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Abstract
Objective We describe the clinical and genetic characteristics of fetuses and infants diagnosed with tuberous sclerosis complex (TSC) in our centre, prenatally or neonatally, for a better understanding of the benefits of early screening.
Methods In this retrospective study, we analysed the data on one fetus and nine infants with a definitive TSC diagnosis by genetic criteria (five patients carrying TSC1 variants and 5 patients carrying TSC2 variants). We explored the differences between phenotypes of patients carrying TSC1 and TSC2 pathogenic variants.
Results The most common initial presenting features of TSC were cardiac rhabdomyomas (CRs) that were observed in nine out of ten patients. The most common postnatal features, besides CR, were presented with subependymal nodules—in five patients, and hypomelanotic macules—in four patients. In total, 10 variants causing TSC were detected in this study, including 5 novel variants. We demonstrated that patients with TSC2 variants had earlier onset and more severe clinical manifestations compared with patients carrying TSC1 variants.
Conclusion Early diagnosis of TSC improves genetic counselling and perinatal management.
- genetic diseases, inborn
- infant, newborn, diseases
- heart
- brain
- skin
Data availability statement
Data sharing not applicable as no datasets generated and/or analysed for this study.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Tuberous sclerosis complex is an autosomal dominant disorder with almost complete penetrance. Most cases of tuberous sclerosis complex (TSC) are caused by de novo TSC1 and TSC2 pathogenic variants and can sometimes be diagnosed prenatally.
WHAT THIS STUDY ADDS
We consider fetal heart examination during second and third trimester of pregnancy to be crucial, especially in cases with family history of TSC. Echocardiography is the preferred method for diagnosing fetuses with cardiac rhabdomyoma. Neuroimaging findings in patients with TSC1/TSC2 variants are usually associated with poor outcome. Subsequent prenatal and/or postnatal genetic testing allows for a better understanding of the prognosis.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The combined approach of echocardiography, MRI and genetic testing allows for the early detection of TSC, the improvement of genetic counselling and perinatal patient management.
Introduction
Tuberous sclerosis complex (TSC (MIM: 191100, 613254)), sometimes called Bourneville’s disease, is a rare inherited disease presenting as phakomatosis (neurocutaneous syndrome) and causing multiple skin, brain, kidney and other organs lesions. The case of a newborn with multiple heart and brain tumours was first described by F. D. von Recklinghausen in 1862. The detailed report of clinical and neuropathological features of TSC was made by Bourneville in 1880.1 The TSC incidence is estimated to be 1:6000–1:10 000 live births, while a population prevalence is 8.8:100 000.2–4
The disease is caused by a heterozygous pathogenic variant in either TSC1 (MIM: 605284) or TSC2 (MIM: 191092) genes, encoding hamartin and tuberin proteins, respectively. Both genes are known to act as tumour suppressors, involved in mTOR (mammalian target of rapamycin) pathway. The proteins inhibit mTORC1 (mTOR complex 1), a serine-threonine kinase considered to be the main nutrient/energy/redox sensor and protein synthesis regulator, providing for its role as an important angiogenesis and metastasis governor.3
About 75%–90% of patients with clinically diagnosed TSC carry pathogenic variants in TSC1 or TSC2 genes. The variants occur sporadically in approximately 60% of cases.4
The characteristic features of TSC, similar to other phakomatoses, are presented by the disturbance of cell proliferation and migration leading to the formation of hamartomas—either benign or malignant tumours occurring in skin, brain, heart, kidneys, lungs and eyes.5
The updated TSC diagnostic criteria were published in 2021 (table 1).2 The diagnosis should be based on either clinical or genetic criteria. The ‘definite’ TSC should be based on 2 major features or 1 major feature with 2 minor features while ‘possible’ TSC is based on either 1 major feature or ≥2 minor features. A pathogenic variant detected in either TSC1 or TSC2 gene is sufficient for the diagnosis to be assured.
