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Herlitz Junctional Epidermolysis Bullosa: Novel and Recurrent Mutations in the LAMB3 Gene and the Population Carrier Frequency
MUTATION REPORT
Herlitz Junctional Epidermolysis Bullosa: Novel and Recurrent Mutations in the LAMB3 Gene and the Population Carrier Frequency Aoi Nakano, Ellen Pfendner, Leena Pulkkinen, Isao Hashimoto,* and Jouni Uitto
Departments of Dermatology and Cutaneous Biology, and Biochemistry and Molecular Pharmacology, Jefferson Medical College, Jefferson Institute of Molecular Medicine, and DebRA Molecular Diagnostics Laboratory, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A.; *Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan
Herlitz junctional epidermolysis bullosa is a heritable bullous disease caused by mutations found primarily in the b3 chain of laminin 5 (LAMB3). In this study, we examined the LAMB3 gene for mutations in 22 Herlitz junctional epidermolysis bullosa families, and identi®ed 15 distinct mutations, eight of them previously unreported, bringing the total number of distinct Herlitz junctional epidermolysis bullosa mutations in LAMB3 to 35. Examination of the mutation database revealed several recurrent mutations that have been reported, as well as six previously unreported. All recurrent mutations may be readily detected by polymerase chain reaction of genomic DNA and restriction endonuclease digestion. Mutation screening and prenatal diagnosis of
families at risk may be expedited by molecular testing for these recurrent mutations prior to screening the entire gene. Finally, the U.S. population carrier risk for Herlitz junctional epidermolysis bullosa and all variants of junctional epidermolysis bullosa was calculated to be one in 781 and one in 350, respectively, while the overall epidermolysis bullosa carrier frequency was calculated to be one in 113. These data allow accurate testing, counseling, and risk calculation for nuclear families, as well as extended family members at risk for junctional epidermolysis bullosa. Key words: basement membrane zone/blistering skin diseases/laminin 5 mutations/prenatal diagnosis. J Invest Dermatol 115:493±498, 2000
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LAMB3, and LAMC2, which encode the laminin 5 subunit polypeptides a3, b3, and g2, respectively. The PTC mutations in H-JEB predict the absence of laminin 5 in the dermal-epidermal junction, which can be veri®ed by negative staining of laminin 5 using speci®c monoclonal antibodies (Heagerty et al, 1986; Verrando et al, 1991; McMillan et al, 1997). Electron microscopy performed on skin biopsies from H-JEB patients reveals poorly developed hemidesmosome/anchoring ®lament complexes (Tidman and Eady, 1986). Mutation detection has been facilitated by sequencing of the LAMA3a, LAMB3, and LAMC2 genes and polymerase chain reaction (PCR) primers have been designed spanning all exons. Among the human laminin 5 genes, LAMA3a has 38 exons and encodes 1713 amino acids (Ryan et al, 1994; Pulkkinen et al, 1998), whereas LAMB3 and LAMC2 both have 23 exons and encode 1172 and 1193 amino acids, respectively (Pulkkinen et al, 1995a; Airenne et al, 1996). Previously, the majority of H-JEB mutations have been disclosed in the LAMB3 gene. Among the 26 distinct mutations characterized thus far in 58 families are two recurrent hotspot mutations, R635X and R42X (Kivirikko et al, 1996). In this study, we have examined 22 new families with H-JEB and identi®ed 15 distinct mutations, including eight novel ones. Examination of the global mutation database also revealed the presence of six previously unrecognized recurrent mutations in the LAMB3 gene. Using the information about speci®c familial mutations in LAMB3, prenatal diagnosis was performed in seven families at risk for recurrence of H-JEB. Carrier risks have been generated from population data taken from Fine et al (1999)
pidermolysis bullosa (EB) is a group of heritable mechanobullous diseases characterized by blistering due to skin fragility that can result from ordinarily minor trauma. Cases of EB are traditionally classi®ed into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone, clinical features, and the inheritance pattern (Fine et al, 1999; 2000). In the EB simplex (EBS) variants, the tissue separation occurs within the epidermis; in junctional EB (JEB), tissue cleavage occurs within the dermal-epidermal basement membrane at the level of the lamina lucida; the dystrophic form of EB (DEB) is characterized by intradermal blistering just beneath the lamina densa. JEB is a relatively rare autosomal recessive genodermatosis presenting at birth with blisters and erosions. There are two variant forms: (i) the Herlitz type (H-JEB) with patients usually surviving only a few weeks or months, and (ii) the non-Herlitz type with patients exhibiting relatively mild skin disease and normal life-span. H-JEB results from mutations, usually premature termination codons (PTC), on both alleles in any of the three genes, LAMA3a, Manuscript received January 18, 2000; revised April 11, 2000; accepted for publication May 30, 2000. Reprint requests to: Dr. Jouni Uitto, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 S 10th Street, Suite 450 BLSB, Philadelphia, PA 19107. Email: [email protected] Abbreviations: CSGE, conformation-sensitive gel electrophoresis; HJEB, Herlitz junctional epidermolysis bullosa; PTC, premature termination codon. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 493
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Table I. Premature termination codon mutations of LAMB3 in families with H-JEB Family
Mutations
Affected exons
Veri®cationc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
R635X/NDa R635X/978delC 617delA/617delA 3228+2T®Ab/ND 957ins77/564+5G®Tb R42X/R635X R635X/R635X 1101delC/957ins77 957ins77/NDd Q243X/Q243X R635X/R635X R972X/R972X R144X/R144X R635X/1629insG R635X/R42X R635X/Q243X 1365delCA/464insT R635X/R635X 957ins77/957ins77 R635X/R635X R635X/R635X R972X/R569X
14/ND 14/10 7/7 21/ND 10/6 3/14 14/14 10/10 10/ND 8/8 14/14 20/20 6/6 14/14 14/3 14/8 12/6 14/14 10/10 14/14 14/14 20/14
BglII/ND BglII/Tsp45 I seq/seq Rsa I/ND PCR/seq Dde I/Bgl II Bgl II/Bgl II Sma I/PCR PCR/ND Bfa I/Bfa I Bgl II/Bgl II AlwN I/AlwN I Hph I/Hph I Bgl II/seq Bgl II/Dde I Bgl II/Bfa I Pml I/Mnl I Bgl II/Bgl II PCR/PCR Bgl II/Bgl II Bgl II/Bgl II AlwN I/Cac8 I
aND,
not detected; PTC, premature termination codon. mutations affect an intron/exon border and are predicted to result in out-of-frame exon skipping leading to a downstream PTC. cVeri®cation of the mutation at the DNA level was performed either by digestion with the restriction endonuclease indicated or by direct nucleotide sequencing (seq). dNo sample was available from the patient or father. bThese
which has facilitated risk determination for extended family members. MATERIALS AND METHODS Clinical material and veri®cation of the diagnosis Twenty-one families with the diagnosis of H-JEB were analyzed. In all families, the proband had a history of skin blistering ®rst noted at birth, and the infant died within 1 y after the birth from complications of the disease. In 17 families DNA was available from the proband and from his/her parents, whereas in ®ve families only the parents as obligate heterozygotes of the mutations were screened (no sample was available from the affected child). Diagnostic skin biopsy specimens were obtained from all probands and subjected to routine histopathologic examination. Furthermore, 13 specimens were subjected to transmission electron microscopy, and immuno¯uorescence staining of frozen skin sections with antibodies recognizing the laminin 5 was performed. A number of antibodies recognizing other basement membrane zone components were used in comparative immuno¯uorescence staining. DNA extraction DNA was isolated from peripheral blood specimens from the probands and their parents by phenol-chloroform extraction using standard procedures (Sambrook et al, 1989). Control DNA was obtained from 50 unrelated individuals with no evidence of a blistering skin disease. DNA was used as a template for PCR ampli®cation of exons for mutation detection analysis. PCR The PCR ampli®cation of LAMB3 exons and of ¯anking intronic sequences was performed using primers previously reported elsewhere (Pulkkinen et al, 1995b). The PCR reactions were performed in a total volume of 50 ml containing 1 3 PCR buffer and 1.25 U of Amplitaq polymerase (Perkin Elmer Cetus, Foster City, CA), in the presence of 4% dimethylsulfoxide, 80 ng of each primer, and 200 ng of genomic DNA isolated from peripheral blood or chorionic villus samples. In some cases, 10 ml of amniotic ¯uid cell aliquot derived from 1 ml of amniotic ¯uid was used as template (see below). The ampli®cation conditions were as follows: 5 min 94°C for one cycle, followed by 38 cycles of 45 s at 94°C, 45 s at the
Figure 1. Schematic illustration of the LAMB3 polypeptide with domain organization and locations of all LAMB3 mutations disclosed thus far in the junctional forms of EB. PTC mutations are shown above the molecule, while missense mutations are shown below. Novel mutations disclosed in this study are shown in bold type. PTC mutations associated with non-Herlitz variants are underlined, while the mutations shown in italics are found in both Herlitz and non-Herlitz JEB.
