Progress in Molecular Genetics of Heritable Skin Diseases: The Paradigms of and Pseudoxanthoma Elasticum

Jouni Uitto, Leena Pulkkinen, and Franziska Ringpfeil Departments of Dermatology and Cutaneous Biology, and Biochemistry and Molecular Pharmacology, Je¡erson Medical College, and Je¡erson Institute of Molecular Medicine,Thomas Je¡erson University, Philadelphia, Pennsylvania, U.S.A.

The 42nd Annual Symposium on the Biology of the this meeting just caught the wave of early pioneering Skin, entitled ‘‘The Genetics of Skin Disease’’, was held studies that have helped us to understand the molecular in Snowmass Village, Colorado, in July 1993. That meet- basis of a large number of genodermatoses. This over- ing presented the opportunity to discuss how modern view presented in the 50th Annual Symposium on the approaches to molecular genetics and molecular biol- biology of the skin, highlights the progress made in ogy could be applied to understanding the mechanisms the molecular genetics of heritable skin diseases over of skin diseases. The published proceedings of this the past decade. Key words: Genodermatoses/epidermolysis meeting stated that ‘‘It is an opportune time to examine bullosa/pseudoxanthoma elasticum JID Symposium Proceed- the genetics of skin disease’’ (Norris et al, 1994). Indeed, ings 7:6^16,2002

he recent progress made in molecular genetics of the basis of clinical, histopathologic, immunohistochemical, and/ heritable skin diseases is abundantly evident from or ultrastructural analysis, to serve as candidate / sys- the present vantage point, as reviewed in the 50th tems. For example, in the case of EB, we initially postulated that Annual Montagna Symposium on the Biology of mutations in the structural expressed within the cutaneous Skin, also held in Snowmass Village, Colorado, in basement membrane zone (BMZ) could harbor mutations that TJuly 2001. At the time of our previous meeting, almost a decade explain the fragility of the skin at the dermal^epidermal junction ago, the molecular basis of the prototypic genodermatoses, such (Uitto and Christiano, 1992).This postulate has now been veri¢ed as epidermolysis bullosa (EB), was beginning to emerge as the by demonstration of a large number of distinct mutations in as ¢rst mutations in the 5 and 14 genes in the simplex forms many as 10 di¡erent genes expressed within the cutaneous BMZ and in the type VII collagen gene in the dystrophic variants (see below). At the same time, a number of mutated genes have had just appeared in the literature (Epstein, 1992; Fuchs, 1992; turned out to be rather surprising, and the exact relationship of Christiano et al, 1993; Hilal et al, 1993). At the same time, there the mutations in the a¡ected genes and their consequences at the was very little understanding of the molecular basis of complex clinical and morphologic level are not well understood. An ex- multisystem disorders, such as pseudoxanthoma elasticum (PXE), ample of such conditions is PXE, in which we and others have the Ehlers^Danlos syndrome, and the . Over recently demonstrated mutations in the ABCC6 gene encoding the ensuing years, this situation has very quickly changed, and the MRP6 protein, a putative ATP-dependent transmembrane there has been tremendous progress towards understanding the pump of unknown function (Bergen et al, 2000; Le Saux et al, molecular basis of di¡erent forms of single-gene heritable disor- 2000; Ringpfeil et al, 2000; Struk et al, 2000). Surprisingly, this ders. In fact, mutations have now been identi¢ed in more than gene is expressed primarily, if not exclusively, in the and 200 distinct genes in a manner that the genetic lesions explain the kidneys, and the pathomechanistic links between the under- the spectrum of phenotypic manifestations encountered in these lying genetic mutations in ABCC6 and calci¢cation of pleio- diseases (Tab l e I , Pulkkinen et al, 2002a). morphic elastic structures in the skin, the eyes, and the arterial blood vessels remain unclear. To illustrate the progress made in understanding the genetic PREDICTABLE AND SURPRISING CANDIDATE GENES basis of heritable skin diseases in general, we will highlight two speci¢c disease entities, in which we have been major con- Examination of the mutation database in these diseases has tributors, namely epidermolysis bullosa and pseudoxanthoma revealed a number of genes that could have been predicted, on elasticum.

Manuscript received July 24, 2002; accepted for publication August 15, 2002 THE PARADIGM OF EB Reprint requests to: Dr. Jouni Uitto, Department of Dermatology and Cutaneous Biology, Je¡erson Medical College, 233S 10th Street, Suite 450 BLSB, Philadelphia, PA 19107; Email: [email protected] An example of the conditions in which spectacular success has Abbreviations: DEB, dystrophic forms of EB; EB, epidermolysis bullo- recently been made is EB, a heterogeneous group of mechano- sa; JEB, junctional variant of EB; MRP, multiple drug resistance-associated bullous disorders manifesting with blistering and erosions of the protein; PGD, preimplantation genetic diagnosis; PXE, pseudoxanthoma skin (Sybert, 1997; Fine et al, 2000; Bruckner-Tuderman, 2002). elasticum. The extent of skin involvement, combined with a number of

0022-202X/02/$15.00 Copyright r 2002 by The Society for Investigative Dermatology, Inc.

