Differential gene expression in chronic inflammatory demyelinating polyneuropathy (CIDP) skin biopsies

Journal of the Neurological Sciences 290 (2010) 115–122

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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Differential gene expression in chronic inflammatory demyelinating polyneuropathy (CIDP) skin biopsies Grace Lee a,⁎, Zhaoying Xiang b, Thomas H. Brannagan III c, Russell L. Chin a, Norman Latov a a b c

Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 525 E. 68th St, New York City, NY 10021, USA Department of Microbiology and Immunology, Weill Medical College of Cornell University, 525 E. 68th St, New York City, NY 10021, USA Neurological Institute, Columbia Presbyterian Medical Center, 710W 168 St, New York City, NY 10021, USA

a r t i c l e

i n f o

Article history: Received 3 July 2009 Received in revised form 14 September 2009 Accepted 7 October 2009 Available online 17 November 2009 Keywords: Chronic inflammatory demyelinating polyneuropathy CIDP Differential gene expression Skin Diabetic neuropathy Charcot–Marie–Tooth disease CMT-1

a b s t r a c t Gene expression analysis previously identified molecular markers that are up-regulated in sural nerve biopsies from patients with chronic inflammatory demyelinating polyneuropathy (CIDP). To determine whether the same or additional genes are also up-regulated in skin, we applied gene microarray profiling and quantitative real-time PCR (qPCR) analysis to skin punch biopsies from patients with CIDP and controls. Five genes, allograft inflammatory factor 1 (AIF-1), lymphatic hyaluronan receptor (LYVE-1/XLKD1), FYN binding protein (FYB), P2RY1 (purinergic receptor P2Y, G-protein-coupled, 1), and MLLT3 (myeloid/ lymphoid or mixed-lineage leukemia translocated to, 3), all associated with immune cells or inflammatory processes, were elevated in punch skin biopsies from patients with CIDP as compared to normal subjects or patients with Charcot–Marie–Tooth Type 1 (CMT1). The average fold change of the 5 genes over normal expression, as determined by qPCR, was significantly elevated in skin biopsies from patients with CIDP in comparison to CMT1 or diabetic neuropathy, and similar to that seen in Lyme disease. The findings indicate the presence of inflammatory changes in the skin of patients with CIDP. © 2009 Elsevier B.V. All rights reserved.

1. Introduction

2. Materials and methods

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an inflammatory, immune-mediated disorder that targets the myelin sheaths of peripheral nerve. The diagnosis is based on the clinical presentation, and evidence for demyelination in the absence of other known causes such as hereditary demyelinating neuropathy or Charcot–Marie–Tooth disease type I (CMT1) [1]. Previous microarray studies identified a panel of genes, including TAC1, NRID1, SCD, AIF1, MSR1, LYVE-1/XKLD1, NQO1, CLCA2, PCSK1, and FYB, that were significantly up-regulated in sural nerve biopsies of CIDP patients [2]. In this study we used microarray analysis and quantitative PCR analysis to determine whether the same or other genes are also expressed in skin biopsies from such patients. Recent studies reported that some pathological changes can also be seen in skin biopsies from patients with demyelinating or inflammatory neuropathies including CIDP [3], CMT [4,5], Guillain Barre syndrome [6], anti-MAG neuropathy [7], vasculitic neuropathy [8], and eosinophilia-associated neuropathy [9].

2.1. Patients

⁎ Corresponding author. E-mail address: [email protected] (G. Lee). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.10.006

Patient and normal volunteers were recruited at The Neuropathy Center of the Cornell Weill Medical College, with IRB approval and the patient informed consent. Tissue from 11 patients with CIDP, 8 patients with Charcot–Marie–Tooth disease type I, and 7 normal controls were used in the microarray studies. CIDP was diagnosed according to the EFNS and PNS Joint Task Force guidelines [10]. The diagnosis of CMT type I, was made on the basis of the clinical presentation, family history, electrodiagnostic studies [11], and genetic testing (Athena Diagnostics Inc, Wooster MA). Of the 11 CIDP patients, 8 were men and 3 were women, ages 39 to 64. Three had classical CIDP with proximal and distal weakness, and 8 had distal CIDP. Skin biopsies were done at the time of initial diagnosis and prior to therapy. Of the 8 CMT-1 patients, 2 were men and 6 were women, ages 18 to 59. Six had PMP22 duplication, 1 PMP22 sequence alteration, and 1 MPZ sequence alteration. Of the 7 healthy subjects, 4 were men and 3 were women, ages 34 to 60. Tissue from 5 patients with diabetic neuropathy and 4 with Lyme disease was additionally examined by quantitative PCR. Patients with diabetic neuropathy had type II diabetes as defined by the American Diabetes Association, and a distal axonal neuropathy. Patients with Lyme disease had axonal neuropathy and positive serologic tests for antibodies to Borrelia burgdorferi by ELISA and Western blot (Quest Diagnostics).

