Supplementary title page · 2012-09-26 · Supplementary Information Loss of function mutations in HINT1 cause axonal neuropathy with neuromyotonia Magdalena Zimoń 1, 2*, Jonathan

Supplementary Information

Loss of function mutations in HINT1 cause axonal neuropathy with neuromyotonia

Magdalena Zimoń 1, 2*, Jonathan Baets 2, 3, 4*, Leonardo Almeida‐Souza 2, 5, Els De Vriendt 1, 2,

Jelena Nikodinovic 6, Yesim Parman 7, Esra Battaloğlu 8, Zeliha Matur 7, Velina Guergueltcheva 9,

Ivailo Tournev 9, 10, Michaela Auer‐Grumbach 11, Peter De Rijk 12, Britt‐Sabina Petersen 13,

Thomas Müller 14, Erik Fransen 15,16, Philip Van Damme17, 18, 19,20, Wolfgang N. Löscher 21, Nina

Barišić 22, Zoran Mitrovic 23, Stefano C. Previtali 24, 25, Haluk Topaloğlu 26, Günther Bernert 27,

Ana Beleza‐Meireles 28, 29, Slobodanka Todorovic 6 , Dusanka Savic‐ Pavicevic 30, Boryana

Ishpekova 9, 10, Silvia Lechner 31, Kristien Peeters 1, 2, Tinne Ooms 1, 2, Angelika F. Hahn 32,

Stephan Züchner 33, Vincent Timmerman 2, 5, Patrick Van Dijck 34, Vedrana Milic Rasic 6, 35,

Andreas R. Janecke 14, 31*, Peter De Jonghe 2, 3, 4* & Albena Jordanova 1, 2, 36*

* ‐ These authors contributed equally to this work

1Molecular Neurogenomics Group, VIB Department of Molecular Genetics, University of Antwerp, Belgium. 2Neurogenetics laboratory, Institute Born‐Bunge, University of Antwerp, Antwerpen, Belgium. 3Neurogenetics

Group, VIB Department of Molecular Genetics, University of Antwerp, Belgium. 4Department of Neurology,

Antwerp University Hospital, Antwerp, Belgium. 5Peripheral Neuropathies Group, VIB Department of Molecular

Genetics, University of Antwerp, Belgium. 6Clinic for Neurology and Psychiatry for Children and Youth, University of

Belgrade, Belgrade, Serbia. 7Department of Neurology, Istanbul Medical Faculty, Istanbul University, Istanbul,

Turkey. 8Department of Molecular Biology and Genetics, Bogazici University, Istanbul, Turkey. 9Department of

Neurology, Medical University‐Sofia, Sofia, Bulgaria. 10Department of Cognitive Science and Psychology, New

Bulgarian University, Sofia, Bulgaria. 11Department of Internal Medicine, Division of Endocrinology and

Metabolism, Medical University of Graz, Graz, Austria. 12Applied Molecular Genomics Unit, VIB Department of

Molecular Genetics, University of Antwerp, Belgium. 13Institute of Clinical Molecular Biology, Christian‐Albrechts‐

University, Kiel, Germany. 14Department of Pediatrics I, Innsbruck Medical University, Innsbruck, Austria. 15Department of Medical Genetics and 16StatUA Center for Statistics, University of Antwerp, Belgium. 17Experimental Neurology, University of Leuven, Leuven, Belgium. 18Leuven Institute for Neurodegenerative

disorders (LIND), University of Leuven, Leuven, Belgium. 19Vesalius Research Center, VIB, Leuven, Belgium. 20Department of Neurology, University Hospital Leuven, University of Leuven, Leuven, Belgium. 21Department of

Nature Genetics: doi:10.1038/ng.2406

Neurology, Innsbruck Medical University, Innsbruck, Austria. 22Department of Paediatrics, University of Zagreb,

Medical School, University Hospital Centre Zagreb, Zagreb,Croatia. 23National Center for Neuromuscular Diseases,

Department of Neurology, University Hospital Center Zagreb, Zagreb, Croatia. 24Institute for Experimental

Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Milano, Italy. 25Department of Neurology,

San Raffaele Scientific Institute, Milano, Italy. 26Department of Paediatric Neurology, Faculty of Medicine,

Hacettepe University, Ankara, Turkey. 27Department of Paediatrics, Gottfried von Preyer'sches Kinderspital,

Vienna, Austria. 28Autonomous Section of Health Sciences, University of Aveiro, Portugal. 29Department of

Genetics, Coimbra Paediatric Hospital, CHUC‐EPE, Portugal. 30PCR Center, Faculty of Biology, University of

Belgrade, Belgrade, Serbia. 31Division of Human Genetics, Innsbruck Medical University, Innsbruck,

Austria.32Department of Clinical Neurological Sciences, London Health Sciences Centre, University of Western

Ontario, London, Ontario, Canada. 33Hussman Institute for Human Genetics, University of Miami Miller School of

Medicine, Miami, Florida, USA. 34VIB Department of Molecular Microbiology, Laboratory of Molecular Cell biology,

University of Leuven, Leuven, Belgium. 35Faculty of Medicine, University of Belgrade, Belgrade, Serbia. 36Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Medical University‐Sofia, Sofia,

Bulgaria


Supplementary Note The patients in this study carrying HINT1 mutations were diagnosed with a ‘motor greater than

sensory neuropathy’ typically with an early disease onset in the first decade of life (Table 1,

Supplementary Table 2). Presenting symptoms were most often gait impairment due to distal

weakness in the legs but several patients also presented with cramps in hands and legs and

muscle stiffness. Upon clinical examination, delayed muscle relaxation of the hands after strong

flexion of the fingers (action myotonia) was observed in 36/50 patients although percussion

myotonia of the thenar eminence was typically absent. Nerve conduction studies were

consistent with an axonal neuropathy, pure motor in some and mixed motor and sensory in the

majority of patients (37/50). If available, nerve biopsy studies confirmed changes in the sural

nerve consistent with an axonal neuropathy in 5/6 patients, even if clinical sensory

abnormalities were absent in some of these patients. In addition to the expected chronic

neurogenic changes, concentric needle EMG showed in 39/50 patients the unusual but striking

feature of spontaneous high‐frequency motor unit action potentials also known as

neuromyotonic or myokymic discharges. In several patients mild to moderate elevation of CK

levels were detected. Muscle pathology in 5 patients however did not reveal myopathic

changes but rather the hallmarks of a chronic neurogenic process consistent with long‐standing

denervation due to a peripheral neuropathy. The mildly elevated CK levels are therefore most

likely a consequence of the chronic neurogenic muscle atrophy in combination with the

neuromyotonia.

Heterozygous HINT1 mutation carriers were all asymptomatic. Detailed studies including in

depth clinical testing, nerve conduction studies, concentric needle EMG recordings and CK

measurements were normal in 28 carrier individuals (Table 1, Supplementary Figure 3,

Supplementary Table 3).

The combination of clinically apparent muscle cramps, delayed muscle relaxation upon

contraction or muscle stiffness with spontaneous discharges of high‐frequency motor unit

action potentials upon needle EMG is known as neuromyotonia. The underlying cause of this

condition is a generalized hyperexcitability of the peripheral nerve, in particular the nerve

terminals of the motor axons1.


Neuromyotonia (NM) as a syndrome can occur in association with a host of other clinical

conditions. In many instances NM is an acquired syndrome with a strong immune mediated

etiology. Patients often develop anti‐voltage‐gated potassium‐channel auto‐antibodies, either

as an auto‐immune entity in itself, as a paraneoplastic syndrome or in association with other

auto‐immune disorders, including immune mediated neuropathies such as Guillain‐Barré

syndrome and chronic inflammatory demyelinating neuropathy. There are however several

non‐immune mediated etiologies where NM is seen in conjunction with neurodegenerative

diseases, toxic neuropathies or focal nerve damage due to radiation. Finally, NM can be

observed in association with hereditary conditions most notably hereditary peripheral

neuropathy1. The clinical association between NM and various types of hereditary peripheral

neuropathy was previously recognized2‐4. A more recent paper reported in detail a kinship with

recessively inherited pure motor axonal neuropathy with associated NM5. Interestingly, in the

current study we found disease‐causing HINT1 mutations in this same family (CMT‐1350) firmly

establishing the link between HINT1 and the disease entity of Autosomal Recessive Axonal

Neuropathy with Neuro‐Myotonia (ARAN‐NM).

Supplementary Methods

Patient Cohort

We studied a cohort of 262 unrelated index patients diagnosed with autosomal recessive (AR)

or sporadic Charcot‐Marie‐Tooth disease (CMT). Mutations in GDAP1, SH3TC2, MTMR2 and

SBF2 were excluded in most patients. A second cohort comprised 31 patients diagnosed with

axonal neuropathy and neuromyotonia, both familial and sporadic.

Genomic DNA of affected and control individuals was isolated from peripheral blood using

standard procedures.

Standard Protocol Approvals, Registrations and Patient Consents

All patients or their legal representatives signed an informed consent form prior to enrolment.

This study was approved by the local institutional review boards.


SNP and STR Genotyping, Homozygosity Mapping and Linkage Analysis

Whole genome SNP genotyping on 11 individuals from family CMT‐68 was done using the

Illumina Human660W‐Quad platform. Homozygosity mapping selecting regions ≥1 Mb

containing ≥100 SNPs was done with PLINK6. Multipoint parametric linkage analysis was

performed with easyLINKAGE7 (fully penetrant AR model, equal female/male recombination

rates, 0.0001 disease frequency and inter‐SNP distance 0.001 cM). Regions with LOD scores

≥0.6 were checked for co‐segregation.

Whole genome SNP genotyping on 6 individuals from family CMT‐1380 was done using the

Affymetrix Human Mapping 50K Xba array (Affymetrix, High Wycombe, UK). Analysis was

performed with GCOS and GRYPE (Affymetrix) for genotyping and quality control, PedCheck8,

GRR9 and Merlin10 for linkage genotype quality testing. Fine‐mapping in all eight available

family members was performed with six STR‐markers (D5S659, D5S471, D5S2120, D5S2002,

D5S500, D5S1480, sequences at www.ncbi.nlm.nih.gov) as described below. Parametric linkage

analysis was conducted using ALLEGRO11 on the hypothesis of homozygosity‐by‐descent of the

disease mutation12 and assuming 8 alleles with equal frequencies at each marker locus.

Next Generation Sequencing

Genomes of both patients from family CMT‐68 were paired‐end sequenced by Complete

Genomics Inc (CGI, http://www.completegenomics.com/)13. Primary data analysis including

image analysis, base calling, alignment and variant calling, copy number variations and

structural variations was performed by CGI. Reads were mapped to NCBI build 36.1 reference

genome. Subsequent annotation and filtering was performed with GenomeComb14

(http://genomecomb.sourceforge.net/). After excluding mutations in known CMT‐causing

genes, we extracted variants shared between CMT‐68.04 and CMT‐68.06 in exons, 3’ and 5’

UTRs or splice‐sites located within the linkage intervals (Supplementary Fig. 1c). All exonic

regions within the linkage intervals not covered by CGI, were Sanger‐sequenced.

We excluded SNPs present in data of 90 in‐house genomes, 69 HapMap genomes sequenced by

CGI and public databases (dbSNP129, 1000 Genomes Project15) if their frequency was >5% or


http://www.ncbi.nlm.nih.gov/

http://www.completegenomics.com/

http://genomecomb.sourceforge.net/

were found homozygous. We eliminated synonymous SNPs from further analysis and selected

variants complying with the AR disease model (Supplementary Table 1).

The exome of individual CMT‐1380.V.4 was enriched from genomic DNA using the TruSeq

Exome Enrichment Kit (Illumina, Essex, UK). HiSeq2000 sequencing produced approximately 10

Gb of paired‐end 101 bp sequence reads that were aligned to the human genome (hg18) with

BWA16, and variants were called with SAMtools17. All variants were submitted to SeattleSeq

(http://snp.gs.washington.edu/SeattleSeqAnnotation/) for annotation. Filtering within the

linked region on chromosome 5q was performed as described above.

Mutation Analysis

All coding exons and exon‐intron boundaries of HINT1 were PCR‐amplified on total genomic

DNA. Primers were designed with Primer318 (available upon request). Purified with Exonuclease

I‐ Shrimp Alkaline Phosphatase (USB, Cleveland, USA), PCR products were bi‐directionally

sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster

City, USA). Fragments were separated on an ABI3730xl DNA Analyzer (Applied Biosystems,

Foster City, USA) and analyzed with SeqMan™II (DNASTAR Inc., Madison, USA) or Sequence

Pilot (JSI Medical systems, Kippenheim, Germany). Codon numbering was based on the

published online protein (NP_005331.1) and mRNA (NM_005340.5) sequences of HINT1

(http://www.ncbi.nlm.nih.gov/) using the first methionine as an initiation codon. Mutations

were described according to the HGVS nomenclature (http://www.hgvs.org/mutnomen). All

sequence variants were confirmed by an independent sequencing of the original or newly

obtained DNA sample. Segregation analysis was performed in all available family members (see

Supplementary Fig. 3). Geographically and ethnically matched control individuals were

screened; for exon 1 (p.R37P) 1239 control individuals (270 Belgian, 202 Turkish, 200 Serbian,

192 Bulgarian, 75 Bulgarian Romas, 300 Austrian) for exon 2 (p.H51R, p.Q62*) 270 Belgian

individuals and for exon 3 (p.C84R, p.G89V, p.G93D, p.H112N, p.W123*) 885 controls (140

Turkish, 270 Belgian, 84 Bulgarian, 91 Italian, 300 Austrian ‐only p.G89V). In silico prediction of

the functional effect of mutations was performed with PolyPhen2 algorithm

(http://genetics.bwh.harvard.edu/pph2/index.shtml)19.


http://snp.gs.washington.edu/SeattleSeqAnnotation/

http://www.ncbi.nlm.nih.gov/

http://www.hgvs.org/mutnomen

http://genetics.bwh.harvard.edu/pph2/index.shtml

Haplotype Sharing and Paternity Testing

Haplotype sharing analysis between families with common mutation was performed using nine

STR‐markers (D5S1495, D5S642, D5S2120, D5S809, D5S666, D5S2110, D5S2057, D5S2002,

D5S2117) surrounding the HINT1 region. We additionally genotyped SNPs in the same region

using MassARRAY® technique (Sequenom). Paternity was tested with 15 STRs (ATA38A05,

D1S1646, D1S1653, D1S1360, D2S2256, D3S3037, D4S2382, D4S3240, D7S509, D8S1759,

D9S1118, D12S1056, D12S2082, D16S2619 and GATA152H04). STRs were PCR‐amplified with

fluorescently‐labeled primers (sequences at www.ncbi.nlm.nih.gov), mixed with a formamide

and GeneScanTM 500 Liz® Size Standard (Applied Biosystems, Foster City, USA) (ratio 1:30) and

size‐separated on an ABI3730xl DNA Analyzer. Results were scored with Local Genotype Viewer,

an in‐house developed software program, http://www.vibgeneticservicefacility.be/). PCR and

extension primers for Sequenom analysis were designed with MassARRAY®. After multiplex

PCR‐amplification of SNP‐specific fragments, iPLEX Gold primer extension assay was performed

using MALDI‐TOF spectrometry.

Lymphoblastoid Cell Cultures – Establishment, Growth and MG‐132 Treatment Conditions

Peripheral blood lymphocytes of patients and control individuals were isolated on a Ficol Paque

gradient. Lymphocytes were transformed with Epstein‐Barr virus and incubated at 37 °C for 2

hrs. After centrifugation, cells were re‐suspended in 4 ml RPMI complete medium (Invitrogen)

supplemented with 1% phytohaemagglutinin. Cells were seeded on a 24‐well plate and

incubated at 37 °C, 5% CO2 for minimum of 3 days. After establishment, cell lines were

cultivated in RPMI1640 medium containing 15% fetal calf serum, 1% natrium pyruvate, 1% 200

M L‐glutamine and 2% penicilin/streptomycin. To inhibit proteasome function, cell suspensions

(1x106 cells/ml) were treated with 10 µM MG‐132 (Calbiochem); or with an equal volume of

DMSO. At 0, 4 and 8 hours 1 ml sample was taken for further immunoblotting analysis.


http://www.vibgeneticservicefacility.be/

RNA Isolation and qPCR Analysis

RNA was isolated from lymphoblasts or yeast cells with the RNeasy Mini Kit (Qiagen,

Germantown, MD) according to the manufacturer’s instructions and subsequently treated with

DNase (TURBO DNA‐free kit, Applied Biosystems). cDNA was synthesized by RT‐PCR with

random hexamers using SuperScript III First‐Strand Synthesis System (Invitrogen, Carlsbad, CA).

The quantitative RT‐PCR was performed with SYBR green. The average ΔΔCt values from three

experiments obtained with HINT1 primers were normalized against HMBS and yeast TBP,

respectively.

Cloning and Site Directed Mutagenesis

Full‐length human HINT1 cDNA was PCR‐amplified from HeLa and yeast HNT1 sequence from

genomic DNA of BY2 strain using Platinum DNA High Fidelity Taq Polymerase (Invitrogen). All

constructs were generated using the Gateway recombination system (Invitrogen) according to

manufacturer’s instructions. Entry vector pDONR221 (Invitrogen) and destination vector

pAG415GPD‐ccdB enabled gene expression in Saccharomyces cerevisiae with constitutive GPD

promoter (Addgene, http://www.addgene.org/yeast‐gateway/). Five HINT1 mutations (R37P,

H51R, C84R, H112R, W123*) were introduced into the human transcript by site‐directed‐

mutagenesis using specially designed primers (available upon request). All constructs were

sequence‐verified.

Protein Extraction and Immunoblotting

Total protein extracts from frozen adult mouse tissues, lymphoblastoid or yeast cultures were

isolated by homogenization in E1a lysis buffer20 containing a Complete Protease Inhibitor

Cocktail tablet (Roche, Basel, Switzerland). Supernatant protein concentrations were

determined using Bradford Assay (Bio‐Rad, München, Germany). Equal amounts of protein

were denatured (5 min at 95 °C) in SDS loading buffer (5x solution): 250 mM Tris‐HCl, pH 6.8,

10% SDS, 30% glycerol, 0.02% bromophenol blue, 100 nM DTT). Samples were size‐separated

by SDS‐PAGE on NuPAGE 4‐12% gradient gels (Invitrogen) and transferred to a nitrocellulose

membrane. Membranes were immunoblotted with polyclonal rabbit anti‐human HINT1


http://www.addgene.org/yeast-gateway/

antibody (1:1000, Sigma Aldrich). Equal loading was demonstrated with mouse monoclonal

anti‐β‐actin antibody (1:20,000, Sigma‐Aldrich), mouse monoclonal anti‐GAPDH antibody

(1:20,000, Sigma‐Aldrich) or mouse monoclonal anti‐Pgk antibody (1:10,000, Invitrogen) for cell,

tissue lysates or yeast extracts respectively. Secondary antibodies were anti‐mouse or anti‐

rabbit horseradish peroxidase (Jackson Immuno Research Laboratories Inc.). Results were

visualized by enhanced chemiluminescence detection (GE Healthcare, Waukesha, USA). Band

intensity was quantified using the ImageJ (http://rsbweb.nih.gov/ij/). Statistical significance of

the differences in HINT1 protein expression levels in different mouse tissues was tested using a

two‐tailed t‐test.

Yeast Strain and Growth Analysis

S. cerevisiae strain BY8‐5c21 (MATα ura3‐52 his3∆200 trp1∆901 lys2‐801 suc2‐∆9 leu2‐3,112

hnt1∆::URA3) was provided by Dr. C. Brenner, University of Iowa, USA. Yeast cells were grown

on rich (YP) or minimal (SD) medium supplemented with 2% glucose or galactose as carbon

source. The yeast strain was transformed22 with pAG415GPD plasmids containing HNT1, empty

vector or human wild‐type, R37P, H51R, C84R, H112R, W123* constructs. Transformants were

cultured on SDglu‐Leu/SDgal‐Leu media at 30 °C. For spot assay liquid pre‐cultures were grown

overnight. After absorbance measurement (600 nm) and dilution to optical density of 0.1, 0.02,

0.004, 0.0008, each dilution (5 μl) was spotted on SDglu‐Leu/SDgal‐Leu plates and incubated at

30 °C or 39 °C for 5 days. Growth curve assays were performed for 55 hrs at 30/39 °C as

described elsewhere23. All yeast cultures were monitored in triplicate.

Protein Structure Modeling

The structure of HINT1 (PDB accession 3TW2) was modeled using PyMOL (www.pymol.org).


Supplementary Figures

Supplementary Figure 1. Linkage analysis and variant filtering from next generation

sequencing data in two initial families.

(a) In Belgian pedigree CMT‐68, SNP‐based multipoint linkage analysis pinpointed three

putative disease‐associated regions reaching maximal predicted LOD score Zmax=1.59 (Θ=0.0)

on chromosomes 5, 7 and 15. Linkage plot of CMT‐68 (b) generated by easyLINKAGE and

representing the parametric LOD score values on the y‐axis in relation to genetic position on

the x‐axis. Human chromosomes are concatenated from p‐ter (left) to q‐ter (right) on the x‐axis,

and the genetic distance is given in cM. Individuals CMT‐68.II.3 and CMT‐68.II.5 were whole

genome sequenced. (c) Variant prioritization steps for CMT‐68; SS – splice‐site; UTR‐

untranslated 5’/3’‐regions; not in DB – not reported in a homozygous state in databases (see

Supplementary Methods); HMZ – homozygous; NS – nonsynonymous. (d) In consanguineous

Austrian family CMT‐1380, (e) combined SNP‐ (with Alohomora19) and STR‐based multipoint

(see inset) linkage analysis revealed a single autosomal candidate region on 5q23‐q31 with

Zmax=3.07 (Θ=0.0). (f) Filtering steps of exome sequencing data in individual CMT‐1380.V.4.



Supplementary Figure 2. Mutations identified in HINT1 and their evolutionary conservation.

(a) Electropherograms illustrating the different mutations in HINT1. Mutated nucleotides are

marked in red. (b) Protein sequence alignment of HINT1 orthologues. Targeted amino acid

residues are highlighted in red.



Supplementary Figure 3. Pedigrees of the CMT families with HINT1 mutations.

Pedigrees are grouped according to the mutation identified. DNA of numbered individuals was

available for segregation analysis. Square = male, circle = female, black filled symbol = affected,

empty symbol = unaffected.



Supplementary Figure 4. Functional analyses in yeast.

(a) Spot assay demonstrating that all transformants expressing wild type or mutant HINT1

proteins have similar growth at non‐permissive conditions (30°C and SD‐Leu media with glucose

as a carbon source). (b) Quantitative transcript analysis in yeast transformants reveals

comparable HINT1 mRNA levels. The mRNA amount is first normalized to the TATA box binding

protein (TBP) expression level. Error bars represent SD of three replicates. (c) Immunoblotting

analysis presenting the HINT1 expression in the studied yeast strains using a polyclonal rabbit

anti‐human HINT1 antibody (1:1000, Sigma Aldrich). Phosphoglycerate kinase 1 (Pgk1) is used

as a loading control.



Supplementary tables

Supplementary table 1. Exonic variations identified in the three linked loci in CMT‐68.

Chr ‐ chromosome; NT ‐ nucleotide; AA – amino acid; htz – heterozygous.



Supplementary table 2. Clinical findings in patients with HINT1 mutations.

Cons. ‐ consanguinity; N° aff ‐ number of affected; UL ‐ upper limb; y‐ year; sympt.‐ symptoms;

EMG‐ electromyography; NCS‐ nerve conduction studies; RS‐ Republic of Serbia; TR‐ Turkey; HR‐

Croatia; AT‐ Austria; Eur‐ European; BE‐ Belgium; BG‐ Bulgaria; CN‐ China; IT‐ Italy; GI‐ gait

impairment; NM‐ not mentioned; “+”‐ present; “‐”‐ absent; CK‐ creatine kinase; HMN‐

hereditary motor neuropathy; HMSN‐ hereditary motor and sensory neuropathy.


Genotype Family

Origin (cons.)

N° aff

Age at onset

Presenting symtpoms UL action myotonia

Ambulatory (y)

Sensory sympt./signs

Neuromyotonia on EMG

Extra Subtype (NCS)

R37P/R37P PN-1281a RS (-) 3 9-10y GI, myotonia hands + + (24-36y) + + - HMSN-II PN-1281b RS (-) 2 7-10y GI + + (26y) + + (1 patient) - HMSN-II PN-1616 RS (-) 2 5-10y GI, foot deformities + + (14-16y) + (1 patient) + - HMSN-II PN-1658 RS (-) 1 15y GI + NM + - - HMSN-II CMT-1287 RS (-) 2 10y GI + + (14y) - + - HMSN-II CMT-1288 RS (-) 1 13y falling + + (15y) - + - HMSN-II CMT-1289 RS (-) 2 10-12y GI, foot deformities + (1 patient) + (14-16y) + (1 patient) + - HMSN-II CMT-1290 RS (-) 2 10y GI, foot deformities + (1 patient) + (17-24y) + (1 patient) + - HMSN-II CMT-302 BG (-) 1 6y GI - + - + CK 146 U/L HMN CMT-732 BG (-) 1 3y GI - + (41y), aids + + - HMSN-II CMT-842 BG (-) 1 5y GI + + (37y) + + CK 309 U/L HMSN-II CMT-899 BG (-) 1 10y GI - + (12y) - + CK 504 U/L HMN CMT-162 TR 2 6-8y falling, GI, myotonia

hands + + - + CK 306-1457 U/L HMN

CMT-579 TR (-) 1 15y GI - + (37y) + NM - HMSN(-II) CMT-1239 TR (+) 2 22-25y GI + + (31-37y) + (1 patient) myokymia CK 272-515 HMSN-II CMT-1284 TR (-) 2 10-12y GI + + (30-36y) + (1 patient) + muscle biopsy: neurogenic changes HMSN-II PN-1334 HR 1 9y GI, cramps + + - + nerve biopsy: axonal neuropathy, CK

474 U/L HMSN-II

CMT-419 HR 2 10y GI, toe contractures NM + + - CK 111U/L HMSN-II PN-492 AT (-) 1 8y GI + + (29y) - NM nerve biopsy: predominantly axonal

neuropathy, muscle biopsy: neurogenic changes

HMSN-II

CMT-1274 TR 2 8-9y GI + + (11-13y) - + CK 463-679 U/L HMN CMT-1315 Eur (-) 1 3y GI, falling NM NM + - CK 242 U/L HMSN-II CMT-329 Roma (-) 1 3y GI - + (35y) - + - HMN CMT-1380 AT (+) 3 10-14y GI, cramps + (1 patient) + (17-28y) + + (1 patient) CK 767-902 U/L HMSN-II R37P/C84R CMT-68 BE (-) 2 10y stiffness legs, GI + + - + muscle biopsy: neurogenic changes,

nerve biopsy: axonal neuropathy HMSN-II

R37P/G89V CMT-1311 AT (-) 1 7y GI, stiffness legs, myotonia hands

+ + (16y) - + CK 214 U/L HMSN-II

CMT-1379 AT (-) 1 12y GI, falling + + (34y) - - nerve biopsy: axonal neuropathy HMSN-II R37P/H112N CMT-1271 BG (-) 2 6-10y GI + + (11-17y) - + CK 380 U/L HMN H51R/C84R PN-1899 BE (-) 1 12y cramps, weakness

thumbs + + - + - HMN

Q62*/G93D CMT-1350 CN (-) 2 10y muscle stiffness, GI + + (36-39y), aids

-? + CK 1248 U/L, muscle biopsy: neurogenic changes

HMN

H112N/H112N CMT-726 IT (+) 1 8y GI, falling + + (25y), aids - + CK 963 U/L, nerve biopsy: axonal neuropathy; muscle biopsy: neurogenic changes

HMSN-II

CMT-1120 Roma (+) 1 6y GI NM + (15y), aids - + muscle biopsy: neurogenic changes; sural nerve biopsy and CK: normal

HMN

CMT-1285 TR (-) 1 4y GI + + (18y) + myokymia CK 440 U/L HMSN-II W123*/W123* CMT-1265 TR (+) 1 12y GI + + (25y) + + HMSN-II 33 families 50 patients


Supplementary table 3. Clinical findings in 28 heterozygous HINT1 mutation

carriers.

CE ‐ clinical exam; NCS – nerve conduction studies; EMG ‐ concentric needle

electromyography; CK ‐ creatine kinase; “‐“ – not available; nl – normal; AAI – age at

investigation; y – years.


Genotype Individual CE NCS EMG CK AAI R37P htz PN-1281a.9 nl - - - 36y PN-1281a.10 nl - - - 35y PN-1281b.8 nl nl nl - 38y PN-2067.2 nl nl - - 44y PN-2067.3 nl nl - - 41y PN-1616.1 nl nl nl - 38y PN-1616.2 nl nl nl - 39y CMT-732.2 nl - - - 89y CMT-732.3 nl - - - 75y CMT-1239.2 nl - - - - PN-1334.7 nl - - nl 46y PN-1334.8 nl - - nl 20y CMT-1380.IV.1 nl nl - nl 52y CMT-1380.IV.2 nl nl - nl 47y CMT-68.II.6 nl - - - 50y CMT-68.II.9 nl - - - 43y C84R htz CMT-68.I.1 nl - - - 85y CMT-68.II.1 nl - - - 59y CMT-68.II.4 nl - - - 54y Q62* htz CMT-1350.3 nl nl nl nl 41y G93D htz CMT-1350.2 nl nl nl nl 46y H112N htz CMT-726.3 nl nl nl - - CMT-1120.2 - - - nl 46y CMT-1120.3 nl - - nl 42y CMT-1271.2 nl nl nl - 39y CMT-1285.2 nl - - - - CMT-1285.3 nl - - - - CMT-1285.5 nl - - - - 28 heterozygous HINT1 mutation carriers


Supplementary table 4. Haplotype sharing analysis for R37P, C84R and H112N mutations in

HINT1.

The alleles of the STRs are sized in base pairs (bp). The position of the SNP and STR markers is

according to the reference assembly of NCBI genome build 36.3. Shading indicates shared

disease haplotype, core ancestral haplotypes are delineated with thick lines. Individuals with

violet genotypes are compound heterozygous for mutations; BG – Bulgaria; EU – Europe.


MutationOrigin Belgium BG Italy

Index patient Marker

CMT‐12

39.03

CMT‐16

2.03

CMT‐12

74.03

CMT‐12

89.01

CMT127

1.03

CMT‐13

11.01

CMT‐13

79.01

CMT‐13

80.01

CMT68.03

CMT‐68

.03

PN‐189

9.1

CMT‐12

71.03

CMT‐72

6.01

D5S1495 386 380 386 386 380 388 380 284 390 380 384 390 380 380 388 387 384 384 380 384 391 384 384 384 380 380 384 380/389 380 384 386 390 380 376 380 388 390D5S642 189 191 195 195 183 189 191 185 187 185 191 195 185 185 185 185 191 191 185 185 185 185 191 187 185 185/195 195/191 191 185 197 187 185 185D5S2120 255 245 255 251 251 259 259 255 255 251 259 253 255 245 255 245 255 255 251 245 255 245 255 249 255 255 255 255 245 245 245 247 245D5S809 105 99 105 105 105 97 105 97 105 105 97 97 105 105 97 99 105 101 97 97/101 97/105 97 105 97 97 97 97rs30706 A A A A A A G G A A G G A A A G A G A/G G A G G G G

