CLINICAL REPORT
High Functioning Male with Fragile X Syndrome and Fragile X-Associated Tremor/Ataxia Syndrome Kirin Basuta,1 Andrea Schneider,2,3 Louise Gane,2 Jonathan Polussa,2,3 Bryan Woodruff,4 Dalyir Pretto,1 Randi Hagerman,2,3 and Flora Tassone1,2* 1
Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Sacramento, California
2
MIND Institute, University of California, Davis, Medical Center, Sacramento, California Department of Pediatrics, University of California at Davis Medical Center, Sacramento, California
3 4
Department of Neurology, Mayo Clinic, Scottsdale, Arizona
Manuscript Received: 17 December 2014; Manuscript Accepted: 6 April 2015
Fragile X syndrome (FXS) affects individuals with more than 200 CGG repeats (full mutation) in the fragile X mental retardation 1 (FMR1) gene. Those born with FXS experience cognitive and social impairments, developmental delays, and some features of autism spectrum disorders. Carriers of a premutation (55–200 CGG repeats) are generally not severely affected early in life; however, are at high risk of developing the late onset neurodegenerative disorder, Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), or Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and may have other medical conditions such as developmental delay, autism spectrum disorders, hypertension, anxiety, and immune-mediated disorders. Here we present a case of a 58-year-old man with a borderline IQ, average memory skills, and executive function deficits. He met criteriafor multiple psychiatric diagnoses and presented with tremor and ataxia, meeting criteria for FXTAS. Molecular testing unveiled a completely unmethylated FMR1 full mutation in peripheral blood mononucleated cells with elevated FMR1 mRNA and premutation alleles of different sizes in two other tissues (primary fibroblasts and sperm), indicating the presence of allele instability based on both inter- and intra-tissue mosaicism. The observation of FXTAS in this case of a full mutation mosaic man suggests that the pathogenic mechanism underlying this disorder is not observed exclusively in premutation carriers as it was originally thought. The concomitant presence of features of FXS and late onset neurological deterioration with probable FXTAS likely result from a combined molecular pathology of elevated FMR1 mRNA levels, a molecular hallmark of FXTAS and low FMRP expression that leads to FXS. Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Basuta K, Schneider A, Gane L, Polussa J, Woodruff B, Pretto D, Hagerman R, Tassone F. 2015. High functioning male with fragile X syndrome and fragile Xassociated tremor/ataxia syndrome. Am J Med Genet Part A 167A:2154–2161.
methylation with consequent silencing expression of the FMR1 protein, FMRP. The lack of FMRP leads to up-regulation of synaptic signaling proteins as FMRP is an essential translational repressor [Chen et al., 2014] ubiquitously expressed throughout many tissues [Pieretti et al., 1991; Hinds et al., 1993; Tassone et al., 1999], most importantly in neural cells [Sidorov et al., 2013; Beebe et al., 2014; Doers et al., 2014]. The absence of FMRP effects neuronal networks of cognition and leads to severe learning disabilities, social and behavioral impairments, as well as the clinical features usually falling under the umbrella of autism spectrum disorders (ASD) [Bagni and Oostra, 2013]. In individuals carrying a premutation allele with shorter expansions of 55–200 CGG repeats, FMR1 typically is not silenced. Instead the gene is excessively transcribed producing 2–6-fold higher mRNA levels as observed in peripheral blood mononucleated cells (PBMCs) [Tassone et al., 2000; Sullivan et al., 2005] and in brain tissue [Pretto et al., 2014a]. However, FMRP levels are reduced with declining expression depending on the length of the
Key words: fragile X syndrome; FXTAS
INTRODUCTION Fragile X syndrome (FXS) is a trinucleotide repeat expansion disorder. More than 200 CGG repeats in the promoter region of the fragile X mental retardation 1 (FMR1) gene trigger hyper-
Ó 2015 Wiley Periodicals, Inc.
Conflict of interest: none. Grant sponsor: NICHD; Grant numbers: HD02274, HD036072. Correspondence to: Flora Tassone, Ph.D., Department of Biochemistry and Molecular Medicine, 2700 Stockton Blvd Suite 2102, Sacramento, CA 95817. E-mail [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 29 April 2015 DOI 10.1002/ajmg.a.37125
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BASUTA ET AL. CGG expanded alleles. Some premutation carriers, especially males with a large premutation, may experience mild cognitive deficits, but they are at increased risk of developing the late onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS) [Hagerman et al., 2001], characterized by intentional tremor, gait ataxia, neuropathy, and parkinsonism [Hagerman and Hagerman, 2013]. The leading hypothesis suggests that FXTAS is caused by RNA toxicity, which results from the elevated FMR1 mRNA, particularly affecting neuronal signaling pathways in the brain [Wang et al., 2010; Kindler and Kreienkamp 2012; Deng et al., 2013; Friedman et al., 2013; Beebe et al., 2014; Colvin and Kwan, 2014; Sidorov et al., 2013]. Although FXTAS was initially thought to affect only premutation carriers, recent studies have reported individuals with an intermediate allele (45–54 CGG repeats) also being diagnosed with FXTAS. Liu et al. [2012] described three individuals (two males and a female) with FXTAS carrying an intermediate allele with elevated levels of FMR1 mRNA. Another study described three additional individuals with an intermediate allele, presenting with FXTAS symptoms of different severities [Hall et al., 2012]. In addition, FXTAS has been reported in individuals with the full mutation. One study reported a full mutation mosaic male with unmethylated alleles ranging from 180 to 410 CGG repeats and a sevenfold increase in FMR1 mRNA [Santa Maria et al., 2013]. Another mosaic male was reported with a fully unmethylated full mutation (297–480 CGG repeats) and a threefold increased FMR1 mRNA levels [Loesch et al., 2011]. Finally, very rare intranuclear inclusions, the neuropathological hallmark of FXTAS, were observed in three males with a full mutation [Hunsaker et al., 2011]. In this study, we present a case of a mosaic male with intra- and inter-tissue mosaicism presenting with clinical features of both FXTAS and FXS.
METHODS Study Subject This participant was recruited through the Genotype–Phenotype study and informed consent, approved by the University of California at Davis, Medical School IRB was signed.
Molecular Measures Genomic DNA (gDNA) was extracted from fibroblasts, blood, and sperm tissue samples utilizing standard protocols (Qiagen, Valencia, CA). PCR amplification and Southern Blot analyses were run on gDNA from the three tissue samples to determine the CGG repeat length and methylation status as previously described [Tassone et al., 2008; Filipovic-Sadic et al., 2010]. mRNA expression levels for the FMR1 gene were quantified using a 7900 sequence detector (Applied Biosystems, Foster City, CA) using conditions previously described in Tassone et al. [2000]. Western Blot analysis using PBMCs and fibroblasts protein extracts derived from five controls and the patient’s samples. Specific antibodies against FMRP (cat# MAB2160, Merck Millipore, Darmstadt, Germany) and GAPDH (cat# AB2302, EMD Millipore, Darmstadt, Germany) were used as previously described [Pretto et al., 2013].
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Neuropsychological Measures The neuropsychological/neuropsychiatric assessments included the following standardized IQ tests: Wechsler Adult Intelligence Scales (WAIS-III and WAIS-IV) [Wechsler, 1997, 2008]. Memory function was assessed using the Wechsler Memory Scales, Fourth Edition (WMS-IV) [Wechsler, 2009]. The Reading subtest from the Wide Range Achievement Test (WRAT 4) [Wilkinson and Robertson, 2006] was administered by an outside psychologist. The MiniMental Status Exam (MMSE) [Folstein et al., 1975], and Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) [First et al., 1997] were administered to evaluate mental status and psychiatric disorders. For the assessment of attention and executive function, the Integrated Visual and Auditory Continuous Performance Test (IVA-CPT) [Sandford et al., 1995] and the Behavioral Dyscontrol Scale 2nd edition (BDS-2) [Grigsby et al., 2002] were administered. The Autism Diagnostic Observation Schedule (ADOS2) [Lord et al., 1999] was used to evaluate for symptoms of ASD.
RESULTS Family History The patient has three obligate carrier daughters who have not been tested for FMR1 related disorders. His oldest daughter is 40 years old and has a daughter reported as being gaze avoidant and shy, and a son, both with unknown FMR1 gene status. The second daughter is 39 years old and has three sons, one who was diagnosed with FXS while the others have not been tested for FXS. The youngest daughter is 38 years old and has four sons and one daughter. One son is reported to have FXS but FMR1 DNA test results were unavailable. The other children have not been tested. The patient had three brothers, one who died neonatally, and one sister. One of these brothers is 60 years old and is reported to have balance problems and vertigo suggestive of FXTAS. A second brother, age 51, is reported to have depression and to live isolated and reclusive from others. His sister is 51 years old and is reported to have migraines, mood instability, and depression. The patient’s siblings have not been tested. The patient’s mother is reported to have died in her 80s. Her cause of death was attributed to a urinary tract infection in addition to a history of gait ataxia with many falls, sleep apnea, thyroid problems, and possible dementia, suggestive of diagnosis of FXTAS.
Medical History The patient was referred to the Mayo clinic and subsequently to the MIND Institute at age 58 with a history of neurological problems. He was diagnosed with the FMR1 full mutation after his grandson was diagnosed with FXS. His childhood medical history includes premature birth at 30 weeks gestation (BW 4.25 pounds) requiring an incubator for several weeks without ventilation needed. He was breastfed for several months. In school he required special education classes for reading, he had problems with eye contact and occasional hand flapping, and a history of hyper-sensitivity to noises. The patient graduated from high school and worked in construction for 17 years before joining the Army. He had chronic
2156 outburst behavior and obsessive-compulsive behavior and was diagnosed with bipolar disorder and treated with lithium. At 39 years old, he required surgery for a neck injury following a parachute jump and experienced chronic cervical pain since that time, associated with degenerative disc disease. At age 44, the patient developed symptoms consistent with cauda equina syndrome with urinary and bowel incontinence as well as erectile dysfunction. This prompted emergent decompressive surgery. He began intermittent use of an electric wheelchair at age 45. He developed hypogonadism and began testosterone replacement therapy. With rehabilitation, his legs grew stronger but began giving out on a regular basis at about age 50 and he began using his wheelchair regularly. At age 50 he developed an intermittent intention tremor and more severe gait problems with frequent falling. He developed painful neuropathy symptoms in his lower legs as well as restless legs syndrome (RLS). He also developed sleep apnea, treated with CPAP. At age 51, he began using opioids for pain control, including oxycodone, lidocaine jelly, and Lidoderm patches. At age 57 he developed migraines with visual aura. His current complaints include migraines, irritable bowel syndrome, RLS, gastroesophageal reflux disease, myalgias of his upper arms and shoulders, arthralgias in his hands, occasional severe leg cramps, severe constipation, subjective memory difficulties, depression/bipolar disorder, and a 30–50% loss of exercise stamina over the past few years. On examination at age 58 the proband had a height of 179.5 cm, weight of 116.2 kg (for a BMI of 36.1), blood pressure of 138/ 89 mmHg, a pulse of 70 bpm, head circumference of 59.4 cm. The patient has a long face with mildly prominent ears and his hearing was mildly decreased bilaterally. Cranial nerve examination demonstrated mild end-gaze nystagmus with lateral gaze. The patient uses dentures, but nose and throat exam was otherwise normal and he has an intact gag reflex. Neck was supple without adenopathy, and thyroid gland was palpably normal. Finger joints were not hyperextensible, with some guarding noted due to arthritis. The patient did not have scoliosis, but did have scars on his spine in the neck and lumbar area due to past surgeries. The patient has a normal phallus and macroorchidism with a testicular volume of 70 ml bilaterally. The patient exhibited diffuse areflexia. Plantar responses were flexor bilaterally. Vibration sense was absent in his left foot and decreased in his right foot. Pinprick sensation was normal. Muscle strength was normal in all four limbs. The patient’s gait was broadbased with listing to one side, and he could not perform tandem gait. He exhibited diminished rate of alternating movements diffusely. The patient’s current medications include Fortesta for hypogonadism several times per day, ondansentron (8 mg) for nausea, omeprazole 20 mg daily for reflux, oxycodone 40 mg twice daily for pain, donepezil 10 mg daily for memory, hydroxyzine 25 mg for panic attacks and stress, Lipozene 15 mg daily for weight loss, lithium 300 mg twice a day for bipolar disorder, gabapentin 600 mg twice daily for neuropathy, nabumetone 500 mg twice daily for arthritis, and Lexapro 40 mg daily for depression.
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Diagnostic Procedure Summary Initial MRI at age 56 demonstrated a few scattered subcortical foci of white matter hyperintensity adjacent to the atrium of the left lateral ventricle (Fig. 1). Repeat MRI two years later demonstrated a thin corpus callosum, dilated ventricles, and scattered white matter hyperintensity similar to his previous MRI. No hyperintensity of the middle cerebellar peduncles was noted. MRI of the lumbar spine at age 57 demonstrated post-operative changes from prior decompressive laminectomy at L2–L3 without residual central canal stenosis at any level. Small far lateral disc protrusion was noted at L3–L4 on the left with potential impingement of the exiting L3 nerve root. Mild bilateral foraminal stenosis was also noted at L4–L5 and L5–S1. EEG at age 56 demonstrated mild to moderate generalized slowing, but no potentially epileptogenic activity. EMG of the upper and lower extremities at age 55 demonstrated a length-dependent primarily axonal sensorimotor peripheral neuropathy with superimposed moderate right median neuropathy at the wrist. Repeat EMG in 2013, at age 58, demonstrated some improvement in the right median neuropathy.
