Background: Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by the development of hamartomas in multiple organs, including the brain, heart, skin, kidney, lung and retina. A diagnosis of TSC is established with a recently revised clinical/radiological set of criteria and/or a causative mutation in TSC1 or TSC2 gene. Case presentation: We report a Chinese TSC family with two siblings presenting with multiple hypomelanotic macules, cardiac rhabdomyomas and cortical tubers associated with a small subependymal nodule. The older child had seizures. A novel heterozygous missense variant in the TSC2 gene (c.899G > T, p.G300 V) was identified and shown to be inherited from their father as well as paternal grandfather, both of whom presented with variable TSC-associated signs and symptoms. Conclusion: We identified a novel heterozygous TSC2 variant c.899G > T as the causative mutation in a Chinese family with TSC, resulting in wide intrafamilial phenotypic variability. Our study illustrates the importance of clinical evaluation and genetic testing for family members of the patient affected with TSC. Keywords: Tuberous sclerosis complex, TSC2, Expressivity, Rhabdomyoma, Subependymal nodule, Cortical tubers Background been recommended and widely accepted in clinical Tuberous sclerosis complex (TSC, OMIM #191100 practices [1, 3–5]. and #613254) is an autosomal dominant genetic dis- An increasing number of studies have directed at iden- ease with an estimated incidence of 1/6000 to 1/10000 tifiying phenotype-genotype correlations of the affected among live births and a population prevalence of patients [4, 6–14]. However, a great phenotypic variabil- around 1 in 20,000 . TSC is a highly variable dis- ity was observed in TSC. This is also evident in the same order characterized by the development of multisys- family and even in monozygotic twins, with the same tem hamartomatous lesions in the brain, kidney, lung, mutation leading to very different clinical expression skin, heart and retina. One-third of TSC cases are [10, 12, 15–17]. The likely contributory factors for intra- familial and two-thirds are sporadic . Since two familial phenotypic variation include specific mutation , causative genes, TSC1 (NM_000368.4) and TSC2 genetic modifiers [10, 11, 19], apparent non-penetrance (NM_000548.4) were discovered, utilization of genetic , “second-hit” mutation in the unaffected TSC1 or testing for TSC along with refined clinical criteria has TSC2 allele in the somatic cells [21, 22], and gene expres- sion . In some cases, somatic and germline mosaicism might be explanations [8, 24]. * Correspondence: firstname.lastname@example.org Feng Wang and Shiyi Xiong contributed equally to this work. In this study, we report a Chinese family with variably Equal contributors affected members caused by a novel missense variant in Department of Cardiology, Children’s Hospital of Fudan University, 399 TSC2 gene (c.899G > T, p.G300 V). Wanyuan Road, Shanghai 201102, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Medical Genetics (2018) 19:90 Page 2 of 6 Case presentation size of 12*10 mm. Two cardiac rhabdomyomas were The proband girl (IV:2) was vaginally delivered at demonstrated by echocardiography (Fig. 1d)and mul- 42 weeks gestation with a birth weight of 3760 g. She tiple cortical tubers associated with a small subepen- was transferred to Neonatal Intensive Care following the dymalnodulebybrain MRI(Fig. 1e-f). Retinal, teeth, development of paroxysmal supraventricular tachycardia nails examination and renal ultrasound were all nor- 6 h after birth. Echocardiography showed multiple small mal for both of the siblings. During every 1 year cardiac rhabdomyomas in both ventricles. The girl was follow-up by echocardiography and brain MRI for the discharged after her paroxysmal supraventricular tachy- proband and her brother, no siginificant change had cardia was well controlled by Propranolol. been noted. The timeline of clinical management for She was lost to follow-up until two years later, when her the affected siblings was described in Fig. 2a. younger brother (IV:3) was born uneventfully with a birth The parents (III:6 and III:7) were apparently healthy, weight of 3700 g. Two cardiac rhabdomyomas were identi- but on detailed examination the father (III:6) was noted fied following the detection of a heart murmur at 2 months to have one hypomelanotic macule of 3*5 mm and a old. The family was referred for genetic