- Email: [email protected]
Ataxia Syndrome
Physiology/Medical Disease Testicular and Pituitary Inclusion Formation in Fragile X Associated Tremor/Ataxia Syndrome Claudia M. Greco, Kultida Soontrapornchai, Juthamas Wirojanan, John E. Gould, Paul J. Hagerman and Randi J. Hagerman* From the Departments of Pathology (CMG) and Biochemistry and Molecular Medicine (PJH), University of California Davis School of Medicine, Davis and Medical Investigation of Neurodevelopmental Disorders Institute (CMG, KS, JW, RJH), Department of Urology (JEG) and Department of Pediatrics (RJH), University of California Davis Medical Center, Sacramento, California
Purpose: We describe the medical course, neuropathology and testicular pathology in 2 men who died with fragile X associated tremor/ataxia syndrome. Fragile X associated tremor/ataxia syndrome, which is a recently described, late onset neurodegenerative disorder, affects up to a third of males and occasionally females older than age 50 years who are carriers of premutation alleles (55 to 200 CGG repeats) of the fragile X mental retardation 1 gene FMR1. Clinical manifestations of premutation status are distinct from those of the full mutation, which is the cause of the fragile X syndrome. Materials and Methods: Standard pathological techniques were used to examine the brain, pituitary gland and testicular tissues of 2 males who had fragile X associated tremor/ataxia syndrome. Results: The clinical course of the 2 cases included impotence before the onset of neurological symptoms of tremor and ataxia. Neuropathological findings included eosinophilic intranuclear inclusions in neurons and astrocytes throughout the central nervous system, and in the anterior and posterior pituitary gland of 1 of the 2 men. Inclusions were also seen in the Leydig and myoid cells in the testicles of these 2 men with fragile X associated tremor/ataxia syndrome. Conclusions: Fragile X associated tremor/ataxia syndrome inclusions are formed in tissues outside of the central nervous system. Involvement of the testicles and the pituitary gland may lead to neuroendocrine dysfunction, including testosterone deficiency. These noncentral nervous system components of fragile X associated tremor/ataxia syndrome require further study. Key Words: testes; FMR1 protein, human; Leydig cells; pituitary gland; ataxia
remutation carrier status (55 to 200 CGG repeats) of the FMR1 gene (OMIM ⫹309550) is found in approximately 1/260 women and 1/800 men1,2 in the general population. The premutation is the cause of premature ovarian failure3 and FXTAS.4 In contrast, full mutation of the FMR1 gene (greater than 200 CGG repeats) causes FXS, a neurodevelopmental disorder that leads to mental retardation and autism or severe learning disabilities. For the latter condition the gene is generally silenced and little or no FMRP is produced. FXTAS is a condition consisting of progressive intention tremor, ataxia, dementia, cognitive decline, Parkinsonism and autonomic dysfunction, including impotence, hypertension, and eventual loss of bowel and bladder control.4 It
P
Submitted for publication July 28, 2006. Study received University of California Internal Review Board approval. Supported by National Institute of Child Health and Human Development Grants HD36071 and HD02274, National Institute of Neurological Disorders and Stroke Grant NS044299, National Institute on Aging Grant AG024488, Collaborative Agreement U10/ CCU925123 with the Center for Disease Control and Prevention, and the Medical Investigation of Neurodevelopmental Disorders Institute, University of California-Davis. * Correspondence: Department of Pediatrics, Medical Investigation of Neurodevelopmental Disorders Institute, University of California Davis Health System, 2825 50th St., Sacramento, California 95817 (telephone: 916-703-0247; FAX: 916-703-0240; e-mail: [email protected]).
