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Diagnosis of mosaic tuberous sclerosis complex utilizing next generation sequencing of subtle or unusual cutaneous findings

Open AccessPublished:January 09, 2023DOI:https://doi.org/10.1016/j.xjidi.2023.100180

      ABSTRACT:

      Skin findings can be critical to determine whether a patient with lymphangioleiomyomatosis (LAM), a progressive pulmonary disease that predominantly affects adult women, has sporadic disease (S-LAM) or LAM in association with tuberous sclerosis complex (TSC-LAM). Three individuals with LAM underwent evaluation for TSC-associated mucocutaneous and internal findings. We used our previously published algorithm to confirm clinical suspicion for mosaicism and guide selection of tissue specimens and genetic workup. Next-generation sequencing (NGS) of cutaneous findings was used to confirm clinical suspicion for mosaic TSC in individuals with LAM. Two individuals previously thought to have S-LAM were diagnosed with mosaic TSC-LAM upon NGS of unilateral angiofibromas in one and an unusual cutaneous hamartoma in the other. A third individual, diagnosed with TSC in childhood, was found to have a mosaic pathogenic variant in TSC2 in cutaneous tissue from a digit with macrodactyly. Accurate diagnosis of mosaic TSC-LAM may require enhanced genetic testing, and is important due to implications regarding surveillance, prognosis, and risk of transmission to offspring.

      Abbreviations:

      TSC (Tuberous sclerosis complex), AML (angiomyolipomas), LAM (lymphangioleiomyomatosis), S-LAM (sporadic lymphangioleiomyomatosis), NVI (no variant identified), NGS (next generation sequencing), VAF (variant allele fraction)

      INTRODUCTION:

      Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that usually presents in utero or early in childhood with cardiac rhabdomyomas, seizures, and hypomelanotic macules.(
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      ) However, about 12% of individuals with TSC are diagnosed in adulthood.(
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      Tuberous sclerosis complex: diagnostic challenges, presenting symptoms, and commonly missed signs.
      ) Those diagnosed in adulthood may present with renal angiomyolipomas (AMLs) or a progressively destructive lung disease called lymphangioleiomyomatosis (LAM).(
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      Recognition of tuberous sclerosis in adult women: delayed presentation with life-threatening consequences.
      ) LAM may occur in association with TSC (TSC-LAM) or sporadically (S-LAM). Distinguishing TSC-LAM and S-LAM is important because of divergent implications with regards to surveillance, prognosis, and risk of transmission to offspring. In many instances, the diagnosis of TSC-LAM is readily apparent, due to the widespread systemic manifestations of TSC. However, in some individuals with undiagnosed TSC, classic manifestations of TSC may be subtle and genetic testing may yield no findings.
      Dermatologists have an important role in the diagnosis of TSC for their ability to identify characteristic skin findings, and increasingly they may also be called upon to provide skin samples for genetic analysis. TSC is caused by a pathogenic variant in either TSC1 (15-23% of individuals) or TSC2 (77-85%).(
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      Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing.
      ) Approximately one-third of cases are inherited from an affected parent, resulting in germline TSC, whereas two-thirds of cases occur sporadically.(
      • Sampson J.R.
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      Genetic aspects of tuberous sclerosis in the west of Scotland.
      ) Individuals with confirmed or suspected TSC often undergo genetic testing of the blood to confirm the diagnosis and provide information about prognosis. However, about 15% of individuals who meet the diagnostic criteria for TSC have no variant identified (NVI) in the blood with conventional genetic testing. Recent studies have found that many of these individuals with NVI have mosaicism; only a proportion of their cells carry the pathogenic variant due to a post-zygotic mutation during development.(
      • Tyburczy M.E.
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      • Chekaluk Y.
      • Thorner A.R.
      • et al.
      Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing.
      ) Detection of mosaic pathogenic variants in these individuals typically requires enhanced genetic testing with next generation sequencing (NGS). Additionally, analysis of TSC skin tumors rather than blood can improve detection as the variant allele fraction (VAF) is typically higher in tumor samples than blood in the mosaic condition and is particularly important in those with unilateral or asymmetrically distributed angiofibromas.(
      • Giannikou K.
      • Lasseter K.D.
      • Grevelink J.M.
      • Tyburczy M.E.
      • Dies K.A.
      • Zhu Z.
      • et al.
      Low-level mosaicism in tuberous sclerosis complex: prevalence, clinical features, and risk of disease transmission.
      ,
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      • Hamieh L.
      • Nathan N.R.
      • Tyburczy M.E.
      • Wang J.A.
      • Oyerinde O.
      • et al.
      Phenotypic distinctions between mosaic forms of tuberous sclerosis complex.
      ,
      • Tyburczy M.E.
      • Dies K.A.
      • Glass J.
      • Camposano S.
      • Chekaluk Y.
      • Thorner A.R.
      • et al.
      Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing.
      )
      In contrast to TSC-LAM, individuals with S-LAM do not have systemic mosaicism, and their genetic variant causing LAM, typically in TSC2, will be restricted to their LAM cells, and is not present in blood cell DNA, even with NGS. Despite this, S-LAM has a similar genetic mechanism for tumor formation, since two-hit pathogenic variants in TSC2, less commonly TSC1, are found in the majority of S-LAM associated AMLs and LAM cells.(
      • Murphy S.J.
      • Terra S.B.
      • Harris F.R.
      • Nasir A.
      • Voss J.S.
      • Smadbeck J.B.
      • et al.
      Genomic rearrangements in sporadic lymphangioleiomyomatosis: an evolving genetic story.
      ,
      • Smolarek T.A.
      • Wessner L.L.
      • McCormack F.X.
      • Mylet J.C.
      • Menon A.G.
      • Henske E.P.
      Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.
      ) Rarely, extreme low-level mosaicism for TSC2 variants in individuals thought to have S-LAM have been detected in peripheral blood(
      • Han M.K.
      • Tyburczy M.E.
      • Darling T.N.
      • Kazerooni E.A.
      • Myers J.L.
      • McCormack F.X.
      • et al.
      Apparent Sporadic Lymphangioleiomyomatosis in a Man as a Result of Extreme Mosaicism for a TSC2 Mutation.
      ) and cell-free DNA(
      • Ogorek B.
      • Hamieh L.
      • Lasseter K.
      • Bagwe S.
      • Machado T.
      • Herranz-Ors C.
      • et al.
      Generalised mosaicism for TSC2 mutation in isolated lymphangioleiomyomatosis.
      ), and subsequent studies confirmed that these individuals had mosaic TSC with minimal features. In these rare instances, LAM cells have arisen from a first TSC2 mutation (or rarely TSC1) occurring during embryogenesis, and a second post-embryonic somatic mutations in TSC2 occurring in a LAM progenitor cell. Thus, the clinical phenotype of LAM exists on a spectrum that ranges from: a) S-LAM with involvement limited to LAM cells in the lung and other sites including kidney, lymphatic system, and uterus; to b) mosaic TSC in which TSC2/TSC1 mutations are present in multiple tissues including blood, skin and/or brain; to c) germline TSC, in which TSC2/TSC1 mutations are present in all cells, and which usually presents in childhood with diffuse multi-organ involvement. Differentiating between S-LAM and LAM associated with mosaic TSC may be challenging when TSC-associated mucocutaneous findings are subtle, cutaneous findings are not classic, neurological findings are absent, or findings present later in adulthood.
      In this case series, three women with LAM were found to have subtle and/or unique mucocutaneous findings potentially consistent with mosaic TSC upon careful examination. They were subsequently proven to have mosaic TSC through biopsy and NGS of suspected cutaneous lesions. Two of the three were previously thought to have S-LAM, while the third was previously diagnosed with TSC but had NVI upon routine genetic testing of the blood.

      MATERIALS AND METHODS:

      Three women previously diagnosed with LAM underwent examination at the National Institutes of Health Clinical Center. They provided written and informed consent under protocol 00-H-0051 and 96-H-0100 which was reviewed and approved by the National Heart, Lung, and Blood Institute Institutional Review Board (ClinicalTrials.gov identifier NCT00001975, NCT00001532). These individuals underwent dermatological evaluation for the presence of mucocutaneous findings of TSC. Internal manifestations were assessed by clinical records and imaging. The presence of sclerotic bone lesions, which occur in greater frequency in TSC-LAM than S-LAM, was also assessed on imaging.(
      • Avila N.A.
      • Dwyer A.J.
      • Rabel A.
      • Darling T.
      • Hong C.H.
      • Moss J.
      CT of sclerotic bone lesions: imaging features differentiating tuberous sclerosis complex with lymphangioleiomyomatosis from sporadic lymphangioleiomymatosis.
      ) An algorithm based on clinical phenotype was used to predict germline TSC, possible mosaic TSC, or low-level mosaic TSC in order to guide tissue selection and genetic workup.(
      • Treichel A.M.
      • Kwiatkowski D.J.
      • Moss J.
      • Darling T.N.
      A diagnostic algorithm for enhanced detection of mosaic tuberous sclerosis complex in adults.
      ) Cutaneous biopsies were routinely processed, sectioned, stained with hematoxylin and eosin, and reviewed by a dermatopathologist (C.R.L).(
      • Giannikou K.
      • Lasseter K.D.
      • Grevelink J.M.
      • Tyburczy M.E.
      • Dies K.A.
      • Zhu Z.
      • et al.
      Low-level mosaicism in tuberous sclerosis complex: prevalence, clinical features, and risk of disease transmission.
      ,
      • Treichel A.M.
      • Hamieh L.
      • Nathan N.R.
      • Tyburczy M.E.
      • Wang J.A.
      • Oyerinde O.
      • et al.
      Phenotypic distinctions between mosaic forms of tuberous sclerosis complex.
      )
      Fresh or frozen tissue samples from skin biopsies, blood, urine pellets and saliva were collected for DNA isolation using QIAamp DNA Kit (Qiagen) according to manufacturer’s protocol. DNA samples were run on agarose gels for quality control, and submitted for NGS using an oligonucleotide capture panel for the entire genomic extent of TSC1, TSC2, and exonic regions of 50 mTOR related genes. A custom pipeline was used to identify both low frequency and heterozygous variants in TSC1 and TSC2.(
      • Ogorek B.
      • Hamieh L.
      • Hulshof H.M.
      • Lasseter K.
      • Klonowska K.
      • Kuijf H.
      • et al.
      TSC2 pathogenic variants are predictive of severe clinical manifestations in TSC infants: results of the EPISTOP study.
      ,
      • Ogorek B.
      • Hamieh L.
      • Lasseter K.
      • Bagwe S.
      • Machado T.
      • Herranz-Ors C.
      • et al.
      Generalised mosaicism for TSC2 mutation in isolated lymphangioleiomyomatosis.
      ) Candidate pathogenic single nucleotide variants had to have a minimum VAF of 0.5%, and be seen in at least three reads including one in each orientation. Candidate pathogenic deletions and insertions (indels) had to have a VAF >0.2%, and be seen in at least two reads from different DNA molecules. Large genomic deletions in TSC1 and TSC2 were identified by calculating the read depth in 100 nucleotide windows, with comparison to other samples from the sequencing batch. Large genomic deletions were also sought by analysis for aberrantly large insert sizes. Candidate variants were reviewed using Integrated Genome Viewer and in silico assessment of functional significance including comparison with the gene databases (https://databases.lovd.nl/shared/genes/TSC2). Candidate pathogenic variants were validated in the original DNA sample and all other available samples from the patient using amplicon NGS.

      RESULTS:

