Ningning Dang1, 9, 10, Sandra Klingberg2, Adam I. Rubin1, Matthew Edwards3, Siegfried Borelli4, John Relic5, Penelope Marr6, Kim Tran6, 10, Anne Turner7, Nicholas Smith8 and Dédée F. Murrell1, 7, 10

1Department of Dermatology, St George Hospital, Sydney, 2Department of Chemical Pathology, Royal Brisbane Hospital, Brisbane, 3Hunter Genetics & University of Newcastle, Newcastle, Australia, 4Triemli Hospital, Zurich, Switzerland, 5John Hunter Hospital, Newcastle, Australia, 6Department of Anatomical Pathology, St George Hospital, 7EB Clinic, Sydney Children’s Hospital, Sydney, 8Pathology Department, Queen Elizabeth II Hospital, Perth, Australia, 9Department of Dermatology, Jinan Central Hospital, Shandong Province, China, and 10University of New South Wales, Sydney, Australia

Junctional epidermolysis bullosa with pyloric atresia (JEB-PA) is an autosomal recessive blistering disease including lethal and non-lethal variants due to mutations in ITGB4 and ITGA6. It is unclear whether PA is caused directly by the mutations in these genes or by other factors. Skin biopsies from patients with JEB were processed for immunofluorescence mapping. When staining for integrin β4 or α6 was absent or reduced, ITGB4 was screened for mutations. A review of known mutations of ITGB4 and the phenotypes of patients with JEB-PA was undertaken. Three novel ITGB4 mutations were identified in 3 families with JEB-PA: 2 splice-site and one insertion mutation. Two families with lethal phenotypes (EB-050 and EB-049) were due to combinations of premature termination codons and missense mutations (658delC/R252C and 3903dupC/G273D, respectively). The third family EB-013 has 2 JEB affected siblings; a brother with PA and a sister without PA. Both were homozygous for ITGB4 264G > A/3111-1G > A. Two cases had no gastrointestinal symptoms or signs of PA. PA is an inconstant feature of the subtype of epidermolysis bullosa known as JEB-PA. It is most likely that multiple factors influence the development of PA and its presence is not predictive of a poor outcome. It is possible that institutions that do not routinely screen immunofluorescence mapping for integrin α6β4 staining in the absence of PA are missing this form of epidermolysis bullosa. Key words: integrins; complications; phenotype; gastrointestinal; aplasia cutis; dysmorphic.

(Accepted March 10, 2008.)

Acta Derm Venereol 2008; 88: 438–448.

Dédée F. Murrell, Department of Dermatology, St George Hospital, University of New South Wales, Gray St, Kogarah, vSydney, NSW 2217, Australia. E-mail: d.murrell@unsw.edu.au

Junctional epidermolysis bullosa with pyloric atresia (JEB-PA) is a clinically and genetically heterogeneous autosomal recessive blistering disease, usually noted in the neonatal period, associated with congenital pyloric atresia (PA). This disease is frequently lethal in early infancy, despite surgical correction of PA (1), but non-lethal variants with diminishing blistering tendency with age have been noted previously (2–6). In addition to skin lesions, there are a variety of extracutaneous manifestations, including corneal erosions, dental, hair and nail abnormalities, as well as tracheal and urinary tract involvement (7). Ultrastructurally, the skin lesions are characterized by blister formation at the level of the lamina lucida with hypoplastic hemidesmosomes (HD) in reduced numbers and without distinct inner and outer plaques (8). Occasionally, a split in the HD in the lower basal layer occurs. Immunofluorescence antigen mapping (IFM) of the affected skin in lethal forms is usually associated with completely negative staining for integrin β4 and/or integrin α6, whereas non-lethal cases are usually associated with positive but attenuated staining (4). Mutations in the ITGB4 gene were first demonstrated in JEB-PA by Vidal et al. (9). Like all integrin β subunits, the extracellular domain of integrin β4 contains 5 putative N-linked glycosylation sites and 4 homologously repeated cysteine-rich domains in which the presence of conserved residues at fixed positions allows protein-protein interactions (10, 11). Lam332 (laminin 5) and type XVII collagen bind to the extracellular domain of integrin β4 (12). Integrin β4 has an unusually large cytoplasmic tail of 1141 amino acids, which contains 2 pairs of fibronectin III-like (FNIII) domains separated by a connecting segment (CS) (10, 13). The second FNIII repeat and CS are essential for the assembly of α6β4 into HD (10, 13–15). This CS harbours a tyrosine activation motif in which residues 1422–1440 are critical for binding to lam332 and for HD assembly (16, 17). The first pair of FNIII repeats and the first 36 amino acids (1320–1355) of the CS are crucial for the recruitment of plectin into HD (14, 18). Sequences within the CS and the second pair of FNIII repeats of ITGB4 are involved in targeting BP230 into HD-like structures (19). The main binding site for BP180 to integrin β4 resides in the segment comprising the carboxy-terminal half of the CS and the third FNIII repeat (19). As integrin β4 is also expressed in the epithelia of the gastrointestinal tract (20), one theory is that the absence of α6β4 integrin could explain the development of congenital PA. Integrin β4-null mice have epithelial detachment in other epithelia, such as the tongue, oesophagus, stomach and bladder as well as the skin (21). In lethal JEB-PA, mutations usually consist of premature termination codons (PTC) affecting both ITGB4 alleles, which result in the complete absence of α6β4 integrin; missense or splice site mutations are more prevalent in non-lethal forms (3, 4, 22). However, it is not only the nature, but also the position, of mutations reflected in the protein functional domains of β4 integrin that affect the phenotype of JEB-PA (23). To extend our knowledge of ITGB4 mutations and to better understand the genotype-phenotype correlation in this form of EB, we studied 3 families, comprising 4 cases, and found 3 novel mutations in ITGB4. Interestingly, we discovered what we believe to be the first reported case of differential expression of PA in one family with 2 siblings bearing the same 2 ITGB4 mutations. The latter suggests that unknown factors, such as epigenetic or environmental factors, may influence whether pyloric atresia occurs, as has been described in variability of pseudosyndactyly in recessive dystrophic EB with metalloproteinase 1 isoforms (24).

FOUR CASE REPORTS

Family 1: EB-050. This case was described briefly in an earlier report (4). A female infant was born at 30 weeks gestation complicated by polyhydramnios. She was the second child of a non-consanguineous union. Her mother presented in preterm labour and was given a course of betamethasone (Celestone) to suppress labour for foetal lung maturation and tocolytics. At birth, the infant had no blisters, but the first blisters appearing on her fingertips and toes when she was 2 days old. Subsequently blisters appeared at the sites of tape attachment or where she had been handled (Fig. 1A). At no stage did the infant have severe or widespread blistering. PA was noted shortly after birth and was surgically repaired. She developed respiratory distress, which was relatively mild but required mechanical ventilation. She was extubated at 6 days of age. She coughed up copious mucous secretions and had apnoeic attacks, requiring ongoing oxygen support from 28 days to 13 weeks of age. A chest radiograph showed widespread reticulonodular opacities consistent with “Northway & Rosen” stage-3 bronchopulmonary dysplasia. She developed anaemia, requiring 4 blood transfusions, neutropaenia and presumed sepsis, although no bacterial organisms were cultured. She was treated with multiple courses of i.v. antibiotics. Ultrasound detected a small subependymal intracranial haemorrhage. Other medical problems included jaundice, patent ductus arteriosus, gastro-oesophageal reflux and candida diaper dermatitis. She was discharged from hospital at post-conceptional age 46 weeks with most of the skin erosions having healed (Fig. 1B). A month later, she suddenly deteriorated and developed a new erosion on her leg. She died after vomiting coffee-ground blood, suggestive of a gastro-intestinal bleed, at 147 days of age. No post-mortem was carried out, but the bleed was not thought to be related to the previous PA.

The infant’s skin biopsy showed separated strips of epidermis, and a small fragment of dermis without overlying epidermis. Transmission electron microscopy (TEM) examination of another skin biopsy showed the basement membrane (BM) to be thin, with reduced numbers of HD along the dermo-epidermal junction (Fig. 2E). In addition, occasional breaks in the lamina densa were seen. IFM revealed type IV collagen monoclonal antibody below the blister, laminin V β3 chain monoclonal antibody K140 positive below the blister; anti-ITGA6 and ITGB4 were negative, although they were positive in normal human skin controls (Fig. 2A and B).

Mutation screening of ITGB4 revealed a then-novel c.658delC mutation in exon 7 of maternal origin and p.R252C in exon 8 of paternal origin. The c.658delC is predicted to cause a downstream stop codon in exon 8. The p.R252C was confirmed by digestion with Narl. This mutation is located in the extracellular domain and has been described previously in association with PA (3, 4). As only one mutation is a PTC, it is unclear why the IFM was negative for both integrin β4 and α6. This family subsequently underwent prenatal diagnosis and an affected pregnancy was terminated. A third pregnancy resulted in a heterozygous carrier, but unfortunately this pregnancy was affected by an unrelated fatal condition, thanatophoric dwarfism, and was also terminated.

Family 2: EB-049. A female infant was born preterm at 33 weeks by caesarean section following spontaneous pre-term labour, due to transverse lie and possible maternal sepsis. Her parents were non-consanguineous. She suffered severe respiratory distress syndrome and sepsis and was noted to have widespread erosions and fragile skin along with dysmorphic ears. As she was anuric, a renal ultrasound was performed which showed the ureters to be dilated. She died on day 2 from hyaline membrane disease due to prematurity and renal failure due to EB.

There was marked aplasia cutis in patches throughout the body, especially over the left and right lateral sides of face, the whole neck (Fig. 1C), the lateral aspect of the right upper limb, the left knee, the dorsal surface of both feet, the left and right shins, the dorsal surface of both hands, and the left upper arm. The head was abnormal with dysmorphic facies, the ears were markedly atrophic, but the external auditory meati were apparent (Fig. 1C), the nasal tip was hypoplastic with overlying aplasia cutis. The nares and choanae were patent (Fig. 1D), the eyes were asymmetrical with an ectropion affecting the left lower eyelid, the globes appeared normal and the palpebral fissures were down-slanting bilaterally, the alveolar plates and tongue appeared normal.

On post-mortem, the external appearance of the stomach was normal and internally, no evidence of pyloric stenosis or atresia was apparent externally; the mucosal surface appeared only slightly congested (Fig. 1G). The urinary bladder was very narrow between the umbilical arteries, but was present; the ureters showed marked tortuosity and dilatation; the kidneys were enlarged but reniform and on section there was marked pelvicalyceal dilatation (Fig. 1H). Histological examination of biopsies from involved skin revealed a small amount of dermis and subcutaneous tissue, the overlying epidermis was largely lost. TEM showed HD on the most proximal layer of the blister roof were few and poorly formed (Fig. 2F). IFM of skin biopsies showed cleavage within the lamina lucida with absence of staining for β4 and α6 integrin. In addition, antibodies to laminin V β3, α3, γ2 chain, type IV collagen, type VII collagen, LAD1 and CD151 were positive below the blister (lamina lucida); and antibodies to BP230, BP180, plectin, and keratin5/6, 14 were positive above the blister.

Molecular analysis of genomic DNA extracted from blood saved after death was performed using the same PCR and sequencing primers as those developed for Family 1. This revealed heterozygous ITGB4 mutations c.3903dupC/p.G273D. The novel mutation c.3903dupC in exon 31 was a single base insertion, which is predicted to cause a frameshift and a downstream stop codon in exon 31, resulting in a prematurely truncated protein product, or nonsense-mediated mRNA decay. The p.G273D was detected in exon 8, resulting in the substitution of aspartate for glycine at a highly conserved codon 273. This mutation has been reported in association with PA and another missense mutation (Case 30, Table II) (4).

Family 3: EB013-1 and 013-2. This Chinese girl (EB013-1), now 8-years-old, was born in mainland China to non-consanguineous parents and had blisters on the hands at birth. She did not have PA. A clinical diagnosis of EB simplex-Weber-Cockayne was made, but no further investigations were performed. The family moved to Australia and her blisters remained mostly localized to the feet (Fig. 1E). The blistering was exacerbated both by extensive walking and during the summer. There was no scarring or atrophy from the blisters and no other health problems noted. Her now 4-year-old brother (EB013-2) was born in Australia and had PA at birth, which was surgically repaired. He developed blisters on the feet, which were not as severe as his sister’s (Fig. 1F). There was no scarring, atrophy or milia from the blisters. IFM of a skin biopsy specimen showed no blistering, and staining for both integrins α6 and β4 was markedly reduced compared with controls (Fig. 2C and D), whereas all other antibodies used routinely, including collagen VII, stained normally. Unusually, GOH3 against integrin α6 stained basal and suprabasal keratinocytes. EM showed a decreased number of HD at the BM zone, which were rudimentary in structure (Fig. 2G and H).

ITGB4 gene mutation studies were performed. The paternal mutation was identified as c.264G > A at the exon/intron 4 splice junction. The paternal grandfather was also found to be a carrier of this mutation. The maternal mutation was identified as c.3111-1G > A at the border of intron 26 and exon 27, predicted to alter the consensus splice sequence. Two years later they still had very mild blistering (Fig. 1E and F).

DISCUSSION

Clinical analysis

We have identified 50 cases of JEB-PA reported in the literature, including the 4 cases reported here. Twenty-three of the previously reported cases had TEM performed, which predominantly showed blistering within the lamina lucida (8, 9, 25, 26). However, blistering within the basal cell and even below the lamina densa had been reported in some patients (8). IFM demonstrated reduced immunoreactivity to integrin β4 in non-lethal cases, and complete absence in lethal cases (Tables I and II). Three cases with JEB caused by an ITGB4 mutations did not have gastrointestinal atresia, including one reported by Inoue et al. (27) and cases 2 and 3 reported here. In addition to PA, other commonly reported complications in these patients included nail dystrophy (n = 7), enamel hypoplasia (n = 4), aplasia cutis congenita or congenital localized absence of skin (n = 6), eye involvement (n = 4), ear or nose hypoplasia or atrophy (n = 4), urinary tract involvement (n = 8) and respiratory involvement (n = 5). Many patients died of this disease owing to the extensive denudation of skin, resultant loss of barrier function, fluid and electrolyte problems and sepsis. Difficulty with oral feeding and the development of diarrhoea seem to be common features in many of these cases.

Identification of novel mutations in this study

In our study of 3 families with JEB-PA, we have described 4 probands with 3 novel ITGB4 mutations: 2 splice-site mutations (c.264G > A/C and c.3111-1G > A) and one insertion mutation (c.3903dupC) (Table II, Fig. 3). Two cases had lethal phenotypes due to the combination of PTC and missense mutations (c.3903dupC/p.G273D and c.658delC/p.R252C). The PTC mutations may cause mRNA decay or result in truncated non-functional β4 integrin polypeptides and the missense mutations will determine the phenotype of patients when non-functional β4 integrin polypeptides are synthesized (4). Because arginine-252 and glycine-273 are located in a highly conserved region that may abolish ligand-binding properties similar to those of β3 integrin (4), the phenotypes are lethal when p.G273D and p.R252C are combined with PTC mutations (c.3903dupC and c.658delC, respectively). Additionally, p.R252C, which creates a new cysteine residue, may change disulphide bonding and alter the secondary structure of the polypeptide, resulting in the loss of the putative ligand-binding function of the β4 integrin. There were 2 novel mutations in our non-lethal cases including c264G > A and c.3111-1G > A, and both mutations are predicted to cause mis-splicing, but we were not able to determine the exact nature of the products.

Fig. 3. Mutations in ITGB4 with JEB-PA. Schematic diagram of the ITGB4 gene above showing the exons in alternating dark and light grey. The representative domains of the integrin β4 protein, are shown below, in different colours indicated by the bar key. The published mutations above are shown above in black if they have occurred in lethal cases, red in non-lethal cases and blue if in both. The mutations in the cases in this report are indicated below.

It is interesting that we found 2 cases without PA, cases 2 (c.3903dupC/p.G273D) and 3 (c.264G > A/c.3111-1G > A). The latter’s affected brother, with the same mutations, did have PA, and we found a similar report of a homozygous c.1856+1G > T mutation in the cytoplasmic domain of ITGA6 in a family with 3 affected individuals with JEB-PA, but one child did not have duodenal atresia (28). Inoue et al. (27) also reported one non-lethal JEB case without PA due to homozygous ITGB4 mutations p.G931D/p.G931D affecting the cytoplasmic tail of integrin β4. Overall, at least one mutation, either in ITGB4 or ITGA6, was located in the cytoplasmic domain; however, it could not provide evidence for a domain with a functional role relevant to the development of PA. Recently, the plectin gene was emphasized to be important in the pathogenesis of EB-PA (29, 30) and perturbed interactions between plectin and α6β4 integrin within attachment structures expressed during gastrointestinal development were proposed as a possible cause of the PA (29). Nevertheless, ITGA6 and ITGB4 knock-out mice expressed the cutaneous JEB phenotype but not the PA, suggesting that there may be other factors involved (30).

Genotype-phenotype correlation from ITGB4 mutations reported in the literature and in this study

We have summarized a total of 49 cases with JEB-PA, 21 being classified as non-lethal cases and 28 as lethal cases (Table II). From the mutation database in ITGB4, there is a predominance of PTC mutations in the lethal forms, whereas compound heterozygous missense and PTC mutations are more common in the non-lethal cases. In fact, 22 (78.6%) of lethal cases of JEB-PA had PTC mutations in at least one allele, 4 cases (14.3%) had missense mutations in both alleles and 2 cases (7.1%) had in-frame deletions. In contrast, in non-lethal forms of JEB-PA, at least 19 of 41 alleles (46.3%) in 13 individuals were PTC mutations, 15 alleles (36.6%) were missense mutations and 7 alleles (17.1%) were splice site mutations. This is in contrast to previous reports suggesting that the presence of a missense or splice mutation in at least one allele predicted a milder phenotype (31). It appears that one PTC mutation is not necessarily predictive of lethality, nor is PA. All reported cases of JEB-PA with a lethal outcome who had IFM performed, had either absent or markedly attenuated staining for integrin ß4, in contrast to the non-lethal cases (Table II).

Prevalence of PTC/PTC mutations in lethal JEB-PA

Nonsense, small out-of-frame insertion or deletion mutations in ITGB4 predicted synthesis of a truncated polypeptide and/or down-regulation of the ITGB4 mRNA levels by nonsense-mediated mRNA decay (32, 33). Thus, no functional integrin β4 polypeptides are synthesized, resulting in the JEB-PA phenotype. The presence of PTC mutations in both alleles, either in a homozygous or compound-heterozygous state, would result in a lethal phenotype (3). For example, in Case 19 (Table II), p.C738X/c.4791delCA, combined 2 PTC mutations. The p.C738X mutation within the large cytoplasmic domain was adjacent to the transmembrane segment and is predicted to cause deletions of the entire intracellular domain of the integrin β4 polypeptide, which could affect HD assembly, but ligand binding is preserved (34). The c.4791delCA mutation is predicted to delete the region spanning the last 278 amino acids, which have been identified to interact with the 180-kD bullous pemphigoid antigen (BP180) (35).

However, some non-lethal cases, for example cases 8, 37 and 38 were homozygous for PTC/PTC mutations: 4790delTC/4790delTC, 4580del2/4580del2 and 5046delC/5046delC, respectively (4, 36). IFM was only reported for case 8, showing reduced but positive staining of both integrins α6β4. Case 2 was heterozygous for a PTC mutation (3793+1G-A/W1478X) with positive but reduced staining for integrin β4 and normal integrin α6 staining (2). All of these mutations predicted truncation of integrin β4 polypeptides close to the carboxyl-terminal end (Fig. 3), and might have minor effects.

Missense/missense mutations resulted different clinical variants in a position-dependent pattern

Four lethal and 4 non-lethal JEB-PA cases with ITGB4 (3, 4, 27, 36) mutations had missense/missense combinations, which suggested that the position of these mutations influenced the phenotype. The mutations in 3 lethal cases were located in the extracellular domain of the integrin β4 protein (cases 20, 23, 47 in Table II) (3, 4, 36). The mutations p.C61Y/p.C61Y and p.C245Y/p.C61Y changed cysteine residues to lysine, which may interfere with the formation of intra- or inter-chain disulphide bonds and subsequently change the conformation and/or ligand-binding affinity of integrin β4 (3). Some of the missense mutations in ITGB4, including these ones, resulted in substitution of highly conserved cysteine residues, most of which were associated with a severe phenotype (22). Alternatively, missense mutations that affect highly conserved residues may have significant effects. For example, in Case 23 (p.D131Y/p.G273D), which was lethal at only 2 months of age, aspartic-131 and glycine-273 are located in a highly conserved region, so these mutations may abolish important ligand-binding sites of integrin β4 (4). Our second case which was also lethal also included this p.G273D mutation.

Previous work has shown that the recruitment of plectin into HD was dependent on a region of the integrin β4 cytoplasmic domain containing the first 2 FNIII repeats and a short region of the CS (14, 18). Two missense mutations, p.R1281W (cases 4 and 5 in Table II) and p.R1225H (case 7 in Table II) in the non-lethal form of JEB-PA were located in the second FNIII repeat (3, 4). R1281W was located in a loop region that connected 2 β strands, whereas R1225H is located at the N-terminal end of the second FNIII repeat (37). Both mutations caused a disruption of the interactions with plectin. Thus, the linkage of the intermediate filaments to HD was likely to be compromised because of an inability of integrin β4 to recruit plectin into HD. This helps to explain why these mutations caused a non-lethal type of JEB-PA.

Collectively, most of the missense mutations and the amino acids deletions described in lethal JEB-PA were located in the extracellular domain of ITGB4. Missense or splice mutations associated with the non-lethal form were frequently located in the cytoplasmic tail (4, 8, 36) (Fig. 3).

Missense/PTC mutations associated with lethal and non-lethal phenotype

The presence of a missense mutation in one allele combining with a PTC mutation could predict a more variable phenotype. Six lethal cases in previous study (case 15, 22, 24 and 27 in Table II) (4, 8, 31) and in this study (case 33, EB-50) and 4 non-lethal cases (case 1, 9, 10 and 39) (2, 5, 36, 38) were compound heterozygotes for PTC and missense mutations. PTC could cause mRNA decay or synthesize truncated non-functional integrin β4 polypeptides. Therefore, the missense mutation would direct the phenotype of patients. For example, p.C245G along with p.R252C highly conserved amino acids located in a putative ligand-binding region in integrin β4 polypeptides in human, rat and mouse (4, 8); these mutations created or abolished cysteine residues changing disulphide bonding and the secondary structure of the integrin β4 polypeptides. Mutations p.V325D and p.G273D also occurring in a conserved position substitute a non-polar for an acidic residue (4). Therefore, these phenotypes were lethal when PTCs were combined with missense mutations, including c.120delTG/p.C245G, c.658delC/p.R252C, c.1874delTCTinsC/p.V325D, c.4298-4299ins4/p.R252C and c.3903dupC/p.G273D (4, 8, 31).

In non-lethal cases, such as Case 1 (p.C38R/c.4776delG), c.4776delG resulting in a downstream PTC also predicted an unstable mRNA transcript. The missense mutation, p.C38R, arose in the part of the extracellular amino-terminal domain, a position in a highly conserved region in terms of different integrin β4 polypeptides and different species. So the mutation might disrupt heterodimer formation with the integrin α6 subunit or interaction with ligands within the lamina lucida. Perturbation rather than abolition of β4 subunit function by p.C38R might explain the mild phenotype with only minimal cutaneous involvement and no evidence of other manifestation (2).

In summary, we have identified a total of 3 novel mutations in all 6 ITGB4 alleles of 4 patients affected with JEB-PA, 2 of them without PA. The results indicated that PTC mutations in both alleles in either a homozygous or compound heterozygous state would be more likely to result in a lethal phenotype. Missense mutations, either in combination with a PTC mutation or in both alleles, could predict either a lethal or non-lethal variety, the latter more likely to be related to positive integrin β4. Missense mutations causing substitutions of highly conserved amino acids might be associated with a lethal phenotype, while less conserved amino acid substitutions caused milder phenotypes. However, in these cases it would be difficult to predict the phenotype from the genotype as this may depend not only on the nature of the mutations but may also be influenced by other, as yet unknown, genetic or environmental factors.

ACKNOWLEDGEMENTS

We acknowledge Jinan Central Hospital, Shandong Province, China for supporting Dr Dang’s postdoctoral studies with DM. Also, a grant from DEBRA Australia to DM to support Dr Dang’s work for an extended time in Australia. This work was selected for oral presentation at the Australasian Society for Dermatological Research and the European Academy of Dermatovenereology in May 2007.

REFERENCES

1 Dank JP, Kim S, Parisi MA, Brown T, Smith LT, Waldhausen J, Sybert VP. Outcome after surgical repair of junctional epidermolysis bullosa-pyloric atresia syndrome: a report of 3 cases and review of the literature. Arch Dermatol 1999; 135: 1243–1247.

2 Mellerio JE, Pulkkinen L, McMillan JR, Lake BD, Horn HM, Tidman MJ, et al. Pyloric atresia-junctional epidermolysis bullosa syndrome: mutations in the integrin beta4 gene (ITGB4) in two unrelated patients with mild disease. Br J Dermatol 1998; 139: 862–871.

3 Pulkkinen L, Rouan F, Bruckner-Tuderman L, Wallerstein R, Garzon M, Brown T, et al. Novel ITGB4 mutations in lethal and nonlethal variants of epidermolysis bullosa with pyloric atresia: missense versus nonsense. Am J Hum Genet 1998; 63: 1376–1387.

4 Nakano A, Pulkkinen L, Murrell D, Rico J, Lucky AW, Garzon M, et al. Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations. Pediatr Res 2001; 49: 618–626.

5 Pulkkinen L, Bruckner-Tuderman L, August C, Uitto J. Compound heterozygosity for missense (L156P) and nonsense (R554X) mutations in the beta4 integrin gene (ITGB4) underlies mild, nonlethal phenotype of epidermolysis bullosa with pyloric atresia. Am J Pathol 1998; 152: 935–941.

6 Chavanas S, Gache Y, Vailly J, Kanitakis J, Pulkkinen L, Uitto J, et al. Splicing modulation of integrin beta4 pre-mRNA carrying a branch point mutation underlies epidermolysis bullosa with pyloric atresia undergoing spontaneous amelioration with ageing. Hum Mol Genet 1999; 8: 2097–2105.

7 Fine JD, Bauer EA, Briggaman RA, Carter DM, Eady RA, Esterly NB, et al. Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. A consensus report by the Subcommittee on Diagnosis and Classification of the National Epidermolysis Bullosa Registry. J Am Acad Dermatol 1991; 24: 119–135.

8 Pulkkinen L, Kim DU, Uitto J. Epidermolysis bullosa with pyloric atresia: novel mutations in the beta4 integrin gene (ITGB4). Am J Pathol 1998; 152: 157–166.

9 Vidal F, Aberdam D, Miquel C, Christiano AM, Pulkkinen L, Uitto J, et al. Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia. Nat Genet 1995; 10: 229–234.

10 Hogervorst F, Kuikman I, von dem Borne AE, Sonnenberg A. Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain. EMBO J 1990; 9: 765–770.

11 Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsakonas S. Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 1991; 67: 687–699.

12 Lee EC, Lotz MM, Steele GD Jr., Mercurio AM. The integrin alpha 6 beta 4 is a laminin receptor. J Cell Biol 1992; 117: 671–678.

13 Suzuki S, Naitoh Y. Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells. EMBO J 1990; 9: 757–763.

14 Spinardi L, Ren YL, Sanders R, Giancotti FG. The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes. Mol Biol Cell 1993; 4: 871–884.

15 Tamura RN, Rozzo C, Starr L, Chambers J, Reichardt LF, Cooper HM, Quaranta V. Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4. J Cell Biol 1990; 111: 1593–1604.

16 Mainiero F, Pepe A, Wary KK, Spinardi L, Mohammadi M, Schlessinger J, Giancotti FG. Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J 1995; 14: 4470–4481.

17 Dellambra E, Prislei S, Salvati AL, Madeddu ML, Golisano O, Siviero E, et al. Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly. J Biol Chem 2001; 276: 41336–41342.

18 Nievers MG, Schaapveld RQ, Oomen LC, Fontao L, Geerts D, Sonnenberg A, et al. Ligand-independent role of the beta 4 integrin subunit in the formation of hemidesmosomes. J Cell Sci 1998; 111: 1659–1672.

19 Schaapveld RQ, Borradori L, Geerts D, van Leusden MR, Kuikman I, Nievers MG, et al. Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180. J Cell Biol 1998; 142: 271–284.

20 van der Neut R, Krimpenfort P, Calafat J, Niessen CM, Sonnenberg A. Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice. Nat Genet 1996; 13: 366–369.

21 Dowling J, Yu QC, Fuchs E. Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J Cell Biol 1996; 134: 559–572.

22 Ashton GH, Sorelli P, Mellerio JE, Keane FM, Eady RA, McGrath JA. Alpha 6 beta 4 integrin abnormalities in junctional epidermolysis bullosa with pyloric atresia. Br J Dermatol 2001; 144: 408–414.