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annealing temperature of the primers (see Pulkkinen et al, 1995b), and 45 s at 72°C in a thermal cycler (Hybaid, Teddington, U.K.). Five microliter aliquots of the PCR products were analyzed on 2% agarose gel electrophoresis. Conformation-sensitive gel electrophoresis (CSGE) Heteroduplex scanning of LAMB3 exons was performed by CSGE in 10% polyacrylamide, 99:1 ratio of acrylamide solution (Bio-Rad, Hercules, CA) to 1,4-bis(acryoyl)piperazine (Sigma, St. Louis, MO), 0.5 3 glyceroltolerant gel buffer (USB, Cleveland, OH), 15% formamide, 10% ethylene glycol, 0.1% ammonium persulfate, and 0.07% N,N,N¢,N¢tetramethylethylenediamine (Ganguly et al, 1993; KoÈrkkoÈ et al, 1998). Prior to loading, 8 ml samples were denatured at 98°C for 5 min and annealed at 68°C for 40 min. The samples were electrophoresed at 40 W for 6 h at room temperature. Heteroduplexes were visualized by staining the gel with ethidium bromide. DNA sequencing PCR products with altered mobility (heteroduplexes) detected by CSGE were directly sequenced by using an ABI PRISM 377 automated sequencing system (Perkin-Elmer Cetus, Foster City, CA). PCR restriction enzyme fragment length polymorphism In each family, the veri®cation of the mutation was performed by digestion of PCR products ampli®ed from DNA from the proband and his/her immediate family members by restriction endonucleases. The PCR products were digested at the appropriate temperature (25°C for Sma I; 37°C for AlwN I, Bfa I, Bgl II, Cac8 I, Dde I, Hph I, Mnl I, Pml I, and Rsa I; 65°C for Tsp45 I)
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for 15 h. The fragments were examined on 2%±3.5% agarose gels, or 8% polyacrylamide gel. Prenatal testing Chorionic villus samples obtained at 10±15 wk of pregnancy or amniotic ¯uid cells at 12±19 wk of pregnancy were used to obtain fetal DNA. For PCR of amniotic ¯uid cells, 1 ml of amniotic ¯uid was centrifuged and the cell pellet was dissolved in 200±500 ml of 50 mM NaOH, boiled for 5 min, and then neutralized by the addition of 1/10 volume of 1 M Tris-HCl, pH 8.0. An aliquot of the sample was used directly for PCR ampli®cation (Christiano et al, 1997). DNA was extracted from dissected chorionic villi in a small volume (»500 ml) using standard phenol-chloroform extraction methods (Sambrook et al, 1989). Maternal contamination was ruled out by using sex-speci®c markers as described by Chong et al (1993) or by follow-up analysis of DNA from cultured fetal cells. Carrier frequency determination The incidence of H-JEB and nonHerlitz JEB was taken from Fine et al (1999). The U.S. population carrier risk was determined assuming Hardy±Weinberg equilibrium of the alleles in the population and using the equation p2 + 2pq + q2 = 1 where 2pq equals the carrier frequency in the population studied.
RESULTS A total of 22 families with H-JEB were analyzed for LAMB3 mutations utilizing the strategy described in Materials and Methods. A total of 15 distinct mutations in 41 alleles representing the
Figure 2. Identi®cation and veri®cation of mutations 1365delCA/464insT in the LAMB3 gene of Family 17. (A) Heteroduplex analysis of the PCR product spanning exon 12 of the LAMB3 gene revealed a heteroduplex band in the mother (lane M, arrow), whereas the father (lane F) showed the homoduplex band only. (B) DNA sequencing of the mother's PCR product revealed the heterozygous deletion of CA at nucleotide position 1365 (1365delCA), as shown in the lower panel; the wild-type sequence is shown in the top panel. (C) Veri®cation of the maternal mutation by digestion with Pml I. The mutation created a new restriction enzyme site for Pml I, which cuts the 455 bp PCR product to 277 bp and 178 bp bands in the mutant allele, whereas the 457 bp PCR product remains uncut in the normal allele (the size difference re¯ects the 2 bp deletion). (D) Heteroduplex analysis of the PCR product spanning exon 6 of the LAMB3 gene revealed one heteroduplex band in the mother (lane M, asterisk), whereas the father (lane F) showed an additional heteroduplex band (arrow). The presence of a common heteroduplex band in both the mother's and the father's DNA (asterisk) could be explained by a T/C polymorphism which was detected in the nucleotide position 384 by sequencing (not shown). (E) DNA sequencing of the father's DNA revealed insertion of T at nucleotide position 464 (464insT), as shown in the lower panel; the wild-type sequence is shown in the top panel. (F) Veri®cation of the paternal mutation by digestion with Mnl I. The mutation abolished the restriction enzyme site for Mnl I, which cuts the 280 bp PCR product in the case of the normal allele to 66 bp, 62 bp, 55 bp, 54 bp, 30 bp, 8 bp, and 5 bp bands, and in the case of the mutant allele to 85 bp, 66 bp, 62 bp, 55 bp, 8 bp, and 5 bp bands (the size difference re¯ects the 1 bp insertion). MW, molecular weight markers jX174/HaeIII; C, control.