      Patient 1

      A 44-year-old woman presented for evaluation of possible TSC-LAM. She had a history of pneumothorax two years earlier, and was diagnosed with LAM based on chest CT findings and an elevated serum VEGF-D of 2126 pg/ml. She denied any history of seizures or birthmarks, but reported that a few small red bumps appeared on her nose as a teenager. Examination revealed the presence of subtle skin-colored papules in or near the right alar crease (Figure 1A). No other mucocutaneous findings of TSC were observed. Chest-abdomen-pelvis CT confirmed the diagnosis of LAM and revealed the presence of two sclerotic bone lesions located within the right scapula and left femur, but no kidney lesions. CT imaging of the brain showed no cortical tubers, subependymal nodules, subependymal giant cell astrocytomas, or radial migration lines. CT of the abdomen-pelvis showed no kidney lesions. The algorithm-predicted genotype was low-level mosaicism.(
      • Treichel A.M.
      • Kwiatkowski D.J.
      • Moss J.
      • Darling T.N.
      A diagnostic algorithm for enhanced detection of mosaic tuberous sclerosis complex in adults.
      ) The patient underwent biopsy of three facial papules on the nose and cheek. Pathology of one of the papules was assessed and indicated angiofibroma, confirming the diagnosis of TSC. All three tissue specimens underwent NGS analysis and revealed the same mosaic pathogenic variant of TSC2 c.4842_4844del p.(Ile1614del) with VAFs of 9.77%, 6.83%, and 5.25% (Table I). Upon amplicon NGS, the same pathogenic variant in TSC2 was identified in the blood (VAF of 3.34%), saliva (2.34%), and urine (1.89%). Two of three angiofibromas also had a unique second inactivating mutation in TSC2, consistent with two-hit mutation to inactivate both alleles of TSC2; the third (sample 1A) did not have a second inactivating TSC2 mutation and may have occurred by a genetic mechanism of non-homologous recombination and loss of the wild-type allele in angiofibroma cells, an event not detectible in this sample with low enrichment for tumor cells.
      Figure thumbnail gr1
      Figure 1Mosaic tuberous sclerosis complex: Dermatologic findings in three individuals. (A) Patient 1: Angiofibromas on the right nasal ala, alar crease, and nasolabial fold along with bilateral scattered lentigines and telangiectasias. (B) Patient 2: A pink papule on the right chest present at birth measuring 4 mm in diameter. (C) Patient 2: Cross section near the level of the hair follicle isthmus revealed expansion of the perifollicular sheath with sclerotic collagen and interstitial fibroblasts, multiple sebaceous ducts, a hair shaft, and emanating epithelial strands. (D) Patient 3: Thickened overgrowth of the left index finger. No other digit showed macrodactyly.

      Patient 2

      A 32-year-old woman with recently diagnosed TSC-LAM was evaluated for possible mosaic TSC. She had been diagnosed with LAM and renal AMLs during a workup for abdominal fullness 2 years previously. She had no history of seizures but an MRI of the brain showed cortical tubers and subependymal nodules consistent with TSC. She also had numerous sclerotic bone lesions noted on CT imaging. Routine genetic testing of the blood did not identify any pathogenic variants in TSC1 or TSC2. She reported the presence of two skin lesions since birth, a white spot on her right calf and a small bump on her chest. Examination revealed a 4 cm hypomelanotic macule on the right calf and 4 mm pink papule on the right chest (Figure 1B). The predicted genotype was possible mosaic TSC.(
      • Treichel A.M.
      • Kwiatkowski D.J.
      • Moss J.
      • Darling T.N.
      A diagnostic algorithm for enhanced detection of mosaic tuberous sclerosis complex in adults.
      ) Shave biopsy of the papule showed expansion of the perifollicular sheath accompanied by hamartomatous changes of the follicular epithelium including several pilosebaceous units converging into a single follicular infundibulum and emanating epithelial strands with connection to a possible eccrine duct (Figure 1C). Genetic analysis of whole tissue from the chest papule revealed a variant of TSC2 c.532_546delinsGGTCAAATTCAAGCTA p.(Leu178Glyfs*10) with a VAF of 4.77% (Table I). The same variant in TSC2 was identified in the blood (VAF 2.45%) by amplicon NGS.

