an unusual n-terminal deletion of the laminin alpha-3a isoform

An unusual N-terminal deletion of the laminina3a isoform leads to the chronic granulationtissue disorder laryngo-onycho-cutaneoussyndrome

W. H. Irwin McLean1,*, Alan D. Irvine2, Kevin J. Hamill1, Neil V. Whittock1,

Carrie M. Coleman-Campbell1, Jemima E. Mellerio3, Gabrielle S. Ashton3,

Patricia J. H. Dopping-Hepenstal3, Robin A. J. Eady3, Tanvir Jamil4, Rodney J. Phillips5,

S. Ghulam Shabbir6, Tahir S. Haroon6, Khawar Khurshid6, Jonathan E. Moore7, Brian Page7,

Jonathan Darling8, David J. Atherton9, Maurice A. M. van Steensel10, Colin S. Munro11,

Frances J. D. Smith1 and John A. McGrath3

1Epithelial Genetics Group, Human Genetics Unit, Department of Molecular and Cellular Pathology, University of

Dundee, Ninewells Medical School, Dundee DD1 9SY, UK, 2Department of Paediatric Dermatology, Our Lady’s

Hospital for Sick Children, Dublin, Ireland, 3Genetic Skin Disease Group, St John’s Institute of Dermatology,

The Guy’s, King’s College and St Thomas’ Hospitals’ Medical School, St Thomas’ Hospital, London, UK, 4Hasham

M. Jamil Trust, Burnham Health Centre, Minniecroft Road, Burnham, Burkinghamshire SL1 7DE, UK, 5Department

of Paediatrics, Royal Children’s Hospital, Melbourne, Australia, 6Department of Dermatology, King Edward Medical

College, Nila Gumbad, Lahore, Pakistan, 7Department of Ophthalmology, Royal Victoria Hospital, Belfast, UK,8Department of Paediatrics, St James’s University Hospital, Leeds, UK, 9Department of Paediatric Dermatology,

Great Ormond Street Hospital for Children, London, UK, 10Department of Dermatology, University Hospital Maastricht,

The Netherlands and 11Department of Dermatology, Southern General Hospital, Glasgow, UK

Received June 25, 2003; Revised and Accepted July 8, 2003

Laryngo-onycho-cutaneous (LOC or Shabbir) syndrome (OMIM 245660) is an autosomal recessive epithelialdisorder confined to the Punjabi Muslim population. The condition is characterized by cutaneous erosions,nail dystrophy and exuberant vascular granulation tissue in certain epithelia, especially conjunctiva andlarynx. Genome-wide homozygosity mapping localized the gene to a 2 Mb region on chromosome 18q11.2with an LOD score of 19.8 at h¼ 0. This region includes the laminin a3 gene (LAMA3), in which loss-of-expression mutations cause the lethal skin blistering disorder Herlitz junctional epidermolysis bullosa.Detailed investigation showed that this gene possesses a further 38 exons (76 exons in total) spanning 318 kbof genomic DNA, and encodes three distinct proteins, designated laminin a3a, a3b1 and a3b2. The causativemutation in 15 families was a frameshift mutation 151insG predicting a stop codon 7 bp downstream in anexon that is specific to laminin a3a. This protein is secreted only by the basal keratinocytes of stratifiedepithelia, implying that LOC is caused by dysfunction of keratinocyte–mesenchymal communication.Surprisingly, the 151insG mutation does not result in nonsense-mediated mRNA decay due to rescue of thetranscript by an alternative translation start site 6 exons downstream. The resultant N-terminal deletion oflaminin a3a was confirmed by immunoprecipitation of secreted proteins from LOC keratinocytes. Thesestudies show that the laminin a3a N-terminal domain is a key regulator of the granulation tissue response,with important implications not only in LOC but in a range of other clinical conditions associated withabnormal wound healing.

*To whom correspondence should be addressed. Tel: þ44 1382425618; Fax: þ44 1382425619; Email: [email protected]

Human Molecular Genetics, 2003, Vol. 12, No. 18 2395–2409DOI: 10.1093/hmg/ddg234

Human Molecular Genetics, Vol. 12, No. 18 # Oxford University Press 2003; all rights reserved

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INTRODUCTION

Laryngo-onycho-cutaneous syndrome (LOC, OMIM 245660)is a chronic granulation tissue disorder seen in the PunjabiMuslim population. The disease was first characterized byShabbir in 1986 (1), in 22 patients, all of whom sharedsignificantly similar clinical features that had not beenpreviously described. Eighteen of the 22 patients were childrenof consanguineous marriages between clinically normalindividuals, strongly indicative of autosomal recessive inheri-tance. Some of the original families examined by Shabbir werestudied here. All LOC families reported to date are of Punjabidescent (1–4), including all subjects in this report, suggesting afounder effect and perhaps an unusual defect that has not arisenin other populations. The term laryngeal and ocular granulationin children originating in the Indian subcontinent (LOGICsyndrome), has been suggested as an alternative name for thedisorder (3).

Patients with LOC syndrome share common clinical features(Fig. 1). The onset is characterized by a hoarse cry soon afterbirth, although the physical appearance of these neonates isnormal. Granulation tissue develops in the mucosal regions,nail bed and the larynx. Beginning in infancy, chronic skinulcers and conjunctival lesions appear (1–4). Although eyeinvolvement in LOC syndrome was not mentioned in theoriginal description (1), ocular granulation tissue resembling aparticularly aggressive pterygium has been reported in allsubsequent patients described (2–4) and was a prominentfeature in the patients studied here. In addition, there is failureof tooth enamel formation (2–4) and marked dental malforma-tions (1) (Fig. 1). Many patients diagnosed with LOCsyndrome do not live beyond childhood, the most commoncause of premature death being acute or chronic respiratoryobstruction with secondary pulmonary sepsis (2–4). However,patients who survive the early neonatal period often survive toadulthood (A.D. Irvine and C.S. Munro, personal observation)and symptoms of hoarseness and ulceration slowly improvewith age (1). As granulation tissue accumulates in the larynx,a permanent tracheostomy often becomes necessary to ensure aclear respiratory passageway. In addition, granulation tissue canspread to the epiglottis, trachea (3) and even the main bronchi(4). Furthermore, most of the older patients studied here areblind due to corneal pterygium formation.

Here, by genomewide homozygosity mapping in consangui-neous families, we have localized the LOC gene to a 2 Mbinterval on 18q11.2 and identified the causative mutation as anN-terminal deletion of the laminin a3a isoform.

RESULTS

Clinical details

All living patients and their families were seen and examinedby one or more of the authors (A.D.I., C.S.M., R.P. or T.J.).Families from a Pakistani Punjabi background were seen inScotland, England, Northern Ireland, France and in Pakistan(Lahore and the surrounding Punjab region). Medical recordsfor living patients and possibly affected deceased relatives wereexamined where available in the local health care setting in

conjunction with local health professionals. Owing to the rangeof health care institutions that the subjects had attended (basicrural health care in some, tertiary referral hospital for others),there was a wide variation in the degree of clinical investiga-tions and medical interventions performed. To ensure aconsistent approach, ascertainment of affected phenotype forthe purposes of this study was based on clinical history andphysical examination and not on the basis of laboratoryinvestigations. After review of the few clinical papers publishedto date (1–4) and discussion with the original authors, subjectswere deemed to be affected with LOC if they fulfilled thefollowing clinical diagnostic criteria: (1) hoarse cry in first fewweeks of life or evidence of laryngeal granulation tissue,scarring or stenosis in the first few years of life; (2) naildystrophy; (3) ocular involvement including aggressive con-junctival pannus formation; (4) dental anomalies (hypodontia,poor enamel formation, excessive caries); (5) slow-healingcutaneous erosions; (6) absence of a history of skin fragility orblistering at any stage. All subjects deemed affected in thestudy filled all of these criteria. The cardinal features of LOCsyndrome are illustrated in Figure 1. Patient DNA and tissuesamples were obtained after informed consent with theapproval of the relevant local Ethics Committees.