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2.2. Skin biopsies and RNA preparation Punch skin biopsies, approximately 2 mm3, were obtained in all cases from the anterior aspect of the forearm and immediately immersed in RNALater solution (Applied Biosystems, Foster City) for RNA preservation. The samples were homogenized mechanically in 1 ml Qiagen RLT lysis solution, extracted at 4 °C with 0.5 ml phenol:chloroform, pH 4.7, then purified and DNAse-treated on a spin column using the RNeasy Mini-kit (Qiagen, Valencia, CA). RNA was quantitated on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and assayed for quality on a 2100 Bioanalyzer (Agilent Technologies, Foster City, CA). 2.3. Microarray gene profiling RNA from 10 CIDP, 6 CMT1 and 5 normal samples were quantitatively reverse transcribed and amplified, then labeled and fragmented with the Nugen Ovation RNA Amplification and the FL-Ovation cDNA Biotin Module V2 kits, respectively (NuGen Technologies, Inc., San Carlos, CA). The samples were hybridized to Affymetrix Human U133 Plus 2.0 microarray chips (Affymetrix, Santa Clara, CA) and signal intensities were read in the Hewlett-Packard G2500A Gene Array Scanner. 2.4. Microarray data analysis Chip data was imported into the GeneSpring GX 7.3 program (Agilent Technologies, Foster City, CA). Signal values less than 0.01 were set to 0.01, arrays were normalized to the 50th percentile, and individual genes normalized to the median. Normalized data was then filtered to retain genes flagged as present or marginal in all of the CIDP samples. One-way ANOVA (variances not assumed to be equal) with p < 0.05, comparing each neuropathy group with the normal group or with each other, followed by filtration for greater or less than 1.5 fold differences, was applied to determine potential differential expression. Classification by Gene Ontology (GO) Consortium assigned biologic processes, pathway and network analyses through the GeneSpring GS and Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City CA) software further identified genes of potential interest. 2.5. Quantitative real-time PCR (qPCR) DNAse-treated RNA was quantitatively reversed transcribed and amplified according to the protocol of the WT-Ovation RNA Amplification kit (NuGen Technologies, Inc., San Carlos, CA). Equal amounts of total RNA from 7 normal subjects were pooled as a single control unit. cDNA was purified though Zymo DCC-25 spin columns (Zymo Research, Orange, CA). Quantitative PCR was performed in triplicate wells of 384-well plates in the ABI 7900HT instrument (Applied Biosystems, Foster City, CA) with 12 ng cDNA, 5 μl TaqMan 2× Gene Expression Master Mix (Applied Biosytems), and 0.5 μl gene-specific 20× TaqMan Gene Expression Assay primer-probe per 10 μl reaction volume. Thermocycling settings were: 50 °C, 2 min; 95 °C, 10 min; 40 cycles of 95 °C, 2 s, 60 °C, 1 min. To identify optimal endogenous gene controls, a panel of 10 Taqman primer-probe sets was tested either individually or in various combinations for normalization of target gene data. Genes with the most stable expression among all tissue samples were determined through the geNorm VBA applet software (http://medgen.ugent.be/~jvdesomp/ genorm/) [12]. 2.6. qPCR data analysis Target gene qPCR data were normalized to endogenous controls and relative mRNA expression was calculated with the ΔΔCt method through the SDS 2.2/RQ Manager software (Applied Biosystems, Foster City, CA) and using the pooled normal samples as the calibrator [13]. Significant differences at p < 0.05 in gene expression were determined with the two-tailed, nonparametric Mann–Whitney test

for unpaired data using the GraphPad Instat software (Instant Statistics, GraphPad Software, San Diego, CA). 3. Results 3.1. Microarray analysis Tissues from patients with CIDP or CMT-1, and from normal subjects, were examined by microarray analysis. From 54,675 genes on the gene chip, through a series of filters, 143 with Genbank assignments were found to be significantly up-regulated (p < 0.05) in at least 6 of 10 CIDP samples with a fold change (FC) of >1.5 relative to both CMT1 and normal samples. Similarly, 145 genes were identified as down-regulated in CIDP. Gene ontology, pathway and network analyses were applied to these genes to identify associated functions, processes and diseases. Genes with GO classifications are broadly categorized in Tables 1 (up-regulated) and 2 (down-regulated) together with the fold change values of CIDP compared to normal, CIDP to CMT1, and CMT1 to normal. As many genes have multiple functions across different categories, these groups are not exclusionary. The top biological or disease processes most significantly associated with up-regulated genes in CIDP include immunological (27% of genes, p = 0.0001–0.0189), cell death (24%, p = 0.0001–0.0189), cell signaling/ interaction (30%, p = 0.0002–0.0189), cellular movement (33%, p = 0.0005–0.0189), cancer (32%, p = 0.0006–0.0189), inflammatory (27%, p = 0.0011–0.0126), and skeletal/muscular system development/ function (30%, p = 0.0063–0.0126). Many of the genes have multiple associations. The processes most significantly associated with down-regulated genes in CIDP include cell growth/proliferation (27% of genes, 0.0001– 0.04), cell death (39%, p = 0.0001–0.05), small molecule biochemistry (34%, 0.0010–0.05), cancer (44%, 0.0052–0.05), gene expression (23%, p = 0.0052–0.05), and neurological (21%, 0.0052–0.05). The microarray study revealed primarily small changes in gene expression in the skin samples, with fold change values predominantly in the 1.5–3 range, with none above 5. A number of potentially relevant genes, for which Taqman primer-probe sets were available, with the highest fold change from normal or CMT1 were selected for validation by qPCR in addition to a panel of 10 genes that were found to be up-regulated in CIDP sural nerve [2]. 3.2. Validation of endogenous control genes for qPCR The high sensitivity of qPCR for assay of gene expression requires one or more stably expressed controls in a given set of tissues for optimal normalization, with housekeeping genes such as GAPDH (glyceraldehyde-3-phosphate dehydrogenase), B-Actin, and 18S ribosomal RNA frequently used for this purpose. However, housekeeping gene expression has been reported to vary considerably and could result in variable target gene expression when used for normalization. We evaluated 10 commonly used housekeeping genes for use as endogenous reference controls for skin samples after first determining from the microarray studies that none showed significant expression above normal levels in the neuropathy patients. These genes, GAPDH, B-Actin, 18S, HMBS, HPRT, PGK1, STST1, TBP, and UBC, were assayed for expression in 32 skin biopsies from normal and patient forearm, thigh or finger. Except for GAPDH and PGK1, which are both in the glycolysis pathway, these genes are not co-regulated. From the qPCR Ct values, the cycle number at which fluorescence crosses a threshold point, the geNorm program calculates the gene expression stability measure, M, for a reference gene as the average pairwise variation for that gene with all other tested reference genes. Stepwise exclusion of the gene with the highest M value allows ranking of the tested genes according to their expression stability [12]. By this measure, GAPDH and PPIA (peptidylprolyl isomerase A (cyclophilin A))

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Table 1 Up-regulated genes in CIDP as compared to CMT1 and normal skin biopsies. Fold change (FC) Gene symbol

CIDP vs. N

CIDP vs. CMT1

CMT1 vs. N

Description

Genbank no.

Signal transduction APBB1IP

4.65

1.87

2.49

AI093231

MS4A7 P2RY13 MS4A4A GRB14 OR7A5 ARHGAP20 P2RY1 GPR34 GNAS FGF13

3.24 2.82 2.67 2.62 2.62 2.12 1.85 1.83 1.60 1.57

1.60 1.64 1.61 1.70 1.54 1.52 2.61 1.56 2.04 2.17

2.02 1.71 1.66 1.54 1.70 1.39 0.71 1.17 0.79 0.73

Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein Membrane-spanning 4-domains, subfamily A, member 7 Purinergic receptor P2Y, G-protein-coupled, 13 Membrane-spanning 4-domains, subfamily A, member 4 Growth factor receptor-bound protein 14 Olfactory receptor, family 7, subfamily A, member 5 Rho GTPase activating protein 20 Purinergic receptor P2Y, G-protein-coupled, 1 G-protein-coupled receptor 34 GNAS complex locus Fibroblast growth factor 13

Immune, inflammatory, defense response LILRB1 4.62 2.71

1.70

AI681260

TLR4 TLR7 FYB CX3CR1 CD163 MS4A2

3.46 3.40 2.50 2.45 2.39 2.34

2.30 2.19 1.50 1.51 1.50 1.55

1.50 1.55 1.67 1.62 1.59 1.51

AIF-1 BCL2 FCGR2A CD69

2.26 2.18 1.97 1.91

1.60 1.71 1.59 1.94

1.41 1.28 1.24 0.98

Leukocyte immunoglobulin-like receptor, subfam. B (with TM, ITIM domains), mem. 3 Toll-like receptor 4 Toll-like receptor 7 FYN binding protein (FYB-120/130) Chemokine (C-X3-C motif) receptor 1 CD163 antigen (macrophage scavenger receptor) Membrane-spanning 4-domains, subfamily A, member 2 (Fc fragment of IgE, high affinity I, receptor for; beta polypeptide) Allograft inflammatory factor 1 B-cell CLL/lymphoma 2 Fc fragment of IgG, low affinity IIa, receptor (CD32) CD69 antigen (p60, early T-cell activation antigen)

Protein synthesis/modification ALPK2 4.06 PELO 3.73 PTPRB 2.37 TTLL5 2.27 RARS 2.17 STYK1 2.12 KIAA0804 2.08 PTPRO 1.89 PDE4DIP 1.88 DMXL2 1.79

2.12 2.98 2.07 1.98 1.71 1.80 2.06 1.68 1.92 1.64

1.91 1.25 1.15 1.15 1.27 1.18 1.01 1.12 0.98 1.09

Alpha-kinase 2 Pelota homolog (Drosophila) Protein tyrosine phosphatase, receptor type, B Tubulin tyrosine ligase-like family, member 5 Arginyl-tRNA synthetase Serine/threonine/tyrosine kinase 1 KIAA0804 (ubiquitin-protein ligase activity) Protein tyrosine phosphatase, receptor type, O Phosphodiesterase 4D interacting protein (myomegalin) Dmx-like 2

BE551416 BG619261 AL080103 AK021879 AW593666 NM_018423 AW270499 NM_002848 R44149 AB020663

Transport SLC2A10 LOC51760 SLC26A7 CLIC6 NBEA COPB2 RINT-1 MLPH ABCA8 SYTL4 AFTIPHILIN CENTG2 KCNAB1 KCNC4 RYR2

3.93 3.06 2.92 2.39 2.33 2.05 1.97 1.97 1.95 1.87 1.82 1.74 1.68 1.60 1.58

1.91 1.59 1.59 1.53 1.56 1.55 1.53 1.61 1.51 1.68 1.70 1.71 1.53 1.85 2.11

2.06 1.92 1.83 1.57 1.49 1.32 1.29 1.22 1.29 1.12 1.07 1.01 1.10 0.87 0.75

Solute carrier family 2 (facilitated glucose transporter), member 10 Synaptotagmin XVII Solute carrier family 26, member 7 Chloride intracellular channel 6 Neurobeachin Coatomer protein complex, subunit beta 2 (beta prime) Rad50-interacting protein 1 Melanophilin ATP-binding cassette, sub-family A (ABC1), member 8 Synaptotagmin-like 4 (granuphilin-a) Aftiphilin protein Centaurin, gamma 2 Potassium voltage-gated channel, shaker-related subfamily, beta member 1 Potassium voltage-gated channel, Shaw-related subfamily, member 4 Ryanodine receptor 2 (cardiac)

NM_030777 AI582818 AI758950 AI638295 NM_015678 AF070618 NM_021930 NM_024101 NM_007168 AI167292 AW195572 AA706753 BF433830 BF969982 BE968750

Enzymatic activity GALNTL2

3.65

2.55

1.43

BF055343

HAS2 TBC1D12

3.24 1.75

1.89 1.54

1.72 1.14

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 2 Hyaluronan synthase 2 TBC1 domain family, member 12

Cell adhesion NRXN1 NEGR1 CLDN23 L1CAM RAPH1 HNT XLKD1/LYVE-1 SSPN

3.38 2.93 2.88 2.77 2.39 2.27 2.01 2.00

2.04 1.74 2.06 1.62 1.59 1.55 1.52 1.87

1.66 1.69 1.40 1.72 1.50 1.46 1.32 1.07

Neurexin 1 Neuronal growth regulator 1 Claudin 23 L1 cell adhesion molecule Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 Neurotrimin Extracellular link domain containing 1 Sarcospan (Kras oncogene-associated gene)

AU146874 AW001754 AW375186 AI653981 BF196252 AW085558 AL574194 AL136756

AF177765 AI301935 NM_023914 NM_024021 NM_017506 AI936560 AF039686 AK054976 AA650558 NM_004114

AF177765 NM_016562 BF679849 AF177765 NM_004244 NM_000139 BF213829 BF003032 NM_021642 L07555

AI374739 N34407

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Table 1 (continued) Fold change (FC) Gene symbol

CIDP vs. N

CIDP vs. CMT1

CMT1 vs. N

Description

Genbank no.

Cell adhesion KIAA1467 PCDHB2 CHL1

1.96 1.73 1.51

1.52 2.26 1.61

1.28 0.77 0.94

KIAA1467 protein (cell-matrix adhesion) Protocadherin beta 2 Cell adhesion molecule with homology to L1CAM (close homolog of L1)

AA035771 NM_018936 AL359583

Transcription ZNF101 RFX2 TFAP2E CRSP6 ZNF441 MLLT3

2.98 2.63 2.63 2.54 2.28 2.18

1.61 1.92 1.57 1.52 1.82 2.04

1.84 1.37 1.67 1.67 1.25 1.07

NM_033204 AA417099

ZFHX1B ZNF642 ZNF302 THRB

2.16 2.16 2.12 2.10

1.52 1.68 1.57 1.51

1.42 1.29 1.35 1.39

LOC114977 ZNF277 POLR1B MBD1 ZNF180 ZNF655 RBM15

2.04 2.01 1.96 1.86 1.85 1.76 1.59

1.51 1.86 1.57 1.64 1.82 1.51 1.69

1.36 1.08 1.25 1.14 1.02 1.16 0.94

Zinc finger protein 101 Regulatory factor X, 2 (influences HLA class II expression) Transcription factor AP-2 epsilon (activating enhancer binding protein 2 epsilon) Cofactor required for Sp1 transcriptional activation, subunit 6, 77 kDa Zinc finger protein 441 Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 3 Zinc finger homeobox 1b Zinc finger protein 642 Zinc finger protein 302 Thyroid hormone receptor, beta (erythroblastic leukemia viral (v-erb-a) oncogene homolog 2, avian) Hypothetical protein BC014148 Zinc finger protein 277 Polymerase (RNA) I polypeptide B, 128 kDa Methyl-CpG binding domain protein 1 Zinc finger protein 180 (HHZ168) Zinc finger protein 655 RNA binding motif protein 15

Binding FLJ11259 ZDHHC11 ZNF533 PCOLCE2 ANTXR2 FLJ31265 ADD3 ABC1 KATNAL2 KIAA1377 TTC17 TTC9C MAGI3 LCP1 KIAA1040 SERAC1 RUFY2 TTC18 ZXDA; ZXDB MSI2

2.97 2.93 2.87 2.66 2.50 2.19 2.07 2.05 2.01 1.95 1.87 1.87 1.84 1.81 1.77 1.77 1.76 1.72 1.70 1.65

1.52 1.56 1.74 1.80 1.52 1.77 1.64 1.72 1.55 1.62 1.51 1.75 1.53 1.64 1.51 1.64 2.06 1.51 1.71 1.72

1.95 1.87 1.65 1.48 1.64 1.24 1.26 1.19 1.29 1.20 1.24 1.07 1.20 1.11 1.18 1.08 0.86 1.14 1.00 0.96

Hypothetical protein FLJ11259 Zinc finger, DHHC-type containing 11 Zinc finger protein 533 Procollagen C-endopeptidase enhancer 2 Anthrax toxin receptor 2 Nudix (nucleoside diphosphate linked moiety X)-type motif 16 Adducin 3 (gamma) Amplified in breast cancer 1 Katanin p60 subunit A-like 2 KIAA1377 protein Tetratricopeptide repeat domain 17 Tetratricopeptide repeat domain 9C Membrane associated guanylate kinase, WW and PDZ domain containing 3 Lymphocyte cytosolic protein 1 (L-plastin) KIAA1040 protein Serine active site containing 1 RUN and FYVE domain containing 2 Tetratricopeptide repeat domain 18 Zinc finger, X-linked, duplicated A; zinc finger, X-linked, duplicated B Musashi homolog 2 (Drosophila)

AA724995 NM_024786 AI694320 NM_013363 BE673665 AI809108 AW450360 BU631635 BC034999 BF057799 AK023161 AF289605 AI692181 J02923 BG548738 AA128978 BF028405 AW024437 AL034396 BF029215

Apoptosis NCKAP1 DOCK1

1.82 1.64

1.64 1.69

1.10 0.97

NCK-associated protein 1 Dedicator of cytokinesis 1

NM_013436 AI939580

Other ETNK1 UTP14C AMOT PROM1

2.24 2.16 1.87 1.86

1.57 1.53 2.32 2.08

1.43 1.41 0.80 0.89

Ethanolamine kinase 1 UTP14, U3 small nucleolar ribonucleoprotein, homolog C (yeast) Angiomotin Prominin 1

AL137750 AA708016 AF286598 NM_006017

showed the least variability for skin tissue, with similar low M values of 0.0777. As there was no advantage to using the averaged Ct values of both genes, and normalization with GAPDH yielded fold change values closest to those determined by microarray, GAPDH was selected as the endogenous control for the skin biopsy qPCR studies. 3.3. qPCR assessment of gene expression and validation microarray results Of a panel of 10 genes with elevated expression in CIDP sural nerve three, AIF-1, LYVE-1, and FYB, were also significantly up-regulated in CIDP skin biopsies (Table 1). Among the additional genes identified from microarray studies of CIDP, CMT1 and normal skin, only MLLT3 and P2RY1 could be validated by qPCR (Fig. 1A) at levels in CIDP that are

AK022156 NM_152355 BC030550 AF086037 BQ433060 NM_018443 BG494007 AV683221 BC020626 BC004882 AI205309 NM_013256 BF219240 AF086464

significantly higher than in CMT and normal samples. The expression of other genes selected in an initial round of screening, NRXN1, TLR4, TLR7, CIRL, LTB4R, LILRB1, VNN1, PROM1, and MARCO was either not elevated sufficiently or varied too widely to reach statistically significant difference at p < 0.05. The myelin protein, P0, was detected in all samples, indicating that myelinated nerve tissue was present in the skin biopsies, although expression was generally lower compared to normals among the CIDP and diabetic neuropathy patients and in half of the CMT patients, possibly reflecting a decrease of myelinated fibers. Each of the 5 genes, AIF-1, LYVE-1, FYB, MLLT3 and P2RY1, was expressed at >1.5 fold (FC) over normal controls in 8–10 of 11 CIDP skin biopsies. With the exception of FYB (p = 0.09) in diabetic neuropathy, the CIDP group had, on average, significantly elevated expression

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Table 2 Down-regulated genes in CIDP as compared to CMT1 and normal skin biopsies. Fold change (FC) Gene symbol

CIDP vs. N

CIDP vs. CMT1

CMT1 vs. N

Description

Genbank no.

Enzymatic activity FADS1 − 3.46 DDT − 2.32 OAZ1 − 2.28 CES2 − 2.00 MORG1 − 1.58 ETHE1 − 1.55

− 5.75 − 1.58 − 1.50 − 1.53 − 1.52 − 1.76

1.66 − 1.47 − 1.52 − 1.31 − 1.04 1.13

Fatty acid desaturase 1 D-dopachrome tautomerase Ornithine decarboxylase antizyme 1 Carboxylesterase 2 (intestine, liver) Mitogen-activated protein kinase organizer 1 Ethylmalonic encephalopathy 1

BE540552 NM_001355 AF090094 AW157619 BC005870 NM_014297

Transcription, RNA processing RORA − 3.01 SRPK2 − 2.69 DPF3 − 2.59 THRAP3 − 2.53 ARL6IP4 − 2.39 MLL3 − 2.00 AES − 1.89 MGC14151 − 1.83 POLR2L − 1.76 PQBP1 − 1.71 NR2F6 − 1.70 GTF3C1 − 1.69 IFI16 − 1.60 TCEB2 − 1.54

− 1.59 − 1.79 − 1.63 − 1.63 − 1.93 − 1.60 − 1.72 − 1.83 − 1.56 − 1.64 − 1.54 − 1.86 − 1.72 − 1.56

− 1.89 − 1.50 − 1.58 − 1.55 − 1.24 − 1.25 − 1.10 1.00 − 1.13 − 1.04 − 1.11 1.10 1.07 1.01

RAR-related orphan receptor A SFRS protein kinase 2 D4, zinc and double PHD fingers, family 3 Thyroid hormone receptor-associated protein 3 ADP-ribosylation-like factor 6 interacting protein 4 Myeloid/lymphoid or mixed-lineage leukemia 3 Amino-terminal enhancer of split Hypothetical protein MGC14151 ; hypothetical protein MGC14151 Polymerase (RNA) II (DNA directed) polypeptide L, 7.6 kDa Polyglutamine binding protein 1 Nuclear receptor subfamily 2, group F, member 6 General transcription factor IIIC, polypeptide 1, alpha 220 kDa Interferon, gamma-inducible protein 16 Transcription elongation factor B (SIII), polypeptide 2 (18 kDa, elongin B)

BC029440 BF954306 AI125562 BE967048 NM_016638 AK025911 NM_001130 BC006407 BC005903 NM_005710 BC002669 NM_001520 BG256677 NM_007108

DNA synthesis/repair HIST2H2AA − 2.93 HIST1H4C − 2.26 HIST1H1C − 1.85 GUK1 − 1.80 APRT − 1.77 NUDT1 − 1.61 HTATIP − 1.54 SRISNF2L − 1.54 KLHDC3 − 1.52

− 1.67 − 1.55 − 2.33 − 1.74 − 1.95 − 1.72 − 1.99 − 1.60 − 1.74

− 1.75 − 1.46 1.26 − 1.04 1.10 1.07 1.29 1.04 1.15

Histone 2, H2aa Histone 1, H4c Histone 1, H1c Guanylate kinase 1 Adenine phosphoribosyltransferase Nudix (nucleoside diphosphate linked moiety X)-type motif 1 HIV-1 Tat interacting protein, 60 kDa KIAA0809 protein Kelch domain containing 3

AI313324 NM_003542 BC002649 AW182892 NM_000485 NM_002452 BC000166 AU159543 BC001793

Apoptosis NDUFA13 LGALS1 LGALS7 BNIP1 TTC11

− 2.92 − 2.58 − 1.95 − 1.81 − 1.79

− 1.92 − 1.51 − 1.61 − 1.59 − 1.66

− 1.52 − 1.71 − 1.21 − 1.14 − 1.08

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 Lectin, galactoside-binding, soluble, 1 (galectin 1) Lectin, galactoside-binding, soluble, 7 (galectin 7) BCL2/adenovirus E1B 19 kDa interacting protein 1 Tetratricopeptide repeat domain 11

NM_015965 NM_002305 NM_002307 NM_013978 NM_016068

Cell adhesion SLURP1 CLDN4 PCDH7

− 2.79 − 2.24 − 1.94

− 1.73 − 1.56 − 2.14

− 1.62 − 1.43 1.10

Secreted LY6/PLAUR domain containing 1 Claudin 4 BH-protocadherin (brain–heart)

NM_020427 NM_001305 AB006757

Metabolism APOC1 IDH2 SLC7A5 SHMT2 MGC3234 RARRES2 COMT

− 2.73 − 2.11 − 2.04 − 1.94 − 1.77 − 1.74 − 1.51

− 5.81 − 1.72 − 2.51 − 1.58 − 2.19 − 1.81 − 1.73

2.12 − 1.23 1.22 − 1.23 1.24 1.04 1.15

Apolipoprotein C-I Isocitrate dehydrogenase 2 (NADP+), mitochondrial Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 Serine hydroxymethyltransferase 2 (mitochondrial) Hypothetical protein MGC3234 Retinoic acid receptor responder (tazarotene induced) 2 Catechol-O-methyltransferase

W79394 U52144 AB018009 AW190316 AF212229 BC000069 BC000419

− 1.50 − 1.80 − 1.91 − 2.11 − 1.87 − 1.61 − 1.72 − 2.12 − 1.63 − 1.74 − 1.56 − 1.56 − 1.58 − 1.67 − 1.52 − 1.98

− 1.75 − 1.27 − 1.18 − 1.04 − 1.14 − 1.32 − 1.16 1.08 − 1.16 − 1.05 − 1.13 − 1.12 − 1.11 1.01 − 1.04 1.30

Ribosomal protein L18a ; similar to 60S ribosomal protein L18a Serine/threonine kinase 4 Phosphomevalonate kinase Peptidylprolyl isomerase B (cyclophilin B) Mitochondrial ribosomal protein L23 Argininosuccinate synthetase Dual adaptor of phosphotyrosine and 3-phosphoinositides HLA class II region expressed gene KE2 FK506 binding protein 2, 13 kDa Activin A receptor, type IB Ribosomal protein L29 Mitochondrial ribosomal protein L4 Adaptor-related protein complex 2, beta 1 subunit Tripartite motif-containing 2 Eukaryotic translation initiation factor 3, subunit 9 eta, 116 kDa Protein phosphatase 2A, regulatory subunit B' (PR 53)

NM_000980 BF433725 NM_006556 NM_000942 AI832239 NM_000050 AI632216 NM_014260 NM_004470 BC000254 AL096829 NM_015956 NM_001282 AA149745 BC001173 X86428

Protein synthesis/modification RPL18A − 2.62 STK4 − 2.29 PMVK − 2.26 PPIB − 2.19 MRPL23 − 2.13 ASS − 2.12 DAPP1 − 2.00 HKE2 − 1.98 FKBP2 − 1.89 ACVR1B − 1.83 RPL29 − 1.77 MRPL4 − 1.75 AP2B1 − 1.75 TRIM2 − 1.66 EIF3S9 − 1.58 PPP2R4 − 1.52

(continued on next page)

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Table 2 (continued) Fold change (FC) Gene symbol

CIDP vs. N

CIDP vs. CMT1

CMT1 vs. N

Description

Genbank no.

