Neurogenetics (2011) 12:6572 DOI 10.

1007/s10048-010-0269-y

ORIGINAL ARTICLE

Genomic duplications mediate overexpression of lamin B1 in adult-onset autosomal dominant leukodystrophy (ADLD) with autonomic symptoms
Jens Schuster & Jimmy Sundblom & Ann-Charlotte Thuresson & Sharon Hassin-Baer & Thomas Klopstock & Martin Dichgans & Oren S. Cohen & Raili Raininko & Atle Melberg & Niklas Dahl

Received: 11 August 2010 / Accepted: 30 November 2010 / Published online: 12 January 2011 # Springer-Verlag 2011

Abstract Adult-onset autosomal dominant leukodystrophy (ADLD) with autonomic symptoms features micturition urgency, constipation, erectile dysfunction, and orthostatic hypotension, usually followed by pyramidal signs and ataxia. Peripheral nerve conduction is normal. The disease is often mistaken for multiple sclerosis in the initial phase. There is a characteristic pattern of white matter changes in the brain and spinal cord on magnetic resonance imaging (MRI), mild atrophy of the brain, and a more marked atrophy of the spinal cord. ADLD is associated with duplications of the lamin B1 (LMNB1) gene but the mechanism by which the rearrangement conveys the phenotype is not fully defined. We analyzed four unrelated families segregating ADLD with autonomic symptoms for duplications of the LMNB1 gene. A single nucleotide polymorphism (SNP) array analysis
Jens Schuster and Jimmy Sundblom contributed equally to this study. J. Schuster : A.-C. Thuresson : N. Dahl (*) Department of Genetics and Pathology, The Rudbeck Laboratory and Science for Life Laboratory, Uppsala University and University Hospital, SE-751 85, Uppsala, Sweden e-mail: niklas.dahl@genpat.uu.se J. Sundblom : A. Melberg Department of Neuroscience, Neurology, Uppsala University and University Hospital, Uppsala, Sweden S. Hassin-Baer : O. S. Cohen Department of Neurology and Parkinsons Disease and Movement Disorders Clinic, Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel Hashomer, Israel S. Hassin-Baer The Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

revealed novel duplications spanning the entire LMNB1 gene in probands from each of the four families. We then analyzed the expression of lamin B1 in peripheral leukocytes by Western blot analysis in five patients from two available families. The protein levels of lamin B1 were found significantly increased. These results indicate that the ADLD phenotype associated with LMNB1 duplications is mediated by increased levels of the lamin B1 protein. Furthermore, we show that a molecular diagnosis for ADLD with autonomic symptoms can be obtained by a direct analysis of lamin B1 in peripheral leukocytes. Keywords LMNB1 . Duplication . Autosomal dominant leukodystrophy (ADLD) . Autonomic symptoms . Lamin B1

T. Klopstock Friedrich Baur Institute, Department of Neurology, Ludwig Maximilians University, Ziemssenstr. 1a, Munich, Germany

M. Dichgans Department of Neurology, Neurologische Klinik, Klinikum Grosshadern, Ludwig Maximilians University, Marchioninistr. 15, Munich, Germany

R. Raininko Department of Radiology, Uppsala University and University Hospital, Uppsala, Sweden

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Abbreviation ADLD Autosomal dominant leukodystrophy CNV Copy number variation MR Magnetic resonance SNP Single nucleotide polymorphism PCR Polymerase chain reaction

(IL) of Arab descent. We identified distinct LMNB1 duplications in each of the four families and we analyzed the expression of LMNB1 mRNA and protein in peripheral nucleated blood cells from patients in the two Swedish families.

Materials/methods Introduction Adult-onset autosomal dominant leukodystrophy (ADLD) with autonomic symptoms is a rare disorder with an onset in the fourth to sixth decade. Autonomic manifestations, such as micturition urgency, bladder retention, erectile dysfunction, orthostatic hypotension, and constipation are usually followed by pyramidal signs and ataxia [17]. Impaired sweating and body temperature regulation may occur and was reported as the possible cause of death in one case [6]. ADLD with autonomic symptoms may be distinguished from primary progressive multiple sclerosis by the family history suggesting autosomal dominant inheritance, the prominent autonomic dysfunction early in the disease course, confluent white matter changes in the brain on neuroimaging, and absence of oligoclonal bands in the cerebrospinal fluid [1]. Peripheral nerve conduction studies are normal in ADLD with autonomic symptoms [1, 36]. Magnetic resonance imaging (MRI) in the initial phase of ADLD reveals T2 hyperintense changes in the upper corticospinal tract progressing along the corticospinal tract down to medulla oblongata, in the upper and the middle cerebellar peduncles, and then to extensive confluent white matter changes in the frontoparietal area with relative sparing of the periventricular white matter [5]. Atrophy of the cerebrum has been described in some individuals [5, 7]. The spinal cord is atrophic [6, 7] and there are diffuse T2 signal intensity changes in the white matter assessed in transverse images [7]. Neuropathological investigations have shown preservation of oligodendroglia and sparse astrogliosis in demyelinated areas of the brain [4, 5]. Genetic mapping of the ADLD gene mutation on chromosome 5q23 [4, 8] was followed by the identification of tandem duplications spanning the gene encoding lamin B1 (LMNB1) in four ADLD families [9]. Subsequently, LMNB1 duplications were confirmed in two other families with ADLD with autonomic symptoms [6, 10] and the disease can thus be defined as a genomic disorder [11, 12]. Increased LMNB1 mRNA levels have also been shown in brain tissue of affected individuals [9] and in transformed lymphoid cells [10]. We report on four non-related families with ADLD with autonomic symptoms including two Swedish (SE1 and SE2) [5, 7, 8], one German (DE), and one Israeli family Patients The families segregate autosomal dominant adultonset autonomic symptoms preceding or, in some cases, together with motor symptoms, imbalance, and white matter changes on MR examination of the brain [5]. The SE1 and SE2 families have been described clinically [5, 7] and genetic linkage to chromosome 5q23 was documented in the SE1 family [8]. The SE1 family includes 31 affected family members [7], and the SE2 family previously reported with two affected [5, 7] now includes three affected members (Fig. 1). Patients from the DE and IL families, not previously reported, were also included (Fig. 1). Two clinically affected patients in each of the DE and IL families (Table 1) underwent clinical neurological examination and MR imaging of the brain. Duplication analysis Samples from one affected patient from each of the families (individuals SE1 VI:1, SE2 II:1, DE III:3, and IL II:1), were analyzed for gene copy number variations (CNVs) using the genome-wide human single nucleotide polymorphism (SNP) array 6.0 (Affymetrix). Data were analyzed with the Genotying Console 3.0.2. Western blot analyses of lamin B1 in nucleated blood cells and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) For western blot analysis, we pelleted nucleated white blood cells from five affected individuals in the two Swedish families (individuals SE1-V:18, SE1-VI:1, SE1-VI:5, SE1-VI:6, and SE2-II:2), as well as from six healthy controls. The cells were dissolved in RIPA buffer containing protease inhibitor cocktail (Invitrogen) or TriZol Reagent (Invitrogen) and stored on ice for 30 min and centrifuged at 13,000g. The supernatant, containing protein, was saved for subsequent analysis. Protein samples were separated on a 412% SDS-PAGE (NuPage, Invitrogen) and transferred to PVDF membranes using the iBLOT transfer system (Invitrogen) according to manufacturer s protocols. Lamin B1 and -actin were detected using primary -lamin B1 and --actin antibodies (both Abcam), respectively. Proteins were visualized using IRD680- or IRD800-labeled secondary antibodies, respectively (LiCor Biosciences), and analyzed using the Odyssey infrared imaging system, determining integrated intensities for lamin B1 following the instructions manual (LiCor Bioscience). -actin was measured in the same

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Fig. 1 Pedigrees of the four families segregating ADLD with autonomic symptoms. Swedish family 1 (SE1), Swedish family 2 (SE2), German family (DE), and Israeli family (IL). Probands analyzed for LMNB1 duplications are denoted with arrows

samples for internal normalization. Each sample was analyzed on four independent blots and the average of the independent measurements was used for further analysis to rule out experimental error. Analyses of all affected individuals were normalized to the controls. For qRT-PCR analysis, we extracted total RNA using TriZol reagent (Invitrogen) from EDTA-treated peripheral blood samples obtained from two available probands (SE1VI:1, SE2-II:1) and healthy controls. RNA quality was assessed by gel electrophoresis and RNA concentration was measured by spectrometry (NanoDrop) before cDNA synthesis using RevertAid cDNA synthesis kit (Fermentas). Primers for qRT-PCR were designed for two different amplicons of the LMNB1 cDNA (primer sequences available upon request). In addition, primers were designed for qRT-PCR analysis of the closest neighboring gene MARCH3. The amplicons were normalized to -actin (BACT) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; primer sequences available upon request). The PCR products were analyzed

in triplicate using a SYBR green RT-PCR kit (Invitrogen) and a Stratagene MX3005 qPCR Thermocycler. The experiment was repeated three times using independent samples from each individual. Data analysis was performed in Microsoft Excel (Microsoft). Statistical analysis Students two-tailed t test was used to calculate the relative and normalized levels of lamin B1 determined by quantitative RT-PCR and Western blot analysis in patients and controls.

Results Clinical and neuroradiological features The clinical and neuroradiological features of families SE1 and SE2 have previously been reported [5, 7]. The

68 Table 1 Clinical features of individuals affected by ADLD with autonomic symptoms in four families Family/individual/ gender SE1/IV:1/M Onset Autonomic symptoms

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Pyramidal Ataxia Cognition Neurophysiological MRI showing Other symptoms/ signs studies leukodystrophy signs + Normal NCS: normal SEP: normal +

SE2/II:2/M

+ 45 year Bladdera symptoms Constipation Postural hypotension Erectile dysfunction 43 year Bladder + symptoms Constipation Postural hypotension Dry skin

Normal

VEP: normal

+ (Fig. 2 c,d)

Increased symptoms with infection

NCS: normal SEP: pathological

DE/III:2/F

DE/III:3/F

IL/II:1/M

IL/III:1/M

53 year Bladder + symptoms Postural hypotension Heat intolerance 58 year Bladder + symptoms Fecal incontinence 54 year Bladder + symptoms Constipation Erectile dysfunction 50 year Bladder + symptoms Constipation Erectile dysfunction

MMT: 24 NCS: normal

MMT: 26 NCS: normal

Normal

Distal sensory loss clinically

Postural tremor

MMT: 23 NCS: normal

+ (Fig. 2 a,b)

Parkinsonism, levodopa responsive No tremor

Bladder, refers to urinary bladder

MMT mini-mental test, NCS nerve conduction study, SEP somatosensory evoked potential, VEP visual evoked potential

clinical details of patients SE1 individual V1:1 and SE2 individual II:2 are presented in Table 1. The family history of the probands DE individual III:3 and IL individual II:1 indicates that four patients were affected in the DE family over two generations and that four patients were affected in the IL family over three generations. Patients from the DE and IL families had an onset in their sixth decade with autonomic symptoms, pyramidal signs, and had brain MRI signs characteristic of this type of leukodystrophy. Peripheral nerve conduction studies in two affected members of the DE family and one affected member in the IL family were normal. Individual III:1 in the IL family had levodopa-responsive parkinsonism. This clinical feature has not been described previously in ADLD with autonomic symptoms but the patients autonomic symptoms, pyramidal signs, ataxia, and MRI signs (Fig. 2ab) are compatible with this type of ADLD (Fig. 2cd and Table 1).

