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Germline Activating MTOR Mutation Arising Through Gonadal Mroske et al. BMC Medical Genetics (2015) 16:102 DOI 10.1186/s12881-015-0240-8 CASE REPORT Open Access Germline activating MTOR mutation arising through gonadal mosaicism in two brothers with megalencephaly and neurodevelopmental abnormalities Cameron Mroske1, Kristen Rasmussen2, Deepali N. Shinde1, Robert Huether1, Zoe Powis1, Hsiao-Mei Lu1, Ruth M. Baxter1, Elizabeth McPherson2 and Sha Tang1* Abstract Background: In humans, Mammalian Target of Rapamycin (MTOR) encodes a 300 kDa serine/ threonine protein kinase that is ubiquitously expressed, particularly at high levels in brain. MTOR functions as an integrator of multiple cellular processes, and in so doing either directly or indirectly regulates the phosphorylation of at least 800 proteins. While somatic MTOR mutations have been recognized in tumors for many years, and more recently in hemimegalencephaly, germline MTOR mutations have rarely been described. Case presentation: We report the successful application of family-trio Diagnostic Exome Sequencing (DES) to identify the underlying molecular etiology in two brothers with multiple neurological and developmental lesions, and for whom previous testing was non-diagnostic. The affected brothers, who were 6 and 23 years of age at the time of DES, presented symptoms including but not limited to mild Autism Spectrum Disorder (ASD), megalencephaly, gross motor skill delay, cryptorchidism and bilateral iris coloboma. Importantly, we determined that each affected brother harbored the MTOR missense alteration p.E1799K (c.5395G>A). This exact variant has been previously identified in multiple independent human somatic cancer samples and has been shown to result in increased MTOR activation. Further, recent independent reports describe two unrelated families in whom p.E1799K co-segregated with megalencephaly and intellectual disability (ID); in both cases, p.E1799K was shown to have originated due to germline mosaicism. In the case of the family reported herein, the absence of p.E1799K in genomic DNA extracted from the blood of either parent suggests that this alteration most likely arose due to gonadal mosaicism. Further, the p.E1799K variant exerts its effect by a gain-of-function (GOF), autosomal dominant mechanism. Conclusion: Herein, we describe the use of DES to uncover an activating MTOR missense alteration of gonadal mosaic origin that is likely to be the causative mutation in two brothers who present multiple neurological and developmental abnormalities. Our report brings the total number of families who harbor MTOR p.E1799K in association with megalencephaly and ID to three. In each case, evidence suggests that p.E1799K arose in the affected individuals due to gonadal mosaicism. Thus, MTOR p.E1799K can now be classified as a pathogenic GOF mutation that causes megalencephaly and cognitive impairment in humans. Keywords: Diagnostic exome sequencing, Family-trio, MTOR, Autism, ASD, Macrocephaly, Megalencephaly, Gonadal mosaicism, Gain-of-function, Missense alteration * Correspondence: [email protected] 1Ambry Genetics Corporation, Aliso Viejo, CA 92656, USA Full list of author information is available at the end of the article © 2015 Mroske et al. 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. Mroske et al. BMC Medical Genetics (2015) 16:102 Page 2 of 11 Background cascade, a simplified version of which is depicted in Autism spectrum disorders (ASDs) are a group of neuro- Fig. 1. At the center of this pathway lies MTOR, a developmental maladies marked by social/communication 300 kDa serine/threonine protein kinase encoded by the impairments, repetitive behaviors, and sensory reactivity MTOR gene, which in humans is situated on chromo- issues [1]. Even though the molecular etiology of autism is some 1p36 [13]. By integrating upstream signals to genetically heterogeneous, up to 10 % of cases are associ- downstream effector molecules like ribosomal P70 S6 ated with a distinct genetic condition such as Fragile X (S6) and ribosomal P70 S6 kinase (S6K), the PI3K-AKT- syndrome, tuberous sclerosis, phenylketonuria, or Rett MTOR axis serves to regulate a myriad of biological syndrome [2–5]. Furthermore, 20 % of autistic patients ex- processes including cell growth and proliferation, apop- hibit macrocephaly [6]. In a number of these cases, germ- tosis, protein synthesis, and transcription [14]. MTOR is line mutations in the tumor suppressor gene PTEN known to mediate normal brain development by playing (Phosphatase and