Development FLNA CLEC3B TAGLN KRT17 GSTP1 KRT16 TRPS1 MYL9 KRT8 TAZ KRT6B

− 2.46 − 2.40 − 2.14 − 2.11 − 2.03 − 1.98 − 1.85 − 1.76 − 1.70 − 1.69 − 1.65

− 1.68 − 1.57 − 2.27 − 2.30 − 1.57 − 2.15 − 1.70 − 1.86 − 1.52 − 1.57 − 1.84

− 1.46 − 1.53 1.06 1.10 − 1.29 1.09 − 1.08 1.06 − 1.12 − 1.08 1.12

Filamin A, alpha (actin binding protein 280) C-type lectin domain family 3, member B Transgelin Keratin 17 Glutathione S-transferase pi Keratin 16 (focal non-epidermolytic palmoplantar keratoderma) Trichorhinophalangeal syndrome I Myosin, light polypeptide 9, regulatory Keratin 8; keratin 8 Tafazzin (cardiomyopathy, dilated 3A (X-link.); endocardial fibroelastosis 2; Barth syndr.) Keratin 6B

AI625550 NM_003278 NM_003186 NM_000422 NM_000852 AF061812 AI086336 NM_006097 U76549 NM_000116 L42612

− 1.23 − 1.22 − 1.01

Calmodulin-like 3 Amyloid beta (A4) precursor-like protein 2 Single immunoglobulin and toll-interleukin 1 receptor (TIR) domain

M58026 BC000373 NM_021805

− 1.30 1.05 1.24 1.66 1.10

Lysosomal trafficking regulator Macrophage migration inhibitory factor (glycosylation-inhibiting factor) Chemokine (C-C motif) ligand 21 Interferon, alpha-inducible protein 27 Peroxiredoxin 5

U84744 NM_002415 NM_002989 NM_005532 AF197952

Signal transduction, signaling pathway CALML3 − 2.31 − 1.88 APLP2 − 1.89 − 1.55 SIGIRR − 1.67 − 1.66 Immune, inflammatory, defense response LYST − 2.29 − 1.76 MIF − 1.88 − 1.97 CCL21 − 1.61 − 2.00 IFI27 − 1.56 − 2.60 PRDX5 − 1.53 − 1.69 Binding LYPD5 BLOC1S1 S100A2 NDUFA11 NUCB1 ZNHIT1 MRPL41 BANF1 AD-003 KIAA1967 ZCCHC7 ZDHHC18

− 2.25 − 2.12 − 2.04 − 2.02 − 1.98 − 1.97 − 1.86 − 1.84 − 1.72 − 1.66 − 1.55 − 1.53

− 2.05 − 1.89 − 1.51 − 1.67 − 1.75 − 1.53 − 1.52 − 1.73 − 1.55 − 1.55 − 1.51 − 1.55

− 1.10 − 1.12 − 1.35 − 1.21 − 1.14 − 1.29 − 1.22 − 1.06 − 1.11 − 1.07 − 1.03 1.01

LY6/PLAUR domain containing 5 Biogenesis of lysosome-related organelles complex-1, subunit 1 S100 calcium binding protein A2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 11, 14.7 kDa Nucleobindin 1 Zinc finger, HIT type 1 Mitochondrial ribosomal protein L41 Barrier to autointegration factor 1 AD-003 protein KIAA1967 Zinc finger, CCHC domain containing 7 Zinc finger, DHHC-type containing 18

N31975 NM_001487 NM_005978 BE741920 BC002356 NM_006349 AV726260 AF044773 BC001396 AK022661 AA872187 BG168720

Electron transport NDUFS8 − 2.13 PDPR − 1.86 ALOXE3 − 1.59 UCRC − 1.52

− 1.84 − 1.58 − 1.51 − 1.65

− 1.16 − 1.18 − 1.06 1.09

NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23 kDa (NADH-coenzyme Q reductase) Pyruvate dehydrogenase phosphatase regulatory subunit Arachidonate lipoxygenase 3 Ubiquinol-cytochrome c reductase complex (7.2 kD)

NM_002496 BE644918 NM_021628 NM_013387

Transport: CLTB CLIC3 ARF5 RYR1 SLC25A6 BCAP31 ARL2 AP2S1 SLC39A7 KCNK7 AP2M1 MPST ATP5D

− 2.02 − 1.90 − 1.86 − 1.85 − 1.83 − 1.82 − 1.80 − 1.79 − 1.69 − 1.69 − 1.67 − 1.59 − 1.59

− 1.72 − 1.63 − 1.64 − 2.09 − 1.53 − 1.55 − 1.54 − 1.98 − 1.53 − 1.64 − 1.69 − 1.81 − 1.70

− 1.18 − 1.17 − 1.13 1.13 − 1.19 − 1.18 − 1.17 1.11 − 1.11 − 1.03 1.01 1.14 1.07

Clathrin, light polypeptide (Lcb) Chloride intracellular channel 3 ADP-ribosylation factor 5 Ryanodine receptor 1 (skeletal) Solute carrier family 25 (mitochondrial carrier; adenine nucleotide ranslocator), mem. 6 B-cell receptor-associated protein 31 ADP-ribosylation factor-like 2 Adaptor-related protein complex 2, sigma 1 subunit Solute carrier family 39 (zinc transporter), member 7 Potassium channel, subfamily K, member 7 Adaptor-related protein complex 2, mu 1 subunit Mercaptopyruvate sulfurtransferase ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit F1 complex, delta subunit

NM_007097 NM_004669 NM_001662 NM_000540 AA916851 NM_005745 NM_001667 BC006337 NM_006979 NM_005714 NM_004068 NM_021126 BE798517