6 VOL. 7, NO. 1 DECEMBER 2002 HERITABLE SKIN DISEASES 7

Table I. Genodermatoses with known gene defects Diseasea Mutated geneb A¡ected protein/function Referencec

Epidermal fragility disorders Dystrophic EBd COL7A1 TypeVII collagen Pulkkinen and Uitto (1999) Junctional EB LAMA3, LAMB3, a3, b3, and g2 chains of laminin 5, Pulkkinen and Uitto (1999) LAMC2, COL17A1 Type XVII collagen EB with pyloric atresia ITGA6, ITGB4 a6b4 integrin Pulkkinen and Uitto (1999) EB with muscular dystrophy PLEC1 Pulkkinen and Uitto (1999) EB simplex ogna PLEC1 Plectin Koss-Harnes et al (2002) EB simplex KRT5, KRT14 5 and 14 Irvine and McLean (1999) with skin PKP1 McGrath (1999) fragility Hailey^Hailey disease ATP2C1 ATP-dependent calcium transporter Hu et al (2000) Keratinization disorders Epidermolytic KRT1, KRT10 Keratins 1 and 10 Irvine and McLean (1999) hystrix KRT1 Sprecher et al (2001a) Epidermolytic PPK KRT9 Irvine and McLean (1999) Non-epidermolytic PPK KRT1, KRT16 Keratins 1 and 16 Irvine and McLean (1999) Ichthyosis bullosa Siemens KRT2e Keratin 2e Irvine and McLean (1999) , types 1 and 2 KRT6a, KRT6b, Keratins 6a, 6b, 16 and 17 Irvine and McLean (1999) KRT16, KRT17 White sponge naevus KRT4, KRT13 Keratins 4 and 13 Irvine and McLean (1999) X-linked recessive ichthyosis STS Steroid sulfatase Bonifas et al (1987) TGM1 Transglutaminase 1 Ishida-Yamamoto and Iizuka (1998) Mutilating with LOR Loricrin Maestrini et al (1996) ichthyosis Non-bullous congenital ALOXE3 Lipoxygenase-3 Jobard et al (2002) ichthyosiform erythroderma (NCIE) ALOX12B 12(R)-lipoxygenase Vohwinkel’s syndrome GJB2 26 Maestrini et al (1999) PPK with deafness GJB2 Connexin 26 Richard (2001) variabilis GJB3, GJB4 31 and 30.3 Richard (2001) Darier disease ATP2A2 ATP-dependent calcium transporter Sakuntabhai et al (1999) Striate PPK DSP, DSG1 , desmoglein 1 Armstrong et al (1999); Rickman et al (1999) Conradi^Hˇnermann^Happle EBP Delta 8-delta 7 sterol isomerase (emopamil Derry et al (1999) syndrome binding protein) Mal de Meleda ARS SLURP-1 (secreted Ly-6/uPAR related Fischer et al (2001) protein 1) Chanarin^Dorfman syndrome CGI 58 Member of esterase/lipase/thioesterase Lefevre et al (2001) subfamily Hair disorders Woolly hair, keratoderma and DSP, PG Desmoplakin, Norgett et al (2000); McKoy et al cardiomyopathy (Naxos disease) (2000) Generalized atrichia with papular VDR Vitamin D receptor Zhu et al (1998) lesions Congenital atrichia HR Hairless (a ) Ahmad et al (1999) HHB1, hHB6 Hair cortex keratins 1 and 6 Korge et al (1998); Winter et al (1997) Familial cylindromatosis CYLD1 Tumor-suppressor protein Bignell et al (2000) Pigmentation disorders (di¡erent forms) TYR Tyrosinase Boissy and Nordlund (1997) p Putative membrane transporter protein Boissy and Nordlund (1997) OA1 Member of -coupled receptors Boissy and Nordlund (1997) TRP1 Tyrosinase-related protein Boissy and Nordlund (1997) OCA4 MATP, a membrane-associated transporter Newton et al (2001) protein Chediak^Higashi syndrome CHS1 Lysosomal tra⁄cking regulator Nagle et al (1996) Hermansky^Pudlak syndrome HPS1 Putative transmembrane protein with Boissy and Nordlund (1997) unknown function ADTB3A Coat protein involved in vesicle formation HPS3 Protein with unknown function Anikster et al (2001) HPS4 Protein with unknown function Suzuki et al (2002) KIT Proto-oncogene, a transmembrane tyrosine Giebel and Spritz (1991) kinase receptor for stem cell factor Tietz syndrome MITF Microphthalmia-associated transcription Nordlund et al (1998) factor PAX3, MITF Transcription factors Nordlund et al (1998) Phenylketonuria PAH Phenylalanine hydroxylase DiLella et al 1986 Richner-Hanbart syndrome TAT Tyrosine aminotransferase Natt et al., 1992 8 UITTO ET AL JID SYMPOSIUM PROCEEDINGS