      Patient 3

      A 26-year-old woman with TSC-LAM was evaluated for possible mosaicism. At birth she had an overgrown 2nd digit on the left hand. This brought her to medical attention and at age five years, she was diagnosed with TSC based on hypomelanotic macules and facial angiofibromas. She had no seizures and brain imaging did not show any TSC-related findings. She underwent embolization of renal AMLs at age 17. One year prior to evaluation, she presented with a pneumothorax and she was diagnosed with LAM. Her facial angiofibromas were treated with laser on multiple occasions, most recently at age 21 years. She had been taking sirolimus 1 mg daily for 6 months. She previously underwent routine genetic testing of the blood with NVI. Examination revealed three hypomelanotic macules and macrodactyly of the left index finger (Figure 1D). On the nose and adjacent cheeks were several tiny pinpoint pink papules that were too small to provide adequate sample for genetic analysis. No other mucocutaneous features diagnostic of TSC were identified. MRI of the left hand demonstrated cortical bone thickening of the second metacarpal bone and proximal phalanx of the index finger along with amorphous soft tissue thickening surrounding the proximal interphalangeal joint. The predicted genotype was low-level mosaicism.(
      • Treichel A.M.
      • Kwiatkowski D.J.
      • Moss J.
      • Darling T.N.
      A diagnostic algorithm for enhanced detection of mosaic tuberous sclerosis complex in adults.
      ) NGS analysis of DNA from a punch biopsy of the overgrown finger revealed a pathogenic variant of TSC2 c.1491del p.(Glu498Argfs*37) with a VAF of 5.78% (Table I). Amplicon NGS assessment of the blood, saliva, and urine showed a VAF < 0.1% for this pathogenic variant, comparable to unrelated control DNA.
      Table ISummary of the clinical and genetic findings of three individuals supporting the diagnosis of mosaic tuberous sclerosis complex.
      Validation with aNGS# (VAF%)
      PtMuco-cutaneous Findings*Internal FindingsAlgorithm PredictionTissue SampleTissue Sample IDTSC2 Pathogenic VariantVariant ClassificationTissue Sample for NGS (VAF%)Tissue SampleBloodSalivaUrineUnrelated Control DNA 1Unrelated Control DNA 2
      1Unilateral AFsLAM, 6 SBLsLow Level MosaicAF1ATSC2 ex.36 c.4842_4844del p.(Ile1614del)In frame deletion9.77%11.17%3.34%2.34%1.89%0.00%0.00%
      AF1BTSC2 ex.36 c.4842_4844del p.(Ile1614del)In frame deletion6.83%10.06%3.34%2.34%1.89%0.00%0.00%
      TSC2 ex.37 c.4949_4959del p.(Tyr1650Trpfs*2)Frame shift deletion2.61%1.00%0.00%0.00%0.00%0.00%0.00%
      AF1CTSC2 ex.36 c.4842_4844del p.(Ile1614del)In frame deletion5.25%7.14%3.34%2.34%1.89%0.00%0.00%
      TSC2 ex.14 c.1507C>T p.(Gln503*)Nonsense6.21%5.22%0.07%0.08%0.08%0.08%0.08%
      TSC2 ex.17 c.1946+2_1946+4delinsCASplice site0.34%0.55%0.00%0.00%0.00%0.00%0.00%
      23 HMs, 1 UF, AFsCortical tubers, SEN, LAM, AML, SBLsPossible MosaicChest Hamartoma2ATSC2 ex.5 c.532_546delinsGGTCAAATTCAAGCTA p.(Leu178Glyfs*10)Frame shift deletion4.77%11.24%2.45%NANA0.00%0.00%
      3AFs, HMs, macrodactylyLAM, AMLLow Level MosaicSkin from digit with macro-dactyly3ATSC2 ex.14 c.1491del p.(Glu498Argfs*37)Frame shift deletion5.78%6.28%0.03%0.03%0.02%0.02%0.02%
      Abbreviations: Pt, Patient; AF, angiofibroma; HM, hypomelanotic macule; UF, ungual fibroma; LAM, lymphangioleiomyomatosis; SBL, sclerotic bone lesion; SEN, subependymal nodule; AML, angiomyolipoma; NGS, next generation sequencing; VAF, variant allele fraction; aNGS, amplicon next generation sequencing; DNA, deoxyribonucleic acid.
      * Mucocutaneous findings assessed included: Angiofibroma, hypomelanotic macule, ungual fibroma, fibrous cephalic plaque, shagreen patch, confetti, dental pitting, and oral fibroma.
      † Internal findings assessed included: lymphangioleiomyomatosis, angiomyolipoma, cortical tuber, subependymal nodules, subependymal giant cell astrocytoma, and sclerotic bone lesions.
      ‡ ACMG interpretation of variant classification was assessed by standard criteria as in Genetics in Medicine (2015) 17:405; with reference to the TSC gene mutation database LOVD, http://chromium.lovd.nl/LOVD2/TSC/home.php?select_db=TSC2.
      # Two independent control samples from healthy individuals were used as controls for every amplicon.