0022-5347/07/1774-1434/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION
affects approximately a third of male carriers older than 50 years, as ascertained through known fragile X families.5,6 Furthermore, FXTAS accounts for 2% to 5% of adult onset cerebellar ataxia in males.7,8 Neuroimaging in FXTAS cases revealed symmetrically increased T2-weighted signal intensity in the MCPs (MCP sign) in approximately 60% of cases, while additional high signal changes were found in contiguous cerebellar white matter, and in subcortical and periventricular white matter.9,10 Cerebral, cerebellar and brainstem atrophy is also a general feature of FXTAS. Neuropathological findings of FXTAS at autopsy include eosinophilic nuclear inclusions in neurons and astrocytes throughout the central nervous system, Purkinje cell loss, and significant cerebral subcortical and cerebellar white matter disease.10,11 The pathogenesis of FXTAS is thought to involve an RNA toxicity mechanism, due in part to expanded-repeat FMR1 mRNA levels that are 2 to 8 times normal in premutation carriers.4 FMR1 mRNA is present in the inclusions along with multiple proteins, including lamin A/C, ␣ B-crystallin and myelin basic protein.12,13 We identified intranuclear inclusions in the testicles of 2 males, and in the available anterior and posterior pituitary gland of 1 who died with autopsy proven FXTAS. MATERIALS AND METHODS Brain tissue, including the pituitary gland, was processed as previously described by Greco et al.10,11 Informed consent
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Vol. 177, 1434-1437, April 2007 Printed in U.S.A. DOI:10.1016/j.juro.2006.11.097
TESTICULAR INCLUSIONS IN FRAGILE X ASSOCIATED TREMOR/ATAXIA SYNDROME was obtained for the cases in this study, as approved by the University of California Internal Review Board. For case 1 the 2 testicles were removed at autopsy with portions of each immediately snap frozen in liquid nitrogen and fixed in 10% formalin. For case 2 a single testicle was fixed in 10% formalin. Testicular tissue from each case was processed for paraffin embedding according to standard protocol, and then examined by hematoxylin and eosin, and trichrome stains. Immunostains using anti-ubiquitin, neurofilament and anti-human muscle (clone HHF35) antibodies were performed using standard methodology, as previously described. Manual counts of inclusions in Leydig and tubular wall myoid cells were done in each case by 1 of us (CMG) using an Olympus® BX40 microscope at 1,000⫻ magnification on ubiquitin immunostained slides in a grid pattern for 60 fields. Histological review of control testicular tissue from 10 men 65 to 75 years old was also performed. Control cases were chosen from the autopsy files of the Department of Pathology at University of California Davis Medical Center. Anti-ubiquitin immunostaining on 5 of the 10 control cases was performed as described.
Case Histories Case 1. This 64-year-old male presented with a 9-year history of progressive tremor, ataxia and behavioral changes. At age 54 years he began to notice tremor in the right hand with activity. Tremor slowly progressed to involve each hand with impaired fine motor ability, leading to retirement as an electrician at age 58 years. At age 61 years the tremor occurred occasionally at rest. He also experienced frequent falls due to postural instability. He had poor memory for recent events and names of family members, disinhibition, distractibility, poor frustration tolerance and perseverative thoughts. He complained of decreased sensation of the lower extremities. Nerve conduction velocity and electromyography were normal. Medical history included hypertension, hypercholesterolemia, type 2 diabetes and impotence that began at age 50 years. CGG repeats were 98 with FMR1 mRNA levels at 3.38 times normal. He was the first patient at our center to be diagnosed with FXTAS. His daughter is a premutation carrier and has 2 children with FXS. Neurological examination at age 64 years showed facial masking, slurred speech and bilateral hearing loss. He had a symmetrical 4 to 6 Hz large amplitude intention tremor bilaterally. He had a wide based, slow and lurching gait. Finger-to-nose and heel-to-shin movements were ataxic and dysmetria was more prominent on the right side. He had an intermittent resting and static postural tremor in the arms, slight cogwheel rigidity and minimal bradykinesia. Sensation to vibration, light touch and pinprick were decreased in each lower extremity. Muscle strength was graded 5/5 in the arms and 4/5 bilaterally in the proximal lower extremities. Deep tendon reflexes were 1⫹ in the upper extremities and they could not be elicited in the lower extremities. Cognitive evaluation at age 64 years included the MiniMental Status Examination with a score of 18/30, consistent with the diagnosis of dementia (full scale IQ 83, verbal IQ 93 and performance IQ 73). Magnetic resonance imaging revealed mild to moderate generalized atrophy with enlarged ventricles and it was without the MCP sign.
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Symptoms of tremor, ataxia, cognitive and psychological problems progressed, leading to use of a cane at age 66 years and then a wheelchair at age 68 years. Urinary and bowel incontinence developed and the patient was bedridden at age 68.5 years. He died at age 69 years. Case 2. This man, whose clinical history was previously reported in 2006 by Greco et al as case 5,11 was a carrier of the fragile X premutation allele and the grandfather of 2 girls with FXS. He had a successful business career before retirement. A history of impotence began at age 68 years, and muscle weakness and ataxia developed beginning at age 71 years. At age 73 years he had memory problems and began using a walker. He had a pacemaker in place, so that magnetic resonance imaging could not be performed. He died of congestive heart failure at age 74 years. Pathological Findings Case 1. Pathological findings for this case have not been reported. Autopsy tissue was harvested within 8 hours of death using previously published protocols.10,11 One hemisphere of the brain was frozen at ⫺70C for further studies. Coronal sections of the formalin fixed hemisphere showed mild ventricular dilatation and no abnormalities of the cerebral cortex, basal ganglia, brainstem or cerebellum. Histological sections showed ubiquitin positive intranuclear inclusions in neurons and astrocytes, as in all previously described affected cases.10,11 Otherwise the brain showed mild to moderate vascular hyalinosis in deep white matter vessels on hematoxylin and eosin, myelin (luxol fast blueperiodic acid-Schiff) and axon (anti-neurofilament antibody) stains. Hematoxylin and eosin stained sections of the pituitary gland showed no abnormalities except intranuclear inclusions in the anterior and posterior components of the gland (fig. 1). The average diameter of inclusions in the anterior and pituitary gland was 2.3 . Inclusions were more easily identifiable on hematoxylin and eosin staining in the posterior than the anterior pituitary gland. Inclusions were obvious in each area on ubiquitin immunostaining. Case 2. As reported as case 8 in 2006 by Greco et al,11 on external examination brain pathology showed frontal, rostral cingulate and superior temporal gyral atrophy. Moderate ventrigulomegaly was present. Histological examination showed intranuclear inclusions in neurons and astrocytes throughout the brain. There was patchy white matter loss in subcortical cerebral white matter and in the MCPs.
FIG. 1. Case 1. A, ubiquitin positive inclusions (arrows) were easily identified in anterior pituitary gland. Immunostaining, reduced from ⫻400. B, inclusions in posterior pituitary gland. Ubiquitin immunostaining, reduced from ⫻400. Inset, arrow indicates inclusion. H & E, reduced from ⫻400.
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Testicular Pathology Gross findings on examination of the testicles from patients 1 and 2 were similar with size and appearance indistinguishable from those of normal age matched controls. General histological features on hematoxylin and eosin staining were similar, showing nonspecific atrophic changes, including a decreased number of Leydig cells, tubular fibrosis and decreased spermatogenesis. Sertoli’s cells were numerous in the tubules, and a small number of germ cells and rare spermatozoa were the remnants of sperm maturation. Eosinophilic intranuclear inclusions indistinguishable from those present in the brain of individuals with FXTAS10,11 were seen in the 2 cases in Leydig and myoid cells of the tubular walls on hematoxylin and eosin staining (fig. 2). The average measurement of inclusions immunostained with ubiquitin antibody in the 2 cell populations was 2.6 . Using the method described case 1 showed 5.1% of Leydig cells and 4.2% of spermatic tubule myoid cells with intranuclear inclusions. Case 2 showed inclusions in 1.6% of Leydig cells and 4.8% of spermatic tubule myoid cells. In case 2 inclusions were not identified in Sertoli’s or sperm cells, although
FIG. 2. A, case 2. Low power view shows nonspecific atrophic changes in testes with decreased spermatogenesis evidenced by little germ cell differentiation and rare to absent spermatid formation (a), decreased number of interstitial Leydig cells (b) and thickened, fibrotic tubular walls (c). H & E, reduced from ⫻100. B, case 1. Medium power view shows spermatic tubule with multiple intranuclear inclusions (arrow) in tubular wall. H & E, reduced from ⫻200. C, case 2. Intranuclear inclusions (arrows) are identified in Leydig cell population. H & E, reduced from ⫻400. D, case 2. positive staining of inclusions in Leydig cells. Ubiquitin immunostaining, reduced from ⫻400. E, case 2. Positive staining of inclusions in myoid cells of tubular walls. Ubiquitin immunostaining, reduced from ⫻400.
they could occasionally be seen in myoid cells of the tunica vascularis and albuginea. The myoid nature of inclusion bearing cells in tunical layers was verified by immunohistochemical staining with anti-muscle actin antibody (clone HHF35). Histological examination of 10 age matched control testicles showed decreased spermatogenesis, a decreased number of Leydig cells and tubular fibrosis, which on average was similar to that seen in our 2 cases. Intranuclear inclusions were not observed on hematoxylin and eosin stained sections in 10 of the 10 control cases or on ubiquitin immunostained sections in 5. DISCUSSION This report documents intranuclear inclusions in the myoid cells of spermatic tubules and Leydig cells of the testicles in 2 men with FXTAS. These inclusions appeared identical to those previously reported in the brain of patients with FXTAS.13,14 In FXS macro-orchidism is thought to be caused by a deficiency of FMRP. Macro-orchidism is not typically seen in men with the premutation except in the occasional carrier who has a mild FMRP deficit. We also saw inclusions typical of FXTAS in the anterior and posterior pituitary gland of case 1. Inclusions at this location were reported previously in 1 patient with FXTAS.15 Pituitary inclusions may be related to dysregulated neuroendocrine function, including luteinizing hormone and follicle-stimulating hormone levels in patients with the premutation. In females with the premutation follicle-stimulating hormone increases are common in those with premature ovarian failure.3 We have also seen intranuclear inclusions in the anterior and posterior pituitary gland of 1 female carrier with FXTAS who showed intranuclear inclusions in the brain (unpublished data). This may be further support for neuroendocrine dysregulation in carriers. The presence of intranuclear inclusions in myoid cells in testicular fibrous