23 Micheloni A, De Luca N, Tadini G, Zambruno G, D’Alessio M. Intracellular degradation of beta4 integrin in lethal junctional epidermolysis bullosa with pyloric atresia. Br J Dermatol 2004; 151: 796–802.

24 Titeux M, Pendaries V, Tonasso L, Décha A, Bodemer C, Hovnanian A. A frequent functional SNP in the MMP1 promoter is associated with higher disease severity in recessive dystrophic epidermolysis bullosa. Hum Mutat 2008; 29: 267–276.

25 Pulkkinen L, Kurtz K, Xu Y, Bruckner-Tuderman L, Uitto J. Genomic organization of the integrin beta 4 gene (ITGB4): a homozygous splice-site mutation in a patient with junctional epidermolysis bullosa associated with pyloric atresia. Lab Invest 1997; 76: 823–833.

26 Takizawa Y, Shimizu H, Nishikawa T, Hatta N, Pulkkinen L, Uitto J. Novel ITGB4 mutations in a patient with junctional epidermolysis bullosa-pyloric atresia syndrome and altered basement membrane zone immunofluorescence for the alpha6beta4 integrin. J Invest Dermatol 1997; 108: 943–946.

27 Inoue M, Tamai K, Shimizu H, Owaribe K, Nakama T, Hashimoto T, McGrath JA. A homozygous missense mutation in the cytoplasmic tail of beta4 integrin, G931D, that disrupts hemidesmosome assembly and underlies Non-Herlitz junctional epidermolysis bullosa without pyloric atresia? J Invest Dermatol 2000; 114: 1061–1064.

28 Pulkkinen L, Kimonis VE, Xu Y, Spanou EN, McLean WH, Uitto J. Homozygous alpha6 integrin mutation in junctional epidermolysis bullosa with congenital duodenal atresia. Hum Mol Genet 1997; 6: 669–674.

29 Pfendner E, Uitto J. Plectin gene mutations can cause epidermolysis bullosa with pyloric atresia. J Invest Dermatol 2005; 124: 111–115.

30 Nakamura H, Sawamura D, Goto M, Nakamura H, McMillan JR, Park S, et al. Epidermolysis bullosa simplex associated with pyloric atresia is a novel clinical subtype caused by mutations in the plectin gene (PLEC1). J Mol Diagn 2005; 7: 28–35.

31 Iacovacci S, Cicuzza S, Odorisio T, Silvestri E, Kayserili H, Zambruno G, et al. Novel and recurrent mutations in the integrin beta 4 subunit gene causing lethal junctional epidermolysis bullosa with pyloric atresia. Exp Dermatol 2003; 12: 716–720.

32 Frischmeyer PA, Dietz HC. Nonsense-mediated mRNA decay in health and disease. Hum Mol Genet 1999; 8: 1893–1900.

33 Hentze MW, Kulozik AE. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 1999; 96: 307–310.

34 Spinardi L, Einheber S, Cullen T, Milner TA, Giancotti FG. A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins. J Cell Biol 1995; 129: 473–487.

35 Aho S, Uitto J. Direct interaction between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and beta 4 integrin, hemidesmosomal components of basal keratinocytes. Biochem Biophys Res Commun 1998; 243: 694–699.

36 Varki R, Sadowski S, Pfendner E, Uitto J. Epidermolysis bullosa. I. Molecular genetics of the junctional and hemidesmosomal variants. J Med Genet 2006; 43: 641–652.

37 Koster J, Kuikman I, Kreft M, Sonnenberg A. Two different mutations in the cytoplasmic domain of the integrin beta 4 subunit in nonlethal forms of epidermolysis bullosa prevent interaction of beta 4 with plectin. J Invest Dermatol 2001; 117: 1405–1411.

38 Masunaga T, Ishiko A, Takizawa Y, Kim SC, Lee JS, Nishikawa T, Shimizu H. Pyloric atresia-junctional epidermolysis bullosa syndrome showing novel 594insC/Q425P mutations in integrin beta4 gene (ITGB4). Exp Dermatol 2004; 13: 61–64.

39 De Jenlis Sicot B, Deruelle P, Kacet N, Vaillant C, Subtil D. Prenatal findings in epidermolysis bullosa with pyloric atresia in a family not known to be at risk. Ultrasound Obstet Gynecol 2005; 25: 607–609.