Analysis of gene CNV The analysis of CNVs using the Affymetrix SNP Array 6.0 showed a pattern consistent with duplications spanning the entire LMNB1 gene on chromosome 5q in all four probands. Each proband has a distinct duplication (Fig. 3). Patient SE1 V1:1 has a duplication extending between nucleotide positions 126.133.787 126.321.325 (CN_1108542 to CN_287299) with an estimated size of 187 kb; patient SE2 II:2 has a duplication between 126.159.058 and 126.266.384 (CN_1108548 to CN_1108578) with an estimated size of 107 kb; patient DE has a duplication between 126.049.850 and 126.267.351 (SNP_A-4283349 to CN_1108579) with an estimated size of 218 kb and patient IL has a duplication between 126.073.820 and 126.267.351 (SNP_A-8383323 to CN_1108579) with an estimated size of 194 kb. The duplicated segments extend over part of the MARCH3-gene in all four cases.

Neurogenetics (2011) 12:6572 Fig. 2 Brain changes on MR images (T2-weighted spin echo sequence) in family IL individual III:1 (a, b) and family SE2 individual II:2 (c, d). a, c Image slices are shown at the level of lateral ventricles and b, d through the brain stem and cerebellum. The less affected area around the lateral ventricles is characteristic as well as large hyper-intensities in the middle cerebellar peduncles. The pyramidal tracts are affected in the pons

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Lamin B1 expression The lamin B1 protein levels were significantly increased in white blood cells from five ADLD patients from the SE1 and SE2 families when compared to healthy controls (p =

0.022; Fig. 4ab). The increase was approximately twofold. Similarly, the levels of LMNB1 mRNA in white blood cells were increased in two available ADLD patients when compared to the levels from healthy controls (p <0.0001 from three independent experiments; Fig. 4c). This pattern

Fig. 3 Schematic presentation of chromosome 5 (top) with extension of duplicated segments spanning the LMNB1 gene on 5q23 in probands from four different ADLD families (bottom). The duplicated

segments associated with ADLD are indicated as black bars and span 187 (SE1), 107 (SE2), 218 (DE), and 194 kb (IL), respectively

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Fig.

4 Levels of lamin B1 protein and its mRNA in ADLD patients carrying LMNB1 duplications. a Representative western blot analysis showing detection of lamin B1 and -actin in peripheral white blood cells from five individuals of two families segregating ADLD (individuals SE1-VI:1, SE1-VI:5, SE1-VI:6, SE1-V:18, and SE2-II:2) and six controls (ctrl 1ctrl 6). b Quantification of western blot analysis of lamin B1 expression normalized to -actin. The values are illustrated as a lamin B1/-actin ratio with average of controls set to 1.0. Results of four independent experiments were combined. ***p value= 0.022 (Students t test). c Diagram showing the relative expression of LMNB1 mRNA in white blood cells from two available ADLD patients (SE1-VI:1 and SE2-II:2) and two controls measured by quantitative real-time PCR using primers spanning exons 6 and 7 of the LMNB1 gene. Mean values are shown from three independent measurements from the two ADLD patients and healthy controls (n =2). The analysis was normalized to GAPDH and average values of controls are set to 1.0. Error bars denote standard deviation (p value <0.0001, Students t test)

was observed for both amplicons detecting the LMNB1 transcript. The levels of MARCH3 mRNA were similar when comparing patients and controls.

Discussion We present four families segregating ADLD with autonomic symptoms associated with novel duplications spanning the LMNB1 gene. Two of the families (DE and IL) have not been reported previously whereas some characteristics of families SE1 and SE2 have been reported elsewhere [5, 7, 8]. LMNB1 duplications have been documented in six families with ADLD with autonomic symptoms [6, 9, 10]. With the exception of two American families having an apparently identical duplication spanning 169 kb, the sizes of the duplications varied between the families indicating recurrent mutational events. Two Japanese families have LMNB1 duplications spanning 341 and >150 kb, respectively [9]. One LMNB1 duplication of approximately 280 kb was reported in a FrenchCanadian family [6] and a 140190-kb duplication in an Italian family [8]. In this study, we identified another four different LMNB1 duplications ranging from 107 to 218 kb in size supporting independent events. Lamins are major components of the nuclear lamina, which is a meshwork of intermediate filament proteins. Lamins are classified into two forms, A- and B-type lamins. The B-type lamins are ubiquitously expressed and further classified into lamin B1 and B2 [1315]. LMNB1 is expressed in a variety of human cell types as judged by the demonstration of lamin B1 mRNA and lamin B1 protein [13]. Lamins interact with each other and with proteins of the inner nuclear membrane, transcription factors, DNA, and chromatin [16]. LMNB1 over-expression leads to a degenerative phenotype in Drosophila melanogaster eye [9]. Overexpression