TSC diagnostic criteria
TSC can be diagnosed prenatally by a routine ultrasound examination in the second and third trimesters (specifically in case of the presence of cardiac nodules and brain lesions6). The variability of clinical features and later onset of certain symptoms may complicate the diagnostics of TSC.7 8
In this study, we included fetuses and infants who underwent whole exome sequencing and were diagnosed with TSC prenatally or within the first months of life to better understand the clinical manifestations and health prognosis.
Materials and methods
Study design and participants
This retrospective study included fetuses and neonates diagnosed with TSC (prenatally or within 1 month after birth) observed in the ‘National Medical Research Center for Obstetrics, Gynecology and Perinatology’ Ministry of Health of Russia from November 2019 to July 2022. Our cohort composed of 10 patients, 9 of them were clinically diagnosed with TSC based on Updated International Tuberous Sclerosis Complex Diagnostic Criteria, while 1 neonate was diagnosed through expanded neonatal screening.2 Pretest counselling was carried out by a trained geneticist, appropriate written informed consent was provided by parents prior to the participation in the study.
Clinical data
The obtained clinical data included family history, clinical presentation, physical and imaging examinations.
Genetic analysis
Genomic DNA from blood leucocytes or amniotic fluid was extracted using PREP-MB MAX DNA Extraction Kit and IGENatal kit, respectively.
TSC1 and TSC2 variants were detected in individuals by whole exome sequencing (WES). Exome capture was accomplished using the IDT xGen Exome Hyb Panel V.2 and sequencing was performed using the Illumina NovaSeq 6000 platform following the manufacturer protocol. The minimum required depth of coverage was ×70. Variant calling was performed using GATK best practices pipeline. The variants were characterised using the pathogenicity prediction algorithms (SIFT, PolyPhen-2, CADD, SpliceAI). The population frequencies of the identified variants were estimated using the Genome Aggregation Database (gnomAD) and the in-house database. The clinical relevance of the identified variants was assessed using the OMIM, ClinVar, LOVD and other specialised databases, as well as available literature. ACMG criteria were applied for variant categorisation.9 The GRCh38 version of the reference genome was used. The de novo status of the variant observed in probands was confirmed by the whole exome trio (using the parental exomes) or Sanger trio sequencing.
Results
Clinical characteristics of the fetus and infants with TSC
Nine infants, six females, three males and one fetus were included. NGS was performed prenatally or on the admission to the neonatal intensive care unit (NICU); in one case the analysis was performed within the framework of expanded neonatal screening. The main indication for testing in clinical cases was heart nodules observed using echocardiography.
Nine out of 10 patients revealed findings consistent with cardiac rhabdomyomas (CRs), 6 of them met diagnostic criteria for TSC with brain involvement prenatally (figure 1). Four patients were suspected for subependymal nodules (SENs) prenatally, which were confirmed postnatally. Fetal ultrasound examinations were unremarkable in patient 10. The most common postnatal features, besides CRs, were SENs in five patients and hypomelanotic macules in four patients.
Prevalence of TSC features in fetus and neonate. SENs, subependymal nodules; EEG, electroencephalography; TSC, tuberous sclerosis complex.
The median turnaround time of molecular genetic diagnosis was 45 days. In prenatal case 9, it was accelerated (21 days) due to ongoing pregnancy.
Genetic findings and phenotypic correlations
All of the nine infants and one fetus were reported to carry TSC pathogenic variants, five had a TSC1 variant and five—a TSC2 variant. Of those nine patients for whom both parents underwent genetic testing, one had an inherited variant from his father, whereas the others had de novo variants. In one case the parents were not available for testing.
It is known that the majority of TSC1 and TSC2 pathogenic variants are truncating and result in a loss of function. Overall, we discovered five novel variants (three in TSC1, two in TSC2); two of the reported variants were missense considered to be likely pathogenic according to the ACMG criteria.