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Table II. Mutation-based prenatal testing in seven families at risk for H-JEB Family
Materiala
Fetal genotypeb
Clinical predictionc
8 10 11 12 16 17 20
CVS CVS CVS CVS AFC CVS AFC (twins)
1101delC/957ins77 (±)/Q243X (±)/(±) (±)/(±) (±)/(±) (±)/464insT (±)/R635X
Affected Unaffected carrier Unaffected Unaffected Unaffected Unaffected carrier Unaffected carriers
aCVS,
chorionic villous sample; AFC, amniotic ¯uid cells. the genotype of previously affected child in each family, see Table I. cIn case of Family 8, the pregnancy was terminated; In case of Families 10, 11, 12, 16 and 17, the prediction was con®rmed by birth of a healthy child; The pregnancy in Family 20 resulted in birth of healthy twins. bFor
Table III. Frequency of recurrent LAMB3 mutations Mutations R635X R42X 957ins77 Q243X R569X 565-2A®G R972X R144X
Frequency (%)a
Restriction endonucleaseb
45.4 5.9 8.6 5.3 3.3 2.6 2.6 2.0
Bgl II Dde I (PCR) Bfa I Cac8 I Nla IV AlwN I Hph I
aFrequency
as a percentage of all mutated LAMB3 alleles in H-JEB. mutations can be veri®ed by digestion with the corresponding restriction enzyme indicated. The mutation 957ins77 results in a PCR product which can be resolved from the normal allele on agarose gel electrophoresis (see Fig 4). bThe
Figure 3. Prenatal testing in Family 20. (A) The fetuses (lanes A and B; dizygotic twins) and parents (lanes M and F; mother and father, respectively) are heterozygous for the R635X mutation which creates a new restriction enzyme site for Bgl II. In the case of the mutant allele, this endonuclease cuts the 593 bp PCR product to 430 bp and 163 bp bands (lane PC; positive control), whereas in case of the normal allele the PCR product remains uncut (lane C; negative control). (B) DNA from both fetuses and the father is digested by Hae III, which cuts the Y chromosome PCR product of 300 bp to 216 bp and 84 bp bands, whereas the X chromosome product remains uncut. Thus, both fetuses are males, excluding maternal contamination of DNA, and consequently both fetuses were predicted to be healthy male carriers of the R635X mutation. MW, molecular weight markers jX174/HaeIII.
probands in the 22 families were identi®ed in the LAMB3 gene (Table I); among these mutations, eight were previously unpublished. The eight novel mutations characterized in the nine alleles of the LAMB3 gene included two splicing mutations (564 + 5G®T and 3228 + 2T®A), one or two bp deletions (617delA, 978delC, 1101delC, and 1365delCA) and one bp insertions (464insT and 1629insG). The six deletion or insertion mutations created a downstream PTC. These results are summarized in Table I and Fig 1. A representative example of a complete mutation detection analysis is shown in Fig 2. Prenatal testing was performed in Families 8, 10, 11, 12, 16, 17, and 20 after identi®cation of their mutations (Table II). Only one fetus was predicted to be affected and, as a result, this pregnancy was terminated (Family 8). Cultured amniocytes were used to con®rm the mutations in Families 16 and 20 (Fig 3). The genotypic prediction was con®rmed by the birth of a healthy child in Families 10, 11, 12, 16, and 17, and the pregnancy in Family 20 resulted in birth of healthy twins. Examination of the LAMB3 mutations in all cases previously reported as well as in the cases described in this report revealed eight recurrent mutations, six of them being previously unrecognized (Table III). At present, the comprehensive H-JEB database consists of a total of 152 mutant alleles in 80 H-JEB cases, including a set of 111 previously reported alleles from 58 families (Pulkkinen et al, 1994, 1995b, 1997a, b, 1999; McGrath et al, 1995; Vailly et al, 1995; Christiano and Uitto, 1996; Kivirikko et al, 1996; Ashton et al, 1997; Christiano et al, 1997; Cserhalmi et al, 1997; McMillan et al, 1997; Kon et al, 1998; Takizawa et al, 1998a, b, c). Analysis of the entire mutation database revealed that R635X and R42X were present in 45.4% and 5.9% of the mutant LAMB3 alleles, whereas 957ins77, Q243X, R569X, 565±2 A®G, R972X, and R144X were present in three or more unrelated families (see Table III). It is notable that the mutation 957ins77 was found only in patients of
African descent. All of these recurrent mutations can be readily detected by PCR and restriction endonuclease digestion (Figs 3, 4, Table III). Multiple requests by clinical geneticists and genetic counselors prompted us to calculate the carrier frequency of H-JEB and all forms of JEB from the incidence data presented by Fine et al (1999). The risk of an individual in the general U.S. population of carrying any type of EB mutation is one in 113, whereas the carrier risk for JEB is one in 350 and for H-JEB one in 781 (Table IV). These numbers re¯ect the relatively small likelihood of having a child with H-JEB. Since the risk of recurrence in families with a previous affected child is one in four, rapid and ef®cient mutation detection strategies, including identi®cation of recurrent mutations as described in this study, are helpful in the prenatal diagnosis of fetuses at risk. DISCUSSION The b3 chain, encoded by LAMB3, is one of the constitutive polypeptides of laminin 5, a trimeric anchoring ®lament protein associated with hemidesmosome/anchoring ®lament complexes. Recently, the detailed structure and function of hemidesmosomes has been elucidated by a number of molecular genetic, biochemical, and ultrastructural studies (see Pulkkinen and Uitto, 1998a, 1999; Aumailley and Rousselle, 1999; Borradori and Sonnenberg, 1999; Nievers et al, 1999). Laminin 5 is localized to the lower lamina lucida/lamina densa region. In addition to established interactions with the a6b4 integrin, laminin 5 interacts with noncollagenous amino-terminal globular (NC1) domain of type VII collagen and plays an important role in cell-matrix adhesion (Borradori and Sonnenberg, 1999). In H-JEB, the genetic mutations comprise primarily PTC mutations found on both alleles of the genes encoding laminin 5
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Figure 4. Detection of recurrent mutations in the LAMB3 gene in patients with H-JEB. (A) The R42X mutation creates a new restriction enzyme site for Dde I, which cuts the 498 bp PCR product in the normal allele to 272 bp, 103 bp, 85 bp, and 38 bp, and in the mutant allele to 240 bp, 103 bp, 85 bp, 38 bp, and 32 bp bands. (B) The 957ins77 mutant allele results in a 428 bp band in PCR, whereas the normal allele is detected as a 351 bp band. (C) The Q243X mutation creates a new restriction enzyme site for Bfa I, which cuts the 407 bp PCR product in the normal allele to 323 bp and 84 bp bands, and in the mutant allele to 218 bp, 105 bp, and 84 bp bands. (D) The R569X mutation abolishes the restriction enzyme site for Cac8 I, which cuts the 593 bp PCR product in the normal allele to 232 bp, 219 bp, 44 bp, 38 bp, 37 bp, and 23 bp bands, and in the mutant allele to 270 bp, 219 bp, 44 bp, 37 bp, and 23 bp bands (the smaller bands are not visible). (E) The 565±2A®G mutation creates a new restriction enzyme site for Nla IV, which cuts the 266 bp PCR product in the normal allele to 192 bp and 74 bp bands, and in the mutant allele to 123 bp, 74 bp, and 69 bp bands. (F) The R972X mutation creates a new restriction enzyme site for AIwN I. The normal allele of 324 bp remains uncut in AIwN I digestion, whereas this enzyme cuts the PCR product to 207 bp and 117 bp bands in the mutant allele. (G) The R144X mutation creates a new restriction enzyme site for Hph I. The normal allele of 451 bp remains uncut in Hph I digestion, whereas this enzyme cuts the PCR product to 234 bp and 217 bp bands in the mutant allele in the heterozygous parents (lanes M and F). MW, molecular weight markers jX174/HaeIII; P, patient; M, mother; F, father; C, control.
