Expanding the Mutational Spectrum of CRLF1 in Crisponi/CISS1 Syndrome

MUTATION UPDATEOFFICIAL JOURNAL

www.hgvs.org

Expanding the Mutational Spectrum of CRLF1 inCrisponi/CISS1 Syndrome

Roberta Piras,1,2 † Francesca Chiappe,1 † Ilaria La Torraca,3 Insa Buers,4 Gianluca Usala,1 Andrea Angius,1,5

Mustafa Ali Akin,6 Lina Basel-Vanagaite,7,8,9 Francesco Benedicenti,10 Elisabetta Chiodin,11 Osama El Assy,12

Michal Feingold-Zadok,7 Javier Guibert,13 Benjamin Kamien,14 Cigdem Seher Kasapkara,15 Esra Kılıc,16 Koray Boduroglu,16

Selim Kurtoglu,6 Adnan Y Manzur,17 Eray Esra Onal,18 Enrica Paderi,19 Carmen Herrero Roche,20 Leyla Tumer,15 Sezin Unal,18

Gulen Eda Utine,16 Giovanni Zanda,19 Andreas Zankl,21,22 Giuseppe Zampino,3 Giangiorgio Crisponi,23 Laura Crisponi,1∗ †

and Frank Rutsch4 †

1Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Cagliari, Italy; 2Department of Public Health and Clinical andMolecular Medicine, University of Cagliari, Cagliari, Italy; 3Istituto di Pediatria, Policlinico “A. Gemelli”, Universita Cattolica del S. Cuore, Rome,Italy; 4Department of General Pediatrics, Munster University Children’s Hospital, Munster, Germany; 5CRS4 Center for Advanced Studies,Research and Development in Sardinia, Laboratorio di Bioinformatica, Parco tecnologico della Sardegna, Pula, Italy; 6Department of Pediatrics,Medical Faculty, Erciyes University, Kayseri, Turkey; 7Pediatric Genetics, Schneider Children’s Medical Center of Israel, and Raphael RecanatiGenetic Institute, Rabin Medical Center, Beilinson Hospital, Petah Tikva 49100, Israel; 8Sackler School of Medicine, Tel Aviv University, Tel Aviv69978, Israel; 9Felsenstein Medical Research Center, Tel Aviv University, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel;10Genetic Counseling Service, Department of Pediatrics, Regional Hospital of Bolzano, Bolzano, Italy; 11Neonatal Intensive Care Unit, Departmentof Pediatrics, Regional Hospital of Bolzano, Bolzano, Italy; 12Pediatric Department-NICU, Al-Hada Military Hospital, Taif, Saudi Arabia; 13Serviciode Pediatrıa, Complejo Hospitalario de Navarra, Pamplona, Spain; 14Queensland Health Pathology, Royal Brisbane Hospital, Herston, Australia;15Gazi University Hospital, Pediatric Metabolism and Nutrition, Ankara, Turkey; 16Hacettepe University School of Medicine, Ihsan DogramaciChildren’s Hospital, Department of Pediatric Genetics, Ankara, Turkey; 17The Dubowitz Neuromuscular Centre, Department of Neurosciences,Great Ormond Hospital for Children, London, United Kingdom; 18Gazi University Hospital, Department of Pediatrics, Division of NeonatologyBesevler, Ankara, Turkey; 19Unita Operativa Pediatria -Neonatologia – Nido, Ospedale San Martino, Oristano, Italy; 20Department of PediatricNeurology, La Paz Hospital, Madrid, Spain; 21Discipline of Genetic Medicine, The University of Sydney, Sydney, Australia; 22Academic Departmentof Medical Genetics, The Children’s Hospital at Westmead, Sydney, Australia; 23Clinica Sant’Anna, Cagliari, Italy

Communicated by Segolene AymeReceived 23 October 2013; accepted revised manuscript 24 January 2014.Published online 1 February 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22522

ABSTRACT: Crisponi syndrome (CS) and cold-inducedsweating syndrome type 1 (CISS1) share clinical char-acteristics, such as dysmorphic features, muscle contrac-tions, scoliosis, and cold-induced sweating, with CS pa-tients showing a severe clinical course in infancy involvinghyperthermia associated with death in most cases in thefirst years of life. To date, 24 distinct CRLF1 mutationshave been found either in homozygosity or in compoundheterozygosity in CS/CISS1 patients, with the highestprevalence in Sardinia, Turkey, and Spain. By reporting11 novel CRLF1 mutations, here we expand the muta-tional spectrum of CRLF1 in the CS/CISS1 syndrome toa total of 35 variants and present an overview of the dif-ferent molecular and clinical features of all of them. Tocatalog all the 35 mutations, we created a CRLF1 muta-

Additional Supporting Information may be found in the online version of this article.†These authors contributed equally to this work.∗Correspondence to: Laura Crisponi, Istituto di Ricerca Genetica e Biomedica, Con-

siglio Nazionale delle Ricerche, Cagliari, Italy. E-mail: laura.crisponi@irgb.cnr.it

Contract grant sponsors: Associazione Sindrome di Crisponi e Malattie Rare ON-

LUS (http://www.sindromedicrisponi.it) and Regione Autonoma della Sardegna, Italy;

The Interdisciplinary Center for Clinical Research and Innovative Medical Research,

Muenster University Medical School, Germany.

tions database, based on the Leiden Open (source) Varia-tion Database (LOVD) system (https://grenada.lumc.nl/LOVD2/mendelian_genes/variants). Overall, the avail-able functional and clinical data support the fact thatboth syndromes actually represent manifestations of thesame autosomal-recessive disorder caused by mutationsin the CRLF1 gene. Therefore, we propose to renamethe two overlapping entities with the broader term ofCrisponi/CISS1 syndrome.Hum Mutat 35:424–433, 2014. C© 2014 Wiley Periodicals, Inc.

KEY WORDS: CRLF1; CNTFR pathway; Crisponi syn-drome; CISS1

IntroductionMutations in the CRLF1 gene (MIM #604237; locus 19p13.11), en-

coding for the cytokine receptor-like factor-1 (CRLF1), account forCrisponi syndrome (CS) as well as cold-induced sweating syndrometype 1 (CISS1) as causative of both these rare autosomal-recessivedisorders (CS/CISS1; MIM #272430).

The first to describe these two syndromes were Sohar et al. (1978)for CISS1 and Crisponi (1996) for CS. Knappskog et al. (2003) re-ported that mutations in the CRLF1 gene are responsible for theCISS1 phenotype, and Crisponi et al. (2007) and Dagoneau et al.

(2007) found that the same gene was mutated in CS. At that time,it was thought that the two phenotypes were allelic syndromes.Successively, both genotype–phenotype correlation and functionalstudies on mutagenized forms of the CRLF1 protein suggested thatCS and CISS1 are actually manifestations of the same disease, tra-ditionally separated due to differences in severity that have led todifferent ages at diagnosis (neonatal for CS and evolutive for CISS1)[Herholz et al., 2011].

The CRLF1 protein is a member of the ciliary neurotrophic fac-tor receptor (CNTFR; MIM #118946; locus 9p13.3) pathway andinteracts with cardiotrophin-like cytokine factor 1 (CLCF1; MIM#607672; locus 11q13.2) to form a stable heterodimeric complex,CRLF1/CLCF1, which belongs to the family of the interleukin-6(IL-6) cytokine family and acts as a second functional ligand for theCNTF receptor alpha [Elson et al., 2000], composed by gp130 (MIM#600694; locus 5q11.2) and leukemia inhibitory factor receptor beta(LIFR; MIM# 151443; locus 5p13.1). This pathway is known to beimportant for the development and maintenance of the nervoussystem and muscles.