A heterozygous variant c.587C>T (p.Pro196Leu, NM_000368.5) in the TSC1 (patient 3) is absent from the dbSNP database and healthy controls in the population databases. The patient’s father diagnosed with TSC and focal epilepsy was heterozygous for the variant, furthermore it is listed as pathogenic in the LOVD3 database.
A heterozygous variant c.2126T>G (p.Val709Gly, NM_000548.5) in the TSC2 (patient 9) is listed in the dbSNP database (rs1326276839), but has not been previously reported in an affected individual and in population databases (gnomAD). The genotyping of parents revealed that this variant originated de novo. The parents opted for termination of pregnancy and unfortunately declined fetal pathological studies.
Table 2 presents pathogenic TSC variants in our study cohort.
TSC variants in our study cohort
Overall, three cases (30%) that were negative for cerebral lesion examined using neurosonography or MRI, had a TSC1 pathogenic/likely pathogenic variants. Only two of TSC1 patients compared with all TSC2 patients in our cohort were found to have brain pathology. Our results (table 3) are consistent with the previous studies: the TSC2 pathogenic variants are associated with a more severe phenotype compared with the TSC1. 10–12 Furthermore, genotype–phenotype correlations of patients with TSC revealed that patients with mutations in TSC2 gene had a higher frequency of renal angiomyolipomas, renal cysts, retinal hamartomas.13–15 In our study, the prenatal findings of renal angiomyolipomas were associated with TSC2 variant in patient 4, multiple renal cysts were diagnosed postnatally in four patients, three of whom carried a TSC2 variant. Retinal hamartomas were identified in five patients, four of whom had a TSC2 variant (online supplemental table).
Supplemental material
Prenatal and neonatal clinical characteristics of infants (total 10) depending on affected gene
Discussion
The aim of this study was to identify and review the early clinical manifestations of TSC in the fetuses and infants, and to determine whether there was an evidence for the correlation between the genotypes and phenotypes.
With the advances of fetal echocardiography, neuroradiology and molecular genomics technologies, the number of prenatally diagnosed TSC cases based on clinical or genetic criteria is growing rapidly. This generates the need for a geneticist to provide parental prenatal counselling not only if termination of pregnancy is considered but also concerning the nature of the disease and for advancing postnatal care.16
It is considered that the genetic diagnosis can assist in clarifying the neonatal outcome. Furthermore, given the possibility of targeted treatment for many clinical symptoms of the disease, early use of mTOR inhibitors could be an option, starting from the prenatal period.17 18 Since in most cases, we expect a de novo variant, it is worth conducting a trio study, especially in prenatal cases.
When CRs are detected in a fetus, both the perinatal outcome dominated by a tumour position and the long-term prognosis related to TSC are critical. In 9 out of 10 patients, the CRs were present as the initial symptom. Rhabdomyomas are the most frequently occurring cardiac tumours in prenatal and neonatal periods.19 Prenatally, CRs are strongly associated with TSC.20 In view of the natural course of tumour regression throughout the first year of life the management of CRs is observational in all cases that do not present severe haemodynamic complications, fetal hydrops or malignant arrhythmias.15 In our group, the arrhythmia presented prenatally in one case and postnatally—in three cases.
SENs are observed in 80% of TSC patients and are often detected prenatally or at birth.21 Cortical tubers have an incidence of about 33%–62%.22–24 Prenatal diagnosis of cerebral lesions is critical, because the association with CR confirms the diagnosis of TSC and it may be crucial for the determination of the neurodevelopmental outcome.
Six cases of TSC were diagnosed prenatally using the detailed ultrasound examination and cerebral MRI.
In one case, hemimegalencephaly (HME) associated with TSC was diagnosed (patient 7). HME is not included in updated TSC diagnostic criteria. HME related to hyperactivation of mTORC1 pathway, which is responsible for unilateral brain and spinal cord overgrowth.25 HME may arise from somatic mutations in the PIK3CA or AKT3 genes, or may be a part of neurocutaneous syndromes, less often TSC. It is generally associated with intractable epilepsy, developmental delay and hemiparesis. In our patient, the severity of neurological symptoms required a transfer to specialised neurological department.