Neuropsychological Findings Table I summarizes the results from the comprehensive neuropsychological assessment at the UCD MIND Institute and previous records from outside sources.
FIG. 1. Axial CUBE MRI demonstrating minimal white matter hyperintensity adjacent to the posterior horn of the left lateral ventricle.
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TABLE I. Overview of Neuropsychological Assessments Assessment Reading Cognitive assessment
Memory
Executive function
Age 55
Age 58
(WRAT) Reading standard score ¼ 83 (WAIS-III) Verbal comprehension ¼ 103 Perceptual reasoning ¼ 82 Full scale IQ ¼ 90 Working memory index ¼ 88 Processing speed index ¼ 84 Auditory verbal learning test ¼ 27 Percent recall ¼ 38
Attention
Trail marking test Trail A - MOANS ¼ 2 Trail B - MOANS ¼ 6 WAIS III digit span ¼ 10
Mental status
(not done)
Autism spectrum disorders
(not done)
(not done) (WAIS-IV) Verbal comprehension ¼ 108 Perceptual reasoning ¼ 79 Full scale IQ ¼ 84 Working memory index ¼ 71 Processing speed index ¼ 84 (WMS-IV) Auditory memory ¼ 94 Visual memory ¼ 98 Visual working memory ¼ 91 Delayed memory score ¼ 98 Immediate memory ¼ 93 (BDS-2) 6* (out of 27), severely impaired (IVA) Response control quotient (RCQ) ¼ 76 Auditory RCQ ¼ 70 Visual RCQ ¼ 90 Attention quotient (AQ) ¼ 82 Auditory AQ ¼ 82 Visual AQ ¼ 84 (MMSE) 24* (out of 30), mildly impaired (ADOS-4) No symptoms of ASD
Scores are reported in the standard score scale with a mean of 100, standard deviation of 20, *score reported as raw score because there are no normative data available. WRAT, wide range achievement test; WAIS-III, Wechsler adult intelligence scale 3rd edition; WAIS-IV, Wechsler adult intelligence scale 4th edition; WMS-IV, Wechsler memory scale 4th edition; BDS-2, behavioral dyscontrol scale 2nd edition; IVA, integrated visual and auditory attention test; MMSE, mini mental state exam; ADOS-4, Autism diagnostic observation schedule module 4. MOANS, Mayo Older American Normative Study.
On the SCID-I (Structured Clinical Interview for DSM-IV Axis I Disorders) the patient meets criteria for Bipolar I Disorder with rapid cycling, most current episode depressive. The patient reports some psychotic symptoms with hearing voices, particularly his wife calling him, and reports religious perseverations and grandiosities. The patient meets criteria for panic disorder and generalized anxiety disorder. He reports significant obsessive-compulsive symptoms with frequent lock checking and sexual obsessions. At his visit, he insisted on donating sperm. The patient also meets criteria for somatization disorder.
Molecular Measures PCR and Southern Blot analyses of the patient’s PBMCs reveal FMR1 alleles ranging from 20 to 800 CGG repeats, >90% unmethylated (Fig. 2a,b); An unmethylated 200 CGG repeat premutation allele was present in primary fibroblasts (Fig. 2a,c) while an 86 CGG premutation allele was present in sperm (Fig. 2a,d) indicating inter-tissue instability. In all samples, allelic instability (intra-tissue mosaicism) was also observed and visualized by a series of peaks on capillary electrophoresis (Fig. 2b,c,d). FMR1 mRNA expression level in PBMCs was threefold higher than normal (2.98 0.06). CGG allele instability and mosaicism were
observed between and among all three tissues. FMRP expression levels in peripheral blood cells (PBMCs) and primary fibroblast cells, measured by Western blot analysis were 75% lower than controls in PBMCs and 50% lower in fibroblasts.
DISCUSSION The term FMR1 mosaicism indicates the presence of FMR1 alleles with either multiple different CGG repeat sizes (i.e., premutation and full mutation alleles, termed as size mosaic), different methylation status (i.e., unmethylated and methylated full mutation size alleles, termed methylation mosaic), or differences in both size and methylation (unmethylated alleles that span both premutation and full mutation ranges and methylated alleles, termed methylation and size mosaic). Differences in methylation and CGG repeat size can be observed between different cell types, this is termed intertissue mosaicism. These differences can also be observed within the same type, and this is termed intra-tissue mosaicism. In this study we present clinical and molecular findings in a mosaic man (for size and methylation). DNA testing revealed the presence of both FMR1 inter- and intra-tissue (PBMCs, primary fibroblasts and sperm) CGG allele size and methylation mosaicism. Alleles of different CGG size were observed in all three tissues analyzed. As
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FIG. 2. Methylation status and CGG size instability as detected by Southern blots (SB) and capillary electrophoresis. The SB analysis (a) demonstrates the presence of a series of unmethylated alleles (smear) spanning from the normal range (2.8 Kb) through the full mutation methylated range (>5.2 Kb) in peripheral blood lymphocytes (PBMCs). The presence of a premutation allele is visible in both primary cultured fibroblasts and sperm. M ¼ 1 Kb DNA size ladder marker; C1 ¼ normal female, negative control. The electrophoregrams show CGG instability as illustrated by the presence of serial peaks, each representing single distinct alleles, with differences between PBMCs (b) fibroblasts (c) and sperm (d). The X-axis indicates the size of the alleles in base pairs and the Y-axis indicates the fluorescence intensity of each allele.