tissue in FXTAS is intriguing. The tunica propria of the testicle is a component of the tunica albuginea and it is the middle of the 3 layers of the fibrous capsule beneath the scrotal skin that protects and supports the testes. Among other cellular components the tunica albuginea contains myofibroblasts. In the tunica propria smooth muscle cells are involved in contractile and transport functions.16 Inclusions in these cells suggests that other structures outside of the central nervous system that contain such cells may also have inclusions. This could be beneficial diagnostically or for monitoring the therapeutic response. Because Leydig cells produce testosterone, we were concerned about possible testosterone deficiency in FXTAS. Although the 2 men reported on did not have testosterone tested during life, we know of 3 with FXTAS in whom testosterone was measured. The 2 men who were deficient in this hormone were treated with intramuscular testosterone with positive effects in energy and mood, while the other had low normal serum testosterone and was not treated. It is known that testosterone treatment can have positive effects on cognition and memory,17 which are problems for individuals with FXTAS.4 The 2 men presented with impotence before the onset of other neurological signs. In general male sexual dysfunction is multifactorial. Hormonal causes are diabetes mellitus, testosterone deficiency, hyperprolactinemia, hyperthyroidism, hypothyroidism, Cushing’s syndrome and Addison’s
TESTICULAR INCLUSIONS IN FRAGILE X ASSOCIATED TREMOR/ATAXIA SYNDROME disease. Low serum testosterone can be caused by pituitary or testicular pathology, prescription or recreational drugs, chromosomal anomalies, environmental toxins and aging. A number of epidemiological studies described the relationship between aging and hypogonadism.18,19 Some data indicate a 110 ng/dl decrease in serum testosterone every 10 years in men between ages 66 and 80 years.20 In this report we describe what is to our knowledge a new histological finding in FXTAS that might provide clues to the etiology of low testosterone in some older males. Furthermore, this report underscores the importance of considering FXTAS in men older than 50 years who complain of erectile dysfunction and neurological symptoms. We recommend evaluating testosterone in males with the FMR1 premutation who have neurological symptoms, FXTAS or impotence. Urologists should be aware of FXTAS and consider testing for the FMR1 premutation in males older than 50 years who present with impotence and neurological findings such as ataxia, tremor or memory problems. Such testing is also important in patients with established impotence who report neurological abnormalities. Clinical impotence may precede neurological symptoms in male premutation carriers, as in the cases reported. Early treatment of testosterone deficiency may help some symptoms, at least transiently.
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Abbreviations and Acronyms FMR1 ⫽ fragile X mental retardation 1 gene FMRP ⫽ fragile X mental retardation 1 gene protein FXS ⫽ fragile X syndrome FXTAS ⫽ fragile X tremor/ataxia syndrome MCP ⫽ middle cerebellar peduncle
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REFERENCES 1.
Dombrowski C, Levesque ML, Morel ML, Rouillard P, Morgan K and Rousseau F: Premutation and intermediate-size FMR1 alleles in 10,572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles. Hum Mol Genet 2002; 11: 371. 2. Rousseau F, Rouillard P, Morel ML, Khandjian EW and Morgan K: Prevalence of carriers of premutation-size alleles of the FMRI gene–and implications for the population genetics of the fragile X syndrome. Am J Hum Genet 1995; 57: 1006. 3. Sullivan AK, Marcus M, Epstein MP, Allen EG, Anido AE, Paquin JJ et al: Association of FMR1 repeat size with ovarian dysfunction. Hum Reprod 2005; 20: 402. 4. Hagerman PJ and Hagerman RJ: The fragile-X premutation: a maturing perspective. Am J Hum Genet 2004; 74: 805. 5. Jacquemont S, Hagerman RJ, Leehey MA, Hall DA, Levine RA, Brunberg JA et al: Penetrance of the fragile X-associated tremor/ataxia syndrome in a premutation carrier population. J Am Med Assoc 2004; 291: 460.