rs2429191 T G T T T T G T G G G/T G T G G T Trs12652539 T T T T A T A A A/T T T T T T Trs1860249 G G G G G A/G A/G G G A A A Ars11948739 C C C C C T/C T/C C C T T T Trs2419939 T T T T T T T T T T T T Trs1363696 A A A A A A A A A A A A Ars60667115 C C C C C C C C C C C C Crs79379477 A A A A A A A A A A A A Ars3852209 C C C C C C C C C C C C Crs2551038 G G G G G G G G G G G G Grs17165675 T T T T T T T T T T T T Trs76872591 C C C C C C C C C C C C Crs13340335 C C C C C C C C C C C C Crs10036296 C C C C C C C C C C C C Crs17689125 G G G G G G G G G G G G Grs73786612 T T T T T T T T T T T T Trs2278058 G G G G G G G G G G G G Grs7735084 C C C C C C C C C C C C CD5S666 247 245 245 247 245 247 245 245 247 247 245 247 247 245 247 245/251 245/251 245 245 245 245 249 249 247 249

rs2549018 T T T T T T T T T T T T T T T C/T C/T T T C C C C C C

D5S2110 290 290 290 290 290 290 290 290 290 290 290 290 290 290 290 290/292 290/292 290 290 290 290 292 292 292 292

D5S2057 116 116 116 116 116 116 116 116 116 116 116 116 116 116 116 116 116 116 116 100 100 116 216 116 116

rs26003 T T T T T T T T T T T T T T T T C T T C C C C C C

rs30487 A A A A A G A G A A A A A G A A A A A G A A A A A A A

D5S2002 176 174 170 172 176 176 170 172 170 174 176 176 174 176 170 176 180 170 176 170 176 174/180 174 170 168 174 174 180 182 180 180

D5S2117 217 209 225 231 225 225 225 219 225 221 219 225 225 225 225 217 223 225 221 225 221 225 225 217 225 221 229 225/219 225/227 227 219 228 228 225 229 219 219

rs4958263 G G G A G A G G G G G A G G G G G G A G G G A G G G G A A G G G G G G G G G G

97

247

185

388

CMT‐11

20.01

Roma

H112NTurkey Serbia Bulgaria Croatia EU Belgium TurkeyAustria

CMT‐12

84.02

CMT‐57

9.01

PN‐121

8.1

PN‐121

8.4

PN‐161

6.3

PN‐165

8.1

CMT‐12

87.04

CMT‐12

88.01

CMT‐12

90.01

G

257/259

97/105

245/255

PN‐492

.1

CMT‐13

15.01

CMT‐12

85.01

384

185 185 195 185

CMT‐30

2.01

CMT‐73

2.01

CMT‐84

2.01

CMT‐89

9.01

PN‐133

4.4

CMT‐41

9.01

380

191

380/398

255 245 251 247

105 97 97 97 99 105 105 97

GT/G T T/G T/G

A G G G A G/A A G/A

T T T T T T T T T

G/A A

T T T T T T T

T G

T/A T T A

G G G G G G

T T A T/A T T/A

G G G G G AG G G G G G

C C TC C C C C C

T T T T T T

C C CC C C C C C

T T T T T TT T T T T T

A A AA A A A A A

C C C C C C

A A AA A A A A A

C C C C C CC C C C C C

C C C

HINT1

A A A A A

T T T T

G G G G G

G G G G G

A A A A A AA A A A A A

G G

C C C C C CC C

A

C C C C C C

G G G G G GG G G G G G

C

G G G G

T T

C C C C C C C C

T T T T T TT T T T T T

C C C C

C C C C C C

C C C C C C

C C C C C CC C C C C C

C C CC C C C C C

G

C C CC C C C C C


T T TT T T T T T

G

T T TT T T T T T


C

245 245 247 245 245

C C CC C C C

249

C

290 290 290 290 290 290 290 290 290 290 290 292

T T T T T T T

C C

T T

116

T T T T T T T T T

116 116 116 116 116 116

T T C

T T

G/A G

A G/A G/A G/A A

227 225 219

A A A A G/A A

G G/A

180174174

216/228

172/174184 176

C

170

388/380

253/259

97/105

116 116 116 116 116

245 245 247 247 247

CC C C C

245

C C

A/G

225

A/G

191/195 185/197

R37P C84R

T

116

290

T

247/245

176

284/390

219

G

217/219

174/170

384/390

189/197

247/249

103/105

G/A

170

G

C

C

T

G

C

G

T

G

C

C

A

C

A

T

T

A

A

G


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Supplementary title page · 2012-09-26 · Supplementary Information Loss of function mutations in HINT1 cause axonal neuropathy with neuromyotonia Magdalena Zimoń 1, 2*, Jonathan