Table I. Continued. Diseasea Mutated geneb A¡ected protein/function Referencec

Porphyrias Congenital erythropoietic porphyria UROS Uroporphyrinogen III synthase Sassa and Kappas (2000) Erythropoietic protoporphyria FECH Ferrochelatase Sassa and Kappas (2000) Familial porphyria cutanea tarda URO-D Uroporphyrinogen decarboxylase Sassa and Kappas (2000) Variegate porphyria PPO Protoporphyrinogen oxidase Sassa and Kappas (2000) Coproporphyria CPO Coproporphyrinogen oxidase Sassa and Kappas (2000) Multisystem disorders Gingival ¢bromatosis type I SOS1 Guanine nucleotide exchange factor Hart et al (2002) Hypotrichosis with juvenile macular CDH3 P-cadherin Sprecher et al (2001b) dystrophy Trichothiodystrophy XPB, XPD Complementation groups B and D Berneburg and Lehmann (2001) Tricho-rhino^phalangeal syndrome TRPSI Zinc ¢nger protein Momeni et al (2000) type I GAN Gigaxonin Bomont et al (2000) Cockayne syndrome CSA/CKN1, CSB Transcription-repair coupling factors Nakura et al (2000) Human nude/SCID WHN Winged-helix transcription factor Frank et al (1999) Fabry’s disease GLA Alpha-galactosidase A Peters et al (1997) Ataxia telangiectasia ATM Protein kinase Spacey et al (2000) Hereditary hemorrhagic ENG, Endoglin Azuma (2000) telangiectasia ALK-1 Activin receptor-like kinase I Papillon^LefeŁ vre syndrome CTSC Cathepsin C Toomes et al (1999); Nakano et al (2001c) Haim^Munk syndrome CTSC Cathepsin C Hart et al (2000) , X-linked, DKC1 Dyskerin Marciniak et al (2000) autosomal dominant hTR RNA component of telomerase Vulliamy et al (2001) SPINK5 LEKTI (lympho-epthelial Kazal-type Chavanas et al (2000) inhibitor) Lipoid proteinosis ECM1 Extracellular matrix protein 1 Hamada et al (2002) Multiple cutaneous and uterine FH Fumarate hydratase Tomlinson et al (2002) leiomyomatosis Sj˛gren^Larsson syndrome FALDH Fatty aldehyde dehydrogenase Rizzo (1998) Refsum disease PHYH Phytanoyl-CoA hydroxylase Gould and Valle (2000) Hyperkeratotic cutaneous capillary- CCM1 KRIT1 (KREV1 interacting protein) Eerola et al (2000) venous malformation Milroy’s disease VEGFR-3 Vascular endothelial growth factor Irrthum et al (2000) receptor Glomuvenous malformations VMBLOM Glomulin Brouillard et al (2002) Waardenburg^Hirschsprung EDN3 Endothelin-3 Edery et al (1996) syndrome EDNRB Endothelin receptor B Attie et al (1995) SOX10 Transcription factor Pingault et al (1998) ELN, FBLN5 , Fibulin 5 Uitto and Pulkkinen (2002); Loeys et al, 2002 Williams syndrome ELN Contiguous gene syndrome, Uitto and Pulkkinen (2002) including elastin Marfan syndrome FBN1 1 Le Parc et al (2000) Pseudoxanthoma elasticum ABCC6 MRP6, a multidrug resistance associated Uitto et al (2001) protein Ehlers^Danlos syndrome COL1A1, COL1A2, Type I, III and V collagens, procollagen Steinmann et al (2002); Mao and (di¡erent variants) COL3A1, COL5A1, N-peptidase, procollagen-lysine 2- Bristow (2001); COL5A2, oxoglutarate 5-dioxygenase (lysyl Okajima et al (1999); Schalkwijk et al ADAMTS2, hydroxylase), xylosylprotein 4-beta- (2001) PLOD, B4GALT7, galactosyl-transferase, tenascin-X TNX Progressive osseous heteroplasia GNAS1 Gsa,ana-subunit of the stimulatory G Shore et al (2002) (POH) protein Menkes’ syndrome ATP7A ATP-dependent copper transporter Kaler (1998) Occipital horn syndrome ATP7A ATP-dependent copper transporter Kaler (1998) Werner syndrome WRN DNA helicase Nakura et al (2000) BLM DNA helicase Nakura et al (2000) Rothmund^Thomson syndrome RECQ4 DNA helicase Nakura et al (2000) Neuro¢bromatosis NF1, NF2 Neuro¢bromins 1 and 2 MacCollin and Kwiatkowski (2001) TSC1,TSC2 Hamartins 1 and 2 MacCollin and Kwiatkowski (2001) RAB27A, MYO5A Ras-associated protein, Va Menasche et al (2000); Pastural et al (2000) Wiskott^Aldrich syndrome WAS WASP (Arp2/3 complex interacting Nonoyama and Ochs (1998) protein) VOL. 7, NO. 1 DECEMBER 2002 HERITABLE SKIN DISEASES 9

Table I. Continued. