MICROSCOPY RESEARCH AND TECHNIQUE 78:731–736 (2015)
Ultra-Structural Hair Alterations in Friedreich’s Ataxia: A Scanning Electron Microscopic Investigation F. PINAR TURKMENOGLU,1* U. BARAN KASIRGA,2 AND H. HAMDI CELIK3 1 2 3
Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey Department of Anatomy, Faculty of Medicine, Maltepe University, Ankara, Turkey Department of Anatomy, Faculty of Medicine, Hacettepe University, Ankara, Turkey
KEY WORDS
Friedreich’s ataxia; scanning electron microscopy; hair; ultra-structure
ABSTRACT Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder involving progressive damage to the central and peripheral nervous systems and cardiomyopathy. FRDA is caused by the silencing of the FXN gene and reduced levels of the encoded protein, frataxin. Frataxin is a mitochondrial protein that functions primarily in iron-sulfur cluster synthesis. Skin disorders including hair abnormalities have previously been reported in patients with mitochondrial disorders. However, to our knowledge, ultra-structural hair alterations in FRDA were not demonstrated. The purpose of this study was to determine ultra-structural alterations in the hairs of FRDA patients as well as carriers. Hair specimen from four patients, who are in different stages of the disease, and two carriers were examined by scanning electron microscope. Thin and weak hair follicles with absence of homogeneities on the cuticular surface, local damages of the cuticular layer, cuticular fractures were detected in both carriers and patients, but these alterations were much more prominent in the hair follicles of patients. In addition, erosions on the surface of the cuticle and local deep cavities just under the cuticular level were observed only in patients. Indistinct cuticular pattern, pores on the cuticular surface, and presence of concavities on the hair follicle were also detected in patients in later stages of the disease. According to our results, progression of the disease increased the alterations on hair structure. We suggest that ultra-structural alterations observed in hair samples might be due to oxidative stress caused by deficient frataxin expression in mitochondria. Microsc. Res. Tech. 78:731–736, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Friedreich’s ataxia (FRDA), which is an autosomal recessive inherited neuro-degenerative disorder, is caused by an expansion mutation of GAA repeats in the first intron within the FXN gene. Patients show severely reduced levels of a FXN-encoded mitochondrial protein called frataxin. Although full function of frataxin remains unclear, reduced frataxin expression results in deficient assembly of iron-sulfur clusters, abnormal accumulation of intra-mitochondrial iron, impaired cellular energy production, and elevated oxidative stress (Campuzano et al., 1996; Pandolfo, 2009; Pearce, 2004) which may play a central role in the disease. The neuropathological condition occurred in FRDA involves spinal cord, cerebellum, and the spinal nerves. In addition to neuropathological disabilities such as progressive ataxia, muscle weakness, and sensory loss, common clinical signs are scoliosis, foot deformity, and hypertrophic cardiomyopathy. Diabetes is the additional signs of this pathological condition (Campuzano et al., 1996; Pandolfo, 2009; Pearce, 2004; Lane et al., 2013; Cnop et al., 2013). Although rare, FRDA is the most common of the early-onset hereditary ataxias, affecting about 1 in every 50,000 people in Europe (Campuzano et al., 1996; Lane et al., 2013; Pandolfo, 2009). Patients with FRDA usually present around the time of puberty with ataxia, dysarthria, C V
2015 WILEY PERIODICALS, INC.
and scoliosis. On average, after 10–15 years of disease, progressive gait and limb ataxia eventually results in the need for a wheelchair. The majority of deaths occurred between ages 16 and 45, and the most common cause of the death for FRDA patients is hypertrophic cardiomyopathy (Lane et al., 2013; Tsou et al., 2011). Mitochondrial diseases, which are a major category of childhood illness, produce a wide variety of symptoms and multisystemic disorders, and can present with signs in almost any organ (Debray et al., 2008). Cutaneous abnormalities, which is associated with primary mitochondrial dysfunction and alterations in mitochondria-related metabolic pathways at different levels were extensively reviewed by Feichtinger et al. (2014). Skin eruptions and hair abnormalities were regarded as a part of the broad spectrum of presenting signs of mitochondrial diseases (Bodemer at al., 1999; Feichtinger et al., 2014; Silengo et al., 2003). In a French study, cutaneous manifestations observed in *Correspondence to: F. Pinar Turkmenoglu, PhD, Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, 06100, Sihhiye, Ankara, Turkey. E-mail: [email protected] Received 1 April 2015; accepted in revised form 22 May 2015 REVIEW EDITOR: Prof. Alberto Diaspro DOI 10.1002/jemt.22531 Published online 3 July 2015 in Wiley Online Library (wileyonlinelibrary.com).