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Loesch DZ, Churchyard A, Brotchie P, Marot M and Tassone F: Evidence for, and a spectrum of, neurological involvement in carriers of the fragile X pre-mutation: FXTAS and beyond. Clin Genet 2005; 67: 412. Jacquemont S, Leehey MA, Hagerman RJ, Beckett LA and Hagerman PJ: Size bias of fragile X premutation alleles in late-onset movement disorders. J Med Genet 2006; 43: 804. Van Esch H, Dom R, Bex D, Salden I, Caeckebeke J, Wibail A et al: Screening for FMR-1 premutations in 122 older Flemish males presenting with ataxia. Eur J Hum Genet 2005; 13: 121. Brunberg JA, Jacquemont S, Hagerman RJ, Berry-Kravis EM, Grigsby J, Leehey MA et al: Fragile X premutation carriers: characteristic MR imaging findings in adult males with progressive cerebellar and cognitive dysfunction. Am J Neuroradiol 2002; 23: 1757. Greco C, Hagerman RJ, Tassone F, Chudley AE, Del Bigio MR, Jacquemont S et al: Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers. Brain 2002; 125: 1760. Greco CM, Berman RF, Martin RM, Tassone F, Schwartz PH, Chang A et al: Neuropathology of fragile X-associated tremor/ataxia syndrome (FXTAS). Brain 2006; 129: 243. Iwahashi CK, Yasui DH, An HJ, Greco CM, Tassone F, Nannen K et al: Protein composition of the intranuclear inclusions of FXTAS. Brain 2006; 129: 256. Arocena DG, Iwahashi CK, Won N, Beilina A, Ludwig AL, Tassone F et al: Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells Hum Mol Genet 2005; 14: 3661. Tassone F, Hagerman RJ, Garcia-Arocena D, Khandjian EW, Greco CM and Hagerman PJ: Intranuclear inclusions in neural cells with premutation alleles in fragile X associated tremor/ataxia syndrome. J Med Genet 2004; 41: e43. Louis E, Moskowitz C, Friez M, Amaya M and Vonsattel JP: Parkinsonism, dysautonomia, and intranuclear inclusions in a fragile X carrier: a clinical-pathological study. Mov Disord 2006; 21: 420. Haider SG, Talati J and Servos G: Ultrastructure of peritubular tissue in association with tubular hyalinization in human testis. Tiss Cell 1999; 31: 90. Cherrier MM, Craft S and Matsumoto AH: Cognitive changes associated with supplementation of testosterone or dihydrotestosterone in mildly hypogonadal men: a preliminary report. J Androl 2003; 24: 568. Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ and McKinlay JB: Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urology 1994; 151: 54. Harman SM, Metter EJ, Tobin JD, Pearson J and Blackman MR: Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrin Metab 2001; 86: 724. Morley JE, Kaiser FE, Perry HM 3rd, Patrick P, Morley PM, Stauber PM et al: Longitudinal changes in testosterone, luteinizing hormone, and follicle-stimulating hormone in healthy older men. Metabolism 1997; 46: 410.
Purpose: We describe the medical course, neuropathology and testicular pathology in 2 men who died with fragile X associated tremor/ataxia syndrome. Fragile X associated tremor/ataxia syndrome, which is a recently described, late onset neurodegenerative disorder, affects up to a third of males and occasionally females older than age 50 years who are carriers of premutation alleles (55 to 200 CGG repeats) of the fragile X mental retardation 1 gene FMR1. Clinical manifestations of premutation status are distinct from those of the full mutation, which is the cause of the fragile X syndrome. Materials and Methods: Standard pathological techniques were used to examine the brain, pituitary gland and testicular tissues of 2 males who had fragile X associated tremor/ataxia syndrome. Results: The clinical course of the 2 cases included impotence before the onset of neurological symptoms of tremor and ataxia. Neuropathological findings included eosinophilic intranuclear inclusions in neurons and astrocytes throughout the central nervous system, and in the anterior and posterior pituitary gland of 1 of the 2 men. Inclusions were also seen in the Leydig and myoid cells in the testicles of these 2 men with fragile X associated tremor/ataxia syndrome. Conclusions: Fragile X associated tremor/ataxia syndrome inclusions are formed in tissues outside of the central nervous system. Involvement of the testicles and the pituitary gland may lead to neuroendocrine dysfunction, including testosterone deficiency. These noncentral nervous system components of fragile X associated tremor/ataxia syndrome require further study. Key Words: testes; FMR1 protein, human; Leydig cells; pituitary gland; ataxia
remutation carrier status (55 to 200 CGG repeats) of the FMR1 gene (OMIM ⫹309550) is found in approximately 1/260 women and 1/800 men1,2 in the general population. The premutation is the cause of premature ovarian failure3 and FXTAS.4 In contrast, full mutation of the FMR1 gene (greater than 200 CGG repeats) causes FXS, a neurodevelopmental disorder that leads to mental retardation and autism or severe learning disabilities. For the latter condition the gene is generally silenced and little or no FMRP is produced. FXTAS is a condition consisting of progressive intention tremor, ataxia, dementia, cognitive decline, Parkinsonism and autonomic dysfunction, including impotence, hypertension, and eventual loss of bowel and bladder control.4 It
P
Submitted for publication July 28, 2006. Study received University of California Internal Review Board approval. Supported by National Institute of Child Health and Human Development Grants HD36071 and HD02274, National Institute of Neurological Disorders and Stroke Grant NS044299, National Institute on Aging Grant AG024488, Collaborative Agreement U10/ CCU925123 with the Center for Disease Control and Prevention, and the Medical Investigation of Neurodevelopmental Disorders Institute, University of California-Davis. * Correspondence: Department of Pediatrics, Medical Investigation of Neurodevelopmental Disorders Institute, University of California Davis Health System, 2825 50th St., Sacramento, California 95817 (telephone: 916-703-0247; FAX: 916-703-0240; e-mail: [email protected]).