BackgroundCS was initially described in 17 patients from 12 different families

in Central and Southern Sardinia [Crisponi, 1996]. The syndromeusually manifests at birth, when patients present with hyperthermiaand abnormal paroxysmal contractions of the facial and oropha-ryngeal muscles, as well as feeding and respiratory difficulties oftenrequiring the use of nasogastric feeding. Physical dysmorphismssuch as a large face, broad nose, and camptodactyly have beendescribed in most of CS patients. Hyperthermia, as well as acuterespiratory crises, is frequently associated with death within the firstmonths of life. Evolution is fatal in most cases due to respiratorydistress or unexplained episodes of hyperthermia. In the rare surviv-ing CS patients, feeding difficulties and hyperthermia often resolveafter infancy, but then patients develop scoliosis and sometimespsychomotor retardation. In preadolescent CS patients, evidence ofcold-induced sweating was reported. CISS1, instead, was first de-scribed in two Israeli sisters [Sohar et al., 1978] and a similar clinicalphenotype was reported later in two Norwegian brothers [Knapp-skog et al., 2003]. It involves paradoxical sweating at cold ambienttemperatures on the upper part of the body, along with progres-sive scoliosis, dysmorphic features including a high-arched palate,nasal voice, and joint contractures. Knappskog et al. (2003) detectedpotentially deleterious sequence variants in the CRLF1 gene.

Initially, it was proposed that CS and CISS1 represented two allelicdiseases [Crisponi et al., 2007] comprised in a new family of “CNTFreceptor-related disorders,” along with cold-induced sweating syn-drome type 2 (CISS2; MIM #610313), and Stuve–Wiedemann syn-drome (SWS; MIM #601559), that shows, in addition to comparableclinical features with CS, CISS1, and CISS2, the characteristic bow-ing of the long bones. CISS2 results from mutations in the CLCF1gene [Hahn et al., 2006; Rousseau et al., 2006], whereas SWS iscaused by mutations in the LIFR gene [Dagoneau et al., 2004].

Successively, both genotype–phenotype correlation and func-tional analysis on mutated CRLF1 protein suggested that CS andCISS1 are actually manifestations of the same disease with differ-ent degrees of severity due to altered kinetics of protein secretion[Herholz et al., 2011]. Locus heterogeneity for CS/CISS1 within theCNTF receptor-related disorders could be assumed just with CISS2,which shows the same phenotype, but is due to mutations in theCLCF1 gene. However, as there have been only three cases of CISS2from two families described so far in literature, this assumption is

still a matter of debate [Hahn et al., 2006; Rosseau et al., 2006; Hahnet al., 2010].

Mutations and Polymorphisms DefinedThe human CRLF1 gene is localized on chromosome 19p13.11.

It consists of nine coding exons, spans for 14 kb, and is transcribedas a 1,824-bp linear mRNA (NM 004750.4). It encodes for a 422amino acids protein (�46 kDa). This protein (NP 004741.1) has adomain structure that includes a signal sequence (positions 1–37)followed by an Ig-like C2-type N-terminal domain (positions38–131), two consecutive fibronectin III–like domains (positions134–229 and 234–334), and a C-terminal domain (positions 335–422). Each fibronectin type III repeat contains a highly conservedamino acid motif: the first has two cysteine doublets while the sec-ond has a WSXWS motif, at position 327. This motif at position 327is probably needed for correct folding and domain orientation ofthe protein [Bazan, 1990]. The C-terminus shows no homology toknown functional domains [Elson et al., 1998]. The complete CRLF1coding sequence has been sequenced in all the patients analyzed,along with exon/intron junctions. All mutations are described hereaccording to the Human Variation Society (HGVS) nomenclature[den Dunnen and Antonarakis, 2003] and were checked employingthe Mutalyzer program [Wildeman et al., 2008].

To date, 24 mutations have already been reported in literatureas associated with CS/CISS1 [Knappskog et al., 2003; Hahn et al.,2006; Crisponi et al., 2007; Dagoneau et al., 2007; Okur et al., 2008;Thomas et al., 2008; Di Leo et al., 2010; Hahn et al., 2010; Yamazakiet al., 2010; Cosar et al., 2011; Hahn and Boman, 2011; Herholz et al.,2011; Benoit et al., 2012; Gonzalez Fernandez et al., 2013; Hakanet al., 2012; Tuysuz et al., 2013; Uzunalic et al., 2013], whereas herewe report for the first time additional 11 novel mutations (Tables 1and 2). The study protocol was approved by the Munster UniversityHospital Ethical Committee in Germany and all subjects involvedin this study gave informed written consent.

Hence, 35 distinct CRLF1 mutations have been found overall ei-ther as homozygous or compound heterozygous sequence changesin 56 patients with diagnosis of CS/CISS1 from 47 families (Supp.Table S1). It seems that all the 35 mutations found so far are inher-ited, although inheritance could not be ascertained in some cases.

Of these 35 mutations, 13 are missense (37.1%), 10 small indels(28.6%), 4 splice-site mutations (11.4%), 4 nonsense (11.4%), and4 large deletions (11.4%) (Fig. 1A). There is no apparent mutationalhot spot in CRLF1, and mutations underlying the CS/CISS1 pheno-type are distributed as follows: 18 in FNIII domains (seven in FNIII1 and 11 in FNIII 2) and 6 in the Ig-like domain (Fig. 1B).

Of the novel 11 mutations found in our patient co-hort, 5 are missense (c.433T>C, p.Ser145Pro; c.935G>C,p.Arg312Pro; c.[803T>C;1018C>T], p.[Phe268Ser; Arg340Cys];c.221T>C, p.Leu74Pro and c.646C>T, p.Arg216Cys), 2 donor splice-site defects (c.115+1G>A; c.527+5G>T), 1 large deletion (exon3 4), 2 small indels (c.721 737dup, p.Gly247Cysfs∗3; c.983dupG,p.Ser328Argfs∗2), and 1 nonsense (c.776C>A; p.Ser259∗). For thesenovel mutations, DNA sequences were compared with the referencesequence NM 004750.4. The sequence variants were confirmed byresequencing of PCR products obtained from a second amplificationreaction. The 2 donor splice-site variants were detected in trans inthe same patient, with c.115+1G>A maternally and c.527+5G>T pa-ternally inherited; although this last variant affects the same residueof another mutation described before in a CS patient from Yemen[Dagoneau et al., 2007], the substitution is different, G>T instead ofG>A.