expected, a premutation allele with a CGG repeat number within the premutation range was observed in sperm cells [Reyniers et al., 1993]. A premutation allele, of different size, was also observed in fibroblasts while, a series of unmethylated alleles, from the normal range to the full mutation range were observed in PBMCs demonstrating the presence of inter- and intra-tissue mosaicism. This finding is significant because it suggests that a double molecular mechanism, derived from both the elevated FMR1 mRNA and lower FMRP expression levels observed in this individual, likely contributed to his clinical phenotype [Pretto et al., 2014a,b]. In particular, the moderate lower FMRP expression, as measured in
PBMC cells, likely contributed to the development of a mild cognitive deficit, as his IQ falls in the low average range. However, he had a successful military career, was married and fathered children. He had the physical features of FXS including macroorchidism and the behavioral problems of ADHD, anxiety and mood instability but he was much higher functioning than a typical male patient with FXS. His FXTAS picture was also atypical and complicated by his early cauda equina syndrome from trauma, but by age 50 the FXTAS neurodegenerative symptoms began to develop as tremor, more severe ataxia, increasing pain symptoms, and memory problems were observed during examination. His
BASUTA ET AL. executive function deficits were likely present since childhood along with his ADHD but the presence of FXTAS has likely enhanced their severity. Additional possible manifestations of premutation involvement include his migraine headaches [Au et al., 2013] and his RLS [Summer et al., 2013]. Sleep apnea is also more common in premutation carriers than controls and if left untreated this can exacerbate FXTAS [Hamlin et al., 2011]. His psychotic thinking is atypical for FXTAS in general but we have reported this in two cases with a double hit of elevated FMR1 mRNA and lowered FMRP [Schneider et al., 2013]. One concerning issue is his heavy use of opioids for pain control because there is the potential for opioids to exacerbate white matter disease [Muzar et al., 2014]. His pain symptoms arise from both neuropathy and arthritis. However, the muscle pain may also be the result of a central pain syndrome, as it is similar to fibromyalgia symptoms described in premutation women [Leehey et al., 2011]. Although he does not have the MCP sign, he has thinning of the corpus callosum, brain atrophy, and scattered white matter disease, which meets criteria for probable FXTAS [Jacquemont et al., 2014]. In the revision of the diagnostic criteria for FXTAS, the constraint of the premutation has been expanded to include those with other allele sizes including the gray zone and full mutation alleles, particularly those that are unmethylated, in which elevated FMR1 mRNA levels are observed [Hall et al., 2014]. We have previously reported a significant association between FMRP expression and IQ in individuals with premutation and partial methylation (size and methylation mosaics) [Pretto et al., 2014a] and also in full mutation methylation mosaic males [Pretto et al., 2014b]. In addition, the number of phenotypic features was associated with the level of expression of FMRP [Pretto et al., 2014a]. Although the proband did not present the typical spectrum of FXS clinical features, he did have macroorchidism, psychiatric problems, and behavioral issues that probably were present since childhood as evidenced by reports of hand flapping and anxiety, likely due to the FMRP deficit. The proband also presented with elevated FMR1 mRNA, resulting in RNA toxicity likely leading to the premutation pathology observed, particularly the current manifestations of FXTAS symptoms. We have previously reported on symptoms, pathological and molecular evidence of FXTAS in three cases of full mutation mosaic males, two with a methylation mosaicism [Loesch et al., 2011; Santa Maria et al., 2013] and one with a size mosaicism [Pretto et al., 2014b]. The latter case also presented with co-morbidity for both FXS and FXTAS. His typical features of FXS included a mildly long face, large and mildly prominent ears, macroorchidism, perseverative speech, and anxiety. The presence of intranuclear inclusions, one of the neuropathological criteria for the diagnosis of FXTAS, was observed in several regions of his brain [Pretto et al., 2013]. This case of FXTAS in a male with mosaicism indicates that the risk of developing pathogenic mechanisms leading to a neurodegenerative disorder may be confined not only to those with expanded premutation alleles but also to those with unmethylated full mutation alleles with elevated levels of toxic FMR1 mRNA. In addition, the clinical presentation of the case presented here, shares the molecular and clinical phenotype of both premutation involvement and FXS. Thus, it is becoming more evident that the genetic makeup of individuals with mosaicism, mostly when high pro-
2159 portions of unmethylated CGG repeat expansions are present, may be very complex due to the presence of both RNA toxicity and lower FMRP expression. This case illustrates how the determination of size and methylation status is not only informative but may also be important in establishing ways of managing the disorder from the clinical perspective. It also highlights a not so uncommon scenario of a patient that was unaware that an FMR1 mutation was present in his genetic background and that this mutation is likely responsible for his clinical symptoms and also of those observed in his family members. A likely reason for this delayed knowledge is that mosaic individuals may present with a milder phenotype during childhood as well as during early adulthood, limited mostly to a lower IQ, anxiety, attention deficit/hyperactivity disorder, and behavioral issues [Aziz et al., 2003; Farzin et al., 2006; Bailey et al., 2008] that do not significantly interfere with a normal life. It is not until symptoms of neurological decline start to develop, generally after age 50, that the patient seeks medical attention and diagnostic testing for an FMR1 mutation may be requested by a physician who is aware of the clinical spectrum of patients suspected of developing FXTAS. Finally, the case presented here illustrates the need to further investigate the molecular background of premutation and full mutation cases presenting complex clinical spectra. The methylation status and CGG repeat size may be equally important for accurate diagnosis.
ACKNOWLEDGMENT This work was supported by NICHD grants HD02274 and HD036072. This work is dedicated to the memory of Matteo. Dr. F. Tassone has received funding from Roche and consulted with Novartis. Dr R. Hagerman has received funding from Novartis, Roche, Alcobra, and Seaside Therapeutics to carry out treatment trials in fragile X syndrome. She has also consulted with Novartis, Roche, and Genentech on treatment of fragile X syndrome.
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Hall DA, Birch RC, Anheim M, Jønch AE, Pintado E, Joanne O, Trollor JN, Stebbins GT, Hagerman RJ, Fahn S. 2014. Emerging topics in FXTAS. J Neurodev Disord 6:31. Hamlin A, Liu Y, Nguyen DV, Tassone F, Zhang L, Hagerman RJ. 2011. Sleep apnea in fragile X premutation carriers with and without FXTAS. Am J Med Genet B 156B:923–928. Hinds HL, Ashley CT, Sutcliffe JS, Nelson DL, Warren ST, Housman DE, Schalling M. 1993. Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome. Nat Genet 3:36–43. Hunsaker MR, Greco CM, Tassone F, Berman RF, Willemsen R, Hagerman RJ, Hagerman PJ. 2011. Rare intranuclear inclusions in the brains of 3 older adult males with fragile x syndrome: Implications for the spectrum of fragile x-associated disorders. J Neuropathol Exp Neurol 70:462–469. Jacquemont S, Coe BP, Hersch M, Duyzend MH, Krumm N, Bergmann S, Beckmann JS, Rosenfeld JA, Eichler EE. 2014. A higher mutational
Sandford JA, Fine AH, Goldman L. 1995. Validity study of IVA: A visual and auditory CPT. Paper presented at the 1995 Annual American psychological association convention. New York, NY. Santa Maria L, Pugin A, Alliende M, Aliaga S, Curotto B, Aravena T, Tang HT, Mendoza-Morales G, Hagerman R, Tassone F. 2013. FXTAS in an unmethylated mosaic male with fragile X syndrome from Chile. Clin Genet 86:378–382. Schneider A, Seritan A, Tassone F, Rivera SM, Hagerman R, Hessl D. 2013. Psychiatric features in high-functioning adult brothers with fragile x spectrum disorders. Prim Care Companion CNS Disord 15: PCC.12l01492. Sidorov MS, Auerbach BD, Bear MF. 2013. Fragile X mental retardation protein and synaptic plasticity. Mol Brain 6:15. Sullivan AK, Marcus M, Epstein MP, Allen EG, Anido AE, Paquin JJ, Yadav-Shah M, Sherman SL. 2005. Association of FMR1 repeat size with ovarian dysfunction. Hum Reprod 20:402–412.