Table IV. Carrier frequency of H-JEB
All EB All JEB H-JEB aIncidence
Incidence per 106 births (q2)a
Carrier risk (2pq)
19.6 2.04 0.41
1/113 1/350 1/781
data taken from Fine et al, 1999.
polypeptides. The majority of these mutations reside in the LAMB3 gene. Recently, two non-Herlitz (nonlethal) JEB cases were reported to have compound heterozygous PTC mutations in the LAMB3 gene (Pulkkinen and Uitto, 1998b; McGrath et al, 1999), whereas all the other reported cases that have PTC/PTC mutations are H-JEB. The presence of PTC/PTC mutations in the nonHerlitz variants can be explained by an in-frame deletion of the coding region harboring the mutation (McGrath et al, 1999). A total of 43 distinct PTC mutations in the LAMB3 gene have been reported thus far, including those disclosed in this study, in both Herlitz and non-Herlitz JEB patients (Fig 1). In this study, we examined the LAMB3 gene of 22 H-JEB families and identi®ed eight novel mutations (Table I). Six of the eight novel mutations create a downstream PTC. Two others, 3228 + 2T®A and 564 + 5G®T, affect the intron-exon borders, potentially resulting in aberrant splicing, although this was not con®rmed by RT-PCR. Mutation 3228 + 2T®A alters the consensus splice donor sequence, however, and 564 + 5G®T was not detected in 50 unrelated healthy controls. The G nucleotide in position +5 is conserved in 84% of cases in primates, and consequently the 564 + 5G®T mutation most probably results in aberrant splicing (Shapiro and Senapathy, 1987). We could not disclose any mutation on one allele in patients 1 and 4. It is possible that a mutation could reside in the promoter region, leading to silencing of this LAMB3 allele. Alternatively, failure to amplify the mutated allele by PCR could prevent detection of the mutation in this analysis.
Previous reports have shown 26 distinct mutations in the LAMB3 gene in H-JEB with R635X and R42X recurrent mutations representing ``hotspots'' in the LAMB3 gene. Additionally, we found that the 957ins77 mutation, which has higher frequency than R42X, can be easily detected as a double band on agarose gel following PCR (Fig 4). Other recurrent mutations can also be readily detected by digestion of PCR products with restriction endonucleases (see Fig 4, Table III). The LAMB3 PTC mutations are present along the entire protein domain of the b3 chain and are not restricted to any particular region (Fig 1). It is therefore useful for mutation detection purposes to screen these recurrent mutations before embarking on CSGE scanning of the whole LAMB3 gene, especially in case of prenatal testing when expedient testing is required. Because of the severity of the H-JEB phenotype and the carrier frequency for H-JEB (one in 781) in the general U.S. population, prenatal screening in families with a history of H-JEB will become more common in the future, and will be facilitated by identi®cation of the recurrent mutations reported in this study. In conclusion, we have identi®ed 15 mutations, eight of them novel, in the LAMB3 gene in 22 H-JEB families. This is a signi®cant addition to the JEB mutation database and will undoubtedly be useful for prenatal testing in families at risk for H-JEB. We thank Dr. John McGrath (St. Thomas's Hospital, London) for providing helpful clinical information. Dr. Hajime Nakano provided helpful suggestions. Ms. Lin Lin provided excellent technical assistance. This study was supported by a grant from the U.S. Public Health Service, National Institutes of Health (PO1AR38923), and by the Dystrophic Epidermolysis Bullosa Research Association of America (DebRA). This paper was submitted in partial ful®llment of the PhD degree to Dr. Nakano by Hirosaki University.
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splicing with different tissue distribution of two transcripts. Genomics 32:54± 64, 1996 Ashton GHS, Mellerio JE, Dunnill MGS, et al: A recurrent laminin 5 mutation in British patients with lethal (Herlitz) junctional epidermolysis bullosa: evidence for a mutational hotspot rather than propagation of an ancestral allele. Br J Dermatol 136:674±677, 1997 Aumailley M, Rousselle P: Laminins of the dermo-epidermal junction. Matrix Biol 18:19±28, 1999 Borradori L, Sonnenberg A: Structure and function of hemidesmosomes: more than simple adhesion complexes. J Invest Dermatol 112:411±418, 1999 Chong SS, Kristjansson K, Cota J, Handyside AH, Hughes MR: Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously ampli®ed ZFX and ZFY sequences. Hum Mol Genet 2:1187±1191, 1993 Christiano AM, Uitto J: Molecular complexity of the cutaneous basement membrane zone: revelations from the paradigms of epidermolysis bullosa. Exp Dermatol 5:1±11, 1996 Christiano AM, Pulkkinen L, McGrath JA, Uitto J: Mutation-based prenatal diagnosis of Herlitz junctional epidermolysis bullosa. Prenat Diagn 17:343±354, 1997 Cserhalmi PB, Horvath A, Boros V, Sapi Z, Kormendi M, Christiano AM, Karpati S: Identi®cation of the LAMB3 hotspot mutation R635X in a Hungarian case of Herlitz junctional epidermolysis bullosa. Exp Dermatol 6:70±74, 1997 Fine J-D, Bauer EA, McGuire J, Moshell A: Epidermolysis bullosa. Clinical, Epidemiologic, and Laboratory Advances and the Findings of the National Epidermolysis Bullosa Registry. Baltimore, MD: The Johns Hopkins University Press, 1999 Fine J-D, Eady RAJ, Bauer EA, et al: Revised classi®cation system for inherited epidermolysis bullosa: report of the second international consensus meeting on diagnosis and classi®cation of epidermolysis bullosa. J Am Acad Dermatol 42:1051±1066, 2000 Ganguly A, Rock MJ, Prockop DJ: Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci USA 90:10325±10329, 1993 Heagerty AHM, Kennedy AR, Eady RAJ, Hsi B-L, Verrando P, Yeh C-J, Ortonne J-P: GB3 monoclonal antibody for diagnosis of junctional epidermolysis bullosa. Lancet 1:860, 1986 Kivirikko S, McGrath JA, Pulkkinen L, Uitto J, Christiano AM: Mutational hotspots in the LAMB3 gene in the lethal (Herlitz) type of junctional epidermolysis bullosa. Hum Mol Genet 5:231±237, 1996 Kon A, Pulkkinen L, Hara M, Tamai K, Tagami H, Hashimoto I, Uitto J: Laminin 5 genes and Herlitz junctional epidermolysis bullosa: novel mutations and polymorphisms in the LAMB3 and LAMC2 genes. Hum Mutat 12:288, 1998 KoÈrkkoÈ J, Annunen S, Pihlajamaa T, Prockop DJ, Ala-Kokko L: Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing. Proc Natl Acad Sci USA 95:1681±1685, 1998 McGrath JA, McMillan JR, Dunnill MGS, et al: Genetic basis of lethal junctional epidermolysis bullosa in an affected fetus: implications for prenatal diagnosis in one family. Prenat Diagn 15:647±654, 1995 McGrath JA, Ashton GHS, Mellerio JE, Salas-Alanis JC, Swensson O, McMillan JR, Eady RAJ: Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations. J Invest Dermatol 113:314±321, 1999 McMillan JR, McGrath JA, Pulkkinen L, et al: Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain speci®c antibodies is of limited value in predicting the underlying gene mutation. Br J Dermatol 136:817±822, 1997 Nievers MG, Schaapveld RQJ, Sonnenberg A: Biology and function of hemidesmosomes. Matrix Biol 18:5±17, 1999 Pulkkinen L, Uitto J: Hemidesmosomal variants of epidermolysis bullosa: mutations in the a6b4 integrin and the 180-kD bullous pemphigoid antigen/type XVII collagen genes. Exp Dermatol 7:46±64, 1998a
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Pulkkinen L, Uitto J: Heterozygosity for premature termination codon mutations in LAMB3 in siblings with non-lethal junctional epidermolysis bullosa. J Invest Dermatol 111:1244±1246, 1998b Pulkkinen L, Uitto J: Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol 18:29±42, 1999 Pulkkinen L, Christiano AM, Gerecke D, Wagman DW, Burgeson RE, Pittelkow MR, Uitto J: A homozygous nonsense mutation in the b3 chain gene of laminin 5 (LAMB3) in Herlitz junctional epidermolysis bullosa. Genomics 24:357±360, 1994 Pulkkinen L, Gerecke DR, Christiano AM, Wagman DW, Burgeson RE, Uitto J: Cloning of the b3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa. Genomics 25:192±198, 1995a Pulkkinen L, McGrath JA, Christiano AM, Uitto J: Detection of sequence variants in the gene encoding the b3 chain of laminin 5 (LAMB3). Hum Mutat 6:77±84, 1995b Pulkkinen L, Bullrich F, Czarnecki P, Weiss L, Uitto J: Maternal uniparental disomy of chromosome 1 with reduction to homozygosity of the LAMB3 locus in a patient with Herlitz junctional epidermolysis bullosa. Am J Hum Genet 61:611± 619, 1997a Pulkkinen L, Meneguzzi G, McGrath JA, et al: Predominance of the recurrent mutation R635X in the LAMB3 gene in European patients with Herlitz junctional epidermolysis bullosa has implications for mutation detection strategy. J Invest Dermatol 109:232±237, 1997b Pulkkinen L, Cserhalmi-Friedman PB, Tang M, Ryan MC, Uitto J, Christiano AM: Molecular analysis of the human laminin a3a chain gene (LAMA3a): a strategy for mutation identi®cation and DNA-based prenatal diagnosis in Herlitz junctional epidermolysis bullosa. Lab Invest 78:1067±1076, 1998 Pulkkinen L, Uitto J, Christiano AM. The molecular basis of the junctional forms of epidermolysis bullosa. In: Fine J-D, Bauer EA, McGuire J, Moshell A (eds). Epidermolysis Bullosa: Clinical, Epidemiologic and Laboratory Advances and the Findings of the National Epidermolysis Bullosa Registry. Baltimore, MD: The Johns Hopkins University Press, 1999, pp 300±325 Ryan MC, Tizard R, VanDevanter DR, Carter WG: Cloning of the LamA3 gene encoding the a3 chain of the adhesive ligand epiligrin. J Biol Chem 269:22779± 22787, 1994 Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: a Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory Press, 1989, pp 9.16±9.19 Shapiro MB, Senapathy P: RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucl Acids Res 15:7155±7174, 1987 Takizawa Y, Shimizu H, Pulkkinen L, et al: Novel mutations in the LAMB3 gene shared by two Japanese unrelated families with Herlitz junctional epidermolysis bullosa, and their application for prenatal testing. J Invest Dermatol 110:174±178, 1998a Takizawa Y, Pulkkinen L, Shimizu H, Lin L, Hagiwara S, Nishikawa T, Uitto J: Maternal uniparental meroisodisomy in the LAMB3 region of chromosome 1 results in lethal junctional epidermolysis bullosa. J Invest Dermatol 110:828±831, 1998b Takizawa Y, Shimizu H, Pulkkinen L, Suzumori K, Kakinuma H, Uitto J, Nishikawa T: Combination of a novel frameshift mutation (1929delCA) and a recurrent nonsense mutation (W610X) of the LAMB3 gene in a Japanese patient with Herlitz junctional epidermolysis bullosa, and their application for prenatal testing. J Invest Dermatol 111:1239±1241, 1998c Tidman MJ, Eady RAJ: Hemidesmosome heterogeneity in junctional epidermolysis bullosa revealed by morphometric analysis. J Invest Dermatol 86:51±56, 1986 Vailly J, Pulkkinen L, Miquel C, et al: Identi®cation of a homozygous one-basepair deletion in exon 14 of the LAMB3 gene in a patient with Herlitz junctional epidermolysis bullosa and prenatal diagnosis in a family at risk for recurrence. J Invest Dermatol 104:462±466, 1995 Verrando P, Blanchet-Bardon C, Pisani A, et al: Monoclonal antibody GB3 de®nes a widespread defect of several basement membranes and a keratinocyte dysfunction in patients with lethal junctional epidermolysis bullosa. Lab Invest 64:85±92, 1991
Herlitz Junctional Epidermolysis Bullosa: Novel and Recurrent Mutations in the LAMB3 Gene and the Population Carrier Frequency Aoi Nakano, Ellen Pfendner, Leena Pulkkinen, Isao Hashimoto,* and Jouni Uitto
Departments of Dermatology and Cutaneous Biology, and Biochemistry and Molecular Pharmacology, Jefferson Medical College, Jefferson Institute of Molecular Medicine, and DebRA Molecular Diagnostics Laboratory, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A.; *Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan
Herlitz junctional epidermolysis bullosa is a heritable bullous disease caused by mutations found primarily in the b3 chain of laminin 5 (LAMB3). In this study, we examined the LAMB3 gene for mutations in 22 Herlitz junctional epidermolysis bullosa families, and identi®ed 15 distinct mutations, eight of them previously unreported, bringing the total number of distinct Herlitz junctional epidermolysis bullosa mutations in LAMB3 to 35. Examination of the mutation database revealed several recurrent mutations that have been reported, as well as six previously unreported. All recurrent mutations may be readily detected by polymerase chain reaction of genomic DNA and restriction endonuclease digestion. Mutation screening and prenatal diagnosis of
families at risk may be expedited by molecular testing for these recurrent mutations prior to screening the entire gene. Finally, the U.S. population carrier risk for Herlitz junctional epidermolysis bullosa and all variants of junctional epidermolysis bullosa was calculated to be one in 781 and one in 350, respectively, while the overall epidermolysis bullosa carrier frequency was calculated to be one in 113. These data allow accurate testing, counseling, and risk calculation for nuclear families, as well as extended family members at risk for junctional epidermolysis bullosa. Key words: basement membrane zone/blistering skin diseases/laminin 5 mutations/prenatal diagnosis. J Invest Dermatol 115:493±498, 2000
E
LAMB3, and LAMC2, which encode the laminin 5 subunit polypeptides a3, b3, and g2, respectively. The PTC mutations in H-JEB predict the absence of laminin 5 in the dermal-epidermal junction, which can be veri®ed by negative staining of laminin 5 using speci®c monoclonal antibodies (Heagerty et al, 1986; Verrando et al, 1991; McMillan et al, 1997). Electron microscopy performed on skin biopsies from H-JEB patients reveals poorly developed hemidesmosome/anchoring ®lament complexes (Tidman and Eady, 1986). Mutation detection has been facilitated by sequencing of the LAMA3a, LAMB3, and LAMC2 genes and polymerase chain reaction (PCR) primers have been designed spanning all exons. Among the human laminin 5 genes, LAMA3a has 38 exons and encodes 1713 amino acids (Ryan et al, 1994; Pulkkinen et al, 1998), whereas LAMB3 and LAMC2 both have 23 exons and encode 1172 and 1193 amino acids, respectively (Pulkkinen et al, 1995a; Airenne et al, 1996). Previously, the majority of H-JEB mutations have been disclosed in the LAMB3 gene. Among the 26 distinct mutations characterized thus far in 58 families are two recurrent hotspot mutations, R635X and R42X (Kivirikko et al, 1996). In this study, we have examined 22 new families with H-JEB and identi®ed 15 distinct mutations, including eight novel ones. Examination of the global mutation database also revealed the presence of six previously unrecognized recurrent mutations in the LAMB3 gene. Using the information about speci®c familial mutations in LAMB3, prenatal diagnosis was performed in seven families at risk for recurrence of H-JEB. Carrier risks have been generated from population data taken from Fine et al (1999)
pidermolysis bullosa (EB) is a group of heritable mechanobullous diseases characterized by blistering due to skin fragility that can result from ordinarily minor trauma. Cases of EB are traditionally classi®ed into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone, clinical features, and the inheritance pattern (Fine et al, 1999; 2000). In the EB simplex (EBS) variants, the tissue separation occurs within the epidermis; in junctional EB (JEB), tissue cleavage occurs within the dermal-epidermal basement membrane at the level of the lamina lucida; the dystrophic form of EB (DEB) is characterized by intradermal blistering just beneath the lamina densa. JEB is a relatively rare autosomal recessive genodermatosis presenting at birth with blisters and erosions. There are two variant forms: (i) the Herlitz type (H-JEB) with patients usually surviving only a few weeks or months, and (ii) the non-Herlitz type with patients exhibiting relatively mild skin disease and normal life-span. H-JEB results from mutations, usually premature termination codons (PTC), on both alleles in any of the three genes, LAMA3a, Manuscript received January 18, 2000; revised April 11, 2000; accepted for publication May 30, 2000. Reprint requests to: Dr. Jouni Uitto, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 S 10th Street, Suite 450 BLSB, Philadelphia, PA 19107. Email: [email protected] Abbreviations: CSGE, conformation-sensitive gel electrophoresis; HJEB, Herlitz junctional epidermolysis bullosa; PTC, premature termination codon. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 493
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Table I. Premature termination codon mutations of LAMB3 in families with H-JEB Family
Mutations
Affected exons
Veri®cationc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
R635X/NDa R635X/978delC 617delA/617delA 3228+2T®Ab/ND 957ins77/564+5G®Tb R42X/R635X R635X/R635X 1101delC/957ins77 957ins77/NDd Q243X/Q243X R635X/R635X R972X/R972X R144X/R144X R635X/1629insG R635X/R42X R635X/Q243X 1365delCA/464insT R635X/R635X 957ins77/957ins77 R635X/R635X R635X/R635X R972X/R569X
14/ND 14/10 7/7 21/ND 10/6 3/14 14/14 10/10 10/ND 8/8 14/14 20/20 6/6 14/14 14/3 14/8 12/6 14/14 10/10 14/14 14/14 20/14
BglII/ND BglII/Tsp45 I seq/seq Rsa I/ND PCR/seq Dde I/Bgl II Bgl II/Bgl II Sma I/PCR PCR/ND Bfa I/Bfa I Bgl II/Bgl II AlwN I/AlwN I Hph I/Hph I Bgl II/seq Bgl II/Dde I Bgl II/Bfa I Pml I/Mnl I Bgl II/Bgl II PCR/PCR Bgl II/Bgl II Bgl II/Bgl II AlwN I/Cac8 I
aND,
not detected; PTC, premature termination codon. mutations affect an intron/exon border and are predicted to result in out-of-frame exon skipping leading to a downstream PTC. cVeri®cation of the mutation at the DNA level was performed either by digestion with the restriction endonuclease indicated or by direct nucleotide sequencing (seq). dNo sample was available from the patient or father. bThese
which has facilitated risk determination for extended family members. MATERIALS AND METHODS Clinical material and veri®cation of the diagnosis Twenty-one families with the diagnosis of H-JEB were analyzed. In all families, the proband had a history of skin blistering ®rst noted at birth, and the infant died within 1 y after the birth from complications of the disease. In 17 families DNA was available from the proband and from his/her parents, whereas in ®ve families only the parents as obligate heterozygotes of the mutations were screened (no sample was available from the affected child). Diagnostic skin biopsy specimens were obtained from all probands and subjected to routine histopathologic examination. Furthermore, 13 specimens were subjected to transmission electron microscopy, and immuno¯uorescence staining of frozen skin sections with antibodies recognizing the laminin 5 was performed. A number of antibodies recognizing other basement membrane zone components were used in comparative immuno¯uorescence staining. DNA extraction DNA was isolated from peripheral blood specimens from the probands and their parents by phenol-chloroform extraction using standard procedures (Sambrook et al, 1989). Control DNA was obtained from 50 unrelated individuals with no evidence of a blistering skin disease. DNA was used as a template for PCR ampli®cation of exons for mutation detection analysis. PCR The PCR ampli®cation of LAMB3 exons and of ¯anking intronic sequences was performed using primers previously reported elsewhere (Pulkkinen et al, 1995b). The PCR reactions were performed in a total volume of 50 ml containing 1 3 PCR buffer and 1.25 U of Amplitaq polymerase (Perkin Elmer Cetus, Foster City, CA), in the presence of 4% dimethylsulfoxide, 80 ng of each primer, and 200 ng of genomic DNA isolated from peripheral blood or chorionic villus samples. In some cases, 10 ml of amniotic ¯uid cell aliquot derived from 1 ml of amniotic ¯uid was used as template (see below). The ampli®cation conditions were as follows: 5 min 94°C for one cycle, followed by 38 cycles of 45 s at 94°C, 45 s at the
Figure 1. Schematic illustration of the LAMB3 polypeptide with domain organization and locations of all LAMB3 mutations disclosed thus far in the junctional forms of EB. PTC mutations are shown above the molecule, while missense mutations are shown below. Novel mutations disclosed in this study are shown in bold type. PTC mutations associated with non-Herlitz variants are underlined, while the mutations shown in italics are found in both Herlitz and non-Herlitz JEB.
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annealing temperature of the primers (see Pulkkinen et al, 1995b), and 45 s at 72°C in a thermal cycler (Hybaid, Teddington, U.K.). Five microliter aliquots of the PCR products were analyzed on 2% agarose gel electrophoresis. Conformation-sensitive gel electrophoresis (CSGE) Heteroduplex scanning of LAMB3 exons was performed by CSGE in 10% polyacrylamide, 99:1 ratio of acrylamide solution (Bio-Rad, Hercules, CA) to 1,4-bis(acryoyl)piperazine (Sigma, St. Louis, MO), 0.5 3 glyceroltolerant gel buffer (USB, Cleveland, OH), 15% formamide, 10% ethylene glycol, 0.1% ammonium persulfate, and 0.07% N,N,N¢,N¢tetramethylethylenediamine (Ganguly et al, 1993; KoÈrkkoÈ et al, 1998). Prior to loading, 8 ml samples were denatured at 98°C for 5 min and annealed at 68°C for 40 min. The samples were electrophoresed at 40 W for 6 h at room temperature. Heteroduplexes were visualized by staining the gel with ethidium bromide. DNA sequencing PCR products with altered mobility (heteroduplexes) detected by CSGE were directly sequenced by using an ABI PRISM 377 automated sequencing system (Perkin-Elmer Cetus, Foster City, CA). PCR restriction enzyme fragment length polymorphism In each family, the veri®cation of the mutation was performed by digestion of PCR products ampli®ed from DNA from the proband and his/her immediate family members by restriction endonucleases. The PCR products were digested at the appropriate temperature (25°C for Sma I; 37°C for AlwN I, Bfa I, Bgl II, Cac8 I, Dde I, Hph I, Mnl I, Pml I, and Rsa I; 65°C for Tsp45 I)
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for 15 h. The fragments were examined on 2%±3.5% agarose gels, or 8% polyacrylamide gel. Prenatal testing Chorionic villus samples obtained at 10±15 wk of pregnancy or amniotic ¯uid cells at 12±19 wk of pregnancy were used to obtain fetal DNA. For PCR of amniotic ¯uid cells, 1 ml of amniotic ¯uid was centrifuged and the cell pellet was dissolved in 200±500 ml of 50 mM NaOH, boiled for 5 min, and then neutralized by the addition of 1/10 volume of 1 M Tris-HCl, pH 8.0. An aliquot of the sample was used directly for PCR ampli®cation (Christiano et al, 1997). DNA was extracted from dissected chorionic villi in a small volume (»500 ml) using standard phenol-chloroform extraction methods (Sambrook et al, 1989). Maternal contamination was ruled out by using sex-speci®c markers as described by Chong et al (1993) or by follow-up analysis of DNA from cultured fetal cells. Carrier frequency determination The incidence of H-JEB and nonHerlitz JEB was taken from Fine et al (1999). The U.S. population carrier risk was determined assuming Hardy±Weinberg equilibrium of the alleles in the population and using the equation p2 + 2pq + q2 = 1 where 2pq equals the carrier frequency in the population studied.