consultation. cerebral white matter radial migration line on brain MRI A detailed clinical review and genetic counseling was (Fig. 1g). Echocardiography and renal ultrasound were completed when the proband and her younger brother was normal. An interesting family history was uncovered on 5 and 3 years old respectively. The sister was intellectually detailed questioning (Fig. 2b). The proband’s paternal normal and seizure-free at this initial review, but later had grandfather (II:4) who declined a medical assessment, re- two seizure episodes at age 7 and was treated with portedly had a history of intellectual disability, probably Oxcarbazepine. Three hypomelonatic macules were found in the mild range. He was never schooled or employed, on her skin with the maximal size of 20*15 mm. On but had basic independent living skills. There was no cardiac ultrasound there were multiple cardiac rhabdo- history of seizures. The elder brother of the grandfather myomas (Fig. 1a), with no compromise of cardiac rhythm (II:3) reportedly had multiple cutaneous fibrous lesions or function. Multiple cortical tubers associated with a small and died in his 40s without any offspring. The sister of subependymal nodule was revealed by brain magnetic the grandfather (II:2) had epilepsy and died of accidental resonance imaging (MRI) (Fig. 1b-c). Her younger brother drowning due to seizure episode in her 40s. None of the was also developmentally appropriate, in a normal kinder- family members in this generation accepted medical as- garten and there was no history of seizures. Two hypome- sessment. Other asymptomatic members of this family lonatic macules were found on his skin with the maximal declined further medical evaluation. Fig. 1 Echocardiogram and magnetic resonance imaging. Echocardiogram indicates multiple cardiac rhabdomyomas (arrows) in the ventricles. (a proband; d younger brother). Brain MRI shows multiple cortical tubers (arrows) and small subependymal nodules (*). (b-c proband; e-f younger brother; b, e T2 weighted imaging; c, f T2-tirm-tra-dark-fluid imaging). Axial T MRI of the brain demonstrates a central white matter radial migration line (arrow) in the father (g) Wang et al. BMC Medical Genetics (2018) 19:90 Page 3 of 6 Fig. 2 Pictures of timeline and pedigree. The timeline picture of clinical management for the affected siblings is listed (a), and the pedigree of the family indicates individual phenotype (b) Sanger sequencing covering all the exons, splicing sites Alignment across different species shows that guanosine is and regulatory regions of TSC1 and TSC2 gene in the highly conserved at the position 899. In silico analysis using proband’s lymphocytes was arranged. The results were multiple softwares and databases including SIFT , Poly- verified by ABI 3500 Dx capillary electrophoresis sequen- phen2 (http://genetics.bwh.harvard.edu/pph2), likelihood cing system. Microdeletions or microduplications within ratio test (LRT), MutationTaster (http://www.muta- the TSC1 and TSC2 genes were excluded by multiplex tiontaster.org/), PROVEAN (http://provean.jcvi.org/ ligation-dependent probe amplification (MLPA). A hetero- index.php)and CADD (http://cadd.gs.washingto- zygous missense variant c.899G > T resulting in an amino n.edu/home), p.G300 V is predicted to be deleterious acid substitution from Glycine to Valine (p.G300 V) for the protein function. A possible new splicing (Fig. 3) was identified and confirmed to also be present in isoform might be induced by activation of an exonic her younger brother and inherited from their father as cryptic donor site according to Human Splicing Finder well as paternal grandfather (who consented to genetic (http://www.umd.be/HSF3/) and MutationTaster. testing despite declining a medical assessment) (Fig. 4). To our knowledge, this variant has never been recorded in the literature of TSC, and the variant c.899G > A, Discussion and conclusions resulting in Glycine to Asparagic acid, presented only in TSC is an autosomal dominant neurocutaneous syn- one among 15,472 alleles from East Asian population drome caused by defect in either one of the two tumor with frequency as low as 0.00006463 in the Genome suppressor genes: TSC1 coding for hamartin and TSC2 Aggregation Database (http://gnomad.broadinstitute.org/). coding for tuberin [26, 27]. Fig. 3 Biochemical structure of tuberin. TSC2 c.899G > T variant resulting a substitution from Glycine to Valine, is located