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children with mitochondrial disorders were classified into four categories: hair abnormalities, rashes and pigmentation disorders, hypertrichosis, and acrocyanosis. Skin alterations such as scaly, pruritic, diffuse erythema with reticular pigmentation, have been reported in some patients with Leigh syndrome, Pearson’s syndrome, Wolfram syndrome, and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome (Bodemer et al., 1999). Hypomelanosis has been shown in a few cases of Kearns Sayre syndrome (Kakourou et al., 1999). Somatic mtDNA mutations have also been suggested to be associated with the development of melanoma and head and neck squamous cell carcinomas of the skin, as mutations have been found in almost every mitochondrial gene in these cancer types (Feichtinger et al., 2014). In addition to skin disorders, Bodemer et al. (1999) reported transverse fractures across the hair shaft through the cuticle and the cortex (trichoshisis), involving hairs with a tiger tail pattern, hairs displaying twists (pili torti) and longitudinal grooving in patients with alopecia. While brittle, longitudinal grooving, flattened hair shaft, and localized loss of cuticle cells were observed in patients with mitochondrial progressive encephalopathy, Wolfram syndrome patients presented dry, thick, and brittle hairs (Bodemer et al., 1999). It was also reported that 34% of the Leigh syndrome patients had presented hypertrichosis and 10% had hirsutism (Feichtinger et al., 2014; Sonam et al., 2013). In another study, 8 out of 25 children with a mitochondrial disorder had slow growing, sparse and fragile hair and microscopic evidence of trichorrhexis nodosa and pili torti (Silengo et al., 2003). Pili torti is also a clinical sign of Bj€ornstad syndrome which is caused by mutations in the BCS1L gene involving in the assembly of complex III of the mitochondrial respiratory chain (Debray et al., 2008; Feichtinger et al., 2014; Hinson et al., 2007). Skin and hair abnormalities were previously thought to be directly related to disorders of the mitochondria energy supply (Bodemer et al., 1999; Silengo et al., 2003). In a later study, Hamanaka and Chandel (2013) reported that mitochondria are not only energy source but also vital regulators of skin physiology. Mitochondrial metabolism regulates keratinocyte differentiation by producing mitochondrial reactive oxygen species (ROS), which are necessary to propagate the Notch and b-catenin signals that promote epidermal differentiation and hair follicle development, respectively. Although neurological and non-neurological symptoms of FRDA were described in detail in the literature (Ackroyd et al., 1984; Bourke and Keane, 2011; Carroll et al., 1980; Connelly et al., 2003), to our knowledge, ultra-structure of hairs of these patients have not been reported previously. Therefore, in this study, we have investigated ultra-structural alterations in the hairs of four patients with FRDA and two carriers by scanning electron microscope (SEM) and demonstrated abnormal features. MATERIAL AND METHODS Hair Samples A set of 12 scalp hair specimen from each of four patients, who have already been diagnosed as FRDA
TABLE 1. Age of first symptoms, age of onset, and age of sample collections of FRDA patients carriers and/or volunteers Duration from the first symptoms Patients/carriers/ Age of first Age of Age of sample (year) volunteers symptoms onset collection Sister 1 Sister 2 Unrelated female Unrelated male Father Mother Volunteer 1 Volunteer 2
13 15 14 10 – – – –
13 21 17 15 – – – –
14 23 18 31 50 48 23 41
1 8 4 21 – – – –
at Hacettepe University Hospitals and Erciyes University Hospital and were in different stages of the diseases, two carriers and two healthy volunteers, was collected. Two of the patients and two carriers were the members of a single family: two sisters, mother, and father. The rest two were unrelated patients. Volunteers were proved not to be carriers by genetic analysis, had no chronic disease, and were not under any chemotherapy. Details about all individuals including age of first symptoms, onset, and sample collection as well as duration from the first symptoms are given in Table 1. All experimental protocols were approved by the Non-interventional Clinical Researches Ethics Board of Hacettepe University (Protocol No: GO 14/ 267). SEM Analysis The tissue samples were fixed in 2.5% gluteraldehyde for 24 h, washed in phosphate buffer (pH: 7.4), post-fixed in 1% osmium tetroxide for 1 h, washed in phosphate buffer (pH: 7.4), dehydrated in increasing concentrations of acetone, critical point dried, and mounted on metal stubs with a double sided adhesive ˚ band. Then, the samples were sputtered with a 100 A thick layer of gold in a BIO-RAD sputter apparatus. Electron microscopy analyses have been carried out on a Carl Zeiss EVO 50 EP SEM in Hacettepe University Department of Geological Engineering. Operating conditions were as follows: 15 kV accelerating voltage, 20– 40 pA beam current, and a working distance of 10 mm(C¸elik et al., 2003, 2004, 2007). Two experimenters remained blind to diagnoses during all morphological analysis. RESULTS This study represents the first detailed morphological investigation of the hair samples obtained from FRDA patients and carriers by light microscope and SEM, and results were evaluated by comparing with the ultra-structure of healthy hair samples (Fig. 1). In SEM examination, healthy hair shafts were observed intact, homogeneous, and regular. Cortex, which is the major site of keratinisation and ensures the shaft rigidity, was intact. Cuticle, which forms the hair surface, was also intact, homogeneous and cuticle cells were typically arranged in a similar fashion to roof tiles. In the examination of the hair follicles of one carrier (father of the family), cuticular surface was found to be Microscopy Research and Technique
HAIR ALTERATIONS IN FRDA
irregular and absence of homogeneities were detected. The hair follicles were very thin and weak. Additionally local damages were seen on the cuticular surface. Cuticle cells were partially lifted from the surface of hair follicle (Figs. 2A and 2B). In the hair follicles of the other carrier (mother of the family), ultra-structural examinations showed cuticular damage in some local areas and the cuticle lost its homogenous structure. Additionally cuticle was very thin, fragile, and partially lifted from the surface of hair follicle. Deposit-like cuticular fractures were detected on the surface of the hair follicle (Figs. 2C and 2D). Sisters of the family with FRDA had thin and weak hair follicles and damages on the surface of the cuticu-
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lar pattern were observed. In these patients, erosions were very prominent on the surfaces of the cuticle and irregular cuticular pattern were distinct. In addition, local deep cavities were detected just under the cuticular level. The periphery of these cavities were irregular and they were coursing vertically in most areas (Figs. 3A and 3B). In the hair follicles of the rest two unrelated patients with FRDA, the same ultra-structural findings with two sisters were detected (Figs. 3C and 3D). It was noteworthy that, in later stages of the disease, additional pathological changes were observed. The cuticular pattern was found to be indistinct and pores were observed prominently on the surface of the cuticle in the patient (unrelated male) who has been suffering from the disease for 21 years (Figs. 3E–3G). In addition, unambiguous concave areas were also detected on some hair follicles of patients who have been suffering from the disease for 8 and 21 years (sister 2 and unrelated male, respectively) (Fig. 3H).