HUMAN MUTATION, Vol. 35, No. 4, 424–433, 2014 425

Table 1. Summary of the 35 Known CRLF1 Mutations in CS/CISS1 Patients

Origin/ethnicity DNA variant Number Exon/intron Effect Domain rs dbSNP Mutation type References

Sardinia c.226 T>G M1 Exon 2 p.Trp76Gly Ig-like rs137853143 Missense Crisponi et al., 2007;Dagoneau et al.,2007

Sardinia c.676 677dupA M2 Exon 4 p.Thr226Asnfs∗104 FNIII 1 Insertion Crisponi et al., 2007;Dagoneau et al.,2007

Turkey c.708 709delCCinsT M3 Exon 5 p.Pro238Argfs∗6 FNIII 2 Deletion/Insertion Crisponi et al., 2007Turkey c.1102 A>T M4 Exon 7 p.Lys368∗ C-terminus rs137853144 Nonsense Crisponi et al., 2007Spain c.223 T>G M5 Exon 2 p.Tyr75Asp Ig-like Missense Herholz et al., 2011Gypsy/Spain/

Turkeyc.713dupC M6 Exon 5 p.Pro239Alafs∗91 FNIII 2 Insertion Dagoneau et al., 2007;

Herholz et al., 2011;Gonzalez Fernandezet al., 2013; thisreport

Libya c.539dupA M7 Exon 4 p.Asp181Glyfs∗5 FNIII 1 Insertion Herholz et al., 2011Italy c.[338 A>T;341 T>C] M8 Exon 2 p.[Asn113I;Leu114Pro] Ig-like Missense Herholz et al., 2011Canada c.538C>T M9 Exon 4 p.Gln180∗ FNIII 1 rs137853926 Nonsense Hahn et al., 2006Canada c.852G>T M10 Exon 5 p.Trp284Cys FNIII 2 rs137853927 Missense Hahn et al., 2006Norway c.844 845delGT M11 Exon 5 p.Val282Glyfsa47 FNIII 2 rs137853928 Deletion Knappskog et al.,

2003; Hahn et al.,2010

Israel c.242G>A M12 Exon 2 p.Arg81His Ig-like rs104894670 Missense Knappskog et al., 2003Israel c.1121T>G M13 Exon 7 p.Leu374Arg C-terminus rs104894668 Missense Knappskog et al., 2003Turkey c.829 C>T M14 Exon 5 p.Arg277∗ FNIII 2 rs137853145 Nonsense Okur et al., 2008;

Benoit et al., 2012India c.1-? c.115+?del M15 Exon 1 p.0? Deletion Thomas et al., 2008Pakistan c.433 T>C M16 Exon 3 p.Ser145Pro FNIII 1 Missense This reportAustralia c.721 737dup M17 Exon 5 p.Gly247Cysfs∗3 FNIII 2 Insertion This reportAustralia c.935 G>C M18 Exon 6 p.Arg312Pro FNIII 2 Missense This reportSpain c.115+1 G>A M19 Exon 1 p.0? Donor splice site This reportYemen c.527+5 G>A M20 Exon 3 p.0? Donor splice site Dagoneau et al., 2007Turkey c.475delG M21 Exon 3 p.Ala159Profs∗75 FNIII 1 Deletion Hakan et al., 2012Turkey c.398–456 c.697 +747del M22 Exon 3–4 p.0? – Deletion This reportJapan/USA/

Israelc.31 53del M23 Exon 1 p.Gln11Valfs∗68 Signal peptide rs137853929 Deletion Hahn et al., 2010;

Yamazaki et al.,2010; this report

USA c.303delC M24 Exon 2 p.Asn102Thrfs∗47 Ig-like rs137853931 Deletion Hahn et al., 2010Spain c.[803 T>C;1018 C>T] M25 Exon 5–6 p.[Phe268>Ser;

Arg340Cys]FNIII 2/C-

terminusMissense This report

Italy c.935 G>A M26 Exon 6 p.Arg312His FNIII 2 rs137853933 Missense Di Leo et al., 2010Italy c.856–? c.1269+?del M27 Exon 6–9 p.0? – Deletion Di Leo et al., 2010Saudi Arabia c.983dupG M28 Exon 6 p.Ser328Argfs∗2 FNIII 2 – Insertion This reportTurkey c.698–? c.1269+?del M29 Exon 5–9 p.0? – Deletion Uzunalic et al., 2013Turkey c.413 C>T M30 Exon 3 p.Pro138Leu FNIII 1 rs137853930 Missense Tuysuz et al., 2013? c.397+1G>A M31 Intron 2 p.0? rs137853932 Donor splice site Hahn and Boman,

2011Turkey c.776 C>A M32 Exon 5 p. Ser259∗ FNIII2 Nonsense This reportSardinia c.221 T>C M33 Exon 2 p.Leu74Pro Ig-like Missense This reportSpain c.527+5 G>T M34 Exon 3 p.0? Donor splice site This reportBritish/Pakistan c.646 C>T M35 Exon 4 p.Arg216Cys FNIII 1 – Missense This report

The GenBank accession number for human CRLF1 is NM_004750.4. The DNA mutation numbering system used is based on cDNA sequence. Nucleotide numbering reflectscDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen).The initiation codon is codon 1.

The 1 new large homozygous deletion (exon 3 4del) was ini-tially supposed by absence of relative PCR products. A long-rangePCR that covered the entire genomic region harboring exons 3–4 in both patient and parents (primer pairs 2F and 5R, Supp.Table S2) revealed that the deletion starts in intron 2 (positionc.398–456) and ends in intron 4 (position c.697+747).

In Table 2, all the polymorphisms found during the mutationalanalysis in the CRLF1 gene are shown.

Assessment of PathogenicityAll the 11 novel variants were demonstrated to cosegregate

with the disorder in the respective families; and for the 5novel missense variants, 100 control chromosomes (of matched

ethnicity where available) were screened by direct sequenceanalysis. Furthermore, the novel variants were not listed inpublic/private databases of genetic variation such as dbSNP(http://www.ncbi.nlm.nih.gov/SNP/) [Sherry et al., 2001],the 1000 Genomes Project (http://www.1000genomes.org/)[Abecasis et al., 2012], the Exome Variant Server(http://evs.gs.washington.edu/EVS/), the Human Gene Mu-tation Database (http://www.hgmd.cf.ac.uk/ac/index.php),nor listed as nonpathogenic variants in the literature. Pre-diction of splice sites was performed with NetGene2 v. 2.4(http://www.cbs.dtu.dk/services/NetGene2/) [Brunak et al.,1991; Hebsgaard et al., 1996], whereas for nonsynonymousSNPs functional prediction we employed dbNSFP v.2.0b4(http://sites.google.com/site/jpopgen/dbNSFP) [Liu et al., 2011],

426 HUMAN MUTATION, Vol. 35, No. 4, 424–433, 2014

Table 2. Summary of the Polymorphisms Found in CRLF1

Fast-SNP

Ex/int DNA variant Effect refSNP Type Effect Transcription regulatory

1 Exon 1 c.73 75delCTG p.Leu25del rs137853925 Deletion Untested2 Exon 2 c.237 C>T p.Asn79 = rs2238647 Synonymous Sense/Synonymous with very low

risk (1)3 Intron 4 c.698–19 T>G – rs7247346 Intronic Untested4 Exon 2 c.266 G>A p.Arg89His rs143326783 Missense Untested5 Intron 2 c.398–57 C>T – rs8108207 Intronic Intronic with no known function6 Intron 4 c.697 + 67 G>A – rs35521276 Intronic Intronic enhancer: lower risk

(very low), upper risk (low)Transcription factor

binding sitea

7 Intron 4 c. 698–19 T>G – rs7247346 Intronic Intronic with no known function8 Intron 2 c.398–50 C>T rs28579583 Intronic Intronic with no known function9 Intron 6 c.1025–65 C>A rs79743774 Intronic Untested

∗GATA-2; ∗NF-kB1FastSNP, (http://fastsnp.ibms.sinica.edu.tw/pages/input_SNPListAnalysis.jsp; Yuan et al., 2006).

Figure 1A/1B. Distribution of the 35 known mutations along theCRLF1 gene/protein.

an integrated database of functional predictions from four new andpopular algorithms (SIFT, Polyphen2, LRT, and MutationTaster),along with a conservation score (PhyloP) multiple algorithms. Allmissense and splice-site changes were predicted to be pathogenetic.Regarding the mutations in cis c.[803 T>C;1018C>T], the predic-tion by dbNSFP suggests that the causative one should be c.803T>Crather than c.1018C>T (Table 3).

Recurrent Mutations and Mutation HotspotMost of the genetic defects described so far are private mutations

being confined to one or few patients. Few additional mutationshave been found to reoccur in unrelated patients from a particu-lar geographical area, so that the presence of identical mutationsprobably derives from a founder effect rather than from mutationalhotspots. This might especially hold true for the c.713dupC mu-tation found in 7 patients from different ethnic origin (Spanish,Turkish, and Gipsy), which might reflect a common Gipsy origin.

To date, the most frequent mutations are found in the Sardinian(c.226T>G and c.676 677dupA), Turkish (c.708 709delinsT), andSpanish (c.713dupC) populations. The c.226T>G mutation resultsin a tryptophan to glycine substitution at position 76 of the Ig-likedomain (p.Trp76Gly). Trp76 is likely buried within the moleculeand a substitution by a glycine would be expected to result in aloss of tight internal side-chain arrangement and, thus, in a consid-erable decrease in stability. The Trp76 is strictly conserved withinCRLF1 homologous proteins from different organisms [Crisponiet al., 2007; Dagoneau et al., 2007].

The c.676 677dupA variant results in a threonine-asparaginechange at position 226, followed by a frameshift, which leads to thedeletion of a complete fibronectin domain as well as the C-terminaldomain (p.Thr226Asnfs∗104). This variant was found either inhomozygous or compound heterozygous state [Crisponi et al.,2007].

These 2 mutations listed in Table 1, c.226T>G and c.676 677dupA,have been found so far only in Sardinian individuals, so deriv-ing from a founder effect in this population (5 homozygous forc.676 677dupA, 3 compound heterozygous, and 1 homozygous forc.226T>G). In Sardinia, we also found a third mutation c.221T>C,but only in 1 patient as compound heterozygote for c.676 677dupA.

In the Turkish families, the most frequent mutation, always foundin homozygosity is c.708 709delinsT, which leads to a frameshift inthe second fibronectin type III domain (p.Pro238Argfs∗6). It wasfound in 7 patients from 6 families.

The c.713dupC variant is very common in the Spanish popula-tion. This mutation is located in the region encoding the secondFNIII domain of the protein, and results in a premature termina-tion of translation (p. Pro239Alafs∗91). It was found in 7 patients(2 Spanish, 1 Turkish, 2 Spanish-Gipsy, and 2 French-Gypsy),either in homozygous or compound heterozygous state, from4 families.

“D(a

”;ot

as“T

d).”

“pro

ing”

];“p

g”if

“ben

“neu

“del

s”if

“;”.

d:“D

”(“

g”),

“P”

(“po

ing”

”(“

”).M

“;”.

is“p

g”if

“pos

ing”

];“b

is“n

by“;

”.Po

”(“

g”),

“P”

(“po

ing”

”(“

”).M

“;”.

on,“

(“di

ic”)

”(“

g”),

“N”

(“po

”),o

”(“

ic”)

Geographical DistributionSo far, 56 patients affected either by CS/CISS1 have been reported

in the literature with 17 originating from Turkey (30%), 12 fromItaly, in particular 10 from Sardinia (21%), 7 from Spain (13%), and20 (36%) deriving from different geographical areas. In Italy, theestimate reaches 39% if we consider also 15 Sardinian patients witha clinical diagnosis of CS [Crisponi, 1996] but without a molecularanalysis for CRLF1, since they died before the discovery of the gene(Supp. Fig. S1).

Considering this higher prevalence in Sardinia (Italy), Turkey,and Spain, the most involved area is the one of the Mediterraneanregion. In this area, other cases have been described in Libya [Her-holz et al., 2011] and Israel [Knappskog et al., 2003; this report].Other CS/CISS1 patients have been identified in Eastern countries,in particular in India [Thomas et al., 2008], Pakistan [this report],Yemen [Dagoneau et al., 2007], Saudi Arabia [this report], and Japan[Yamazaki et al., 2010], whereas in the Western states, 1 patient hasbeen identified in Canada [Hahn et al., 2006], 1 in Australia [thisreport], and 1 in USA [Hahn et al., 2010].

In Sardinia, where the syndrome is more common than in the restof Italy, with 3 mutations found so far (c.676 677dupA, c.226T>G,and c.221T>C), we estimated the allele frequency for the 2 mostfrequent mutations (c.676 677dupA, c.226T>G) of 1% and 0.4%,respectively, which resulted in a joint carrier frequency of 1.4% withan expected incidence of about 1 case per 20,700 newborns (i.e.,0.005%), calculated on 15,000 live newborns per year in Sardinia.The assay used was the Custom TaqMan R©SNP Genotyping Assaysprovided by Applied Biosystem (Applied Biosystems, Foster City,CA, USA), and the allelic discrimination was conducted by 7900HTFast Real-Time PCR System (Applied Biosystem). In details, wefound 5 carriers for c.226T>G and 12 for c.676 677dupA on about1,200 anonymous DNA from the SardiNIA Medical Sequencing Dis-covery Project [Kwong et al., 2012]. These findings approximatelyconfirm the epidemiological data collected in 40 years, during which25 CS/CISS1 patients were identified. In fact, according to our re-sults, we would have expected about 28 affected individuals.

Genotype–Phenotype CorrelationWith the exception of SWS caused by LIFR mutations and with

the characteristic bowing of the long bones, manifestations of theCNTF receptor-related disorders are very similar either when causedby mutations in CRLF1, as in CS/CISS1, or by mutations in CLCF1,as in the case of CISS2. In particular, CISS1 and CISS2 are clinicallyindistinguishable, although only three cases of CISS2 have beendescribed so far.

In Supp. Table S1, we show the major clinical findings relative tomost of the CS/CISS1 patients with mutations in the CRLF1 gene.Clinical data and patient history were gathered through a standard-ized questionnaire, which was sent to the responsible clinicians,and through the review of the clinical data presented in the litera-ture. Unfortunately, detailed clinical description was not availablefor some patients. We considered four distinct features of CS/CISS1(hyperthermia and feeding difficulties in the neonatal period, sco-liosis [cut off 2 years] and cold-induced sweating [cut off 6 years]in the evolutive period) and classified patients as follows: (1) mildphenotype: patients, who show no episodes of hyperthermia and/orfeeding difficulties but only scoliosis and cold-induced sweating inadolescence; (2) intermediate phenotype: patients who show eitherhyperthermia or feeding difficulties in infancy and who later developscoliosis and cold-induced sweating; and (3) severe or “complete”

phenotype: patients who show hyperthermia and feeding difficul-ties in infancy and die in the first months of life or survive andlater develop scoliosis and cold-induced sweating. There is no ev-ident correlation between the phenotype and the type/localizationof CRLF1 mutation found. A functional study on the mutated formsof CRLF1 associated with CS/CISS1 [Herholz et al., 2011] showedthat CS and CISS1 are actually the same disease and that the pheno-typic severity of CRLF1-associated disorders depends on defectiveand altered kinetics in the secretion of the mutated CLRF1 protein.In particular, deficient CRLF1 secretion is associated with a severeclinical phenotype, whereas high level of secretion is associated witha milder clinical phenotype [Herholz et al., 2011]. We can assumethat different mutations affecting the folding of the CRLF1 proteinand its interaction with CLCF1 might lead to different secretion ofthe protein, which then might affect phenotypic severity. Neverthe-less, other genetic factors including mutations in additional genesmight also play a role in modulating phenotypic severity.

Missing Heritability in CS/CISS1A fraction of suspected CS/CISS1 cases remains genetically unex-

plained after the CRLF1 gene has been sequenced. The hypothesesthat can explain this are the following: (1) a variant in the pro-moter or other regulatory/splicing element not detected or rec-ognized by current sequencing strategies; (2) unrecognized largedeletions/duplications that are not detected by Sanger sequencing;(3) digenic inheritance; and (4) locus heterogeneity.

Additional Molecular Tests in Cases Lacking TwoMutations

Two patients from our cohort were found to be heterozygotes for1 mutation (case 52 and 53), see Supp. Table S1) so lacking a secondmutation.

Case 52 is a 2-year-old Spanish female. She was positive to CRLF1analysis, but we found only the maternally inherited c.713dupCmutation. The phenotype was clearly attributable to CS; hyperther-mia, contraction of facial muscles, trismus, swallowing and feedingdifficulties, chubby cheeks, and camptodactyly were evident.

Case 53 is an 11-year-old boy of British-Pakistan origin, who pre-sented with contractions of facial muscles particularly in responseto tactile stimuli since early infancy. He also showed marked feed-ing difficulties in the first 18 months of life. Although episodes ofhyperthermia were not noted, CS was suspected. On examination,he shows anteverted nostrils, micrognathia, and a nasal voice. DNAanalysis of the boy was positive for the heterozygous CRLF1 muta-tion c.646C>T. DNA from the parents was not available for mutationanalysis.

We investigated these patients with additional techniques to lookfor mutations, which would not be detected by standard Sangersequencing.

Long-Range PCR

Hypothesizing possible deletion/duplication in heterozygosity,we performed a long-range PCR using the following primers pairs(Supp. Table S2): 1F-1R intronic (3.8 kb); 2F intronic-2R (3.7 kb);2F-6R (3.5 kb), and 5F-9R (3.8 kb), but no additional bands wereseen.

Genome-Wide Human SNP Array 6.0 (Affymetrix)

For case 52, we decided to proceed by SNP array. The analy-sis was processed using the genome-wide human SNP array 6.0(Affymetrix). The reference sets used contained 57 samples. The re-sults showed a loss of heterozygosity of 120 kb on chromosome19p12 (20,596,194–20,716,377 in reference hg19) (start markerCN 795771- end marker CN 165017), about 2 Mb 5′ upstreamof the CRLF1 gene. We were not able to assess whether this variationwas de novo or transmitted, since the parents could not be ana-lyzed. In this region, genes encoding zinc finger proteins are mainlypresent. Although the analysis has not been exhaustive, these datadeserve to be further evaluated.

Screening of Other Potential CS/CISS1 Gene Candidates

Because of the functional link between CRLF1 and CLCF1 andthe clinical overlap between CS/CISS and CISS2 (to date, only fourcausative mutations in the CLCF1 gene have been described in CISS2patients [Hahn et al., 2006; Rousseau et al., 2006]), we sequencedthe CLCF1 gene in both cases hypothesizing a possible digenic in-heritance but no coding sequence variant was found.

Evaluation of Locus HeterogeneityThirty-five patients with suspected diagnosis of CS/CISS1 were

negative to molecular analysis of the CRLF1 gene. As previously dis-cussed, considering the involvement of the CRLF1 with the CNTFRcomplex, we extended the analysis to the CLCF1 and CNTFR genesto evaluate possible locus heterogeneity for CS/CISS1. All the 35negative patients have been sequenced for CLCF1 and only 19 forCNTFR; we did not find pathogenic variants for the phenotypeCS/CISS1, but only polymorphisms (Supp. Tables S3 and S4).

Biological RelevanceThe CNTFR pathway supports the differentiation and survival

of a wide range of neural cell types during development and inadulthood. CRLF1 and CLCF1 are soluble cytokines that form thestable heterodimeric complex CRLF1/CLCF1, which acts as a secondfunctional ligand to CNTFR. Binding of CRLF1/CLCF1 to CNTFRresults in the recruitment, binding, and dimerization of glycopro-tein 130 (gp130) and of LIFR-beta isoform. In turn, this inducesdownstream signaling events and the activation of the JAK1-STAT3signaling pathway [Heinrich et al., 2003]. Furthermore, the key roleof the CNTFRα pathway in the function of the autonomic nervoussystem and in the embryonic development of motor neurons hasbeen described and confirmed both in vitro and in vivo [Forgeret al., 2003]. Dysautonomic symptoms seem to predominate in theCS/CISS1 syndrome, suggesting that CRLF1 functions in the devel-opment of the central and peripheral autonomic nervous systems.

Newborn mice lacking Crlf1, Cntfr, and Clcf1 all mirror the samephenotype, with the inability to suckle, decreased facial motility, sig-nificant reductions in motor neuron number, with perinatal death,whereas mice and humans deficient of CNTF, the primary ligandto CNTFR, are healthy. [De Chiara et al., 1995; Alexander et al.,1999; Zou et al., 2009]. Reduction in motor neuron number, de-creased facial motility, and perinatal death were also seen in thegp130 and Lifr-beta null mice [Li et al., 1995; Nakashima et al.,1999]. Mice lacking Crlf1, Clcf1, and Lifr are animal models forthe respective human syndromes (CS/CISS1, CISS2, and SWS) withassociated severe oral-facial weakness, impaired suckling, and com-

plete disinterest in food and water. The observations illustrate theimportance of the CNTFR/gp130/LIFR tripartite receptor and itsligand CLCF1/CRLF1 for development and maintenance of the ner-vous system, in particular for the embryonic development of facialmotor neurons. It is known that the IL-6 cytokines acting throughgp130 receptors are required for the cholinergic differentiation ofsympathetic neurons innervating sweat glands [Stanke et al., 2006].CRLF1 and CLCF1 are cytokines expressed on the surface of devel-oping sweat-gland tissue, and currently this complex could be oneof the most likely candidates to mediate the switch from the no-radrenergic to the cholinergic phenotype of sympathetic neuronsvia the gp130/LIFR pathway [Stanke et al., 2006]. Also, choliner-gic sympathetic neurons innervate, as additional target tissues, theskeletal muscle vasculature and the periosteum, the connective tis-sue covering bone [Francis and Landis, 1999]. Skin biopsies of anindividual with CS/CISS1, which were derived from areas of hyper-hidrosis, illustrate that the sweat glands lacked cholinergic innerva-tion while adrenergic supply was amply maintained [Di Leo et al.,2010]. If confirmed, these results would indirectly support a rolefor CLCF1/CRLF1 in mediating the switch from noradrenergic tocholinergic properties of sympathetic neurons that innervate sweatglands and periosteum during development.

Although the defects observed in mice and humans suggest vitallyimportant functions of CRLF1 expression in developmental path-ways, new evidence suggests that changes in CRLF1 expression maybe also associated with several postnatal disease processes [Kass,2011].

A paper published in 2009 suggests that the CRLF1/CLCF1 com-plex disrupts cartilage homeostasis and promotes the progress ofosteoarthritis by enhancing the proliferation of chondrocytes andsuppressing the expression level of cartilage structural proteins[Tsuritani et al., 2010]. Furthermore, a potentially important antifi-brotic role for CRLF1 expression in idiopathic pulmonary fibrosishas also been recently reported, suggesting that CRLF1 expressionin the lung could be a potentially reparative response to fibrotic lunginjury [Kass et al., 2012]. Both of these studies show that CRLF1 isinvolved in other more common diseases. This could further explainthe complexity of the CS/CISS1 phenotype.

Crabe et al. (2009) found that similar to CLCF1, the p28 subunitof IL-27 could associate with CRLF1 to form a new complex thatcan bind IL-6R, a tripartite receptor of IL-6Ra, WSX-1, and gp130.Activation of this receptor leads to downstream signaling eventsvia the JAK/STAT pathway (particularly STAT3), MEK/ERK, andPI3K/AKT. This recent discovery suggests that CRLF1 can stimulatecell populations that may not express CNTFR. Up to now, the fullrange of cells, which are potentially responsive to CRLF1 stimulationis unknown, as well as the biological activity of CRLF1 on these cells.These data highlight that the physiological role of CRLF1 is stilluncertain. Furthermore, a role of CRLF1 in immune response wasimplied by its similarity to IL-6. These new findings could furtherelucidate the complexity of the phenotype in CS/CISS1 and may leadto a better understanding of genotype and phenotype correlations.

Clinical and Diagnostic RelevanceBefore 2007, it was thought that CS and CISS1 were different

disorders, with CS and CISS1 reported respectively for the neona-tal and for the evolutive phenotype. In 2007, the identification ofmutations in the CRLF1 gene led to the conclusion that CS andCISS1 were allelic forms of the same disease. Functional studies onmutated forms of CRLF1 gave the hint that the two syndromes, CSand CISS1, represent manifestations of one disorder, with different

degrees of severity. Based on the available data, we propose here torename the two overlapping entities CS and CISS1 with the broaderterm of CS/CISS1 syndrome and the unified MIM #272430.

Since 2007, molecular genetic testing for CRLF1 mutations hasbeen offered allowing for reliable genetic counseling. It comprisesthe sequence analysis of all nine exons and exon–intron boundaries.If a variant is found in a patient, several types of analyses are per-formed to determine its pathogenicity; cosegregation of the variantwith the disorder in the family, absence in mutation databases andpublished data, and absence in at least 100 alleles from control sam-ples (with matched ethnicity where possible). The functional effectof the variant is also checked by prediction software such as dbSNFPand Netgene2.

The results of our study have shown that mutations in the CRLF1gene are responsible for CS/CISS1, representing a single genetic en-tity with varying degrees of severity. Functional studies have shownthat altered kinetics of protein secretion associated with mutatedCRLF1 is related to various degrees of severity of CS/CISS1. How-ever, there is currently no clear genotype–phenotype correlation forboth type and location of mutations in CRLF1. The distinctions arefurther complicated by the frequent presence of the combinationof different mutations in patients. According to the available data,there seems to be no correlation between the type and localization ofany mutation in CRLF1 and the severity of the patient’s phenotype.However, there seems to exist a correlation at the functional level as-sociated with the secretion of the CRLF1 protein. Further extensionof the mutational spectrum of CRLF1 and of the functional studieson mutated CRLF1 proteins will be needed to define their role inthe pathogenesis of CS/CISS1.

Management and Diagnostic StrategiesThere is no treatment available for this syndrome at the mo-

ment. Management is mostly symptomatic. Infants with CS/CISS1require intervention for feeding difficulties, for episodes of respira-tory distress with laryngospasm, and for hyperthermia, which maylead to seizures or sudden death. Bracing, occupational therapy, orplastic surgery may be necessary to correct congenital finger andhand deformities. Surgical intervention or prolonged bracing maybe required to treat the progressive thoracolumbar kyphoscolio-sis. Sweating triggered by cold or apprehension can be effectivelytreated with clonidine/amitriptyline or moxonidine [Hahn et al.,2006, 2010; Herholz et al., 2010]. Heat exposure and prolongedphysical activity in a hot climate are to be avoided.

Keratopathy is often present in these patients, and the use ofartificial tears or lubricating gel from birth could prevent the onsetof surface erosion or more severe corneal damage.

This disease is still poorly understood and often not diagnosedcorrectly because the phenotype is relatively new and extremelycomplex, similar to other disease such as tetanus of the newborn,and the phenotype changes with age. The identification of mutationsin the CRLF1 gene provides a definite diagnosis in patients withsuspected diagnosis of CS/CISS1 syndrome. This can be carriedout with genetic testing for individuals and families with a historyof disease, but also with carrier and prenatal testing if the diseasecausing mutations in the family have been identified.

DatabaseIn order to catalog all the mutations found so far in

the CRLF1 gene, as well as of their functional conse-quences, we created a CRLF1 mutations database, based on

the Leiden Open (source) Variation Database (LOVD) sys-tem, upgraded to the latest version, LOVD 2.0 build 35(https://grenada.lumc.nl/LOVD2/mendelian_genes/variants). Thedatabase (http://www.lovd.nl/CRLF1) comprises the 35 distinctpathogenetic mutations reported so far, distributed as follows: 13missense, four nonsense, 14 deletions and insertions, and foursplice-site defects. All mutations are described according to the Hu-man Variation Society (HGVS) nomenclature [den Dunnen andAntonarakis, 2003] and are checked by the Mutalyzer program[Wildeman et al., 2008].

Future ProspectsThe functions of CRLF1 need to be further explored. Little is

known about other interacting proteins and receptors involved.Future research will be directed toward a better understanding ofmolecular disease mechanisms, including genotype–phenotype cor-relations and modifiers of the phenotype, making use of recombi-nant systems, proteomics approaches, or mouse models. In particu-lar, since Crlf1 null mice die on postnatal day 1, a conditional modelusing the Cre-loxP system may be effective in dissecting the organ-specific effects of Crlf1 deficiency. A deeper understanding of CRLF1signaling pathways would be critical to the development of noveltherapeutic strategies for CS/CISS1 as well as for other diseases. Theconsequences of some missense mutations and in-frame deletionsremain to be elucidated. The determination of the complete crys-tal structure of the CRLF1/CLCF1 complex would potentially clarifythe role of certain mutations in affecting CRLF1 folding. Thus, somemutations might prevent the course of CRLF1 through the qualitycontrol mechanisms of the secretory pathway and its subsequentsecretion. In addition, it would also be useful to test the novel muta-tions reported here in the secretion assays described by Herholz et al.(2011), to determine whether the model already proposed of corre-lating secretion with phenotypic severity holds true also for those.The most involved area for CS/CISS1 is the Mediterranean region.Many possibilities (chance phenomenon, migration, high mutationrates, digenic model, or selective advantage to carriers) have beenproposed to explain the observation that multiple mutations areresponsible for a single disease in an isolated population or area.Selection is a mechanism that conferring the advantage to survive ina particular environment to heterozygous carriers of recessive traitsmay explain this observation. However, this is difficult to prove andthe type of selection may be different in each case (Zlotogora, 2007).Considering the higher prevalence of the syndrome in the Mediter-ranean region, we could hypothesize a positive selection to malaria.Although highly speculative, future studies (i.e., a haplotype-basedstudy in Sardinia) could get more clues whether this disorder is, inpart, due to the evolutionary pressure that malaria exerted on ourancestors.