Genetic linkage analysis

To search for the LOC gene, genomewide homozygositymapping was carried out using the ABI HD5 linkage mappingpanel, consisting of 800 fluorescent microsatellite markers, asdescribed previously (5). Affected individuals were genotypedfrom six apparently unrelated Punjabi families (Fig. 2A). Fiveof these kindreds are UK citizens of Punjabi ancestry (families1–4 and 6) and the other family resides in the Lahore region ofPakistan (family 5). Initial screening identified potential loci onchromosomes 5q in the vicinity of D5S400 and 16q, in theregion of D16S3061. However, these loci were suspected asfalse linkage due to lack of shared haplotypes between thefamilies. Analysis of additional LOC families completelyexcluded these loci (data not shown). No other suspect lociwere identified from the initial screen. In case the locus hadbeen missed, the larger gaps in the HD5 panel were filled bysynthesis of an additional 60 microsatellites. One of theseadditional markers, D18S869, showed homozygosity for thesame allele (195 bp) in all but three of our 15 LOC families,consistent with the anticipated founder effect (Fig. 2B). Furthergenotyping was carried out with an additional nine closelyspaced microsatellites in the vicinity of D18S869. Thisrevealed an area of shared haplotype for three markers,LAMA3GT1, LAMA3GT2 (see below) and D18S1101 in 17individuals from 14 LOC families (Fig. 2B). One familyremained unlinked (family 15), however, in this case one parentcarried the linked haplotype (Fig. 2B). There was no evidenceof consanguinity in family 15 and therefore it was assumed thatthe affected individual in this case might represent a compoundheterozygote. All parents of affected individuals in families1–14 were heterozygous for the linked haplotype and nounaffected siblings of LOC patients were homozygous for thishaplotype. For the kindreds where details of consanguinitywere known (families 1–7), markers within the region of sharedhaplotype gave a highly significant two-point LOD score of 9.6

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at y¼ 0. The combined two-point LOD score for all kindredsshowing linkage (families 1–14) was 19.8 at y¼ 0 for the threemarkers within the region of shared haplotype.

The limits of the LOC locus were defined by visiblerecombinations seen in families 2, 7 and 12 (individuals 2-1,7-1 and 12-2; Fig. 2B) with markers D18S869 (centromeric)and D18S480 (telomeric). This gave a critical interval ofapproximately 2 Mb of genomic DNA (UCSC Genome Map).A total of 12 known and strongly predicted genes were locatedin this interval. Of these, LAMA3, located centrally, wasconsidered by far the most likely candidate gene on the basis ofits biological functions. Laminin a3 is one of the threepolypeptides comprising laminin 5, the major cutaneousbasement membrane laminin with a critical function inadhesion of the epidermis to the dermis. Loss-of-expressionmutations in LAMA3 cause the skin blistering disorder Herlitzjunctional epidermolysis bullosa (H-JEB) (6–8). In addition tomediating cell-substratum adhesion, laminins in general andlaminin 5 in particular have important additional functions inthe control of cell migration and behaviour (9,10). Therefore,since the main phenotype in LOC is uncontrolled production ofgranulation tissue in the skin and other epithelial tissues,LAMA3 was considered a prime candidate. Two microsatelliteswithin introns 12 and 41 of the extended LAMA3 gene(described in detail below), designated LAMA3GT1 and

LAMA3GT2, respectively, were found to be highly poly-morphic in the Punjabi Muslim population. Importantly, therelevant haplotype for these two markers (alleles 290 and 195,respectively) was shared by all LOC affected individualsstudied (Fig. 2B), including one copy carried by thepresumptive compound heterozygote family 15. Therefore,the LAMA3 gene falls within the region of shared haplotypewithin the LOC locus.

LAMA3 gene structure and alternate splicing

The LAMA3 gene was initially reported as having 38 exonsencoding the laminin a3a polypeptide (11). However, partialcDNAs have been reported encoding a longer alternatetranscript which encodes the larger laminin a3b polypeptide,whose functions and protein interactions are less well under-stood (11,12). Since recessive loss-of-function mutations inLAMA3A had already been demonstrated in the severe/lethalskin blistering disorder H-JEB, we initially focused onLAMA3B. Analysis of the human genome sequence withreference to the reported laminin a3b cDNAs and ESTsrevealed an additional 38 exons upstream of the existing 38LAMA3A exons, giving a total of 76 exons spanning 318 kb ofgenomic DNA. The full gene structure and transcripts thereofare shown in Figure 3. Exons 1–38 plus 40–76 encode the

Figure 1. Clinical features of LOC syndrome in patients from families 2 and 3. (A) Proband in family 3, showing cutaneous erosions on the face, which are slow toheal. Healed lesions often leave behind atrophic, pigmented scars as seen on the right cheek of this patient. (B) Nail dystrophy is common in LOC; in infantserosions are often seen but older patients develop a thickened hyperkeratotic appearance, seen here in right great toe of the proband from family 2. (C) Oculargranulation tissue with lateral symblepharon formation in the proband from family 2. This patient, in his 20s, is blind due to almost complete closure of the eyelidsand deep corneal scarring secondary to the invasive fibrovascular growth. (D) Hypoplastic enamel and subsequent carious teeth are common findings in LOC.(proband of family 2).

Human Molecular Genetics, 2003, Vol. 12, No. 18 2397


Figure 2. (A) Pedigrees of consanguineous LOC families 1–6, used for genome-wide homozygosity mapping. Arrows indicate probands and numbers refer to individual probands whose haplotypes are shownin (B), below. Asterisks show individuals from whom DNA was available for study. (B) Haplotypes of affected LOC individuals for microsatellite markers in the vicinity of the LOC locus. Numbers abovehaplotypes refer to individual LOC patients and are in the format family–patient number. Boxes indicate areas of homozygosity-by-descent.

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Figure 3. (A) Schematic diagram (to scale) showing the genomic organization of the 76 exons of the LAMA3 gene, which spans �318 kb on chromosome 18q. Exons involved in transcription initiation and/oralternate splicing are indicated by arrows. (B) Transcripts identified from the LAMA3 gene (spliced exons to scale). These transcripts encode three laminin a3 polypeptides: a3a, a3b1 and a3b2. The predictedlaminin a3a polypeptide consists of 1724 amino acids and has a calculated molecular weight of 190.3 kDa. The predicted laminin a3b1 protein consists of 3333 amino acids and has a calculated molecularweight of 366.3 kDa. The shorter laminin a3b2 polypeptide comprises 3289 amino acids and has a calculated molecular weight of 361.4 kDa. (C) Excerpts of the 50 regions of LAMA3 transcripts showing thestart of the predicted open reading frames. The 50UTR sequences are shown in lower case; and codons are shown in upper case with amino acid translation underneath.

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longer LAMA3B transcript (i.e. exon 39 is skipped in theLAMA3B isoform). In addition, RT–PCR performed on cDNAextracted from normal control keratinocytes revealed that about20% of the LAMA3B mRNA also lacks exon 10. We proposethat the longer LAMA3B isoform should be designatedLAMA3B1 (encoding laminin a3b1) and that the shorter versionlacking exon 10 be designated LAMA3B2 (encoding laminina3b2). The predicted laminin a3b1 polypeptide consists of3333 amino acids and has a calculated molecular weightof 366.3 kDa. The shorter laminin a3b2 polypeptide comprises3289 amino acids and has a calculated molecular weight of361.4 kDa. Promoter sequences immediately upstream of exon 1(B-isoforms) were strongly predicted by both the TSSG andTSSW algorithms (13). The transcriptional start site of the Bisoforms was mapped by RT–PCR (data not shown) and theentire LAMA3B1 and LAMA3B2 cDNAs were amplified in aseries of overlapping RT–PCR fragments and fully sequenced.Primers used for RT–PCR are listed in Table 1.

The LAMA3A transcript starts with exon 39, which includesthe initiation codon and 50-UTR of the LAMA3A transcript, andincludes exons 39–78 of the LAMA3 gene. However,the LAMA3A 50-UTR derived from the human genomesequence (UCSC Browser) was found to have slight differencesfrom the published mRNA sequence and contained anadditional in-frame ATG codon (Fig. 4). The sequencessurrounding the new ATG conform better to the Kozakconsensus sequence than the previously proposed initiationcodon (11). This sequence was verified by sequencing of bothgenomic and RT–PCR fragments, and the likely transcriptionalstart site predicted by both the TSSG and TSSW algorithmswas determined by RT–PCR of keratinocyte mRNA using aseries of primers across the region (Fig. 4). The LAMA3Apromoter was strongly predicted by both the TSSG and TSSWalgorithms to be within intron 38, immediately upstream ofexon 39. The entire LAMA3A cDNA was amplified in a seriesof overlapping fragments and was fully sequenced. Primersused for RT–PCR are listed in Table 1. The predicted laminina3a polypeptide consists of 1724 amino acids and has acalculated molecular weight of 190.3 kDa. By RT–PCR, theA-isoform was found to be both specifically and highlyexpressed by epidermal keratinocytes but not by dermalfibroblasts, whereas the two B-isoforms were expressedby both these cell types (data not shown). Relative expressionwas not assessed in other tissues. The GenBank accessionnumbers for the LAMA3 transcripts determined here areAY327114 (LAMA3A) AY327115 (LAMA3B1) and AY327116(LAMA3B2).

Isoform-specific LAMA3 mutation in LOC

PCR conditions were developed to amplify the individualexons of the LAMA3 gene using intronic primers to facilitatemutation detection based on genomic DNA (primers detailed inTable 2). All 76 exons were screened for mutations in LOCaffected individuals by direct sequencing of genomic PCRproducts, with comparison to normal unrelated controls. InLOC patients from families 1–14, a homozygous 1 bp insertionmutation was detected in exon 39, the exon found exclusivelyin the keratinocyte-specific LAMA3A transcript encoding thelaminin a3a polypeptide (Fig. 5). The mutation was designated

151insG, numbering from the initiating ATG of our revisedLAMA3A sequence (Fig. 4; GenBank accession numberAY327114) and leads to a premature termination codon(PTC) 7 bp after the insertion, within this first LAMA3A exon.Numbering from the previously reported mRNA sequences

Table 1. LAMA3 RT–PCR and sequencing primers

Primer Sequence 50 ! 30 PCR size Anneal

L3B50UTR1 GAC GGC TCA GGC GGG AGG AC 1705 bp 65�Ca

L3CDNA3R GAC ATT CAC ACT GTC CAG TC

L3CDNA1F CGA CTT ACT TCA ACC TGG CC 476 bp 56�CL3CDNA1R GGT GAC AGC CAT ATT TGC CT

L3CDNA2F GAA GCA CCT ACT CAC CAT GG 690 bp 56�CL3CDNA2R CCT TAG CAC ACT GTT CAC AG

L3CDNA3F CAG CAG GCA AGC TTG AAT AC 553 bp 56�CL3CDNA3R GAC ATT CAC ACT GTC CAG TC

L3CDNA4F GCT CTG GTT TCT ACT CAT TCC 638 bp 56�CL3CDNA4R GCC GTC TTC AAT CTC ATA CTT C

L3CDNA5F CAT TGT CCT CCA TGT GCA GTG 578 bp 56�CL3CDNA5R GTA GGC ACA TGG TTC TGT GAC

L3CDNA6F GAG TCC TTC TGG ATT ACC TGG 586 bp 58�CL3CDNA6R GGT CTC ACA TAC TCA GCT GAG

L3CDNA7F GAT GCA GAC ATT CAG CTC AAG 633 bp 56�CL3CDNA7R CTC GAG TGA CTT GTC CAT GG

L3CDNA8F CGC AAC TGT GAA GGT TCC AG 618 bp 56�CL3CDNA8R GAA GCA CAT CGG TCA CAC TG

L3CDNA9F GAA GGC TGC AAC TGT TCC AG 630 bp 56�CL3CDNA9R CTG ACC AGT GAG CTG TAC ATC

L3CDNA10F GTG GTT ACC TCA CTT ACC AAG 764 bp 56�CL3CDNA10R GCT GTT AGT GTG AGG ACA TGG

L3CDNA11F CTC AGG CAT ATG TGT TAA CTG TC 543 bp 56�CL3CDNA11R GCA GAA CGG TAG TTG AGC AAC

L3CDNA12F CCT GGT CAA GTC TCA GCT GC 553 bp 56�CL3CDNA12R CCG GAT ACT GTT AGC AAG CC

L3CDNA13F GCA CCT CAG AGA AGC AGA AG 593 bp 56�CL3CDNA13R CAT TGA GGA TGT TCT CGT AGG

L3CDNA14F GCA GCT GGA AGA GAT CAA GAG 610 bp 60�CL3CDNA14R GTT GAT ACT TTC AAT CTT GCG CC

L3CDNA15F GCT CTG ACT GAT GCA GAT AAC 574 bp 56�CL3CDNA15R CAT CCA TGT CAT AGA CTC CGG

L3CDNA16F CCA AGA GTG AGA CTA AGG AGG 620 bp 56�CL3CDNA16R GAT CTG AAT CGA ATG GTC TCT G

L3CDNA17F GGC AAG CTC ATG GTG AGA TAC 630 bp 60�CL3CDNA17R CCG TAG TCC AGA GTT GTC GC

L3CDNA18F GAA TTG AGC ACC AGC GAT AGC 597 bp 56�CL3CDNA18R CAG TCC TCT GGA GGA TGT TG

L3CDNA19F CCA GCC ACT TGC TAT TCA AGC 598 bp 56�CL3CDNA19R CCA GTG AGA CTT CTT GGG CG

L3CDNA20F CTC TGA AGA AGG AGG TCA TGT C 629 bp 56�CL3CDNA20R CTG TCT GGT TTG TGT CCT GAG

LAMA3B transcription start site testingFollowing used with L3CDNA3R (above)L3B50UTR1 GAC GGC TCA GGC GGG AGG AC 1705 bp 65�Ca

Following used with L3CDNA1R (above)L3BP1 GCT GTC AGT TCC TGA TCC GG (858 bp) 50–65�Ca

L3BP3 AAC GCC TCT CCC ATT CCC C (940 bp) 50–65�Ca

LAMA3A transcription start site testingL3AP4R CCA TTC ACC ACA CAG CCA GT

Following used with L3AP4R (above)L3AP1F AGA GGA AGA GGC AGA GGT TC (473 bp) 50–65�Ca

L3AP2F CAG CCA GCG GAC GTC CAG 448 bp 55�Ca

L3AP3F GGG ATG CCT CCA GCA GTG A 424 bp 55�Ca

Product sizes in brackets are predicted sizes only and were tested over thetemperature range indicated.aBoehringer Long-Range buffer and enzyme mix used in place of standard PCRconditions.



(GenBank accession numbers NM_000227 and X77598), thismutation would be designated 118insG. All parents in families1–14 were heterozygous carriers of the mutation (Fig. 5). Thus,individuals with LOC are homozygous for a frameshiftmutation leading to a PTC within the only exon specific tothe LAMA3A isoform. This mutation should have no effect onexpression of the LAMA3B isoforms and therefore our initialhypothesis was that LOC is caused by a specific, differentialloss of laminin a3a expression. In contrast, all the LAMA3mutations reported in H-JEB patients are PTC mutations inexons that are common to both LAMA3A and LAMA3Btranscripts (6,8). Therefore, we hypothesized that loss of bothlaminin a3a and a3b expression might cause H-JEB, whereasloss of a3a alone would cause LOC.

The father in family 15, who carried the shared LOChaplotype (Fig. 2B), was also found to carry the ancestral151insG mutation, which he had passed on to his affected son,but this was not detected in the mother’s DNA. All other exonsof the LAMA3 gene were analysed in the family 15 proband andhis parents. Both the proband and his mother were found to beheterozygous for nonsense mutation R943X (DNA change2827C>T) in exon 60, the 22nd exon of the LAMA3Atranscript (Fig. 6). This mutation would be numbered R932X inthe previously reported sequence. The mutation ablates a SalIrestriction enzyme site which was used to confirm the mutationin the proband and his mother and exclude it from his father(Fig. 6). Thus, the proband in family 15 is a compoundheterozygote for mutations 151insG and R943X in LAMA3A.This individual would be predicted to lack expression oflaminin a3a but would express 50% of the normal amount oflaminin a3b, again consistent with the hypothesis that LOC iscaused by loss of laminin a3a alone.

Figure 4. Map of exon 39 of the LAMA3 gene, showing the additionaltranscribed sequences compared to the previously reported mRNA sequence.A predicted TATA box is shown in bold type, identified by the TSSG andTSSW programs. The predicted start of transcription is shown in bold.Primers used to map the transcription start site are shown as arrows.Primers P2F and P3F were found to amplify RT–PCR keratinocyte cDNAin combination with reverse primers in downstream exons, whereas P1F didnot (data not shown).

Table 2. LAMA3 genomic PCR


L3A1F GCT GTC AGT TCC TGA TCC GG 710 bp 55�Ca

L3A1R GTG CTT CCA CGA GAT CGC TC

L3A2F CAG CTG ACT TCT CTA AGC TG 477 bp 58�CL3A2R CAG GCA TGA GCC ACT GTG C

L3A3F GTC CAA TAG ACA GGA ATC AGG 425 bp 56�CL3A3R CTG ACC TCA GGT AAT CCA CC

L3A4F GTT AGC ACT GCA TCT CCA AG 460 bp 56�CL3A4R TGA TCT ATG TTA GAT GTC CTT G

L3A5F GGA ACC AGG AAG TGT GAG G 482 bp 56�CL3A5R CAC ACC TAC ATC ATT ACA GGC

L3A6F GAA GCA GAG GCA GCA GAT G 292 bp 56�CL3A6R CTA TTG AGG ACA GGA ACC TAG

L3A7F TGC GAA GTG TTG TGT TCT CC 372 bp 56�CL3A7R CCT AAC ACT GAC ATG ACC AC

L3A8F TGT ATC TTA GTA TAT CCT GGT C 442 bp 56�CL3A8R CCT TGT GTG AGC AAC AGT GG

L3A9F TCC AGA GCC AAG ATT AAG CC 444 bp 60�CL3A9R GAG ACT GTG TAA GTG ACT GAG

L3A10F ACC ATC AAT CAG GTG TGA GG 377 bp 56�CL3A10R CTG TCC ACC ATG CCT CAG AG

L3A11F GGA ATG TCT GGC CAG TCT G 399 bp 56�CL3A11R CAA CTA GGA GAC ACC TGC AAG

L3A12F CAA CGC AGT GTA GTC TTC TG 434 bp 56�CL3A12R GGA CCA TCA CAT GTA ACA TCC

L3A13F CTC ATA AGA AGT GGA GCT GG 412 bp 56�CL3A13R CCA AGC AGA CTA GAT CTG TG

L3A14F CAT GCA GAC TTT GCC ACA CA 331 bp 56�CL3A14R TTT GAA AAA CAA CAA TGA CCT CT

L3A15F GCA ATT AGC AGA CTG TTG AAA TC 380 bp 60�CL3A15R CTT GTC AAA TTG AAG TGA TTC AAC

L3A16F CAA CTG CAC ACG TTG TGT TG 257 bp 56�CL3A16R CCA TGC ACT GTA GAA TGA CAG

L3A17F CTG TCA TTC TAC AGT GCA TGG 459 bp 56�CL3A17R GTG TGC TGG TAC AAG AGA AG

L3A18F CAC TTG CCA GAC AGA TGT GG 338 bp 56�CL3A18R GCT GCT TCT CTC TTC TCC AG

L3A19F GTA CTC TGT CAA TGT GGT GG 319 bp 56�CL3A19R GGA AGT ATC TGA GCT ACT CAG

L3A20F CAA GTC CAG TAG TGA CAC TTG 363 bp 56�CL3A20R CTA GAG AAC CGT CTA ACC AG

L3A21F CCA GTG TAG ATA GCC ATT GC 455 bp 56�CL3A21R GGT CGC TGT GGA TTG TAG TC

L3A22F GGT GAG TCG GGA TGA AGA AC 347 bp 58�CL3A22R GTG ATC CTG AGA CAC CTC ATG

L3A23F GTG AAC TGA ATG ACT GAG AGT C 339 bp 56�CL3A23R GAG AAG CAA CTG TGG CCA AG

L3A24F GCA TCT CAA CCT GTA GGA CC 454 bp 60�CL3A24R CAC AAC TAG TCA CCT GCT TG

L3A25F GAG CTG CAT CCT TCT TCA AC 432 bp 56�CL3A25R TGA AGA GGC AAG TCA GGT TC

L3A26F GAT GCC TGA GGA CAT GCC ATC 471 bp 56�CL3A26R GGT CAG CTT CCT GAT ATT ACG

L3A27F CTA GTT CAA TGG AAC AGG TGG 470 bp 56�CL3A27R CAA CTC AGT AGG TCT GTA GTG

L3A28/29F GTA TGT GTG AAG GCT TCT GG 730 bp 56�CL3A28/29R CTC TCT TCT GTC CTA GTT CC

L3A30F TGA GCA TCA TGT CAG TGC TC 401 bp 56�CL3A30R GAC TCA CTG ACA CTC TCT TG

L3A31F CCT GAC TCC AGA ACC ATG AG 439 bp 60�CL3A31R GTG AGT CTC TAG CAC AGT GC

L3A32F CAT CCA CAC TGT CTG CCA TG 469 bp 61�CL3A32R GTG ACG ATG ACC AGT GTG TG

L3A33F CTC TCC ATT CTG CCT TGG AC 410 bp 56�CL3A33R CAC TAT CAA CAT CTC TTA CGG

L3A34F CTC ACA TCC TCA TCC TAA CAT C 459 bp 56�CL3A34R GTC AGC AAG AGT GTC CAA GG

L3A35F GAG GGC AGG TGC AGG AGT AG 416 bp 56�CL3A35R TAA CCT GAA CTG CTG GAT GCT



By direct DNA sequencing, the incidence of the commonLOC mutation was investigated in 50 normal unrelated BritishPunjabi Muslims with no family history of LOC syndrome aswell as 50 normal unrelated British Caucasians. The mutationwas not detected in the British Caucasian control population.However, one Punjabi control was found to be heterozygous forthe 151insG mutation, indicating that this mutant LAMA3Aallele may be fairly common in the Punjabi population.

Rescue of the LOC mutant transcript

To see if the 151insG mutation would lead to nonsense-mediated mRNA decay, we obtained a skin biopsy from aheterozygous carrier of the LOC mutation; mRNA wasextracted and subjected to RT–PCR using a forward primerwithin exon 39 and reverse primer in exon 42, so that only theLAMA3A transcript would be amplified. mRNA was pre-treatedwith RNAse-free DNAse I and phenol–chloroform extracted tosafeguard against genomic DNA contamination. Direct sequenc-ing of this RT-PCR product (Fig. 7A) was compared to agenomic exon 39 fragment from the same individual sequencedwith the same primer (Fig. 7B). Surprisingly, the sequencetraces obtained from genomic DNA and cDNA were essentiallyidentical, with the overlapping sequence traces due to theinsertion clearly visible in both samples and present at verysimilar levels. Therefore, this demonstrates that the 151insGmutation does not lead to appreciable nonsense-mediated decayof the LAMA3A transcript.

Close examination of the LAMA3A mRNA sequence revealedthat the wild-type 50-UTR does not contain any in-frame stopcodons (Fig. 4), whereas many 50-UTRs do, especially longerones (14,15). We hypothesized that the translation machinerymay regard this premature stop codon near the start of themRNA as a natural feature of the 50-UTR and thereforetranslation might occur from another ATG downstream ifone existed within the context of a suitable Kozak consensusmotif, which is required for efficient translation. The next ATGcodon downstream of the frameshift occurs in exon 45(methionine 218 in the revised protein sequence); however,this codon is a very poor fit for the Kozak consensus (Fig. 7C).In contrast, the next ATG, also in exon 45 (methionine 227) is aperfect fit for the Kozak sequence (14), in fact conforming tothe consensus much better than the natural initiation codon ofthe LAMA3A transcript (Fig. 7C). Thus, it seemed plausible thatprotein translation would initiate from this codon leading toa truncated laminin a3a polypeptide lacking the first 226 aminoacids. To look for this truncated protein and to see if it wouldbe assembled into trimeric laminin 5 and secreted from

Table 2. (Continued )


L3A36F CTC AAC CCC ACC TCC TAG AT 400 bp 56�CL3A36R ACA TGC TTT CAA CTT TCA TTT AG

L3A37F TTT TAC CAG TCA GTT AAT TCT GAA 377 bp 56�CL3A37R ATT AAG GAT GCT CAA CCT GTA TAT

L3A38F GCA GGT ATT TCC CCG TCT TA 503 bp 56�CL3A38R AGC CCT GCA GAA ACT TGA AC

L3A39F GCC TTC CTC ACC TGA GTC AG 419 bp 59�CL3A39R CAC TGC CAG TCA AGG TGA C

L3A40F GAT GTG AAT GCT TCA CAG TGA G 357 bp 56�CL3A40R CAG CCT GAT AAT GGA GCC TC

L3A41F GTC AGA GGA CTT GAG TTG TAG 280 bp 56�CL3A41R CCT AGT CCT AGG CAG CCA C

L3A42F CTC ACT TAA TAC AAG CCA GTC 260 bp 55�CL3A42R TAG CTC TGT GCC AGC ACC TG

L3A43F CTT GCA GAT CTA TGT CGG TC 328 bp 56�CL3A43R CAT CCT GAA GAT GTG TGC TTG

L3A44F GGT GTC TAT TAG GTT GGT GC 399 bp 56�CL3A44R GGT CAT ACA GAG AGA AGC TG

L3A45F GTG ACG ATC TGC GTG CAT GC 329 bp 56�CL3A45R CCT GCT TAT GGA GAA TCC AC

L3A46F CCT GTT GAC CAG GTT GAT AG 861 bp 59�CL3A48R CAT TGC ACT CTG CAC GTT GTG

L3A49F GCA GGT CTG TGT CAA TGT G 460 bp 56�CL3A49R GAG ATG TAC AGA CAG GTG C

L3A50F CTA GAC AAT GTT CAG GTT CAA G 369 bp 56�CL3A50R CTC TTC CAT CTA AGA GAC TAT AC

L3A51F GAC CAA GGA TTA ACT GGT ATG G 481 bp 56�CL3A51R CCT TAT TGC TGC TCT GCT TC

L3A52F GGA TGT CAG CTG CTG GCA G 388 bp 60�CL3A52R CTG TGC TAC AGA CAT GAA ATA G

L3A53F GGG CTT CAG ACT AGG ATA G 303 bp 56�CL3A53R ATC TGC ATT GAC ACT GAG ATT C

L3A54F GCA GAC ACT GGC TAC TTC AG 611 bp 56�CL3A55R CCT GTA TCA GGT GGA GCT G

L3A56F GAC CAT GGT TGA TAG CAA GTA C 534 bp 56�CL3A56R GGA TGA AGG TCA TGT ACA CTT G

L3A57F GAT ACA TGG CTA CGA GTC AC 368 bp 56�CL3A57R CTG CTG TGC TAA TGG CAG TG

L3A58F GAG CAG AGA CAG CTC TCA C 713 bp 56�CL3A59R CAT GAG GTC ATC ACC ATA CC

L3A60F CCC TTA TGC CAT AGT GAC TG 335 bp 60�CL3A60R CAA CAT CAA TAG CAT CTC ACT G

L3A61F GCA CAC TGG CAT GGT GAT C 332 bp 60�CL3A61R GGA TGC AGG TAA CAT TTA CAC

L3A62F GAG TTC CAG TGC TCT GCC TC 314 bp 56�CL3A62R CCC TCA GTT AAC ATC CAT AAG

L3A63F GCT ACT GCT CAG AGC CTT CC 472 bp 56�CL3A63R CCT CCT TCT TTC CAC TGC C

L3A64F GAG CAC AGC GTT TCA GCT C 270 bp 56�CL3A64R GTT GCT GCG TTA ATG GAT ACG

L3A65F GTG AGA TAG GGT AAG GTA CTG 464 bp 56�CL3A65R ATG AGT CCG CGA GTA CAC AC

L3A66F TAC AGG CAT AAT CCT TAT ATG C 389 bp 56�CL3A66R GAG CCA CCA TGT GTG GCA G

L3A67F GAC AGT GAG TTG GCC ACA TG 320 bp 60�CL3A67R GTC TCG TGC AGC ACA TGT TG

L3A68F GAG CTC TCT CTA CCA GAG G 297 bp 58�CL3A68R CCA CAA GGC AGG CTC AGG

L3A69F GAA TTC CTC ACT TAG GAT CAA G 353 bp 56�CL3A69R GTG TCA TGG CAG TCA GAC TTC

L3A70F CTG TCT CAG TGC TGA GAT TCG 383 bp 56�CL3A70R CTG CAA GCA CAC AAG CTC TC

L3A71F GAG CTC AGT TTG TTC AGT ATT TC 500 bp 56�CL3A71R CTT ACT CTA TGT GGT TGT GAA C

L3A72F GCT GAC AAG CTG CAG TGC C 616 bp 56�CL3A73R TAG CAC TGA AGT CAA GGC TC

L3A74F GGA TGC AGC AGA GAG ATC TC 422 bp 56�CL3A74R GAA TGG ACA CGT TAG CCT CG

Table 2. (Continued )


L3A75F CAT GTA CGT GGA AGC ACT AC 400 bp 58�CL3A75R CAC TGT CCA GCC AAG AGC TG

L3A76F GAA CAG TCC CTG GAC AGT ATG 314 bp 56�CL3A76R CCT GTC TGG TTT GTG TCC TG

aBoehringer Long-Range buffer and enzyme mix used in place of standard PCRconditions.



keratinocytes, we performed immunoprecipitation of radiola-belled secreted proteins from control and LOC keratinocytesusing an antibody specific for the C-terminus of laminin b3(16,17). This analysis revealed that indeed a truncated laminina3a is synthesized, assembled and secreted by LOC keratino-cytes (Fig. 8). The reduction in size of the truncated proteincompared to wild-type laminin a3a was consistent with loss of226 amino acids (�24 kDa). Thus, the LOC mutant transcriptis rescued by a novel mechanism leading to expression of anN-terminally truncated protein. We propose the term ‘delayedinitiation codon’ for this novel class of mutation.

Subtle morphological changes in LOC skin

Using frozen skin biopsy material from an LOC patient(proband in family 1, homozygous for the 151insG mutation)and a normal unrelated control, we performed immunofluore-scence staining with a variety of antibodies against all threea3, b3 and g2 chains of the laminin 5 trimer and othercomponents of the cutaneous basement membrane zone.Representative results are shown in Figure 9. Essentially, allantigens examined, including laminin a3, were present innormal amounts and were distributed normally. The laminin a3antibodies used are not able to distinguish laminin a3a and a3bisoforms, nevertheless, there was no evidence of a 50%reduction in staining intensity that one might expect if the a3aisoform was absent. Importantly, normal staining was obtainedin LOC skin with monoclonal antibody GB3, which detectstrimeric laminin 5 (18), confirming the assembly and secretionof this protein complex.