Fig. 1. Scanning electron micrograph showing the ultra-structure of healthy hair shaft. Scale: 10 lm.
DISCUSSION In the examination of the literature; only a very few histological studies were found related to the FRDA. Rizzuto et al. (1981) examined the sural and superficial peroneal nerve biopsies of patients with clinical diagnosis of FRDA. They observed loss of fibers accompanied by axonal atrophy and segmental demyelination in the patients. Caruso et al. (1983) studied biopsies of the sural nerves taken from nine patients. Their quantitative examinations revealed a reduced number of total myelinated fibres with a severe loss of large fibers and a moderate loss of fibers of less than
Fig. 2. Scanning electron micrographs showing hair alterations in FRDA carriers. A: Thin and weak hair follicle with absence of homogeneities on the cuticular surface (Scale: 10 mm). B: Damages on the
cuticular surface (Scale: 2 mm). C: Cuticular damages in some local areas and loss of the homogeneity of cuticle (Scale: 10 mm). D: Cuticular fractures on the surface of the hair follicle (Scale: 2 mm).
Microscopy Research and Technique
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Fig. 3. Scanning electron micrographs showing hair alterations in FRDA patients. A: Thin and weak hair follicle with absence of homogeneities and prominent damages on the cuticular surface (Scale: 10 mm). B, C: Damages on cuticular surface and local deep cavities under the cuticular level (Scale: 2 mm and 10 mm, respectively). D: Erosions
on the surface of the cuticle, cuticular damage, and fractures (Scale: 2 mm). E, F: Indistinct cuticular pattern (Scale: 10 mm and 2 mm, respectively). G: Pore on the cuticular level (Scale: 2 mm). H: Concavity of hair follicle (Scale: 10 mm).
seven microns in diameter. Willers et al. (1993) performed immunocytochemical studies on the vimentin distribution and cell proliferation of fibroblasts in patients with FRDA. These patients’ cells showed a slower outgrowth of vimentin filaments in comparison to normal controls. Nolano et al. (2001) demonstrated
an involvement of cutaneous unmyelinated sensory and autonomic nerve fibres in patients with FRDA. Hair abnormalities and skin disorders are thought to be signs of mitochondrial diseases and various hair abnormalities were reported in children with mitochondrial disorders (Bodemer et al., 1999; Feichtinger Microscopy Research and Technique
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TABLE 2. Ultra-structure of hair of healthy volunteers, carriers and FRDA patients Healthy volunteers
Patients (1,4)a
Carriers
Patients (8,21)a
Hair shaft
Intact
Intact
Intact
Cortex Cuticle
Homogeneous Intact Intact Regular Homogeneous Absent
Homogeneous Intact Locally damaged, partially lifted or broken Irregular Heterogeneous Absent
Homogeneous Intact Prominently damaged Prominently irregular Heterogeneous Present
Prominently irregular Heterogeneous Present
Absent Normal
Absent Thin and weak
Absent Thin and weak
Present Thin and weak
Local deep cavities under cuticle Pores on the cuticle Follicle a
Unambiguous concave areas present Heterogeneous Damaged Indistinct
Duration from the first symptoms (year).
et al., 2014; Silengo et al., 2003). Literature survey revealed that ultra-structural alterations on hair in FRDA have not been investigated before. To our knowledge, this is the first report demonstrating the ultra-structural alterations of the hair of patients with FRDA in details. Beside FRDA patients, we also detected hair alterations in FRDA carriers. As shown in Table 2, in patients, ultra-structural damages were detected much more prominent than carriers. Hair abnormalities observed in patients with other mitochodrial diseases were mentioned in introduction (Bodemer et al. 1999; Debray et al., 2008; Feichtinger et al., 2014; Hinson et al., 2007; Silengo et al., 2003; Sonam et al., 2013). Considering the previous data on hair alterations (trichoshisis, pili torti, trichorrhexis nodosa, longitudinal grooving, flattened hair shaft, localized loss of cuticle cells and dry, thick, and brittle hair structure of hair follicle), thin and weak hair follicles with prominent damages of the cuticular layer, cuticular fractures and erosions on the surface of the cuticle, local deep cavities just under the cuticular level, and irregular vertically coursing cuticular pattern observed in this study are distinctive findings of hair in FRDA patients. Moreover in later stages of the disease, additional pathognomonic ultra-structural findings were added to hair pathology such as indistinct cuticular pattern, pores on the cuticular surface, and the presence of concave areas on the hair follicle (Table 2). FRDA is a progressive disease and according to our results, it seems that progression of the disease increased the alterations on hair structure. FRDA is a hereditary disease caused by deficient frataxin expression. This mitochondrial protein is related to iron homeostasis, energy metabolism, and oxidative stress. In vitro studies have demonstrated that frataxin deficient cells not only generate more free radicals, but also show a reduced capacity to mobilize antioxidant defences (Al-Mahdawi et al., 2006; Marmolino et. al., 2010; Seguin et al., 2009). Oxidative stress not only caused inadequate energy supply but also retrograde mitochondrial signaling mechanisms give rise to functional and structural alterations in the skin and hair (Feichtinger et al., 2014; Hamanaka and Chandel 2013). Negative effects of oxidative stress on hair structure, especially on cuticle layer, were also well established (Kim, 2011; Tr€ ueb, 2006). Therefore, we suggest that ultra-structural alterations demonstrated in this study might be due to oxidative stress caused by frataxin deficiency in FRDA patients. Microscopy Research and Technique