0022-5347/07/1774-1434/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION
affects approximately a third of male carriers older than 50 years, as ascertained through known fragile X families.5,6 Furthermore, FXTAS accounts for 2% to 5% of adult onset cerebellar ataxia in males.7,8 Neuroimaging in FXTAS cases revealed symmetrically increased T2-weighted signal intensity in the MCPs (MCP sign) in approximately 60% of cases, while additional high signal changes were found in contiguous cerebellar white matter, and in subcortical and periventricular white matter.9,10 Cerebral, cerebellar and brainstem atrophy is also a general feature of FXTAS. Neuropathological findings of FXTAS at autopsy include eosinophilic nuclear inclusions in neurons and astrocytes throughout the central nervous system, Purkinje cell loss, and significant cerebral subcortical and cerebellar white matter disease.10,11 The pathogenesis of FXTAS is thought to involve an RNA toxicity mechanism, due in part to expanded-repeat FMR1 mRNA levels that are 2 to 8 times normal in premutation carriers.4 FMR1 mRNA is present in the inclusions along with multiple proteins, including lamin A/C, ␣ B-crystallin and myelin basic protein.12,13 We identified intranuclear inclusions in the testicles of 2 males, and in the available anterior and posterior pituitary gland of 1 who died with autopsy proven FXTAS. MATERIALS AND METHODS Brain tissue, including the pituitary gland, was processed as previously described by Greco et al.10,11 Informed consent
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Vol. 177, 1434-1437, April 2007 Printed in U.S.A. DOI:10.1016/j.juro.2006.11.097
TESTICULAR INCLUSIONS IN FRAGILE X ASSOCIATED TREMOR/ATAXIA SYNDROME was obtained for the cases in this study, as approved by the University of California Internal Review Board. For case 1 the 2 testicles were removed at autopsy with portions of each immediately snap frozen in liquid nitrogen and fixed in 10% formalin. For case 2 a single testicle was fixed in 10% formalin. Testicular tissue from each case was processed for paraffin embedding according to standard protocol, and then examined by hematoxylin and eosin, and trichrome stains. Immunostains using anti-ubiquitin, neurofilament and anti-human muscle (clone HHF35) antibodies were performed using standard methodology, as previously described. Manual counts of inclusions in Leydig and tubular wall myoid cells were done in each case by 1 of us (CMG) using an Olympus® BX40 microscope at 1,000⫻ magnification on ubiquitin immunostained slides in a grid pattern for 60 fields. Histological review of control testicular tissue from 10 men 65 to 75 years old was also performed. Control cases were chosen from the autopsy files of the Department of Pathology at University of California Davis Medical Center. Anti-ubiquitin immunostaining on 5 of the 10 control cases was performed as described.
Case Histories Case 1. This 64-year-old male presented with a 9-year history of progressive tremor, ataxia and behavioral changes. At age 54 years he began to notice tremor in the right hand with activity. Tremor slowly progressed to involve each hand with impaired fine motor ability, leading to retirement as an electrician at age 58 years. At age 61 years the tremor occurred occasionally at rest. He also experienced frequent falls due to postural instability. He had poor memory for recent events and names of family members, disinhibition, distractibility, poor frustration tolerance and perseverative thoughts. He complained of decreased sensation of the lower extremities. Nerve conduction velocity and electromyography were normal. Medical history included hypertension, hypercholesterolemia, type 2 diabetes and impotence that began at age 50 years. CGG repeats were 98 with FMR1 mRNA levels at 3.38 times normal. He was the first patient at our center to be diagnosed with FXTAS. His daughter is a premutation carrier and has 2 children with FXS. Neurological examination at age 64 years showed facial masking, slurred speech and bilateral hearing loss. He had a symmetrical 4 to 6 Hz large amplitude intention tremor bilaterally. He had a wide based, slow and lurching gait. Finger-to-nose and heel-to-shin movements were ataxic and dysmetria was more prominent on the right side. He had an intermittent resting and static postural tremor in the arms, slight cogwheel rigidity and minimal bradykinesia. Sensation to vibration, light touch and pinprick were decreased in each lower extremity. Muscle strength was graded 5/5 in the arms and 4/5 bilaterally in the proximal lower extremities. Deep tendon reflexes were 1⫹ in the upper extremities and they could not be elicited in the lower extremities. Cognitive evaluation at age 64 years included the MiniMental Status Examination with a score of 18/30, consistent with the diagnosis of dementia (full scale IQ 83, verbal IQ 93 and performance IQ 73). Magnetic resonance imaging revealed mild to moderate generalized atrophy with enlarged ventricles and it was without the MCP sign.