      DISCUSSION:

      The three individuals in this case series exhibited a negative family history of TSC, no history of seizures, few major features of TSC, and, in two who had previous genetic analysis, no pathogenic variant identified by routine testing of the blood cell DNA. All three women had findings suggestive of mosaic TSC based on our clinical diagnostic algorithm. NGS of cutaneous findings confirmed the clinical suspicion for mosaic TSC.(
      • Treichel A.M.
      • Kwiatkowski D.J.
      • Moss J.
      • Darling T.N.
      A diagnostic algorithm for enhanced detection of mosaic tuberous sclerosis complex in adults.
      ) Thus, when classic mucocutaneous findings of TSC are absent or inadequate in size for genetic analysis, NGS of subtle (few unilateral angiofibromas) or unusual TSC-associated cutaneous findings (cutaneous hamartoma or macrodactyly) may be used to help distinguish S-LAM from mosaic TSC-LAM and enhance detection of low-level mosaicism. This is especially true in sporadic TSC without seizure or other neurological history, normal brain MRI, and disease onset later in adulthood. It remains important for adults presenting with LAM or AMLs to undergo careful dermatological examination and imaging studies to determine if other clinical findings are present, meeting diagnostic criteria for TSC. In addition, singleton or variant cutaneous findings may be subject to biopsy to enable molecular genetic diagnosis.
      Patient 1 manifested only unilateral angiofibromas and LAM, fewer TSC features than previously reported in genetically confirmed cases of mosacism. (
      • Treichel A.M.
      • Hamieh L.
      • Nathan N.R.
      • Tyburczy M.E.
      • Wang J.A.
      • Oyerinde O.
      • et al.
      Phenotypic distinctions between mosaic forms of tuberous sclerosis complex.
      ) It is possible that some individuals with unilateral angiofibromas have a mosaic form of TSC limited to the skin. However, this individual and others we previously reported had unilateral or asymmetrically distributed angiofibromas associated with internal tumors of TSC. Therefore, apparent isolated unilateral angiofibromas should be considered as potential evidence of mosaic TSC, and evaluation for internal TSC manifestations should be performed.
      In Patient 2, the only classic TSC-associated mucocutaneous finding was a solitary hypomelanotic macule. Identifying pathogenic variants within tissue obtained from hypomelanotic macules has been challenging, likely reflecting the low frequency of the cell population (melanocytes) with a mutation in TSC1 or TSC2.(
      • Cao J.
      • Tyburczy M.E.
      • Moss J.
      • Darling T.N.
      • Widlund H.R.
      • Kwiatkowski D.J.
      Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation.
      ) Thus, genetic analysis was pursued on a chest papule that was present at birth. Histologically, the papule exhibited findings of perifollicular fibroma with additional follicular changes similar to trichofolliculoma and fibrofolliculoma, an unusual combination of histological findings in TSC.(
      • Treichel A.M.
      • Pithadia D.J.
      • Lee C.R.
      • Oyerinde O.
      • Moss J.
      • Darling T.N.
      Histopathological features of fibrous cephalic plaques in tuberous sclerosis complex.
      ) The presence of the same mosaic TSC2 pathogenic variant in the skin hamartoma and blood further supported its categorization as a TSC-associated finding. Thus, cutaneous hamartomas with less classic clinical and/or histological findings can be useful for the identification of pathogenic variants in TSC1/TSC2.
      In Patient 3, the patient’s angiofibromas were insufficient in size for tissue analysis due to prior treatment with lasers and oral sirolimus. Low-level mosaicism was suspected so NGS analysis was performed on a skin sample obtained from a digit with macrodactyly, a finding associated with TSC, though very rare.(
      • Soeiro E.S.M.
      • Moldovan O.
      • Sousa A.B.
      Macrodactyly in tuberous sclerosis complex: Case report and review of the literature.
      ) Ultimately, a mosaic deletion in TSC2 was detected in the skin, but was not detected in the blood. Additional reports of TSC-associated macrodactyly in individuals who underwent genetic testing of the blood found variants in TSC1(
      • Soeiro E.S.M.
      • Moldovan O.
      • Sousa A.B.
      Macrodactyly in tuberous sclerosis complex: Case report and review of the literature.
      ) and TSC2(
      • Sharma S.
      • Maino A.P.
      • Husain S.M.
      • Adams G.G.
      A newborn with unilateral limb enlargement.
      ), or NVI(

      Sasongko TH, Ismail NF, Nik Mohd Ariff NA, Zabidi-Hussin ZA. Macrodactyly and poliosis in tuberous sclerosis complex. Jpn J Clin Oncol 2014;44(11):1130.