Because of the complexity of mitochondrial genetics and biochemistry, clinical manifestations of mitochondrial diseases are extremely heterogeneous and difficult to diagnose in early stages. Cutaneous alterations including hair abnormalities associated with unrelated disorders were reported to be a presenting sign which could be a clinical clue to the early diagnosis (Bodemer et al., 1999; Hinson et al., 2007; Silengo et al., 2003). Discriminating ultra-structural hair alterations determined in this study will make a noteworthy contribution to cutaneous alterations associated with mitochondrial diseases. This SEM approach, which is a three dimensional examination technique revealing easily comparable images, is a very spesific, effective, and fast diagnostic tool in the detection of hair alterations in FRDA. As it is used in Chediak–Higashi syndrome (C¸elik et al., 2003), dyskeratosis congenita (C¸elik et al., 2007), and hereditary trichodysplasia (C¸elik et al., 2004), its routine usage in hair analysis of FRDA patients may result in valuable contribution to early diagnosis of the disease. Additionally, considering the psychology of the patients, analysis of hair follicles has some advantages. Hair sample collection is non-invasive, less embarrassing, and can easily be performed. ACKNOWLEDGMENTS The authors would like to thank Prof. Yasemin G€ ursoy € Ozdemir, MD, PhD, who is former professor in Department of Neurology, Medical School, Hacettepe University, for her contribution to the study with re-evaluation of the patients and providing insight and expertise that greatly assisted the research, Prof. Mustafa F. Sargon, MD, PhD, who is professor in Department of Anatomy, Medical School, Hacettepe University, for his great contributions to anatomic evaluations, and Assoc. Prof. H. Evren C¸ubukc¸u, PhD, who is assistant professor in Department of Geological Engineering, Faculty of Engineering, Hacettepe University, for his great help during SEM studies. REFERENCES Ackroyd RS, Finnegan JA, Green SH. 1984. Friedreich’s ataxia: A clinical review with neurophysiological and echocardiographic findings. Arch Dis Child 59:217–221. Al-Mahdawi S, Pinto RM, Varshney D. 2006. GAA repeat expansion mutation Mouse models of Friedreich ataxia exhibit oxidative stres leading to progressive neuronal and cardiacpathology. Genomics 88:580–590.
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Bodemer C, R€otig A, Rustin P, Cormier V, Niaudet P, Saudubray JM, Rabier D, Munnich A, de Prost Y. 1999. Hair and skin disorders as signs of mitochondrial disease. Pediatrics 103:428–433. Bourke T, Keane D. 2011. Friedreich’s ataxia: A review from a cardiology perspective. Ir J Med Sci 180:799–805. Campuzano V, Montermini L, Molt o MD, Pianese L, Coss ee M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A, Zara F, Ca~ nizares J, Koutnikova H, Bidichandani SI, Gellera C, Brice A, Trouillas P, De Michele G, Filla A, De Frutos R, Palau F, Patel PI, Di Donato S, Mandel JL, Cocozza S, Koenig M, Pandolfo M. 1996. Friedreich’s ataxia: Autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271:1423–1427. Carroll WM, Kriss A, Baraitser M. 1980. The incidence and nature of visual pathway involvement in Friedreich’s ataxia: A clinical and visual evoked potential study of 22 patients. Brain 103:4013–4034. Caruso G, Santoro L, Perretti A, Serlenga L, Crisci C, Ragno M, Barbieri F, Filla A. 1983. Friedreich’s ataxia: Electrophysiological and histological findings. Acta Neurol Scand 67:26–40. C¸elik HH, Tore HG, Balcı S, Ba gc¸ic¸ek G, Ozdemir B. 2003. Scanning electron microscopic examination of the hair in Ch ediak-Higashi syndrome. Case Rep Clin Pract Rev 4:252–255. € C¸elik HH, S€ ur€ uc€ u SH, Aldur MM, Ozdemir BM, Karaduman AA, Cumhur MM. 2004. Light and scanning electron microscopic examination of late changes in hair with hereditary trichodysplasia (Marie Unna hypotrichosis). Saudi Med J 25:1648–1651. Celik HH, Erbil H, Tatar I, Ozdemir MB. 2007. Light and scanning electron microscopic investigation of the changes in hair with Dyskeratosis congenita. Saudi Med J 28:144–145. Cnop M, Mulder H, Igoillo-Esteve M. 2013. Diabetes in Friedreich ataxia. J Neurochem 126:94–102. Connelly T, Farmer JM, Lynch DR, Doty RL. 2003. Olfactory dysfunction in degenerative ataxias. J Neurol Neurosurg Psychiatry 74: 1435–1437. Debray FG, Lambert M, Grant AM. 2008. Disorders of mitochondrial function. Curr Opin Pediatr 20:471–482. Feichtinger RG, Sperl W, Bauer JW, Kofler B. 2014. Mitochondrial dysfunction: A neglected component of skin diseases. Exp Dermatol 23:607–614. Hamanaka RB, Chandel NS. 2013. Mitochondrial metabolism as a regulator of keratinocyte differentiation. Cell Logist 3:e25456. Hinson TJ, Fantin VR, Sch€ onberger J, Breivik N, Siem G, McDonough B, Sharma P, Keogh I, Godinho R, Santos F, Esparza A, Nicolau Y, Selvaag E, Cohen BH, Hoppel CL, Tranebjærg L, Eavey RD, Seidman JD, Seidman CE. 2007. Missense mutations in the BCS1L gene as a cause of the Bj€ ornstad syndrome. N Engl J Med 356:809–819.