Furthermore, a short-time goal will be the clinical and geneticdelineation of CS/CISS1-like phenotypes, which are not causedby CRLF1/CLCF1 mutations. In such cases, the identification ofnew disease-causing genes, after exclusion of rearrangements bySNP/CGH arrays would be achievable by whole-exome sequenc-ing, and will help in better dissecting pathways and networks whereCRLF1 is involved and function.

Accession NumbersThe GenBank accession number for human CRLF1 is

NM 004750.4.

Acknowledgments

We are grateful to all the patients and their family members who withtheir enthusiastic, continuous, and generous participation made this studypossible. We thank Prof. Francesco Cucca for the help and support providedin preparing the manuscript.

Disclosure statement: The authors have no conflict of interest to declare.

References

Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, KangHM, Marth GT, McVean GA. 2012. An integrated map of genetic variation from1,092 human genomes. 1000 Genomes Project Consortium. Nature 491:56–65.

Alexander WS, Rakar S, Robb L, Farley A, Willson TA, Zhang JG, Hartley L, Kikuchi Y,Kojima T, Nomura H, Hasegawa M, Maeda M, et al. 1999. Suckling defect in micelacking the soluble haemopoietin receptor NR6. Curr Biol 9:605–608.

Bazan JF. 1990. Structural design and molecular evolution of a cytokine receptor su-perfamily. Proc Natl Acad Sci USA 87:6934–6938.

Benoit V, Hilbert P, Deprez M, Charon A, Maystadt I, Moortgat S. 2012. Crisponisyndrome in a Turkish newborn: a possible founder mutation in the CRLF1 gene?Abstract ASHG 3181W.

Brunak S, Engelbrecht J, Knudsen S. 1991. Prediction of human mRNA donor andacceptor sites from the DNA sequence. J Mol Biol 220:49–65.

Cosar H, Kahramaner Z, Erdemir A, Turkoglu E, Kanik A, Sutcuoglu S, Onay H, AlpmanA, Ozkinay F, Ozer EA. 2011. Homozygous mutation of CRLF-1 gene in a Turkishnewborn with Crisponi syndrome. Clin Dysmorphol 20:187–189.

Crabe S, Guay-Giroux A, Tormo AJ, Duluc D, Lissilaa R, Guilhot F, Mavoungou-Bigouagou U, Lefouili F, Cognet I, Ferlin W, Elson G, Jeannin P, Gauchat JF. 2009.The IL-27 p28 subunit binds cytokine-like factor 1 to form a cytokine regulatingNK and T cell activities requiring IL-6R for signaling. J Immunol 183:7692–7702.

Crisponi G. 1996. Autosomal recessive disorder with muscle contractions resemblingneonatal tetanus, characteristic face, camptodactyly, hyperthermia, and suddendeath: a new syndrome? Am J Med Genet 62:365–371.

Crisponi L, Crisponi G, Meloni A, Toliat MR, Nurnberg G, Usala G, Uda M, MasalaM, Hohne W, Becker C, Marongiu M, Chiappe F, et al. 2007. Crisponi syndromeis caused by mutations in the CRLF1 gene and is allelic to cold-induced sweatingsyndrome type 1. Am J Hum Genet 80:971–981.

Dagoneau N, Scheffer D, Huber C, Al-Gazali LI, Di Rocco M, Godard A, Marti-novic J, Raas-Rothschild A, Sigaudy S, Unger S, Nicole S, Fontaine B, et al.2004. Null leukemia inhibitory factor receptor (LIFR) mutations in Stuve-Wiedemann/Schwartz-Jampel type 2 syndrome. Am J Hum Genet 74:298–305.

Dagoneau N, Bellais S, Blanchet P, Sarda P, Al-Gazali LI, Di Rocco M, Huber C,Djouadi F, Le Goff C, Munnich A, Cormier-Daire V. 2007. Mutations in cytokinereceptor-like factor 1 (CRLF1) account for both Crisponi and cold-induced sweat-ing syndromes. Am J Hum Genet 80:966–970.

DeChiara TM, Vejsada R, Poueymirou WT, Acheson A, Suri C, Conover JC, FriedmanB, McClain J, Pan L, Stahl N, Ip NY, Yancopoulos GD. 1995. Mice lacking the CNTFreceptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits atbirth. Cell 83:313–322.

den Dunnen JT, Antonarakis SE. 2003. Mutation nomenclature. Curr Protoc HumGenet 7:7.13.1–7.13.8.

Di Leo R, Nolano M, Boman H, Pierangeli G, Provitera V, Knappskog PM, CortelliP, Vita G, Rodolico C. 2010. Central and peripheral autonomic failure in cold-induced sweating syndrome type 1. Neurology 75:1567–1569.

Elson GC, Graber P, Losberger C, Herren S, Gretener D, Menoud LN, Wells TN, Kosco-Vilbois MH, Gauchat JF. 1998. Cytokine-like factor-1, a novel soluble protein,shares homology with members of the cytokine type I receptor family. J Immunol161:1371–1379.

Elson GC, Lelievre E, Guillet C, Chevalier S, Plun-Favreau H, Froger J, Suard I, deCoignac AB, Delneste Y, Bonnefoy JY, Gauchat JF, Gascan H. 2000. CLF associateswith CLC to form a functional heteromeric ligand for the CNTF receptor complex.Nat Neurosci 3:867–872.

Forger NG, Prevette D, deLapeyriere O, de Bovis B, Wang S, Bartlett P, Oppenheim RW.2003. Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophicfactor for lumbar and facial motoneurons in vivo. J Neurosci 23:8854–8858.

Francis NJ, Landis SC. 1999. Cellular and molecular determinants of sympatheticneuron development. Annu Rev Neurosci 22:541–566.

Gonzalez Fernandez D, Lazaro Perez M, Santillan Garzon S, Alvarez Martınez V,Encinas Madrazo A, Fernandez Toral J, Perez Oliva N. 2013. Cold-induced sweatingsyndrome type 1, with a CRLF1 level mutation, previously associated with Crisponisyndrome. Dermatology 227:126–129.

Hahn AF, Jones DL, Knappskog PM, Boman H, McLeod JG. 2006. Cold-induced sweat-ing syndrome: a report of two cases and demonstration of genetic heterogeneity.J Neurol Sci 250:62–70.

Hahn AF, Waaler PE, Kvistad PH, Bamforth JS, Miles JH, McLeod JG, Knapp-skog PM, Boman H. 2010. Cold-induced sweating syndrome:CISS1 and CISS2:manifestations from infancy to adulthood. Four new cases. J Neurol Sci 293:68–75.

Hahn AF, Boman H. 2011. Cold-induced sweating syndrome including Crisponi

syndrome. GeneReviewsTM

[Internet]. Seattle (WA): University of Washington,Seattle; 1993–2013.

Hakan N, Eminoglu FT, Aydin M, Zenciroglu A, Karadag NN, Dursun A, OkumusN, Ceylaner S. 2012. Novel CRLF1 gene mutation in a newborn infant diagnosedwith Crisponi syndrome. Congenit Anom (Kyoto) 52:216–218.

Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouze P, Brunak S. 1996.Splice site prediction in Arabidopsis thaliana DNA by combining local and globalsequence information. Nucleic Acids Res 24:3439–3452.

Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F.2003. Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J 374(Pt 1):1–20.