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71 Ethical standards The study was approved by the local Ethics Committee of Uppsala University Hospital and individuals who entered the study gave oral and written consent.

of LMNB1 mRNA has also been demonstrated in brain tissue of patients with ADLD with autonomic symptoms [9] and in transformed lymphoid cells in ADLD with autonomic symptoms [10]. The precise mechanism by which LMNB1 duplications mediate ADLD is not clear. It has been hypothesized that the neuropathological process in ADLD is caused by a selective effect of LMNB1 overexpression on the transcriptional regulation of genes expressed in myelinogenesis, alternatively by a toxic effect of large amounts of lamin B1 protein on specific cell types [9]. The levels of lamin B1 are regulated by the microRNA miR-23 and this has been shown to be important for normal oligodendroglia development and myelin formation in vitro [17]. The LMNB1 mRNA overexpression in families with LMNB1 duplications suggests that the miR-23 regulation of LMNB1 mRNA is eliminated. This would then lead to the increased levels of lamin B1 protein observed in our study. With the novel results presented here LMNB1 duplications have to date been identified in ten families and exclusively in families with ADLD with autonomic symptoms. A previous study of eight families with adultonset leukoencephalopathy showed that ADLD with autonomic symptoms was only observed in the single family with an LMNB1 duplication [10]. Interestingly, a unique ADLD family without autonomic dysfunction [18, 19] linked to chromosome 5q23 did not show any rearrangement or mutation in the coding regions of LMNB1 [20]. However, LMNB1 mRNA expression was increased in lymphoblastoid cells from these patients and similar to levels in patients with LMNB1 duplications, suggesting a mutation in a LMNB1 regulatory sequence [20]. In combination, our results and previous studies strongly suggest that ADLD with autonomic symptoms is genetically homogeneous and related to LMNB1 duplications, although the sizes of the duplications vary between families. We also show for the first time that the LMNB1 duplication is associated with increased levels of lamin B1 protein as part of the disease mechanism and that the increased lamin B1 levels can be detected in peripheral leukocytes for diagnostic purposes. We recommend that screening for LMNB1 duplications can be combined with, or replaced for, analysis of lamin B1 expression in peripheral leukocytes when ADLD with autonomic symptoms is suspected. Both analyses may also be used in order to discriminate between ADLD with or without autonomic symptoms.