Kakourou T, Garoufi A, Polyxeni N, Dafni E, Tsamouri M, Papadimitriou A, Karpathios T. 1999. Kearns Sayre syndrome initially presenting as hypomelanosis of Ito. Arch Dis Child 81:280– 281. Kim MM. 2011. Effect of procyandin oligomers on oxidative hair damage. Skin Res Technol 17:108–118. Lane DJR, Huang ML, Ting S, Sivagurunathan S, Richardson DR. 2013. Biochemistry of cardiomyopathy in the mitochondrial disease Friedreich’s ataxia. Biochem J 453:321–336. Marmolino D, Manto M, Acquaviva F. 2010. PGC-1alpha downregulation affects the antioxidant response in Friedreich’s ataxia. PLoS One 5:10025. Nolano M, Provitera V, Crisci C, Saltalamacchia AM, WendelschaferCrabb G, Kennedy WR, Filla A, Santoro L, Caruso G. 2001. Small fibers involvement in Friedreich’s ataxia. Ann Neurol 50:17–25. Pandolfo M. 2009. Friedreich ataxia: The clinical picture. J Neurol 256:3–8. Pearce JMS. 2004. Friedreich’s ataxia. J Neurol Neurosurg Psychiatr 75:688. Rizzuto N, Monaco S, Moretto G, Galiazzo-Rizzuto S, Fiaschi A, Forti A, De Maria R. 1981. Friedreich’s ataxia. A light- and electron microscopic study of peripheral nerve biopsies. Acta Neuropathol Suppl 7:344–347. Seguin A, Bayot A, Dancis A. 2009. Overexpression of theyeastfrataxin homolog (Yfh1): Contrastin geffects on iron–sulfur cluster assembly, heme synthesis and resistance to oxidative stress. Mitochondrion 9:130–138. Silengo M, Valenzise M, Spada M, Ferrero GB, Ferraris S, Dassi P, Jarre L. 2003. Hair anomalies as a sign of mitochondrial disease. Eur J Pediatr 162:459–461. Sonam K, Khan NA, Bindu PS, Taly AB, Gayathri N, Srinivas Bharath MM, Govindaraju C, Arvinda HR, Nagappa M, Sinha S, Thangaraj K. 2013. Clinical and magnetic resonance imaging findings in patients with Leigh syndrome and SURF1 mutations. Brain Dev 36:807–812. Tr€ ueb RM. 2006. Pharmacologic interventions in aging hair. Clin Interv Aging 1:121–129. Tsou AY, Paulsen EK, Lagedrost SJ, Perlman SL, Mathews KD, Wilmot GR, Ravina B, Koeppen AH, Lynch DR. 2011. Mortality in Friedreich ataxia. J Neurol Sci 307:46–49. Willers I, Ressler B, Singh S, Koeppen AH. 1993. Immunocytochemical studies on the vimentin distribution and cell proliferation of fibroblasts in patients with Friedreich’s ataxia. J Neurol Sci 117: 159–163.
Microscopy Research and Technique
Ultra-Structural Hair Alterations in Friedreich’s Ataxia: A Scanning Electron Microscopic Investigation F. PINAR TURKMENOGLU,1* U. BARAN KASIRGA,2 AND H. HAMDI CELIK3 1 2 3
Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey Department of Anatomy, Faculty of Medicine, Maltepe University, Ankara, Turkey Department of Anatomy, Faculty of Medicine, Hacettepe University, Ankara, Turkey
KEY WORDS
Friedreich’s ataxia; scanning electron microscopy; hair; ultra-structure
ABSTRACT Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder involving progressive damage to the central and peripheral nervous systems and cardiomyopathy. FRDA is caused by the silencing of the FXN gene and reduced levels of the encoded protein, frataxin. Frataxin is a mitochondrial protein that functions primarily in iron-sulfur cluster synthesis. Skin disorders including hair abnormalities have previously been reported in patients with mitochondrial disorders. However, to our knowledge, ultra-structural hair alterations in FRDA were not demonstrated. The purpose of this study was to determine ultra-structural alterations in the hairs of FRDA patients as well as carriers. Hair specimen from four patients, who are in different stages of the disease, and two carriers were examined by scanning electron microscope. Thin and weak hair follicles with absence of homogeneities on the cuticular surface, local damages of the cuticular layer, cuticular fractures were detected in both carriers and patients, but these alterations were much more prominent in the hair follicles of patients. In addition, erosions on the surface of the cuticle and local deep cavities just under the cuticular level were observed only in patients. Indistinct cuticular pattern, pores on the cuticular surface, and presence of concavities on the hair follicle were also detected in patients in later stages of the disease. According to our results, progression of the disease increased the alterations on hair structure. We suggest that ultra-structural alterations observed in hair samples might be due to oxidative stress caused by deficient frataxin expression in mitochondria. Microsc. Res. Tech. 78:731–736, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Friedreich’s ataxia (FRDA), which is an autosomal recessive inherited neuro-degenerative disorder, is caused by an expansion mutation of GAA repeats in the first intron within the FXN gene. Patients show severely reduced levels of a FXN-encoded mitochondrial protein called frataxin. Although full function of frataxin remains unclear, reduced frataxin expression results in deficient assembly of iron-sulfur clusters, abnormal accumulation of intra-mitochondrial iron, impaired cellular energy production, and elevated oxidative stress (Campuzano et al., 1996; Pandolfo, 2009; Pearce, 2004) which may play a central role in the disease. The neuropathological condition occurred in FRDA involves spinal cord, cerebellum, and the spinal nerves. In addition to neuropathological disabilities such as progressive ataxia, muscle weakness, and sensory loss, common clinical signs are scoliosis, foot deformity, and hypertrophic cardiomyopathy. Diabetes is the additional signs of this pathological condition (Campuzano et al., 1996; Pandolfo, 2009; Pearce, 2004; Lane et al., 2013; Cnop et al., 2013). Although rare, FRDA is the most common of the early-onset hereditary ataxias, affecting about 1 in every 50,000 people in Europe (Campuzano et al., 1996; Lane et al., 2013; Pandolfo, 2009). Patients with FRDA usually present around the time of puberty with ataxia, dysarthria, C V