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Symptoms of tremor, ataxia, cognitive and psychological problems progressed, leading to use of a cane at age 66 years and then a wheelchair at age 68 years. Urinary and bowel incontinence developed and the patient was bedridden at age 68.5 years. He died at age 69 years. Case 2. This man, whose clinical history was previously reported in 2006 by Greco et al as case 5,11 was a carrier of the fragile X premutation allele and the grandfather of 2 girls with FXS. He had a successful business career before retirement. A history of impotence began at age 68 years, and muscle weakness and ataxia developed beginning at age 71 years. At age 73 years he had memory problems and began using a walker. He had a pacemaker in place, so that magnetic resonance imaging could not be performed. He died of congestive heart failure at age 74 years. Pathological Findings Case 1. Pathological findings for this case have not been reported. Autopsy tissue was harvested within 8 hours of death using previously published protocols.10,11 One hemisphere of the brain was frozen at ⫺70C for further studies. Coronal sections of the formalin fixed hemisphere showed mild ventricular dilatation and no abnormalities of the cerebral cortex, basal ganglia, brainstem or cerebellum. Histological sections showed ubiquitin positive intranuclear inclusions in neurons and astrocytes, as in all previously described affected cases.10,11 Otherwise the brain showed mild to moderate vascular hyalinosis in deep white matter vessels on hematoxylin and eosin, myelin (luxol fast blueperiodic acid-Schiff) and axon (anti-neurofilament antibody) stains. Hematoxylin and eosin stained sections of the pituitary gland showed no abnormalities except intranuclear inclusions in the anterior and posterior components of the gland (fig. 1). The average diameter of inclusions in the anterior and pituitary gland was 2.3 . Inclusions were more easily identifiable on hematoxylin and eosin staining in the posterior than the anterior pituitary gland. Inclusions were obvious in each area on ubiquitin immunostaining. Case 2. As reported as case 8 in 2006 by Greco et al,11 on external examination brain pathology showed frontal, rostral cingulate and superior temporal gyral atrophy. Moderate ventrigulomegaly was present. Histological examination showed intranuclear inclusions in neurons and astrocytes throughout the brain. There was patchy white matter loss in subcortical cerebral white matter and in the MCPs.
FIG. 1. Case 1. A, ubiquitin positive inclusions (arrows) were easily identified in anterior pituitary gland. Immunostaining, reduced from ⫻400. B, inclusions in posterior pituitary gland. Ubiquitin immunostaining, reduced from ⫻400. Inset, arrow indicates inclusion. H & E, reduced from ⫻400.
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TESTICULAR INCLUSIONS IN FRAGILE X ASSOCIATED TREMOR/ATAXIA SYNDROME
Testicular Pathology Gross findings on examination of the testicles from patients 1 and 2 were similar with size and appearance indistinguishable from those of normal age matched controls. General histological features on hematoxylin and eosin staining were similar, showing nonspecific atrophic changes, including a decreased number of Leydig cells, tubular fibrosis and decreased spermatogenesis. Sertoli’s cells were numerous in the tubules, and a small number of germ cells and rare spermatozoa were the remnants of sperm maturation. Eosinophilic intranuclear inclusions indistinguishable from those present in the brain of individuals with FXTAS10,11 were seen in the 2 cases in Leydig and myoid cells of the tubular walls on hematoxylin and eosin staining (fig. 2). The average measurement of inclusions immunostained with ubiquitin antibody in the 2 cell populations was 2.6 . Using the method described case 1 showed 5.1% of Leydig cells and 4.2% of spermatic tubule myoid cells with intranuclear inclusions. Case 2 showed inclusions in 1.6% of Leydig cells and 4.8% of spermatic tubule myoid cells. In case 2 inclusions were not identified in Sertoli’s or sperm cells, although
FIG. 2. A, case 2. Low power view shows nonspecific atrophic changes in testes with decreased spermatogenesis evidenced by little germ cell differentiation and rare to absent spermatid formation (a), decreased number of interstitial Leydig cells (b) and thickened, fibrotic tubular walls (c). H & E, reduced from ⫻100. B, case 1. Medium power view shows spermatic tubule with multiple intranuclear inclusions (arrow) in tubular wall. H & E, reduced from ⫻200. C, case 2. Intranuclear inclusions (arrows) are identified in Leydig cell population. H & E, reduced from ⫻400. D, case 2. positive staining of inclusions in Leydig cells. Ubiquitin immunostaining, reduced from ⫻400. E, case 2. Positive staining of inclusions in myoid cells of tubular walls. Ubiquitin immunostaining, reduced from ⫻400.