BASUTA ET AL. Summer SM, Cogswell J, Goodrich JE, Mu Y, Nguyen DV, Brass SD, Hagerman RJ. 2013. Prevalence of restless legs syndrome and sleep quality in carriers of the fragile X premutation. Clin Genet 86:181–184. Tassone F, Hagerman RJ, Gane LW, Taylor AK. 1999. Strong similarities of the FMR1 mutation in multiple tissues: Postmortem studies of a male with the full mutation and a male carrier of a premutation. J Med Genet 84:240–244. Tassone F, Hagerman RJ, Taylor AK, Gane LW, Godfrey TE, Hagerman PJ. 2000. Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet 66:6–15. Tassone F, Pan R, Amiri K, Taylor AK, Hagerman PJ. 2008. A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and highrisk populations. J Mol Diagn 10:43–49.
2161 Wang H, Kim SS, Zhuo M. 2010. Roles of fragile X mental retardation protein in dopaminergic stimulation-induced synapse-associated protein synthesis and subsequent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor internalization. J Biol Chem 285:21888–21901. Wechsler D. 1997. Wechsler adult intelligence scale 3rd edition (WAISIII). 3rd ed. San Antonio, TX: Harcourt Assessment. Wechsler D. 2008. Wechsler adult intelligence scale 4th edition (WAIS-IV). 4th ed. San Antonio, TX: Harcourt Assessment. Wechsler D. 2009. Wechsler memory scale 4th edition (WMS-IV). 4th ed. San Antonio, TX: Harcourt Assessment. Wilkinson GS, Robertson G. 2006. Wide Range Achievement Test (WRAT4).
High Functioning Male with Fragile X Syndrome and Fragile X-Associated Tremor/Ataxia Syndrome Kirin Basuta,1 Andrea Schneider,2,3 Louise Gane,2 Jonathan Polussa,2,3 Bryan Woodruff,4 Dalyir Pretto,1 Randi Hagerman,2,3 and Flora Tassone1,2* 1
Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Sacramento, California
2
MIND Institute, University of California, Davis, Medical Center, Sacramento, California Department of Pediatrics, University of California at Davis Medical Center, Sacramento, California
3 4
Department of Neurology, Mayo Clinic, Scottsdale, Arizona
Manuscript Received: 17 December 2014; Manuscript Accepted: 6 April 2015
Fragile X syndrome (FXS) affects individuals with more than 200 CGG repeats (full mutation) in the fragile X mental retardation 1 (FMR1) gene. Those born with FXS experience cognitive and social impairments, developmental delays, and some features of autism spectrum disorders. Carriers of a premutation (55–200 CGG repeats) are generally not severely affected early in life; however, are at high risk of developing the late onset neurodegenerative disorder, Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), or Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and may have other medical conditions such as developmental delay, autism spectrum disorders, hypertension, anxiety, and immune-mediated disorders. Here we present a case of a 58-year-old man with a borderline IQ, average memory skills, and executive function deficits. He met criteriafor multiple psychiatric diagnoses and presented with tremor and ataxia, meeting criteria for FXTAS. Molecular testing unveiled a completely unmethylated FMR1 full mutation in peripheral blood mononucleated cells with elevated FMR1 mRNA and premutation alleles of different sizes in two other tissues (primary fibroblasts and sperm), indicating the presence of allele instability based on both inter- and intra-tissue mosaicism. The observation of FXTAS in this case of a full mutation mosaic man suggests that the pathogenic mechanism underlying this disorder is not observed exclusively in premutation carriers as it was originally thought. The concomitant presence of features of FXS and late onset neurological deterioration with probable FXTAS likely result from a combined molecular pathology of elevated FMR1 mRNA levels, a molecular hallmark of FXTAS and low FMRP expression that leads to FXS. Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Basuta K, Schneider A, Gane L, Polussa J, Woodruff B, Pretto D, Hagerman R, Tassone F. 2015. High functioning male with fragile X syndrome and fragile Xassociated tremor/ataxia syndrome. Am J Med Genet Part A 167A:2154–2161.
methylation with consequent silencing expression of the FMR1 protein, FMRP. The lack of FMRP leads to up-regulation of synaptic signaling proteins as FMRP is an essential translational repressor [Chen et al., 2014] ubiquitously expressed throughout many tissues [Pieretti et al., 1991; Hinds et al., 1993; Tassone et al., 1999], most importantly in neural cells [Sidorov et al., 2013; Beebe et al., 2014; Doers et al., 2014]. The absence of FMRP effects neuronal networks of cognition and leads to severe learning disabilities, social and behavioral impairments, as well as the clinical features usually falling under the umbrella of autism spectrum disorders (ASD) [Bagni and Oostra, 2013]. In individuals carrying a premutation allele with shorter expansions of 55–200 CGG repeats, FMR1 typically is not silenced. Instead the gene is excessively transcribed producing 2–6-fold higher mRNA levels as observed in peripheral blood mononucleated cells (PBMCs) [Tassone et al., 2000; Sullivan et al., 2005] and in brain tissue [Pretto et al., 2014a]. However, FMRP levels are reduced with declining expression depending on the length of the
Key words: fragile X syndrome; FXTAS
INTRODUCTION Fragile X syndrome (FXS) is a trinucleotide repeat expansion disorder. More than 200 CGG repeats in the promoter region of the fragile X mental retardation 1 (FMR1) gene trigger hyper-
Ó 2015 Wiley Periodicals, Inc.
Conflict of interest: none. Grant sponsor: NICHD; Grant numbers: HD02274, HD036072. Correspondence to: Flora Tassone, Ph.D., Department of Biochemistry and Molecular Medicine, 2700 Stockton Blvd Suite 2102, Sacramento, CA 95817. E-mail [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 29 April 2015 DOI 10.1002/ajmg.a.37125
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BASUTA ET AL. CGG expanded alleles. Some premutation carriers, especially males with a large premutation, may experience mild cognitive deficits, but they are at increased risk of developing the late onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS) [Hagerman et al., 2001], characterized by intentional tremor, gait ataxia, neuropathy, and parkinsonism [Hagerman and Hagerman, 2013]. The leading hypothesis suggests that FXTAS is caused by RNA toxicity, which results from the elevated FMR1 mRNA, particularly affecting neuronal signaling pathways in the brain [Wang et al., 2010; Kindler and Kreienkamp 2012; Deng et al., 2013; Friedman et al., 2013; Beebe et al., 2014; Colvin and Kwan, 2014; Sidorov et al., 2013]. Although FXTAS was initially thought to affect only premutation carriers, recent studies have reported individuals with an intermediate allele (45–54 CGG repeats) also being diagnosed with FXTAS. Liu et al. [2012] described three individuals (two males and a female) with FXTAS carrying an intermediate allele with elevated levels of FMR1 mRNA. Another study described three additional individuals with an intermediate allele, presenting with FXTAS symptoms of different severities [Hall et al., 2012]. In addition, FXTAS has been reported in individuals with the full mutation. One study reported a full mutation mosaic male with unmethylated alleles ranging from 180 to 410 CGG repeats and a sevenfold increase in FMR1 mRNA [Santa Maria et al., 2013]. Another mosaic male was reported with a fully unmethylated full mutation (297–480 CGG repeats) and a threefold increased FMR1 mRNA levels [Loesch et al., 2011]. Finally, very rare intranuclear inclusions, the neuropathological hallmark of FXTAS, were observed in three males with a full mutation [Hunsaker et al., 2011]. In this study, we present a case of a mosaic male with intra- and inter-tissue mosaicism presenting with clinical features of both FXTAS and FXS.
METHODS Study Subject This participant was recruited through the Genotype–Phenotype study and informed consent, approved by the University of California at Davis, Medical School IRB was signed.
Molecular Measures Genomic DNA (gDNA) was extracted from fibroblasts, blood, and sperm tissue samples utilizing standard protocols (Qiagen, Valencia, CA). PCR amplification and Southern Blot analyses were run on gDNA from the three tissue samples to determine the CGG repeat length and methylation status as previously described [Tassone et al., 2008; Filipovic-Sadic et al., 2010]. mRNA expression levels for the FMR1 gene were quantified using a 7900 sequence detector (Applied Biosystems, Foster City, CA) using conditions previously described in Tassone et al. [2000]. Western Blot analysis using PBMCs and fibroblasts protein extracts derived from five controls and the patient’s samples. Specific antibodies against FMRP (cat# MAB2160, Merck Millipore, Darmstadt, Germany) and GAPDH (cat# AB2302, EMD Millipore, Darmstadt, Germany) were used as previously described [Pretto et al., 2013].
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Neuropsychological Measures The neuropsychological/neuropsychiatric assessments included the following standardized IQ tests: Wechsler Adult Intelligence Scales (WAIS-III and WAIS-IV) [Wechsler, 1997, 2008]. Memory function was assessed using the Wechsler Memory Scales, Fourth Edition (WMS-IV) [Wechsler, 2009]. The Reading subtest from the Wide Range Achievement Test (WRAT 4) [Wilkinson and Robertson, 2006] was administered by an outside psychologist. The MiniMental Status Exam (MMSE) [Folstein et al., 1975], and Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) [First et al., 1997] were administered to evaluate mental status and psychiatric disorders. For the assessment of attention and executive function, the Integrated Visual and Auditory Continuous Performance Test (IVA-CPT) [Sandford et al., 1995] and the Behavioral Dyscontrol Scale 2nd edition (BDS-2) [Grigsby et al., 2002] were administered. The Autism Diagnostic Observation Schedule (ADOS2) [Lord et al., 1999] was used to evaluate for symptoms of ASD.
RESULTS Family History The patient has three obligate carrier daughters who have not been tested for FMR1 related disorders. His oldest daughter is 40 years old and has a daughter reported as being gaze avoidant and shy, and a son, both with unknown FMR1 gene status. The second daughter is 39 years old and has three sons, one who was diagnosed with FXS while the others have not been tested for FXS. The youngest daughter is 38 years old and has four sons and one daughter. One son is reported to have FXS but FMR1 DNA test results were unavailable. The other children have not been tested. The patient had three brothers, one who died neonatally, and one sister. One of these brothers is 60 years old and is reported to have balance problems and vertigo suggestive of FXTAS. A second brother, age 51, is reported to have depression and to live isolated and reclusive from others. His sister is 51 years old and is reported to have migraines, mood instability, and depression. The patient’s siblings have not been tested. The patient’s mother is reported to have died in her 80s. Her cause of death was attributed to a urinary tract infection in addition to a history of gait ataxia with many falls, sleep apnea, thyroid problems, and possible dementia, suggestive of diagnosis of FXTAS.
Medical History The patient was referred to the Mayo clinic and subsequently to the MIND Institute at age 58 with a history of neurological problems. He was diagnosed with the FMR1 full mutation after his grandson was diagnosed with FXS. His childhood medical history includes premature birth at 30 weeks gestation (BW 4.25 pounds) requiring an incubator for several weeks without ventilation needed. He was breastfed for several months. In school he required special education classes for reading, he had problems with eye contact and occasional hand flapping, and a history of hyper-sensitivity to noises. The patient graduated from high school and worked in construction for 17 years before joining the Army. He had chronic