2015 WILEY PERIODICALS, INC.
and scoliosis. On average, after 10–15 years of disease, progressive gait and limb ataxia eventually results in the need for a wheelchair. The majority of deaths occurred between ages 16 and 45, and the most common cause of the death for FRDA patients is hypertrophic cardiomyopathy (Lane et al., 2013; Tsou et al., 2011). Mitochondrial diseases, which are a major category of childhood illness, produce a wide variety of symptoms and multisystemic disorders, and can present with signs in almost any organ (Debray et al., 2008). Cutaneous abnormalities, which is associated with primary mitochondrial dysfunction and alterations in mitochondria-related metabolic pathways at different levels were extensively reviewed by Feichtinger et al. (2014). Skin eruptions and hair abnormalities were regarded as a part of the broad spectrum of presenting signs of mitochondrial diseases (Bodemer at al., 1999; Feichtinger et al., 2014; Silengo et al., 2003). In a French study, cutaneous manifestations observed in *Correspondence to: F. Pinar Turkmenoglu, PhD, Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, 06100, Sihhiye, Ankara, Turkey. E-mail: [email protected] Received 1 April 2015; accepted in revised form 22 May 2015 REVIEW EDITOR: Prof. Alberto Diaspro DOI 10.1002/jemt.22531 Published online 3 July 2015 in Wiley Online Library (wileyonlinelibrary.com).
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children with mitochondrial disorders were classified into four categories: hair abnormalities, rashes and pigmentation disorders, hypertrichosis, and acrocyanosis. Skin alterations such as scaly, pruritic, diffuse erythema with reticular pigmentation, have been reported in some patients with Leigh syndrome, Pearson’s syndrome, Wolfram syndrome, and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome (Bodemer et al., 1999). Hypomelanosis has been shown in a few cases of Kearns Sayre syndrome (Kakourou et al., 1999). Somatic mtDNA mutations have also been suggested to be associated with the development of melanoma and head and neck squamous cell carcinomas of the skin, as mutations have been found in almost every mitochondrial gene in these cancer types (Feichtinger et al., 2014). In addition to skin disorders, Bodemer et al. (1999) reported transverse fractures across the hair shaft through the cuticle and the cortex (trichoshisis), involving hairs with a tiger tail pattern, hairs displaying twists (pili torti) and longitudinal grooving in patients with alopecia. While brittle, longitudinal grooving, flattened hair shaft, and localized loss of cuticle cells were observed in patients with mitochondrial progressive encephalopathy, Wolfram syndrome patients presented dry, thick, and brittle hairs (Bodemer et al., 1999). It was also reported that 34% of the Leigh syndrome patients had presented hypertrichosis and 10% had hirsutism (Feichtinger et al., 2014; Sonam et al., 2013). In another study, 8 out of 25 children with a mitochondrial disorder had slow growing, sparse and fragile hair and microscopic evidence of trichorrhexis nodosa and pili torti (Silengo et al., 2003). Pili torti is also a clinical sign of Bj€ornstad syndrome which is caused by mutations in the BCS1L gene involving in the assembly of complex III of the mitochondrial respiratory chain (Debray et al., 2008; Feichtinger et al., 2014; Hinson et al., 2007). Skin and hair abnormalities were previously thought to be directly related to disorders of the mitochondria energy supply (Bodemer et al., 1999; Silengo et al., 2003). In a later study, Hamanaka and Chandel (2013) reported that mitochondria are not only energy source but also vital regulators of skin physiology. Mitochondrial metabolism regulates keratinocyte differentiation by producing mitochondrial reactive oxygen species (ROS), which are necessary to propagate the Notch and b-catenin signals that promote epidermal differentiation and hair follicle development, respectively. Although neurological and non-neurological symptoms of FRDA were described in detail in the literature (Ackroyd et al., 1984; Bourke and Keane, 2011; Carroll et al., 1980; Connelly et al., 2003), to our knowledge, ultra-structure of hairs of these patients have not been reported previously. Therefore, in this study, we have investigated ultra-structural alterations in the hairs of four patients with FRDA and two carriers by scanning electron microscope (SEM) and demonstrated abnormal features. MATERIAL AND METHODS Hair Samples A set of 12 scalp hair specimen from each of four patients, who have already been diagnosed as FRDA
TABLE 1. Age of first symptoms, age of onset, and age of sample collections of FRDA patients carriers and/or volunteers Duration from the first symptoms Patients/carriers/ Age of first Age of Age of sample (year) volunteers symptoms onset collection Sister 1 Sister 2 Unrelated female Unrelated male Father Mother Volunteer 1 Volunteer 2
13 15 14 10 – – – –
13 21 17 15 – – – –
14 23 18 31 50 48 23 41
1 8 4 21 – – – –