Tensin Homologue) have been revealed key roles in axonal and dendritic growth, and in the es- to be the underlying cause of disease [7–11]. It has been tablishment and maintenance of synaptic plasticity [15]. estimated that the rate of PTEN disruption is up to 5 % in As evidenced by the association of PTEN dysfunction ASD patients with macrocephaly [12]. with ASD and macrocephaly, hyper-activation of the PTEN functions as a negative regulator of the PI3K PI3K-AKT-MTOR signaling cascade is an underlying (Phosphoinositide 3-Kinase)–AKT (AKT/Protein Kinase factor for neurological disease and abnormal develop- B)-MTOR (Mammalian Target of Rapamycin) signaling ment. Such ‘TORopathies’ include neurological ailments Fig. 1 Simplified representation of the MTOR signaling network. Green arrows indicate the specific substrates shown to be phosphorylated/ over-stimulated by p.E1799K-hyperactivated MTOR kinase. Other symbols and abbreviations used: stimulatory effects ( ); inhibitory effects ( ); 4E-BP1 (Eukaryotic Translation Initiation Factor 4E-Binding Protein 1); AKT (AKT/ Protein Kinase B); MTOR (Mammalian Target of Rapamycin); PI3K (Phosphoinositide 3-Kinase); PTEN (Phosphatase and Tensin Homolog); RAS (Rat Sarcoma Viral Oncogene Homolog); S6K (Ribosomal P70 S6 Kinase 1); TORC1 (MTOR Complex 1); TORC2 (MTOR Complex 2) TSC1 (Tuberous Sclerosis 1); TSC2 (Tuberous Sclerosis 2) Mroske et al. BMC Medical Genetics (2015) 16:102 Page 3 of 11 like focal cortical malformations (FCM), tuberous scler- MTOR p.E1799K likely originated as a consequence osis complex (TSC), and hemimegalencephaly, all of of gonadal mosaicism. which are highly associated with epilepsy [16, 17]. Fur- thermore, hyperactivation of the MTOR axis has also Case presentation been associated with somatic cancer [18]. Clinical description Although specific molecular lesions have been defined Table 1 lists the major clinical features displayed by the for some TORopathies–for example, in cases where PTEN proband and his brother, as well as the features of add- is disrupted–in many instances it remains difficult to pin- itional patients who harbor MTOR p.E1799K but who point the exact molecular defect due to the complexity of are unrelated to the family described herein. The pro- the MTOR signaling network [17]. Even so, the emer- band, who presented to medical genetics clinic at age 3 gence of next generation sequencing (NGS) has greatly with macrocephaly, bilateral iris colobomas and gross facilitated the search for genes and pathogenic variants motor delay, had a mildly unusual appearance, which that underlie neurological disease. Several recent reports was primarily due to macrocephaly with a prominent have detailed the successful use of whole exome sequen- forehead (Fig. 2a, left panels). The proband was the cing (WES) to uncover both somatic and germline activat- product of an uncomplicated pregnancy and was large ing alterations within MTOR itself. [19–23] These clinical for his gestational age, with a weight of 2950 g (97 %) at studies describe damaging MTOR alleles in unrelated pro- 35 weeks; birth head circumference was not recorded. bands who present overlapping neurological symptoms Since infancy, his linear growth and weight have been and brain defects [19–23]. In a recent cancer study, Grabi- normal; however, macrocephaly was noted by 6 months, ner and colleagues accessed publicly available sequencing which prompted a head ultrasound. The result showed data from tumor samples to uncover 33 MTOR alterations no evidence of hydrocephalus. Since 3 years of age, the that conferred hyperactivation of the MTOR signaling proband’s head growth has been tracking parallel to, and pathway within cancer cells. These alterations clustered 6 standard deviations above the mean. Investigation by around six distinct regions in the C-terminal half of the magnetic resonance imaging revealed megalencephaly. protein [18]. Bilateral iris colobomas with normal posterior poles Herein, we present the clinical and genetic investi- were noted at birth, and the proband wears glasses for gation of two brothers affected with macrocephaly strabismus. During infancy, he displayed hyperinsuline- and ASD. Diagnostic Exome Sequencing (DES) identi- mia, which was managed through frequent feedings and fied a GOF MTOR alteration, p.E1799K, to be the administration of diazoxide. He continues to display causative mutation in this family. Co-segregation and asthma, as well as persistent food allergies that manifest short tandem repeat (STR) analyses revealed
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