2156 outburst behavior and obsessive-compulsive behavior and was diagnosed with bipolar disorder and treated with lithium. At 39 years old, he required surgery for a neck injury following a parachute jump and experienced chronic cervical pain since that time, associated with degenerative disc disease. At age 44, the patient developed symptoms consistent with cauda equina syndrome with urinary and bowel incontinence as well as erectile dysfunction. This prompted emergent decompressive surgery. He began intermittent use of an electric wheelchair at age 45. He developed hypogonadism and began testosterone replacement therapy. With rehabilitation, his legs grew stronger but began giving out on a regular basis at about age 50 and he began using his wheelchair regularly. At age 50 he developed an intermittent intention tremor and more severe gait problems with frequent falling. He developed painful neuropathy symptoms in his lower legs as well as restless legs syndrome (RLS). He also developed sleep apnea, treated with CPAP. At age 51, he began using opioids for pain control, including oxycodone, lidocaine jelly, and Lidoderm patches. At age 57 he developed migraines with visual aura. His current complaints include migraines, irritable bowel syndrome, RLS, gastroesophageal reflux disease, myalgias of his upper arms and shoulders, arthralgias in his hands, occasional severe leg cramps, severe constipation, subjective memory difficulties, depression/bipolar disorder, and a 30–50% loss of exercise stamina over the past few years. On examination at age 58 the proband had a height of 179.5 cm, weight of 116.2 kg (for a BMI of 36.1), blood pressure of 138/ 89 mmHg, a pulse of 70 bpm, head circumference of 59.4 cm. The patient has a long face with mildly prominent ears and his hearing was mildly decreased bilaterally. Cranial nerve examination demonstrated mild end-gaze nystagmus with lateral gaze. The patient uses dentures, but nose and throat exam was otherwise normal and he has an intact gag reflex. Neck was supple without adenopathy, and thyroid gland was palpably normal. Finger joints were not hyperextensible, with some guarding noted due to arthritis. The patient did not have scoliosis, but did have scars on his spine in the neck and lumbar area due to past surgeries. The patient has a normal phallus and macroorchidism with a testicular volume of 70 ml bilaterally. The patient exhibited diffuse areflexia. Plantar responses were flexor bilaterally. Vibration sense was absent in his left foot and decreased in his right foot. Pinprick sensation was normal. Muscle strength was normal in all four limbs. The patient’s gait was broadbased with listing to one side, and he could not perform tandem gait. He exhibited diminished rate of alternating movements diffusely. The patient’s current medications include Fortesta for hypogonadism several times per day, ondansentron (8 mg) for nausea, omeprazole 20 mg daily for reflux, oxycodone 40 mg twice daily for pain, donepezil 10 mg daily for memory, hydroxyzine 25 mg for panic attacks and stress, Lipozene 15 mg daily for weight loss, lithium 300 mg twice a day for bipolar disorder, gabapentin 600 mg twice daily for neuropathy, nabumetone 500 mg twice daily for arthritis, and Lexapro 40 mg daily for depression.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
Diagnostic Procedure Summary Initial MRI at age 56 demonstrated a few scattered subcortical foci of white matter hyperintensity adjacent to the atrium of the left lateral ventricle (Fig. 1). Repeat MRI two years later demonstrated a thin corpus callosum, dilated ventricles, and scattered white matter hyperintensity similar to his previous MRI. No hyperintensity of the middle cerebellar peduncles was noted. MRI of the lumbar spine at age 57 demonstrated post-operative changes from prior decompressive laminectomy at L2–L3 without residual central canal stenosis at any level. Small far lateral disc protrusion was noted at L3–L4 on the left with potential impingement of the exiting L3 nerve root. Mild bilateral foraminal stenosis was also noted at L4–L5 and L5–S1. EEG at age 56 demonstrated mild to moderate generalized slowing, but no potentially epileptogenic activity. EMG of the upper and lower extremities at age 55 demonstrated a length-dependent primarily axonal sensorimotor peripheral neuropathy with superimposed moderate right median neuropathy at the wrist. Repeat EMG in 2013, at age 58, demonstrated some improvement in the right median neuropathy.
Neuropsychological Findings Table I summarizes the results from the comprehensive neuropsychological assessment at the UCD MIND Institute and previous records from outside sources.
FIG. 1. Axial CUBE MRI demonstrating minimal white matter hyperintensity adjacent to the posterior horn of the left lateral ventricle.
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TABLE I. Overview of Neuropsychological Assessments Assessment Reading Cognitive assessment
Memory
Executive function
Age 55
Age 58
(WRAT) Reading standard score ¼ 83 (WAIS-III) Verbal comprehension ¼ 103 Perceptual reasoning ¼ 82 Full scale IQ ¼ 90 Working memory index ¼ 88 Processing speed index ¼ 84 Auditory verbal learning test ¼ 27 Percent recall ¼ 38
Attention
Trail marking test Trail A - MOANS ¼ 2 Trail B - MOANS ¼ 6 WAIS III digit span ¼ 10
Mental status
(not done)
Autism spectrum disorders
(not done)
(not done) (WAIS-IV) Verbal comprehension ¼ 108 Perceptual reasoning ¼ 79 Full scale IQ ¼ 84 Working memory index ¼ 71 Processing speed index ¼ 84 (WMS-IV) Auditory memory ¼ 94 Visual memory ¼ 98 Visual working memory ¼ 91 Delayed memory score ¼ 98 Immediate memory ¼ 93 (BDS-2) 6* (out of 27), severely impaired (IVA) Response control quotient (RCQ) ¼ 76 Auditory RCQ ¼ 70 Visual RCQ ¼ 90 Attention quotient (AQ) ¼ 82 Auditory AQ ¼ 82 Visual AQ ¼ 84 (MMSE) 24* (out of 30), mildly impaired (ADOS-4) No symptoms of ASD
Scores are reported in the standard score scale with a mean of 100, standard deviation of 20, *score reported as raw score because there are no normative data available. WRAT, wide range achievement test; WAIS-III, Wechsler adult intelligence scale 3rd edition; WAIS-IV, Wechsler adult intelligence scale 4th edition; WMS-IV, Wechsler memory scale 4th edition; BDS-2, behavioral dyscontrol scale 2nd edition; IVA, integrated visual and auditory attention test; MMSE, mini mental state exam; ADOS-4, Autism diagnostic observation schedule module 4. MOANS, Mayo Older American Normative Study.
On the SCID-I (Structured Clinical Interview for DSM-IV Axis I Disorders) the patient meets criteria for Bipolar I Disorder with rapid cycling, most current episode depressive. The patient reports some psychotic symptoms with hearing voices, particularly his wife calling him, and reports religious perseverations and grandiosities. The patient meets criteria for panic disorder and generalized anxiety disorder. He reports significant obsessive-compulsive symptoms with frequent lock checking and sexual obsessions. At his visit, he insisted on donating sperm. The patient also meets criteria for somatization disorder.
Molecular Measures PCR and Southern Blot analyses of the patient’s PBMCs reveal FMR1 alleles ranging from 20 to 800 CGG repeats, >90% unmethylated (Fig. 2a,b); An unmethylated 200 CGG repeat premutation allele was present in primary fibroblasts (Fig. 2a,c) while an 86 CGG premutation allele was present in sperm (Fig. 2a,d) indicating inter-tissue instability. In all samples, allelic instability (intra-tissue mosaicism) was also observed and visualized by a series of peaks on capillary electrophoresis (Fig. 2b,c,d). FMR1 mRNA expression level in PBMCs was threefold higher than normal (2.98 0.06). CGG allele instability and mosaicism were
observed between and among all three tissues. FMRP expression levels in peripheral blood cells (PBMCs) and primary fibroblast cells, measured by Western blot analysis were 75% lower than controls in PBMCs and 50% lower in fibroblasts.