in the coiled-coil harmartin interaction region within N terminus of tuberin (number represents the location of amino acid) Wang et al. BMC Medical Genetics (2018) 19:90 Page 4 of 6 Fig. 4 Sequence chromatograms of the TSC2 gene. A same heterozygous TSC2 variant c.899G > T (arrow) is identified in the proband, her younger brother, father and paternal grandfather TSC2 gene is located at 16p13 and comprises of 41 is located in the coiled-coil region within N terminus of exons. Until now, more than 2000 unique DNA variants tuberin, and substitution on the adjancent amino acid have been recorded in Leiden Open Variation Database V299G has been demonstrated to affect intramolecular (LOVD) including all types such as nonsense, missense, packing [33, 34]. Therefore, the variant of c.899G > T insertions and deletions (http://www.lovd.nl/TSC2). (p.G300 V) is a good candidate for further functional assays Variants have been reported within the GAP domain in the future, and its pathogenicity indicates the importance (1517–1674th amino acid) of tuberin, which catalyzes of variants in this region for the diagnosis of TSC. the dydrolysis of RhebGTP to GhebGDP [28–31], and The identification of a pathogenic mutation in TSC1 the hamartin-binding domain (1–418th amino acid) or TSC2 was added as a major diagnostic criterion in . Function and structure investigations have proved 2012 . This TSC-family illustrates the diagnostic the importance of the N terminal of tuberin interaction value of a complete family history and clinical/radio- with harmatin by adopting a HEAT repeat fold . The logical evaluation, together with segregation testing of a c.899G > T variant in TSC2 gene identified in our study variant identified in an affected individual. In this fam- has not been previously reported as associated to TSC. ily, the father’s very subtle manifestations were only Although the complete clinical details of all the relatives identified after the diagnosis of his children. Ideally, to of the family are not available, the co-segregation of the complete the assessment process, the paternal grand- TSC2 mutation within three-generations, all of whom father ought to have a clinical/radiological evaluation, presented with TSC-associated signs and symptoms, but unfortunately he declined. This TSC2 variant is highly supports the pathogenicity of this variant, as does noteworthy for the mild, subclinical phenotype in the the silico prediction modelling. Regarding the possible father. Ideally, other at-risk asymptotic family members effect on protein structure, the novel TSC2 c.899G > T would be offered the opportunity for testing of the variant resulting in a substitution from Glycine to Valine TSC2 mutation. Wang et al. BMC Medical Genetics (2018) 19:90 Page 5 of 6 Clinical manifestations in TSC present in an Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in age-dependent manner. The presence of multiple cardiac published maps and institutional affiliations. rhabdomyomas is highly specific for TSC and often the first noted manifestation [2, 5]. These lesions are now Author details Department of Cardiology, Children’s Hospital of Fudan University, 399 being detected with increasing frequency in the prenatal Wanyuan Road, Shanghai 201102, People’s Republic of China. Fetal setting. The identification of multiple cardiac rhabdomyo- Medicine Unit & Prenatal Diagnosis Center, Shanghai First Maternity and mas in the index case should have raised suspicion of Infant Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China. Department of Medical Genomics, Royal Prince Alfred TSC, and prompted a full clinical/neuroradiological as- Hospital, Missenden Road, Sydney, NSW, Australia. Department of Radiology, sessment and the offer of genetic testing. Earlier diagnosis Children’s Hospital of Fudan University, Shanghai, People’s Republic of China. would have enabled genetic counselling for the family, the Institute of Pediatrics, Children’s Hospital of Fudan University, Shanghai, People’s Republic of China. diagnosis of the father and the institution of the recom- mended surveillance for all affected family members. In Received: 17 December 2017 Accepted: 18 May 2018 summary, we identified a novel heterozygous TSC2 variant c.899G > T as the causative mututation in a Chinese family with TSC. Our report demonstrates the wide intrafamilal References 1. Northrup H, Krueger DA. 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