Herholz J, Crisponi L, Mallick BN Rutsch F. 2010. Successful treatment of cold-inducedsweating in Crisponi syndrome and its possible mechanism of action. Dev MedChild Neurol 52:494–497.

Herholz J, Meloni A, Marongiu M, Chiappe F, Deiana M, Herrero CR, Zampino G,Hamamy H, Zalloum Y, Waaler PE, Crisponi G, Crisponi L, Rutsch F. 2011.Differential secretion of the mutated protein is a major component affectingphenotypic severity in CRLF1-associated disorders. Eur J Hum Genet 19:525–533.

Kass DJ. 2011. Cytokine-like factor 1 (CLF1): life after development? Cytokine 55:325–329.

Kass DJ, Yu G, Loh KS, Savir A, Borczuk A, Kahloon R, Juan-Guardela B, Deiuliis G,Tedrow J, Choi J, Richards T, Kaminski N, Greenberg SM. 2012. Cytokine-likefactor 1 gene expression is enriched in idiopathic pulmonary fibrosis and drivesthe accumulation of CD4+ T cells in murine lungs: evidence for an antifibroticrole in bleomycin injury. Am J Pathol 180:1963–1978.

Knappskog PM, Majewski J, Livneh A, Nilsen PT, Bringsli JS, Ott J, Boman H. 2003.Cold-induced sweating syndrome is caused by mutations in the CRLF1 gene. AmJ Hum Genet 72:375–383.

Kwong A, Sidore C, Sanna S, Kang HM, Jun G, Trost M, Anderson P, Gliedt T, Cusano R,Pitzalis M, Zoledziewska M, Maschio A, et al. 2012. Managing computing resourcesin large sequencing studies: strategies and lessons from sequencing 2,120 Sardiniangenomes. Abstract ASHG 3664W.

Li M, Sendtner M, Smith A. 1995. Essential function of LIF receptor in motor neurons.Nature 378:724–727.

Liu X, Jian X, Boerwinkle E. 2011. dbNSFP: a lightweight database of human nonsyn-onymous SNPs and their functional predictions. Hum Mutat 32:894–899.

Nakashima K, Wiese S, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T, YoshidaK, Kishimoto T, Sendtner M, Taga T. 1999. Developmental requirement ofgp130 signaling in neuronal survival and astrocyte differentiation. J Neurosci 19:5429–5434.

Okur I, Tumer L, Crisponi L, Eminoglu FT, Chiappe F, Cinaz P, Yenicesu I, HasanogluA. 2008. Crisponi syndrome: a new case with additional features and new mutationin CRLF1. Am J Med Genet A 146A:3237–3239.

Rousseau F, Gauchat JF, McLeod JG, Chevalier S, Guillet C, Guilhot F, Cognet I,Froger J, Hahn AF, Knappskog PM, Gascan H, Boman H. 2006. Inactivationof cardiotrophin-like cytokine, a second ligand for ciliary neurotrophic factorreceptor, leads to cold-induced sweating syndrome in a patient. Proc Natl AcadSci USA 103:10068–10073.

Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K.2001. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29:308–311.

Sohar E, Shoenfeld Y, Udassin R, Magazanik A, Revach M. 1978. Cold-induced profusesweating on back and chest. A new genetic entity? Lancet 2:1073–1074.

Stanke M, Duong CV, Pape M, Geissen M, Burbach G, Deller T, Gascan H, OttoC, Parlato R, Schutz G, Rohrer H. 2006. Target-dependent specification of theneurotransmitter phenotype: cholinergic differentiation of sympathetic neuronsis mediated in vivo by gp 130 signaling. Development 133:141–150.

Thomas N, Danda S, Kumar M, Jana AK, Crisponi G, Meloni A, Crisponi L. 2008.Crisponi syndrome in an Indian patient: a rare differential diagnosis for neonataltetanus. Am J Med Genet A 146A:2831–2834.

Tsuritani K, Takeda J, Sakagami J, Ishii A, Eriksson T, Hara T, Ishibashi H, KoshiharaY, Yamada K, Yoneda Y. 2010. Cytokine receptor-like factor 1 is highly expressedin damaged human knee osteoarthritic cartilage and involved in osteoarthritisdownstream of TGF-beta. Calcif Tissue Int 86:47–57.

Tuysuz B, Kasapcopur O, Yalcınkaya C, Isık Hasıloglu Z, Knappskog PM, Boman H.2013. Multiple small hyperintense lesions in the subcortical white matter on cranialMR images in two Turkish brothers with cold-induced sweating syndrome causedby a novel missense mutation in the CRLF1 gene. Brain Dev 35:596–601.

Uzunalic N, Zenciroglu A, Beken S, Piras R, Dilli D, Aydin B, Chiappe F, Okumus N,Crisponi L. 2013. Crisponi syndrome: a new mutation in CRLF1 gene associatedwith moderate outcome. Genet Couns 24:161–166.

Wildeman M, van Ophuizen E, den Dunnen JT, Taschner PE. 2008. Im-proving sequence variant descriptions in mutation databases and literatureusing the Mutalyzer sequence variation nomenclature checker. Hum Mutat 29:6–13.

Yamazaki M, Kosho T, Kawachi S, Mikoshiba M, Takahashi J, Sano R, Oka K, YoshidaK, Watanabe T, Kato H, Komatsu M, Kawamura R, et al. 2010. Cold-inducedsweating syndrome with neonatal features of Crisponi syndrome: longitudinalobservation of a patient homozygous for a CRLF1 mutation. Am J Med Genet A152A:764–769.

Yuan HY, Chiou JJ, Tseng WH, Liu CH, Liu CK, Lin YJ, Wang HH, Yao A, Chen YT,Hsu N. 2006. FASTSNP: an always up-to-date and extendable service for SNPfunction analysis and prioritization. Nucleic Acids Res 34:W635–W641.

Zlotogora J. 2007. Multiple mutations responsible for frequent genetic diseases inisolated populations. Eur J Hum Genet 15:272–278.

Zou X, Bolon B, Pretorius JK, Kurahara C, McCabe J, Christiansen KA, Sun N, DuryeaD, Foreman O, Senaldi G, Itano AA, Siu G. 2009. Neonatal death in mice lackingcardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia. VetPathol 46:514–519.

Expanding the Mutational Spectrum of CRLF1 in Crisponi/CISS1 Syndrome

Documents

The expanding RNA polymerase III transcriptome

Expanding the porthole

Celestial ephemerides in an expanding universe

Gravitomagnetic Instabilities in Anisotropically Expanding Fluids

Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: Expanding the spectrum of Timothy syndrome

Tikkurila expanding to the East

EXPANDING INVESTMENT FINANCE TO NORTHERN

Mutational profiling of kinases in glioblastoma

Expanding the palette: Metamorphic strategies over multiple lattice constant ranges for extending the spectrum of accessible photovoltaic materials

Mutational spectrum of the oral-facial-digital type I syndrome: a study on a large collection of patients

Mutational landscape of aggressive cutaneous squamous cell carcinoma

EXPANDING HORIZONS - Cholamandalam

Article Expanding Innovation Through Challenging

Mutational Dynamics of Aroid Chloroplast Genomes

Workers' Compensation: Expanding the Intentional Tort

Electromagnetic Spectrum

Expanding into South Korea

WATER STORIES: EXPANDING OPPORTUNITIES IN SMALL

Expanding Global Philanthropy - IssueLab

Expanding Access to Injectable Contraception - Toolkits