Other: BZRP WFDC1 C6orf108 TMPRSS13 MAP1LC3A

− 2.45 − 2.02 − 1.65 − 2.06 − 1.96

− 2.22 − 1.83 − 1.76 − 2.36 − 1.89

− 1.10 − 1.10 1.07 1.14 − 1.04

Benzodiazapine receptor (peripheral) WAP four-disulfide core domain 1 Chromosome 6 open reading frame 108 Transmembrane protease, serine 13 Microtubule-associated protein 1 light chain 3 alpha

NM_000714 NM_021197 AA523444 AB048797 AF276658

(p < 0.01 to 0.03) for each gene as compared to normals, and the generally non-inflammatory CMT and diabetic neuropathy groups (Fig. 1A and B). AIF-1 and FYB were also elevated in 1 of 5 diabetic neuropathy skin biopsies, where mild inflammatory changes have been described [14,15]. Of the CMT samples, one had mild elevation of AIF-1 and another of FYB, but none had elevation of more than 1 or 2 markers.

In contrast, in Lyme disease, which is associated with inflammatory changes, the skin biopsies showed elevated expression of the 5 genes similar to that in CIDP (Fig. 1A and B). While expression differences are apparent between individual genes among the study groups, a greater differential may seen with the sum of the qPCR FC values for all 5 genes for each patient (Fig. 1A and B). The

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Fig. 1. A: Confirmation by qPCR (black bars) of gene expression levels detected by microarray (gray bars) for the CIDP and CMT groups. Each gene for individual patients is normalized to the normal group value and expressed as the averaged fold change (FC) over normal. For clarity, the microarray FC standard deviations are not drawn but are as follows for AIF-1, LYVE-1, FYB, MLLT3, and P2RY1, respectively: CIDP (1.17, 1.04, 0.96, 0.89, 1.28), CMT (0.17, 0.43, 1.23, 0.45, 0.36). A and B: Gene expression levels assayed by qPCR (black bars) for CIDP (n = 11) as compared to CMT (n = 8), Lyme disease (n = 4) and Diabetic Neuropathy (n = 5). The Ave. Index is the group average of the sum of FC values for all 5 genes for each patient. *Significantly different at p < 0.05 from values for CIDP. The qPCR FC standard deviations are as follows for AIF-1, LYVE-1, FYB, MLLT3, P2RY1, and ave. index, respectively: CIDP (7.37, 4.91, 4.41, 2.14, 4.06, 21.52), CMT (0.71, 0.17, 1.23, 0.43, 0.96, 3.03), Lyme (6.30, 3.17, 3.21, 5.23, 5.11, 22.90), Diabetic Neuropathy (0.49, 0.56, 1.52, 0.26, 0.56, 2.23).

average index for CIDP (20.84, range 33.37–80.47) is significantly higher than that for CMT1 (4.84, range 1.24–10.25; p = 0.0018) or diabetic neuropathy (4.82, range 2.81–8.53; p = 0.0133), and similar to that for Lyme (21.40, range 6.95–55.48; p > 0.05). 4. Discussion All 5 of the up-regulated genes in CIDP are reported to be involved, directly or indirectly, in inflammatory, immune or defense processes. AIF-1 is produced by activated macrophages in transplant rejection and autoimmune disorders [16,17], and LYVE-1 is a marker for lymphatic endothelium which is also expressed by activated bone marrow and tissue macrophages [18]. FYB, also called ADAP (Adhesion and Degranulation Promoting Adaptor Protein), mediates signaling from T-cell antigen receptors to integrins, leading to enhanced cellular adhesion [19]. P2RY1 is a member of a family of G-protein-coupled receptors that are expressed on monocytes and macrophages and are involved in inflammatory and immunity pathways [20]. MLLT3 is known to regulate erythrocyte and megakaryocyte differentiation [21], and is also induced by ligation of CD44, a cell surface receptor for hyaluronan (HA) the concentration of which is increased in inflammatory and immune responses, and which also binds to LYVE-1 [22]. Mutation of the MLLT3 gene has also been linked to a patient with neuromotor development delay, cerebellar ataxia and epilepsy [23]. In immunocytochemical studies, the expression of AIF-1 has been shown to be increased in T cells, macrophages, and smooth muscle endothelial cells in sural nerve biopsies from patients with vasculitis

or CIDP [24], but its distribution in skin of such patients has not been investigated. Although CIDP targets the myelin sheaths of the peripheral nerves, there is evidence for systemic immune dysregulation including impairment of circulating CD4+CD25+ regulatory T cells [25], reduced expression of inhibitory Fc-gamma receptor IIB expression on B-cells [26], and defective FAS-mediated T-cell apoptosis [27]. Accordingly, the elevated expression of these 5 genes in CIDP skin may represent an inflammatory reaction to myelinated nerve fibers in skin, or a heightened systemic immune state that might predispose to the development of CIDP. It is unlikely, however, that it is a nonspecific reaction to demyelination, as it is not seen in patients with hereditary demyelinating neuropathies. Further investigation of the localization and distribution of these gene products in skin of patients with CIDP and controls would be needed to address these issues. The diagnosis of CIDP can be difficult to make in patients with other causes for neuropathy, and in particular diabetic or hereditary neuropathies [28,29]. Further studies with a larger number of patients, exhibiting different phenotypes or genetic defects would be needed to determine whether differential gene expression could have diagnostic utility in identifying CIDP in such patients. Acknowledgements This study was supported by a research grant from Talecris Biotherapeutics, Inc, and by a generous donation from the Alma and Morris Schapiro Fund.

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