Financial disclosure The authors report no financial competing interests

Conflict of interest The authors report no conflict of interest

References
1. Eldridge R, Anayiotos CP, Schlesinger S, Cowen D, Bever C, Patronas N, McFarland H (1984) Hereditary adult-onset leukodystrophy simulating chronic progressive multiple sclerosis. N Engl J Med 311:948953 2. Schwankhaus JD, Patronas N, Dorwart R, Eldridge R, Schlesinger S, McFarland H (1988) Computed tomography and magnetic resonance imaging in adult-onset leukodystrophy. Arch Neurol 45:10041008 3. Schwankhaus JD, Katz DA, Eldridge R, Schlesinger S, McFarland H (1994) Clinical and pathological features of an autosomal dominant, adult-onset leukodystrophy simulating chronic progressive multiple sclerosis. Arch Neurol 51:757766 4. Coffeen CM, McKenna CE, Koeppen AH, Plaster NM, Maragakis N, Mihalopoulos J, Schwankhaus JD, Flanigan KM, Gregg RG, Ptcek LJ, Fu YH (2000) Genetic localization of an autosomal dominant leukodystrophy mimicking chronic progressive multiple sclerosis to chromosome 5q31. Hum Mol Genet 9:787793 5. Melberg A, Hallberg L, Kalimo H, Raininko R (2006) MR characteristics and neuropathology in adult-onset autosomal dominant leukodystrophy with autonomic symptoms. AJNR Am J Neuroradiol 27:904911 6. Meijer IA, Simoes-Lopes AA, Laurent S, Katz T, St-Onge J, Verlaan DJ, Dupr N, Thibault M, Mathurin J, Bouchard JP, Rouleau GA (2008) A novel duplication confirms the involvement of 5q23.2 in autosomal dominant leukodystrophy. Arch Neurol 65:14961501 7. Sundblom J, Melberg A, Kalimo H, Smits A, Raininko R (2009) MR imaging characteristics and neuropathology of the spinal cord in adult-onset autosomal dominant leukodystrophy with autonomic symptoms. AJNR Am J Neuroradiol 30:328335 8. Marklund L, Melin M, Melberg A, Giedraitis V, Dahl N (2006) Adult-onset autosomal dominant leukodystrophy with autonomic symptoms restricted to 1.5 Mbp on chromosome 5q23. Am J Med Genet B Neuropsychiatr Genet 141B:608614 9. Padiath QS, Saigoh K, Schiffmann R, Asahara H, Yamada T, Koeppen A, Hogan K, Ptcek LJ, Fu YH (2006) Lamin B1 duplications cause autosomal dominant leukodystrophy. Nat Genet 38:11141123 10. Brussino A, Vaula G, Cagnoli C, Mauro A, Pradotto L, Daniele D, Di Gregorio E, Barberis M, Arduino C, Squadrone S, Abete MC, Migone N, Calabrese O, Brusco A (2009) A novel family with Lamin B1 duplication associated with adult-onset leucoencephalopathy. J Neurol Neurosurg Psychiatry 80:237240 11. Lupski JR, Stankiewicz P (2005) Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet 1(49):627633 12. Padiath QS, Fu YH (2010) Autosomal dominant leukodystrophy caused by lamin B1 duplications: a clinical and molecular case study of altered nuclear function and disease. Meth Cell Biol 98:337357

Acknowledgments We thank the patients and families who participated in this study. This project was financially supported by the Swedish Research Council, the Svstaholm Society, Hedberg foundation for Medical Research, Selander Foundation, the Swedish Association for the Neurologically Disabled, Uppsala University and Uppsala University Hospital.

72 13. Lin F, Worman HJ (1997) Expression of nuclear lamins in human tissues and cancer cell lines and transcription from the promoters of the lamin A/C and B1 genes. Exp Cell Res 236:378384 14. Gruenbaum Y, Margalit A, Goldman RD, Shumaker DK, Wilson KL (2005) The nuclear lamina comes of age. Nat Rev Mol Cell Biol 6:2131 15. Worman HJ, Fong LG, Muchir A, Young SG (2009) Laminopathies and the long strange trip from basic cell biology to therapy. J Clin Invest 119:18251836 16. Schirmer EC, Foisner R (2007) Proteins that associate with lamins: many faces, many functions. Exp Cell Res 313:21672179 17. Lin ST, Fu YH (2009) miR-23 regulation of lamin B1 is crucial for oligodendrocyte development and myelination. Dis Model Mech 2:178188

Neurogenetics (2011) 12:6572 18. Bergui M, Bradac GB, Leombruni S, Vaula G, Quattrocolo G (1997) MRI and CT in an autosomal-dominant, adult-onset leukodystrophy. Neuroradiology 39:423426 19. Quattrocolo G, Leombruni S, Vaula G, Bergui M, Riva A, Bradac GB, Bergamini L (1997) Autosomal dominant late-onset leukoencephalopathy. Clinical report of a new Italian family. Eur Neurol 37:5361 20. Brussino A, Vaula G, Cagnoli C, Panza E, Seri M, Di Gregorio E, Scappaticci S, Camanini S, Daniele D, Bradac GB, Pinessi L, Cavalieri S, Grosso E, Migone N, Brusco A (2010) A family with autosomal dominant leukodystrophy linked to 5q23.2q23.3 without lamin B1 mutations. Eur J Neurol 17:541549