The ultrastructure of the dermal–epidermal junction (DEJ)was generally normal, but there were some subtle qualitative

changes (Fig. 10). The number of hemidesmosomes andanchoring fibrils, appeared to be normal. However, some of thehemidesmosome plaques were small and lacked normalsub-basal dense plates. These diminutive cell–stromal junctionswere associated with reduced numbers of anchoring filamentsand focal widening of the lamina lucida, the site of laminin5 localization within the DEJ. There were no overt splits in thelamina lucida, as would be expected in the junctional forms ofepidermolysis bullosa, such as H-JEB.

DISCUSSION

LOC syndrome is a rare recessive disorder thus far onlydescribed in the Punjabi Muslim population (1–4,19). Thedisease is characterized by chronic production of vasculargranulation tissue which invades and disrupts specific epithelialtissues, notably the conjunctiva and anterior corneal epithelium(4,19), as well as the respiratory mucosae. Here, by genome-wide homozygosity mapping the gene causing LOC syndromewas localized to a small interval on human chromosome 18q.The causative mutation in all but one family was found to behomozygosity for a novel class of mutation in the laminina3 gene (LAMA3). The remaining case was a compoundheterozygote for this mutation and a complementarynull-allele. The ancestral genetic defect is a delayed initiationcodon mutation in exon 39 of LAMA3, designated 151insG.Exon 39 is the first exon of the LAMA3A transcript, whichencodes the laminin a3a polypeptide, a component of thelaminin 5 heterotrimer, a basement membrane lamininspecifically secreted by basal keratinocytes in the skin andother stratified epithelia including airway and ocular epithelia(18,20). Furthermore, the mutated exon is the only one specific

Figure 5. The homozygous ancestral LOC mutation in exon 39 of LAMA3 (151insG) shared by all affected persons in families 1–14. In addition, the proband infamily 15 was heterozygous for this mutation. (A) Direct sequence of a LAMA3 exon 39 PCR product from a normal control. Codons 49–53 of the LAMA3Atranscript are shown. (B) Equivalent region as shown in (a) derived from the proband in LOC Family 1 (LOC patient 1-1, Fig. 2), showing homozygous insertionof G at position 151 (151insG in the LAMA3A transcript). This frameshift mutation leads to a premature termination codon 7 bp downstream of the insertion (notseen here). (C) Equivalent region as shown in (a) derived from the father of the proband in LOC family 1. Overlapping sequence traces can be seen due to hetero-zygosity for the insertion mutation. (D) Equivalent region as shown in (a) derived from the mother of the proband in LOC family 1. Again, overlapping sequencetraces can be seen due to heterozygosity for the 151insG mutation.



to laminin a3a and is spliced out of laminin a3b1 and a3b2described here, whose functions are currently unknown.Unexpectedly, we found that this frameshift mutation doesnot lead to appreciable nonsense-mediated decay of theLAMA3A mRNA. The mutant transcript escapes accelerateddegradation by utilising an alternative initiation codon

Figure 6. Compound heterozygous mutation in the proband from LOC family15. This patient was heterozygous for the shared haplotype at the LOC locus(Fig. 2) and was subsequently found to be heterozygous for the 151insG muta-tion (data not shown). (A) Direct sequence of a LAMA3 exon 60 PCR productfrom a normal control. Codons 941–945 of the LAMA3A transcript are shown.Box indicates arginine codon 943. (B) Equivalent region as shown in (A)derived from the proband in LOC family 15 (LOC patient 15-1, Fig. 2), show-ing heterozygous nonsense mutation R943X (boxed). (C) Mutation R943Xdestroys a single SalI restriction enzyme site present in the exon 60 PCR frag-ment. Lane 1 shows undigested 335 bp exon 60 PCR product. Lanes 2–3 showSalI digests of exon 60 PCR products derived from the mother, father and pro-band of family 15, respectively. The father’s DNA digests fully, giving bands of230 and 105 bp (lane 3). In contrast, both the mother and the proband’s DNAshow an additional undigested 335 bp band due to the mutation. Thus, theR943X mutation was inherited maternally, consistent with the haplotype data(Fig. 2).

Figure 7. Mutant LAMA3A transcript escapes nonsense-mediated mRNA decay.(A) Direct sequencing of a LAMA3A-specific RT–PCR product generated withprimers L3AP3F and L3AP4R (Table 1) derived from epidermal mRNA froman LOC heterozygote. The forward L3AP3F sequence beginning at codon 49(AGT) is shown. Heterozygosity for the 151insG mutation is clearly seen asoverlapping sequence traces. (B) Direct sequencing of a LAMA3 exon 39 geno-mic PCR product derived from the same individual as in (A), also sequencedwith primer L3AP3F. Note that the heterozygous bases are very similar in sig-nal intensity between the cDNA and genomic DNA samples, for example,where indicated by arrows in each case. Thus, there is no appreciable decayof the mutant message. (C) Genomic sequence in the region of exon 45, down-stream of the LOC mutation 151insG in exon 39. The first two in-frame ATGcodons that follow the mutation are shown in bold. The first of these, methio-nine codon 218 is a poor match for the Kozak consensus sequence, a featurerequired for eukaryotic translation initiation (shown above with matching basesboxed). In contrast, the next ATG, codon 227, is a perfect match for the Kozaksequence (boxed). Therefore, it was predicted that translation might take placefrom ATG codon 227, leading to an N-terminal truncation of 226 amino acids.Efficient translation of the mutant transcript would prevent nonsense-mediatedmRNA decay.



downstream (in exon 45) which occurs in the context of aparticularly good Kozak sequence (14). Effectively, this leadsto an N-terminal deletion of 226 amino acids from the laminina3a isoform. Here, we have shown that the mutant polypeptideis synthesized, assembled and secreted efficiently by keratino-cytes from LOC patients. In general, PTC mutations locatedtowards the 50 end of mRNA species would be expected to givenear-complete absence of protein expression (21). However, weshow that, in certain cases at least, PTCs located at the extreme50 end may escape nonsense-mediated decay if a suitablealternative initiation codon exists within the message, even at aconsiderable distance downstream. These observations haveimportant implications for predicting the effects of future 50

frameshift or nonsense mutations.Laminin 5 is a heterotrimer composed of a3a, b3 and g2

chains (18,22–24). It has been shown that these molecules mustundergo trimerisation in order to be secreted by keratinocytes(25). We showed here by immunoprecipitation that a mutantform of laminin a3a lacking the first 226 amino acids(�24 kDa) is able to assemble with laminin b3 and g2 to formlaminin 5 trimers that are capable of being secreted by cultured

keratinocytes. This in vitro result is borne out by the fact thatimmunohistochemical analysis of LOC patient skin showed noobvious qualitative or quantitative aberrations with antibodiesagainst all three laminin 5 polypeptides. Importantly, stainingfor antibody GB3 was normal in LOC skin. The GB3 epitope isconformation-dependent and is completely lost in cases of H-JEB when any of the three laminin 5 chains are absent (26,27)and therefore positive staining for GB3 provides furtherevidence for laminin 5 trimerization and secretion.Ultrastructural analysis was again unremarkable. In particular,there was no overt separation of the skin, consistent with therelatively mild erosive skin phenotype in LOC, rather thanthe extremely fragile skin as is the case in H-JEB (8,28).Furthermore, anchoring filaments, which are composed largelyof laminin 5 and generally absent in H-JEB, were clearlyvisible in the skin of an LOC patient. From this, we concludethat N-terminally truncated laminin a3a is both assembly-competent and capable of making anchoring filaments extra-cellularly in the cutaneous basement membrane zone. Onlyvery subtle ultrastructural changes were seen, such as focalwidening of the lamina lucida, demonstrating that the truncatedprotein is largely able to perform its primary function inmediating keratinocyte–stromal adhesion. In contrast, a geneticmutation that reduces the levels of laminin 5 expression hasbeen shown to lead to a skin blistering phenotype (29). Itshould be noted that excessive granulation tissue production isin fact seen in H-JEB and non-lethal forms of JEB (30,31),particularly affecting facial skin as is the case in LOC (Fig. 1).Thus, complete or partial ablation of the entire laminin 5 trimeralso leads to exuberant granulation tissue as would be expectedfrom our results. However, the severe or lethal skin fragility inJEB patients tends to mask this aspect of the phenotype.