they could occasionally be seen in myoid cells of the tunica vascularis and albuginea. The myoid nature of inclusion bearing cells in tunical layers was verified by immunohistochemical staining with anti-muscle actin antibody (clone HHF35). Histological examination of 10 age matched control testicles showed decreased spermatogenesis, a decreased number of Leydig cells and tubular fibrosis, which on average was similar to that seen in our 2 cases. Intranuclear inclusions were not observed on hematoxylin and eosin stained sections in 10 of the 10 control cases or on ubiquitin immunostained sections in 5. DISCUSSION This report documents intranuclear inclusions in the myoid cells of spermatic tubules and Leydig cells of the testicles in 2 men with FXTAS. These inclusions appeared identical to those previously reported in the brain of patients with FXTAS.13,14 In FXS macro-orchidism is thought to be caused by a deficiency of FMRP. Macro-orchidism is not typically seen in men with the premutation except in the occasional carrier who has a mild FMRP deficit. We also saw inclusions typical of FXTAS in the anterior and posterior pituitary gland of case 1. Inclusions at this location were reported previously in 1 patient with FXTAS.15 Pituitary inclusions may be related to dysregulated neuroendocrine function, including luteinizing hormone and follicle-stimulating hormone levels in patients with the premutation. In females with the premutation follicle-stimulating hormone increases are common in those with premature ovarian failure.3 We have also seen intranuclear inclusions in the anterior and posterior pituitary gland of 1 female carrier with FXTAS who showed intranuclear inclusions in the brain (unpublished data). This may be further support for neuroendocrine dysregulation in carriers. The presence of intranuclear inclusions in myoid cells in testicular fibrous tissue in FXTAS is intriguing. The tunica propria of the testicle is a component of the tunica albuginea and it is the middle of the 3 layers of the fibrous capsule beneath the scrotal skin that protects and supports the testes. Among other cellular components the tunica albuginea contains myofibroblasts. In the tunica propria smooth muscle cells are involved in contractile and transport functions.16 Inclusions in these cells suggests that other structures outside of the central nervous system that contain such cells may also have inclusions. This could be beneficial diagnostically or for monitoring the therapeutic response. Because Leydig cells produce testosterone, we were concerned about possible testosterone deficiency in FXTAS. Although the 2 men reported on did not have testosterone tested during life, we know of 3 with FXTAS in whom testosterone was measured. The 2 men who were deficient in this hormone were treated with intramuscular testosterone with positive effects in energy and mood, while the other had low normal serum testosterone and was not treated. It is known that testosterone treatment can have positive effects on cognition and memory,17 which are problems for individuals with FXTAS.4 The 2 men presented with impotence before the onset of other neurological signs. In general male sexual dysfunction is multifactorial. Hormonal causes are diabetes mellitus, testosterone deficiency, hyperprolactinemia, hyperthyroidism, hypothyroidism, Cushing’s syndrome and Addison’s
TESTICULAR INCLUSIONS IN FRAGILE X ASSOCIATED TREMOR/ATAXIA SYNDROME disease. Low serum testosterone can be caused by pituitary or testicular pathology, prescription or recreational drugs, chromosomal anomalies, environmental toxins and aging. A number of epidemiological studies described the relationship between aging and hypogonadism.18,19 Some data indicate a 110 ng/dl decrease in serum testosterone every 10 years in men between ages 66 and 80 years.20 In this report we describe what is to our knowledge a new histological finding in FXTAS that might provide clues to the etiology of low testosterone in some older males. Furthermore, this report underscores the importance of considering FXTAS in men older than 50 years who complain of erectile dysfunction and neurological symptoms. We recommend evaluating testosterone in males with the FMR1 premutation who have neurological symptoms, FXTAS or impotence. Urologists should be aware of FXTAS and consider testing for the FMR1 premutation in males older than 50 years who present with impotence and neurological findings such as ataxia, tremor or memory problems. Such testing is also important in patients with established impotence who report neurological abnormalities. Clinical impotence may precede neurological symptoms in male premutation carriers, as in the cases reported. Early treatment of testosterone deficiency may help some symptoms, at least transiently.
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Abbreviations and Acronyms FMR1 ⫽ fragile X mental retardation 1 gene FMRP ⫽ fragile X mental retardation 1 gene protein FXS ⫽ fragile X syndrome FXTAS ⫽ fragile X tremor/ataxia syndrome MCP ⫽ middle cerebellar peduncle
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