at Hacettepe University Hospitals and Erciyes University Hospital and were in different stages of the diseases, two carriers and two healthy volunteers, was collected. Two of the patients and two carriers were the members of a single family: two sisters, mother, and father. The rest two were unrelated patients. Volunteers were proved not to be carriers by genetic analysis, had no chronic disease, and were not under any chemotherapy. Details about all individuals including age of first symptoms, onset, and sample collection as well as duration from the first symptoms are given in Table 1. All experimental protocols were approved by the Non-interventional Clinical Researches Ethics Board of Hacettepe University (Protocol No: GO 14/ 267). SEM Analysis The tissue samples were fixed in 2.5% gluteraldehyde for 24 h, washed in phosphate buffer (pH: 7.4), post-fixed in 1% osmium tetroxide for 1 h, washed in phosphate buffer (pH: 7.4), dehydrated in increasing concentrations of acetone, critical point dried, and mounted on metal stubs with a double sided adhesive ˚ band. Then, the samples were sputtered with a 100 A thick layer of gold in a BIO-RAD sputter apparatus. Electron microscopy analyses have been carried out on a Carl Zeiss EVO 50 EP SEM in Hacettepe University Department of Geological Engineering. Operating conditions were as follows: 15 kV accelerating voltage, 20– 40 pA beam current, and a working distance of 10 mm(C¸elik et al., 2003, 2004, 2007). Two experimenters remained blind to diagnoses during all morphological analysis. RESULTS This study represents the first detailed morphological investigation of the hair samples obtained from FRDA patients and carriers by light microscope and SEM, and results were evaluated by comparing with the ultra-structure of healthy hair samples (Fig. 1). In SEM examination, healthy hair shafts were observed intact, homogeneous, and regular. Cortex, which is the major site of keratinisation and ensures the shaft rigidity, was intact. Cuticle, which forms the hair surface, was also intact, homogeneous and cuticle cells were typically arranged in a similar fashion to roof tiles. In the examination of the hair follicles of one carrier (father of the family), cuticular surface was found to be Microscopy Research and Technique
HAIR ALTERATIONS IN FRDA
irregular and absence of homogeneities were detected. The hair follicles were very thin and weak. Additionally local damages were seen on the cuticular surface. Cuticle cells were partially lifted from the surface of hair follicle (Figs. 2A and 2B). In the hair follicles of the other carrier (mother of the family), ultra-structural examinations showed cuticular damage in some local areas and the cuticle lost its homogenous structure. Additionally cuticle was very thin, fragile, and partially lifted from the surface of hair follicle. Deposit-like cuticular fractures were detected on the surface of the hair follicle (Figs. 2C and 2D). Sisters of the family with FRDA had thin and weak hair follicles and damages on the surface of the cuticu-
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lar pattern were observed. In these patients, erosions were very prominent on the surfaces of the cuticle and irregular cuticular pattern were distinct. In addition, local deep cavities were detected just under the cuticular level. The periphery of these cavities were irregular and they were coursing vertically in most areas (Figs. 3A and 3B). In the hair follicles of the rest two unrelated patients with FRDA, the same ultra-structural findings with two sisters were detected (Figs. 3C and 3D). It was noteworthy that, in later stages of the disease, additional pathological changes were observed. The cuticular pattern was found to be indistinct and pores were observed prominently on the surface of the cuticle in the patient (unrelated male) who has been suffering from the disease for 21 years (Figs. 3E–3G). In addition, unambiguous concave areas were also detected on some hair follicles of patients who have been suffering from the disease for 8 and 21 years (sister 2 and unrelated male, respectively) (Fig. 3H).
Fig. 1. Scanning electron micrograph showing the ultra-structure of healthy hair shaft. Scale: 10 lm.
DISCUSSION In the examination of the literature; only a very few histological studies were found related to the FRDA. Rizzuto et al. (1981) examined the sural and superficial peroneal nerve biopsies of patients with clinical diagnosis of FRDA. They observed loss of fibers accompanied by axonal atrophy and segmental demyelination in the patients. Caruso et al. (1983) studied biopsies of the sural nerves taken from nine patients. Their quantitative examinations revealed a reduced number of total myelinated fibres with a severe loss of large fibers and a moderate loss of fibers of less than
Fig. 2. Scanning electron micrographs showing hair alterations in FRDA carriers. A: Thin and weak hair follicle with absence of homogeneities on the cuticular surface (Scale: 10 mm). B: Damages on the
cuticular surface (Scale: 2 mm). C: Cuticular damages in some local areas and loss of the homogeneity of cuticle (Scale: 10 mm). D: Cuticular fractures on the surface of the hair follicle (Scale: 2 mm).
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Fig. 3. Scanning electron micrographs showing hair alterations in FRDA patients. A: Thin and weak hair follicle with absence of homogeneities and prominent damages on the cuticular surface (Scale: 10 mm). B, C: Damages on cuticular surface and local deep cavities under the cuticular level (Scale: 2 mm and 10 mm, respectively). D: Erosions
on the surface of the cuticle, cuticular damage, and fractures (Scale: 2 mm). E, F: Indistinct cuticular pattern (Scale: 10 mm and 2 mm, respectively). G: Pore on the cuticular level (Scale: 2 mm). H: Concavity of hair follicle (Scale: 10 mm).