The main phenotype in LOC syndrome is chronic production ofvascular granulation tissue often with activation and invasivemigration of fibroblasts resulting in neovascularization. However,the defective molecule, laminin a3a, is synthesized only by basalkeratinocytes of stratified epithelia, implying that LOC is adisorder of keratinocyte–mesenchymal communication. Since thestructural consequences of this laminin a3a defect are very subtle,it is likely that the aberrant mesenchymal cell behaviour is due to alost keratinocyte signalling function that resides in the truncatedN-terminal domain of laminin a3a. Thus, identification of theLOC defect may shed some light on how the granulation tissueresponse is normally controlled. It has been shown previously thatthe C-terminus of laminin a3a interacts with the extracellulardomains of the integrin a6b4 through a series of G-domainrepeats (32,33). These C-terminal repeats can be proteolyticallycleaved (34,35) in a manner that affects cell behaviour andmigration during wound healing (32). Thus, in the basementmembrane of skin and other stratified epithelia, the laminin 5trimer exists in an inverted cruciform configuration with theN-terminus of laminin a3a orientated towards the mesenchyme(32). It is therefore possible that the N-terminal domain normallyacts as a signal to the underlying mesenchyme that the basementmembrane is intact. When a stratified epithelium is damaged suchthat the basement membrane is breached, cytokines and othersoluble factors derived from the epithelium will activate amesenchymal wound healing response in addition to initiatingepithelial repair activity. When the wound is closed and re-epithelialized, the basement membrane is re-established and at

Figure 8. Radioimmunoprecipitation of [35S]-labelled secreted proteins fromcontrol and LOC keratinocytes with laminin 5 monoclonal antibody K140.Approximate molecular weight values are shown (in kDa). Lane 1, immunopre-cipitation from control keratinocytes showing two high molecular weight bands,assumed to be the laminin a3a and a3b isoforms (marked A and B). Laminina3a normally migrates at � 165 kDa on SDS gels (16). The upper band couldalso be due to co-immunoprecipitation of other laminin alpha chains. Lowerbands may include the laminin b3 and g2 chains and other associated proteins.Lane 2, immunoprecipitation from LOC keratinocytes, showing a size reduc-tion in the presumptive laminin a3a band (marked Da), of � 24 kDa, the sizepredicted by loss of the N-terminal 226 amino acids. The fainter levels of thedeleted a3a and the a3b bands in the LOC sample are likely to be becausethe LOC keratinocyte culture was much less confluent than the normal control.These results were replicable but lack of LOC cells prevented further labellingexperiments to adjust loading.



this stage, the laminin a3a N-terminal domain may act as a‘brake’ to deactivate fibroblasts and/or endothelial cells toterminate the mesenchymal wound healing response. In thismodel, the negative signal is absent in LOC syndrome, and sothe granulation tissue response, once activated by physical traumain certain delicate epithelia, such as the mucosae or ocular epithelia,cannot be deactivated and continues in an uncontrolled manner.Proteolytic cleavage of the C-terminal G-domains of laminin a3ahas been shown to be important in the control of keratinocytegrowth and migration (9,33). Furthermore, synthetic peptidesanalogous to sequences in the N-terminal domains of a number ofclassical full-length laminin alpha chains, including equivalentsequences specific to the laminin a3b chains described here, havebeen shown to modulate endothelial cell attachment, migrationand growth (36). However, similar investigations of the specificN-terminus of laminin a3a have not yet been performed. Here, thisdomain is shown to be important in control of mesenchymal cellgrowth and migration. Thus, the cutaneous basement membrane,which has functions in mediating epithelial–stromal adhesion, canalso be regarded as having a double-sided signalling functionmediated by the N- and C-termini of laminin a3a.

The critical interactions through which the N-terminus oflaminin a3a might mediate mesenchymal cell signalling are asyet unknown. Possibilities include a direct interaction with acell surface receptor expressed by mesenchymal cells oralternatively the domain might interact with other extracellularmatrix molecules and the resultant macromolecular complexmay influence cell behaviour. Granulation tissue is a majorclinical problem, not only in LOC syndrome but in a range ofmuch more common conditions such as rheumatoid arthritis,chronic venous leg ulcers, transplant surgery and others. Afuller understanding of the role of laminin a3a in thissometimes undesirable biological process might lead to theidentification of novel targets for therapeutic intervention.

MATERIALS AND METHODS

Homozygosity mapping

Patient and control lymphocyte DNA was extracted by standardtechniques. Genome-wide homozygosity mapping was carriedout essentially as described previously (5). Briefly, linkageanalysis was initially carried out using four affected personsfrom consanguineous kindreds (families 1–4) using micro-satellite markers from the ABI PRISM Linkage Mapping Set,Version 2 (LMS2) and the ABI HD5 5 cM Mapping Set (PEBiosystems, Foster City, CA, USA). Microsatellite PCR wasperformed according to the manufacturer’s recommendedprotocol, using AmpliTaq Gold polymerase (PE Biosystems)in a 7.5 ml reaction volume, without multiplexing. Markerswere sized on an ABI 377 automated DNA sequencer and dataextracted and analysed using Genescan and Genotyper version2.0 software, as previously described (37). When linkage wasnot detected using families 1–4, the probands from families 5and 6 were analysed and an additional 60 fluorescentmicrosatellites were synthesised to fill gaps in the ABI panel(MWG Biotech AG, Ebersberg, Germany). Genetic linkagewas initially scored ‘by eye’ and where significant homo-zygosity was observed, additional families were analysed andtwo-point LOD scores were calculated with MLINK algorithmof LINKAGE version 5.1. The mutant allele frequency wasassumed to be 0.00001 with 100% penetrance and markerallele frequencies were determined experimentally from apopulation of 50 unrelated unaffected Punjabi Muslims residentin the UK. Homozygosity was confirmed at the LOC locus inan additional family with documented consanguinity (family 7).In families 8–15, where the nature of the consanguinity wasvery distant or uncertain, calculations were done assuming thatthe parents were second cousins to simulate ‘fuzzy’ inheritance

Figure 9. Indirect immunofluorescence staining of LOC skin (A and C), compared with normal control skin (B and D). Monoclonal antibodies used were GB3against the laminin 5 trimer (A and B) and K140 against laminin b3 (C and D). No significant changes in the intensity or distribution of staining was seen betweenthe samples, showing that the mutated laminin a3a chain is capable of assembly and secretion, confirming the immunoprecipitation data (Fig. 8).



(5). Recalculation assuming these parents to be more distantlyrelated (third cousins) did not affect the resultant LOD scores.The two intragenic microsatellite markers within the LAMA3gene, designated LAMA3GT1 and LAMA3GT2 were ampli-fied using the PCR conditions above and the following primerpairs: LAMA3GT1.L (50 TTC TGG TCC TCTAAG AATAC 30),LAMA3GT1.R (50CTT GAC TGC ATC TCC AAC C 30); andLAMA3GT2.L (50CAG GCA TTC TCT TAC CAG C 30),LAMA3GT2.R (50GGC TGA GAA GCA ATA AGT G 30). Theproduct sizes for the LAMA3GT1 and LAMA3GT2 markerswere �284 and �192 bp, respectively.

Cell culture

Keratinocytes and dermal fibroblasts were cultured from skinbiopsy material derived from an LOC patient (proband infamily 1) and a normal unrelated control, as previouslydescribed (38).

cDNA synthesis

mRNA was extracted from cultured cells or skin biopsymaterial using the Quick prep micro mRNA purification kit(Amersham-Pharmacia Biotech, St Albans, UK) and reverse-transcribed using oligo dT and AMV reverse transcriptase(Promega, Southampton, UK) for 1 h at 45�C. For reversetranscription of long templates, samples were subjected to

multiple rounds of reverse transcription, each time heatingto 70�C for 10 min to break up RNA secondary structure andthen cooling to 45�C with addition of more reversetranscriptase and further incubation at 45�C for 1 h.