DISCUSSION The term FMR1 mosaicism indicates the presence of FMR1 alleles with either multiple different CGG repeat sizes (i.e., premutation and full mutation alleles, termed as size mosaic), different methylation status (i.e., unmethylated and methylated full mutation size alleles, termed methylation mosaic), or differences in both size and methylation (unmethylated alleles that span both premutation and full mutation ranges and methylated alleles, termed methylation and size mosaic). Differences in methylation and CGG repeat size can be observed between different cell types, this is termed intertissue mosaicism. These differences can also be observed within the same type, and this is termed intra-tissue mosaicism. In this study we present clinical and molecular findings in a mosaic man (for size and methylation). DNA testing revealed the presence of both FMR1 inter- and intra-tissue (PBMCs, primary fibroblasts and sperm) CGG allele size and methylation mosaicism. Alleles of different CGG size were observed in all three tissues analyzed. As
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FIG. 2. Methylation status and CGG size instability as detected by Southern blots (SB) and capillary electrophoresis. The SB analysis (a) demonstrates the presence of a series of unmethylated alleles (smear) spanning from the normal range (2.8 Kb) through the full mutation methylated range (>5.2 Kb) in peripheral blood lymphocytes (PBMCs). The presence of a premutation allele is visible in both primary cultured fibroblasts and sperm. M ¼ 1 Kb DNA size ladder marker; C1 ¼ normal female, negative control. The electrophoregrams show CGG instability as illustrated by the presence of serial peaks, each representing single distinct alleles, with differences between PBMCs (b) fibroblasts (c) and sperm (d). The X-axis indicates the size of the alleles in base pairs and the Y-axis indicates the fluorescence intensity of each allele.
expected, a premutation allele with a CGG repeat number within the premutation range was observed in sperm cells [Reyniers et al., 1993]. A premutation allele, of different size, was also observed in fibroblasts while, a series of unmethylated alleles, from the normal range to the full mutation range were observed in PBMCs demonstrating the presence of inter- and intra-tissue mosaicism. This finding is significant because it suggests that a double molecular mechanism, derived from both the elevated FMR1 mRNA and lower FMRP expression levels observed in this individual, likely contributed to his clinical phenotype [Pretto et al., 2014a,b]. In particular, the moderate lower FMRP expression, as measured in
PBMC cells, likely contributed to the development of a mild cognitive deficit, as his IQ falls in the low average range. However, he had a successful military career, was married and fathered children. He had the physical features of FXS including macroorchidism and the behavioral problems of ADHD, anxiety and mood instability but he was much higher functioning than a typical male patient with FXS. His FXTAS picture was also atypical and complicated by his early cauda equina syndrome from trauma, but by age 50 the FXTAS neurodegenerative symptoms began to develop as tremor, more severe ataxia, increasing pain symptoms, and memory problems were observed during examination. His
BASUTA ET AL. executive function deficits were likely present since childhood along with his ADHD but the presence of FXTAS has likely enhanced their severity. Additional possible manifestations of premutation involvement include his migraine headaches [Au et al., 2013] and his RLS [Summer et al., 2013]. Sleep apnea is also more common in premutation carriers than controls and if left untreated this can exacerbate FXTAS [Hamlin et al., 2011]. His psychotic thinking is atypical for FXTAS in general but we have reported this in two cases with a double hit of elevated FMR1 mRNA and lowered FMRP [Schneider et al., 2013]. One concerning issue is his heavy use of opioids for pain control because there is the potential for opioids to exacerbate white matter disease [Muzar et al., 2014]. His pain symptoms arise from both neuropathy and arthritis. However, the muscle pain may also be the result of a central pain syndrome, as it is similar to fibromyalgia symptoms described in premutation women [Leehey et al., 2011]. Although he does not have the MCP sign, he has thinning of the corpus callosum, brain atrophy, and scattered white matter disease, which meets criteria for probable FXTAS [Jacquemont et al., 2014]. In the revision of the diagnostic criteria for FXTAS, the constraint of the premutation has been expanded to include those with other allele sizes including the gray zone and full mutation alleles, particularly those that are unmethylated, in which elevated FMR1 mRNA levels are observed [Hall et al., 2014]. We have previously reported a significant association between FMRP expression and IQ in individuals with premutation and partial methylation (size and methylation mosaics) [Pretto et al., 2014a] and also in full mutation methylation mosaic males [Pretto et al., 2014b]. In addition, the number of phenotypic features was associated with the level of expression of FMRP [Pretto et al., 2014a]. Although the proband did not present the typical spectrum of FXS clinical features, he did have macroorchidism, psychiatric problems, and behavioral issues that probably were present since childhood as evidenced by reports of hand flapping and anxiety, likely due to the FMRP deficit. The proband also presented with elevated FMR1 mRNA, resulting in RNA toxicity likely leading to the premutation pathology observed, particularly the current manifestations of FXTAS symptoms. We have previously reported on symptoms, pathological and molecular evidence of FXTAS in three cases of full mutation mosaic males, two with a methylation mosaicism [Loesch et al., 2011; Santa Maria et al., 2013] and one with a size mosaicism [Pretto et al., 2014b]. The latter case also presented with co-morbidity for both FXS and FXTAS. His typical features of FXS included a mildly long face, large and mildly prominent ears, macroorchidism, perseverative speech, and anxiety. The presence of intranuclear inclusions, one of the neuropathological criteria for the diagnosis of FXTAS, was observed in several regions of his brain [Pretto et al., 2013]. This case of FXTAS in a male with mosaicism indicates that the risk of developing pathogenic mechanisms leading to a neurodegenerative disorder may be confined not only to those with expanded premutation alleles but also to those with unmethylated full mutation alleles with elevated levels of toxic FMR1 mRNA. In addition, the clinical presentation of the case presented here, shares the molecular and clinical phenotype of both premutation involvement and FXS. Thus, it is becoming more evident that the genetic makeup of individuals with mosaicism, mostly when high pro-
2159 portions of unmethylated CGG repeat expansions are present, may be very complex due to the presence of both RNA toxicity and lower FMRP expression. This case illustrates how the determination of size and methylation status is not only informative but may also be important in establishing ways of managing the disorder from the clinical perspective. It also highlights a not so uncommon scenario of a patient that was unaware that an FMR1 mutation was present in his genetic background and that this mutation is likely responsible for his clinical symptoms and also of those observed in his family members. A likely reason for this delayed knowledge is that mosaic individuals may present with a milder phenotype during childhood as well as during early adulthood, limited mostly to a lower IQ, anxiety, attention deficit/hyperactivity disorder, and behavioral issues [Aziz et al., 2003; Farzin et al., 2006; Bailey et al., 2008] that do not significantly interfere with a normal life. It is not until symptoms of neurological decline start to develop, generally after age 50, that the patient seeks medical attention and diagnostic testing for an FMR1 mutation may be requested by a physician who is aware of the clinical spectrum of patients suspected of developing FXTAS. Finally, the case presented here illustrates the need to further investigate the molecular background of premutation and full mutation cases presenting complex clinical spectra. The methylation status and CGG repeat size may be equally important for accurate diagnosis.
ACKNOWLEDGMENT This work was supported by NICHD grants HD02274 and HD036072. This work is dedicated to the memory of Matteo. Dr. F. Tassone has received funding from Roche and consulted with Novartis. Dr R. Hagerman has received funding from Novartis, Roche, Alcobra, and Seaside Therapeutics to carry out treatment trials in fragile X syndrome. She has also consulted with Novartis, Roche, and Genentech on treatment of fragile X syndrome.
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