RESULTS A total of 22 families with H-JEB were analyzed for LAMB3 mutations utilizing the strategy described in Materials and Methods. A total of 15 distinct mutations in 41 alleles representing the
Figure 2. Identi®cation and veri®cation of mutations 1365delCA/464insT in the LAMB3 gene of Family 17. (A) Heteroduplex analysis of the PCR product spanning exon 12 of the LAMB3 gene revealed a heteroduplex band in the mother (lane M, arrow), whereas the father (lane F) showed the homoduplex band only. (B) DNA sequencing of the mother's PCR product revealed the heterozygous deletion of CA at nucleotide position 1365 (1365delCA), as shown in the lower panel; the wild-type sequence is shown in the top panel. (C) Veri®cation of the maternal mutation by digestion with Pml I. The mutation created a new restriction enzyme site for Pml I, which cuts the 455 bp PCR product to 277 bp and 178 bp bands in the mutant allele, whereas the 457 bp PCR product remains uncut in the normal allele (the size difference re¯ects the 2 bp deletion). (D) Heteroduplex analysis of the PCR product spanning exon 6 of the LAMB3 gene revealed one heteroduplex band in the mother (lane M, asterisk), whereas the father (lane F) showed an additional heteroduplex band (arrow). The presence of a common heteroduplex band in both the mother's and the father's DNA (asterisk) could be explained by a T/C polymorphism which was detected in the nucleotide position 384 by sequencing (not shown). (E) DNA sequencing of the father's DNA revealed insertion of T at nucleotide position 464 (464insT), as shown in the lower panel; the wild-type sequence is shown in the top panel. (F) Veri®cation of the paternal mutation by digestion with Mnl I. The mutation abolished the restriction enzyme site for Mnl I, which cuts the 280 bp PCR product in the case of the normal allele to 66 bp, 62 bp, 55 bp, 54 bp, 30 bp, 8 bp, and 5 bp bands, and in the case of the mutant allele to 85 bp, 66 bp, 62 bp, 55 bp, 8 bp, and 5 bp bands (the size difference re¯ects the 1 bp insertion). MW, molecular weight markers jX174/HaeIII; C, control.
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Table II. Mutation-based prenatal testing in seven families at risk for H-JEB Family
Materiala
Fetal genotypeb
Clinical predictionc
8 10 11 12 16 17 20
CVS CVS CVS CVS AFC CVS AFC (twins)
1101delC/957ins77 (±)/Q243X (±)/(±) (±)/(±) (±)/(±) (±)/464insT (±)/R635X
Affected Unaffected carrier Unaffected Unaffected Unaffected Unaffected carrier Unaffected carriers
aCVS,
chorionic villous sample; AFC, amniotic ¯uid cells. the genotype of previously affected child in each family, see Table I. cIn case of Family 8, the pregnancy was terminated; In case of Families 10, 11, 12, 16 and 17, the prediction was con®rmed by birth of a healthy child; The pregnancy in Family 20 resulted in birth of healthy twins. bFor
Table III. Frequency of recurrent LAMB3 mutations Mutations R635X R42X 957ins77 Q243X R569X 565-2A®G R972X R144X
Frequency (%)a
Restriction endonucleaseb
45.4 5.9 8.6 5.3 3.3 2.6 2.6 2.0
Bgl II Dde I (PCR) Bfa I Cac8 I Nla IV AlwN I Hph I
aFrequency
as a percentage of all mutated LAMB3 alleles in H-JEB. mutations can be veri®ed by digestion with the corresponding restriction enzyme indicated. The mutation 957ins77 results in a PCR product which can be resolved from the normal allele on agarose gel electrophoresis (see Fig 4). bThe
Figure 3. Prenatal testing in Family 20. (A) The fetuses (lanes A and B; dizygotic twins) and parents (lanes M and F; mother and father, respectively) are heterozygous for the R635X mutation which creates a new restriction enzyme site for Bgl II. In the case of the mutant allele, this endonuclease cuts the 593 bp PCR product to 430 bp and 163 bp bands (lane PC; positive control), whereas in case of the normal allele the PCR product remains uncut (lane C; negative control). (B) DNA from both fetuses and the father is digested by Hae III, which cuts the Y chromosome PCR product of 300 bp to 216 bp and 84 bp bands, whereas the X chromosome product remains uncut. Thus, both fetuses are males, excluding maternal contamination of DNA, and consequently both fetuses were predicted to be healthy male carriers of the R635X mutation. MW, molecular weight markers jX174/HaeIII.
probands in the 22 families were identi®ed in the LAMB3 gene (Table I); among these mutations, eight were previously unpublished. The eight novel mutations characterized in the nine alleles of the LAMB3 gene included two splicing mutations (564 + 5G®T and 3228 + 2T®A), one or two bp deletions (617delA, 978delC, 1101delC, and 1365delCA) and one bp insertions (464insT and 1629insG). The six deletion or insertion mutations created a downstream PTC. These results are summarized in Table I and Fig 1. A representative example of a complete mutation detection analysis is shown in Fig 2. Prenatal testing was performed in Families 8, 10, 11, 12, 16, 17, and 20 after identi®cation of their mutations (Table II). Only one fetus was predicted to be affected and, as a result, this pregnancy was terminated (Family 8). Cultured amniocytes were used to con®rm the mutations in Families 16 and 20 (Fig 3). The genotypic prediction was con®rmed by the birth of a healthy child in Families 10, 11, 12, 16, and 17, and the pregnancy in Family 20 resulted in birth of healthy twins. Examination of the LAMB3 mutations in all cases previously reported as well as in the cases described in this report revealed eight recurrent mutations, six of them being previously unrecognized (Table III). At present, the comprehensive H-JEB database consists of a total of 152 mutant alleles in 80 H-JEB cases, including a set of 111 previously reported alleles from 58 families (Pulkkinen et al, 1994, 1995b, 1997a, b, 1999; McGrath et al, 1995; Vailly et al, 1995; Christiano and Uitto, 1996; Kivirikko et al, 1996; Ashton et al, 1997; Christiano et al, 1997; Cserhalmi et al, 1997; McMillan et al, 1997; Kon et al, 1998; Takizawa et al, 1998a, b, c). Analysis of the entire mutation database revealed that R635X and R42X were present in 45.4% and 5.9% of the mutant LAMB3 alleles, whereas 957ins77, Q243X, R569X, 565±2 A®G, R972X, and R144X were present in three or more unrelated families (see Table III). It is notable that the mutation 957ins77 was found only in patients of
African descent. All of these recurrent mutations can be readily detected by PCR and restriction endonuclease digestion (Figs 3, 4, Table III). Multiple requests by clinical geneticists and genetic counselors prompted us to calculate the carrier frequency of H-JEB and all forms of JEB from the incidence data presented by Fine et al (1999). The risk of an individual in the general U.S. population of carrying any type of EB mutation is one in 113, whereas the carrier risk for JEB is one in 350 and for H-JEB one in 781 (Table IV). These numbers re¯ect the relatively small likelihood of having a child with H-JEB. Since the risk of recurrence in families with a previous affected child is one in four, rapid and ef®cient mutation detection strategies, including identi®cation of recurrent mutations as described in this study, are helpful in the prenatal diagnosis of fetuses at risk. DISCUSSION The b3 chain, encoded by LAMB3, is one of the constitutive polypeptides of laminin 5, a trimeric anchoring ®lament protein associated with hemidesmosome/anchoring ®lament complexes. Recently, the detailed structure and function of hemidesmosomes has been elucidated by a number of molecular genetic, biochemical, and ultrastructural studies (see Pulkkinen and Uitto, 1998a, 1999; Aumailley and Rousselle, 1999; Borradori and Sonnenberg, 1999; Nievers et al, 1999). Laminin 5 is localized to the lower lamina lucida/lamina densa region. In addition to established interactions with the a6b4 integrin, laminin 5 interacts with noncollagenous amino-terminal globular (NC1) domain of type VII collagen and plays an important role in cell-matrix adhesion (Borradori and Sonnenberg, 1999). In H-JEB, the genetic mutations comprise primarily PTC mutations found on both alleles of the genes encoding laminin 5
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Figure 4. Detection of recurrent mutations in the LAMB3 gene in patients with H-JEB. (A) The R42X mutation creates a new restriction enzyme site for Dde I, which cuts the 498 bp PCR product in the normal allele to 272 bp, 103 bp, 85 bp, and 38 bp, and in the mutant allele to 240 bp, 103 bp, 85 bp, 38 bp, and 32 bp bands. (B) The 957ins77 mutant allele results in a 428 bp band in PCR, whereas the normal allele is detected as a 351 bp band. (C) The Q243X mutation creates a new restriction enzyme site for Bfa I, which cuts the 407 bp PCR product in the normal allele to 323 bp and 84 bp bands, and in the mutant allele to 218 bp, 105 bp, and 84 bp bands. (D) The R569X mutation abolishes the restriction enzyme site for Cac8 I, which cuts the 593 bp PCR product in the normal allele to 232 bp, 219 bp, 44 bp, 38 bp, 37 bp, and 23 bp bands, and in the mutant allele to 270 bp, 219 bp, 44 bp, 37 bp, and 23 bp bands (the smaller bands are not visible). (E) The 565±2A®G mutation creates a new restriction enzyme site for Nla IV, which cuts the 266 bp PCR product in the normal allele to 192 bp and 74 bp bands, and in the mutant allele to 123 bp, 74 bp, and 69 bp bands. (F) The R972X mutation creates a new restriction enzyme site for AIwN I. The normal allele of 324 bp remains uncut in AIwN I digestion, whereas this enzyme cuts the PCR product to 207 bp and 117 bp bands in the mutant allele. (G) The R144X mutation creates a new restriction enzyme site for Hph I. The normal allele of 451 bp remains uncut in Hph I digestion, whereas this enzyme cuts the PCR product to 234 bp and 217 bp bands in the mutant allele in the heterozygous parents (lanes M and F). MW, molecular weight markers jX174/HaeIII; P, patient; M, mother; F, father; C, control.
Table IV. Carrier frequency of H-JEB
All EB All JEB H-JEB aIncidence
Incidence per 106 births (q2)a
Carrier risk (2pq)
19.6 2.04 0.41
1/113 1/350 1/781
data taken from Fine et al, 1999.
polypeptides. The majority of these mutations reside in the LAMB3 gene. Recently, two non-Herlitz (nonlethal) JEB cases were reported to have compound heterozygous PTC mutations in the LAMB3 gene (Pulkkinen and Uitto, 1998b; McGrath et al, 1999), whereas all the other reported cases that have PTC/PTC mutations are H-JEB. The presence of PTC/PTC mutations in the nonHerlitz variants can be explained by an in-frame deletion of the coding region harboring the mutation (McGrath et al, 1999). A total of 43 distinct PTC mutations in the LAMB3 gene have been reported thus far, including those disclosed in this study, in both Herlitz and non-Herlitz JEB patients (Fig 1). In this study, we examined the LAMB3 gene of 22 H-JEB families and identi®ed eight novel mutations (Table I). Six of the eight novel mutations create a downstream PTC. Two others, 3228 + 2T®A and 564 + 5G®T, affect the intron-exon borders, potentially resulting in aberrant splicing, although this was not con®rmed by RT-PCR. Mutation 3228 + 2T®A alters the consensus splice donor sequence, however, and 564 + 5G®T was not detected in 50 unrelated healthy controls. The G nucleotide in position +5 is conserved in 84% of cases in primates, and consequently the 564 + 5G®T mutation most probably results in aberrant splicing (Shapiro and Senapathy, 1987). We could not disclose any mutation on one allele in patients 1 and 4. It is possible that a mutation could reside in the promoter region, leading to silencing of this LAMB3 allele. Alternatively, failure to amplify the mutated allele by PCR could prevent detection of the mutation in this analysis.
Previous reports have shown 26 distinct mutations in the LAMB3 gene in H-JEB with R635X and R42X recurrent mutations representing ``hotspots'' in the LAMB3 gene. Additionally, we found that the 957ins77 mutation, which has higher frequency than R42X, can be easily detected as a double band on agarose gel following PCR (Fig 4). Other recurrent mutations can also be readily detected by digestion of PCR products with restriction endonucleases (see Fig 4, Table III). The LAMB3 PTC mutations are present along the entire protein domain of the b3 chain and are not restricted to any particular region (Fig 1). It is therefore useful for mutation detection purposes to screen these recurrent mutations before embarking on CSGE scanning of the whole LAMB3 gene, especially in case of prenatal testing when expedient testing is required. Because of the severity of the H-JEB phenotype and the carrier frequency for H-JEB (one in 781) in the general U.S. population, prenatal screening in families with a history of H-JEB will become more common in the future, and will be facilitated by identi®cation of the recurrent mutations reported in this study. In conclusion, we have identi®ed 15 mutations, eight of them novel, in the LAMB3 gene in 22 H-JEB families. This is a signi®cant addition to the JEB mutation database and will undoubtedly be useful for prenatal testing in families at risk for H-JEB. We thank Dr. John McGrath (St. Thomas's Hospital, London) for providing helpful clinical information. Dr. Hajime Nakano provided helpful suggestions. Ms. Lin Lin provided excellent technical assistance. This study was supported by a grant from the U.S. Public Health Service, National Institutes of Health (PO1AR38923), and by the Dystrophic Epidermolysis Bullosa Research Association of America (DebRA). This paper was submitted in partial ful®llment of the PhD degree to Dr. Nakano by Hirosaki University.
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