PCR

Unless otherwise stated, PCR was performed as follows.Approximately 50 ng of cDNA or 200 ng of genomic DNAwere added to a premix containing PCR buffer [67 mM Tris–HCl pH 8.8, 16.6 mM (NH4)2SO4, 1.5 mM MgCl2, 0.17 mg/mlbovine serum albumin (Sigma, Poole, England), and 10 mM

2-mercaptoethanol], 10 nmol of each dNTP, 20 pmol of eachprimer in a total volume of 50 ml. After an initial denaturation at95�C for 2 min, 1 unit of Taq polymerase (Promega, Crawley,UK) was added followed by 35 cycles of 95�C for 10 s,annealing temperature for 10 s, 72�C for 30 s, with a finalincubation of 72�C for 5 min. For RT–PCR of LAMA3transcripts, a series of overlapping PCR fragments weregenerated using the primer pairs listed in Table 1. For directamplification of exons, primer pairs were positioned within theintrons flanking the exonic sequences (Table 2). The annealingtemperatures for each primer pair are displayed in Tables 1 and2. The PCR products were examined by 1% agarose gelelectrophoresis, purified and directly sequenced as above. Insome cases, particularly fragments involving the GC-rich 50

end of the gene, PCR was performed using Boehringer Long-Range buffer 1 (Roche Diagnostics, Mannheim, Germany)containing 1.75 mM MgCl2. Reactions were subjected to a hotstart with 0.2 ml of Boehringer Long-Range DNA polymerasemix (Roche Diagnostics).

DNA sequencing and mutation detection

PCR products were purified for sequencing using QIA quickPCR purification kit (Qiagen, Crawley, UK). Purified fragmentswere directly sequenced with either the amplification primers orinternal primers as appropriate using the Big-Dye Terminatorchemistry on ABI 377 or ABI 3100 automated sequencers(Applied Biosystems, Foster City, CA, USA). Mutationdetection was carried out by manual comparison of patientsequences with normal control samples. Direct sequencing alsowas used to look for mutation 151insG in 50 normal unrelatedBritish Caucasians and 50 unaffected unrelated British PunjabiMuslims resident in the UK with no family history of LOCsyndrome. Mutation R943X (see Results) was confirmed inmembers of Family 15 by SalI restriction enzyme digestion ofexon 60 PCR products.

Electronic sequence analysis

DNA sequences were aligned, assembled and analysed usingGeneworks version 2.5.1 (Oxford Molecular Systems, Oxford,UK). Promoter and transcriptional start site predictions wereperformed using the TSSG and TSSW algorithms, accessed viathe Medical Research Council Human Gene Mapping Projectwebsite (http://www.hgmp.mrc.ac.uk). BLAST analysis ofDNA sequences was carried out via the National Center forBiotechnology Information website (http://www.ncbi.nlm.nih.gov). Recent assemblies of the human genome data were

Figure 10. Electron microscopy of a skin biopsy from the proband in LOCfamily 1. The ultrastructure of the dermal–epidermal junction (DEJ) was gen-erally normal, but there were some qualitative changes. The number of hemi-desmosomes (HD) and anchoring fibrils (AFB) appear normal. However,some of the hemidesmosome plaques are small and lack normal sub-basaldense plates (HDs). These diminutive cell-stromal junctions are associated withreduced numbers of anchoring filaments (AFL) and focal widening (asterisk) ofthe lamina lucida (LL), although the lamina densa (LD) appeared normal. Thereare no frank splits in the lamina lucida, as would be expected in the junctionalforms of epidermolysis bullosa to laminin 5 ablation mutations, where anchor-ing filaments are absent.



accessed through the University of California Santa Cruzwebsite (http://genome.ucsc.edu). Additional microsatellitemarker primer sequences and genetic mapping data wereobtained from Genethon, Evry, France (http://www.genethon.fr) and The Center for Medical Genetics,Marshfield, WN, USA (http://research.marshfieldclinic.org).

Radioimmunoprecipitation assay

Keratinocytes from control and LOC patients were cultured asabove in 24-well plates. Keratinocyte culture media wasremoved and cells were washed four times with PBS beforethe medium was replaced with 200 ml methionine/cysteine-deficient DMEM (Life Technologies, Paisley, UK) supplemen-ted with 50 mCi [35S]methionine (Amersham Biosciences UKLtd, Buckinghamshire, UK) and incubated for 16 h at 37�C.Radiolabelled conditioned medium was removed and micro-centrifuged for 5 min at 14 000 rpm to remove cellular debrisbefore storage in aliquots at �20�C. Monoclonal antibodyK140 against laminin b3 (1 mg) was added to 20 mlradiolabelled keratinocyte conditioned medium and incubatedovernight at 4�C with rotation. Twelve micrograms per sampleof Protein G sepharose beads (Sigma) were swollen in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl,150 mM NaCl, 0.5% NP-40) and incubated with samples for 3 hat 4�C. Beads with bound antibody–protein complexes weresedimented at 5000 rpm for 5 min and washed three times inRIPA buffer. Beads were resuspended in NuPAGE gel loadingbuffer plus reducing agent (InVitrogen, Groningen, TheNetherlands) and heated to 70�C for 10 min before loadingon 4–12% NuPAGE Bis-Tris gels (InVitrogen). Gels were fixedin 10% acetic acid : 50% methanol for 10 min and incubated in‘Amplify’ (Amersham Biosciences) for 30 min before dryingand fluorography with Kodak Hyperfilm for 9 days at �80�C.

Immunohistochemistry

Part of a 4 mm punch biopsy from non-lesional thigh skin wasembedded in OCT compound (Agar Scientific, Stansted, UK),snap frozen in liquid nitrogen-cooled isopentane and sectionedin a cryostat for subsequent indirect immunofluorescencemicroscopy, as described previously (39). Sections of normalskin were incubated with the same primary and secondaryantibodies as controls. The primary antibodies included thefollowing: GB3 (anti-laminin 5 trimer, SeraLabs,Loughborough, UK); SE144 (anti-laminin 5 g2 chain, a giftfrom Gim Meneguzzi, Nice, France); BM2 (anti-laminin a3, agift from Bob Burgeson, Boston, USA); K140 (anti-lamininb3, a gift from Peter Markinovich, Stanford, CA, USA); PKA1(anti-laminin a3, gift from Peter Markinovich, Stanford, CA,USA); GOH3 (anti-a6 integrin, gift from Stephen Kennel, OakRidge, TN, USA); 450-9D (anti-b4 integrin, gift from ArnoudSonnenberg, Amsterdam, The Netherlands); HD4-233 (anti-type XVII collagen, gift from Katsushi Owaribe, Nagoya,Japan). Appropriate anti-species immunoglobulin secondaryFITC antibody conjugates were obtained from Dako, Glostrup,Denmark.

Electron microscopy

Another part of the skin biopsy (above) was fixed in half-strength Karnovsky solution and osmium tetroxide, as detailedelsewhere (40). Ultrathin sections were stained with uranylacetate and lead citrate and examined in a JEOL 100CXelectron microscope.

ACKNOWLEDGEMENTS

This manuscript is dedicated to the memory of our co-authorProfessor Syed Ghulam Shabbir, who sadly passed away in2002. The authors would like to express their thanks to thepatients, their families and healthy volunteers from the BritishPunjabi community without whom this research would nothave been possible. Thanks also to Andrew J. Cassidy, DNAAnalysis Facility, Human Genetics Unit, Ninewells MedicalSchool, for DNA sequencing and genotyping support services.This work was supported by grants from The DystrophicEpidermolysis Bullosa Research Association (to W.H.I.M. andJ.A.M.); Action Research (J.A.M.); The Wellcome Trust(Senior Research Fellowship in Basic Biomedical Sciences toW.H.I.M. supporting W.H.I.M. and F.J.D.S. and a Prize PhDStudentship supporting K.J.H.); and the Hasham M. JamilTrust (W.H.I.M. and A.D.I.).

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an unusual n-terminal deletion of the laminin alpha-3a isoform

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