      ). Another individual was reported to have a mosaic TSC2 variant in tissue from TSC-associated macrodactyly.(
      • Tessarech M.
      • Malinge M.C.
      • Carmignac V.
      • Vabres P.
      • Petit F.
      Limb overgrowth associated with a mosaic TSC2 second-hit in tuberous sclerosis complex.
      ) These findings are consistent with the hypothesis that macrodactyly in TSC occurs due to bi-allele inactivation of TSC1 or TSC2 in the embryonic tissues participating in digit formation and development, that leads to a significant fraction of cells without a functional TSC1-TSC2 protein complex and impairs normal digit formation.
      Our study is limited by a small sample size. Diagnosis of low-level mosaic TSC is challenging due to the inability of routine genetic testing to detect mosaic pathogenic variants in TSC1/TSC2 in the blood and subtle clinical findings associated with mosaic TSC. However, as NGS becomes increasingly available for commercial use, it is likely to play an important role in personalized medicine in future clinical practice and may enhance detection of mosaicism.(
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      Evaluation of Commercial Next-Generation Sequencing Bioinformatics Software Solutions.
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      • Nikiforov Y.E.
      • Nikiforova M.N.
      • Routbort M.J.
      • Pfeifer J.
      • et al.
      Next-Generation Sequencing Informatics: Challenges and Strategies for Implementation in a Clinical Environment.
      )
      Although TSC and S-LAM are both treated with mTOR inhibitors (sirolimus or everolimus), distinguishing between the two conditions is important. Individuals with TSC-LAM, unlike those with S-LAM, are at risk for involvement of the brain, skin, and eyes. Thus, routine surveillance for the development of these findings is advised for those with TSC.(Krueger et al., 2013) Accurate diagnosis is also important for potential heritability. A parent with germline TSC has a 50% risk of transmission to offspring. The risk of transmission in mosaic TSC appears to be much less, particularly with lower levels of mosaicism, and occurred at a frequency of 1/12 live births (8%) in our mosaic TSC cohort (VAF blood median = 1.35%, range = 0-19%).(
      • Treichel A.M.
      • Hamieh L.
      • Nathan N.R.
      • Tyburczy M.E.
      • Wang J.A.
      • Oyerinde O.
      • et al.
      Phenotypic distinctions between mosaic forms of tuberous sclerosis complex.
      ) Therefore, distinguishing between S-LAM, mosaic TSC-LAM, and germline TSC-LAM is important for assessing prognosis, guiding disease surveillance, and providing counseling on transmission risk of TSC to their offspring.

      DATA AVAILABILITY STATEMENT:

      Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs003108.v1.p1, hosted at database of Genotypes and Phenotypes (dbGaP), under the accession number phs003108.v1.p1.

      CONFLICT OF INTEREST:

      The authors have no conflicts of interest to declare.

      DISCLAIMER:

      The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Uniformed Services University, the Department of Defense or the National Institutes of Health.

      Uncited reference

      Krueger DA, Northrup H, International Tuberous Sclerosis Complex Consensus G. Tuberous sclerosis complex surveillance and management: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol 2013;49(4):255-265.

      .

      ACKNOWLEDGEMENTS:

      Research reported in this publication was supported in part by the Intramural Research Program, National Institutes of Health (NIH), National Heart, Lung, and Blood Institute (NHLBI); the NIH, NHLBI, under Award Number 5U01HL131022; the Doris Duke Charitable Foundation Clinical Research Mentorship grant #2018042. Additionally, this work was made possible through the NIH Medical Research Scholars Program, a public-private partnership supported jointly by the NIH and generous contributions to the Foundation for the NIH from the Doris Duke Charitable Foundation, the American Association for Dental Research, the Colgate-Palmolive Company, Genentech, and other private donors. For a complete list, visit the foundation website at http://www.fnih.org. We thank Katarzyna Klonowska for assistance with data submission to dbGaP.
      AUTHOR CONTRIBUTIONS:
      Conceptualization: AT, DJK, TD, JM; Data curation: AT, BO, CRRL, DJK, JM, TD; Formal Analysis: AT, BO, DJK, TD; Funding Acquisition: DJK, TD, JM; Investigation: AT, BO, CCRL, DJK, JM, TD; Methodology: AT, BO, DJK, TD; Project Administration: DJK, TD, JM; Resources: DJK, JM, TD; Software: DJK; Supervision: DJK, JM, TD; Validation: BO, DJK; Visualization: AT, CCRL; Writing – Original Draft Preparation: AT, TD; Writing- Review and Editing: AT, BO, CCRL, DJK, JM, TD. DJK and TD are co-Senior Authors for this publication.

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