seven microns in diameter. Willers et al. (1993) performed immunocytochemical studies on the vimentin distribution and cell proliferation of fibroblasts in patients with FRDA. These patients’ cells showed a slower outgrowth of vimentin filaments in comparison to normal controls. Nolano et al. (2001) demonstrated
an involvement of cutaneous unmyelinated sensory and autonomic nerve fibres in patients with FRDA. Hair abnormalities and skin disorders are thought to be signs of mitochondrial diseases and various hair abnormalities were reported in children with mitochondrial disorders (Bodemer et al., 1999; Feichtinger Microscopy Research and Technique
HAIR ALTERATIONS IN FRDA
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TABLE 2. Ultra-structure of hair of healthy volunteers, carriers and FRDA patients Healthy volunteers
Patients (1,4)a
Carriers
Patients (8,21)a
Hair shaft
Intact
Intact
Intact
Cortex Cuticle
Homogeneous Intact Intact Regular Homogeneous Absent
Homogeneous Intact Locally damaged, partially lifted or broken Irregular Heterogeneous Absent
Homogeneous Intact Prominently damaged Prominently irregular Heterogeneous Present
Prominently irregular Heterogeneous Present
Absent Normal
Absent Thin and weak
Absent Thin and weak
Present Thin and weak
Local deep cavities under cuticle Pores on the cuticle Follicle a
Unambiguous concave areas present Heterogeneous Damaged Indistinct
Duration from the first symptoms (year).
et al., 2014; Silengo et al., 2003). Literature survey revealed that ultra-structural alterations on hair in FRDA have not been investigated before. To our knowledge, this is the first report demonstrating the ultra-structural alterations of the hair of patients with FRDA in details. Beside FRDA patients, we also detected hair alterations in FRDA carriers. As shown in Table 2, in patients, ultra-structural damages were detected much more prominent than carriers. Hair abnormalities observed in patients with other mitochodrial diseases were mentioned in introduction (Bodemer et al. 1999; Debray et al., 2008; Feichtinger et al., 2014; Hinson et al., 2007; Silengo et al., 2003; Sonam et al., 2013). Considering the previous data on hair alterations (trichoshisis, pili torti, trichorrhexis nodosa, longitudinal grooving, flattened hair shaft, localized loss of cuticle cells and dry, thick, and brittle hair structure of hair follicle), thin and weak hair follicles with prominent damages of the cuticular layer, cuticular fractures and erosions on the surface of the cuticle, local deep cavities just under the cuticular level, and irregular vertically coursing cuticular pattern observed in this study are distinctive findings of hair in FRDA patients. Moreover in later stages of the disease, additional pathognomonic ultra-structural findings were added to hair pathology such as indistinct cuticular pattern, pores on the cuticular surface, and the presence of concave areas on the hair follicle (Table 2). FRDA is a progressive disease and according to our results, it seems that progression of the disease increased the alterations on hair structure. FRDA is a hereditary disease caused by deficient frataxin expression. This mitochondrial protein is related to iron homeostasis, energy metabolism, and oxidative stress. In vitro studies have demonstrated that frataxin deficient cells not only generate more free radicals, but also show a reduced capacity to mobilize antioxidant defences (Al-Mahdawi et al., 2006; Marmolino et. al., 2010; Seguin et al., 2009). Oxidative stress not only caused inadequate energy supply but also retrograde mitochondrial signaling mechanisms give rise to functional and structural alterations in the skin and hair (Feichtinger et al., 2014; Hamanaka and Chandel 2013). Negative effects of oxidative stress on hair structure, especially on cuticle layer, were also well established (Kim, 2011; Tr€ ueb, 2006). Therefore, we suggest that ultra-structural alterations demonstrated in this study might be due to oxidative stress caused by frataxin deficiency in FRDA patients. Microscopy Research and Technique
Because of the complexity of mitochondrial genetics and biochemistry, clinical manifestations of mitochondrial diseases are extremely heterogeneous and difficult to diagnose in early stages. Cutaneous alterations including hair abnormalities associated with unrelated disorders were reported to be a presenting sign which could be a clinical clue to the early diagnosis (Bodemer et al., 1999; Hinson et al., 2007; Silengo et al., 2003). Discriminating ultra-structural hair alterations determined in this study will make a noteworthy contribution to cutaneous alterations associated with mitochondrial diseases. This SEM approach, which is a three dimensional examination technique revealing easily comparable images, is a very spesific, effective, and fast diagnostic tool in the detection of hair alterations in FRDA. As it is used in Chediak–Higashi syndrome (C¸elik et al., 2003), dyskeratosis congenita (C¸elik et al., 2007), and hereditary trichodysplasia (C¸elik et al., 2004), its routine usage in hair analysis of FRDA patients may result in valuable contribution to early diagnosis of the disease. Additionally, considering the psychology of the patients, analysis of hair follicles has some advantages. Hair sample collection is non-invasive, less embarrassing, and can easily be performed. ACKNOWLEDGMENTS The authors would like to thank Prof. Yasemin G€ ursoy € Ozdemir, MD, PhD, who is former professor in Department of Neurology, Medical School, Hacettepe University, for her contribution to the study with re-evaluation of the patients and providing insight and expertise that greatly assisted the research, Prof. Mustafa F. Sargon, MD, PhD, who is professor in Department of Anatomy, Medical School, Hacettepe University, for his great contributions to anatomic evaluations, and Assoc. Prof. H. Evren C¸ubukc¸u, PhD, who is assistant professor in Department of Geological Engineering, Faculty of Engineering, Hacettepe University, for his great help during SEM studies. REFERENCES Ackroyd RS, Finnegan JA, Green SH. 1984. Friedreich’s ataxia: A clinical review with neurophysiological and echocardiographic findings. Arch Dis Child 59:217–221. Al-Mahdawi S, Pinto RM, Varshney D. 2006. GAA repeat expansion mutation Mouse models of Friedreich ataxia exhibit oxidative stres leading to progressive neuronal and cardiacpathology. Genomics 88:580–590.
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