer revealed an identical intragenic TSC1 deletion with the breakpoints on intron 15 and exon 19 in the affected siblings, but not in the apparently unaffected sibling and parents. Subsequently, PCR-based analyses were performed using primers flanking the deletion, showing that the deletion was also present in the father and that the deletion occurred between chr9:135,777,038 (bp) and chr9:135,780,540 (bp) in association with a one bp overlap. Furthermore, RT-PCR analyses were carried out using lymphoblastoid cell lines, revealing a major in-frame insertion/deletion transcript produced by aberrant splicing using a cryptic ″ag″ splice acceptor motif at intron 15 (r.1998_2438delinsTTCATTAGGTGG) and a minor frameshift transcript generated by aberrant splicing between exon 15 and exon 20 (r.1998_2502del, p.Lys666Asnfs*15) in the affected siblings. These findings imply that the intragenic deletion producing two aberrant transcripts was generated as a somatic pathogenic variant involving the germline in the father and was transmitted to the affected siblings.

Introduction

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by non-cancerous tumors in multiple organs including the brain, kidney, lung, heart, eyes, liver, and skin (Curatolo et al., 2008). Developmental delay and seizure are also observed frequently. The prevalence is ~ one in 6000 to 10,000 subjects, and ~ two-thirds of patients are identified as sporadic cases born to apparently healthy parents (Curatolo et al., 2008; Peron et al., 2018). TSC is caused by pathogenic variants of either TSC1 encoding hamartin or TSC2 encoding tuberin. Hamartin and tuberin act as tumor suppressors, and the hamartin-tuberin complex regulates cell growth and division by suppressing the function of the mammalian target of rapamycin complex 1 (mTORC1) (Curatolo et al., 2008). Thus, pathogenic variants of TSC1 or TSC2 result in the tumor development because of the activation of the mTORC1 function. Furthermore, since a second event including loss of heterozygosity is frequently found in tumor tissues (Rosset et al., 2017), it is likely that biallelic pathogenic variants of TSC1/TSC2 lead to the development of non-cancerous tumors.

Here, we report two Japanese siblings with cardiac tumors as the sole recognizable abnormality and a TSC1 intragenic deletion producing two aberrant transcripts which was inherited from their clinically unaffected mosaic father.

We encountered a Japanese family with two siblings who received the possible diagnosis of TSC because of the presence of multiple cardiac rhabdomyomas. The male proband (case II-1 in Fig. 1A) was noticed to have cardiac tumors by fetal ultrasonography at 31 weeks of gestation. He was born at 37 weeks of gestation by vaginal delivery without neonatal asphyxia. At birth, his length was 47.5 cm (~50 percentile), and his weight 2947 g (50–75 percentile). Physical examination at birth showed no abnormalities including the skin, although a wood lamp examination was not performed (Yates, 2006). Postnatal echocardiography confirmed multiple cardiac tumors of 6.3–15.8 mm in diameter in right ventricle (RV) and left ventricle (LV). Hemodynamics remained stable without ventricular inflow or outflow obstruction by the tumors. Electrocardiogram (ECG) was normal. Since the tumors were suspected as rhabdomyomas characteristic of TSC (Rosset et al., 2017), we performed brain magnetic resonance imaging (MRI), ophthalmologic examinations, and visceral ultrasound studies, detecting no other features reported in TSC (Krueger and Northrup, 2013). Echocardiography was performed at a ~ two months interval, showing spontaneous regression of the tumors, as has been reported for rhabdomyomas in TSC (Curatolo et al., 2008). On the last examination at four years of age, while he still had small cardiac tumors, he showed apparently normal growth and development with no episode of seizures.

The third female child (case II-3 in Fig. 1A) was also found to have cardiac tumors by echocardiography performed shortly after birth because of the history of cardiac tumors in case II-1. She was born at 32 weeks of gestation, with a birth length of 43.0 cm (50–75 percentile) and a birth weight of 1820 g (50 percentile). Her cardiac tumors were 6.2–15.2 mm in diameter and were identified at multiple locations including the RV free wall to interventricular septum, RV apex, LV free wall to interventricular septum, and the vicinity of anterior papillary muscle of LV (Fig. 1B). There was no ventricular inflow or outflow obstruction. ECG was normal. Physical examination at birth was non-remarkable including the skin, although a wood lamp examination was not performed (Yates, 2006). She was also suspected to have cardiac rhabdomyomas characteristic of TSC, but brain MRI, ophthalmologic examinations, and visceral ultrasound studies showed no other abnormal findings described in TSC (Krueger and Northrup, 2013). The cardiac tumors gradually diminished in size. On the last examination at one year of age, she remained healthy with normal growth and development.

The two-year-old second child (case II-2 in Fig. 1A) had no clinically discernible TSC features including cardiac masses on echocardiograms. The non-consanguineous parents had no history of cardiac, renal, brain, and skin diseases.

After the genetic diagnosis, we planned to perform electroencephalographic studies and the TSC-Associated-Neuropsychiatric-Disorders evaluation in the affected siblings before entering a junior school, as has been recommended (Krueger and Northrup, 2013). Although we suggested to the father to receive detailed examinations for TSC features, he refused such examinations primarily because of lack of clinical symptoms. Thus, it was unknown whether the father had cryptic clinical features of TSC.

Section snippets

Methods

This study was approved by the Institutional Review Board Committee at Hamamatsu University School of Medicine, and was performed after obtaining written informed consent. We performed whole exome sequencing (WES) using leukocyte genomic DNA (gDNA) samples, to examine two possibilities: (1) the siblings had a monoallelic dominant pathogenic variant in TSC1 or TSC2 that was transmitted from either of the parents with germline mosaicism or intra-familial clinical variability, and (2) they had

Results

WES was performed for the affected siblings (II-1 and II-3) and the parents, identifying apparently de novo sequence variants at exon 19 of TSC1 which were initially evaluated as three successive 4–6 bp insertions, i.e., c.2433_2434insTATC, c.2437_2438insTTCATT, and c.2438_2439insGGTGG, in both the affected siblings (reference sequence: TSC1 transcript variant 1, Genbank NM_000368.4) (http://www.ncbi.nlm.nih.gov/). Since such a complicated finding indicated genomic alteration affecting exon 19

Discussion

Molecular studies identified a heterozygous intragenic deletion of TSC1 in two Japanese siblings with cardiac rhabdomyomas. Unpredictably, RT-PCR analyses revealed a major transcript-1 produced with a cryptic splice acceptor motif at intron 15, as well as a minor transcript-2 generated with a wildtype splice acceptor motif at intron 19. Both transcript-1 and transcript-2 were missing 5′-region of the coiled coil domain and tuberin interaction domain (Peron et al., 2018), and transcript-2 was

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Ms. Fumiko Kato, Ms. Aya Kitamoto, Mr. Naoki Adachi, and Mr. Ryo Horiguchi for their technical support.

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