Finally, one fetus was diagnosed with renal angyomyolipoma during the third trimester (patient 4). Renal angiomyolipomas are observed in about 80% of patients with TSC.26 Although benign, a large angyomyolipoma causes a risk of fatal haemorrhage, spontaneous or with minimal trauma.27
It is important to detect familial forms (one cases in the current series), allowing TSC1 or TSC2 variants to be screened in future pregnancies. Wide phenotypic variation is recognised in TSC, making a postnatal prognosis difficult. The penetrance is high, but not absolute.28
The fact is, that in patients with sporadic TSC, the proportion between TSC1 and TSC2 variants ranges from 1:3 to 1:7, and in familial TSC, it is nearly 1:1.29 It may be due to the fact that patients with TSC2 variants have earlier onset and more severe clinical manifestations, compared with patients with TSC1 variants.30 In our small case series, both cardiac and cerebral lesions were diagnosed prenatally in four patients with TSC2 variants and in two patients with TSC1 variants. However, because of the limited number of individuals (n=10), further investigation is required.
Next-generation sequencing has been rapidly developing and is now being used as a routine molecular diagnostics in clinical genetics. In prenatal settings, whole exome sequencing allows for obtaining a rapid molecular diagnosis of monogenic disease in case of fetal anomalies observed on ultrasound. For example, in a prospective cohort study from the USA, Petrovski et al reported a diagnostic yield of 10% in 234 prenatal trios.31 In selected cohorts (particularly in cases with CRs), previous studies reported a significantly high diagnostic yield—up to 60%.32
In our study, a total of 10 pathogenic/likely pathogenic TSC variants were detected, 5 of them were novel.
As a result, early diagnosis of TSC nowadays is less challenging and provides not only more bases for parental choice regarding a possible termination of pregnancy, but also supports advances in the treatment strategy.
Conclusions
TSC is a dominantly inherited disorder characterised by the development of benign tumours in many tissues and organs. Clinical features are highly variable, and rapid diagnosis of TSC is important for planning the appropriate perinatal care. The early detection of CRs, SENs, cortical tubers and renal lesions are now possible using the fetal ultrasound and MRI, these findings are often revealed as incidental on a routine prenatal examination. Molecular genetic testing using whole exome sequencing can effectively confirm the diagnosis. Recently, our understanding of TSC pathophysiology and new possibilities for the treatment of its complications with targeted therapy have grown; and still more research is required for the evaluation of the outcome of prenatally diagnosed TSC and the benefits of presymptomatic therapy.
We consider the fetal heart examination during second and third trimester of pregnancy to be crucial, especially in cases with a family history of TSC. Echocardiography is the preferred method for diagnosing fetuses with CRs. The neuroimaging findings in patients with TSC1/TSC2 variants are usually associated with poor outcome. The combined approach of MRI, genetic analysis and echocardiography provides for early detection of TSC, improves genetic counselling and perinatal patient management.
Data availability statement
Data sharing not applicable as no datasets generated and/or analysed for this study.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by FSBI National Medical Research Center for Obstetrics Gynecology and Perinatology named after Academician V I Kulakov, Ethics Commission Approval 9 (in Russian). Participants gave informed consent to participate in the study before taking part.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Handling editor Vikram Deshpande.
Contributors ASB, GSV and NVZ consulted the patients and collected the clinical data, ISM, TOK and EEA performed the samples preparation and laboratory research, DNM, JS, ISM, TOK, ERT and IOS performed the bioinformatic analysis, IYB contributed to the genetic analysis, JS, DND and DT contributed to the conception and study design, ASB, DNM and AAB wrote the article and prepared it to publication. JS is responsible for the overall content as the guarantor.
Funding The work was supported by Ministry of Health of the Russian Federation, state assignment # 122030300377-6.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.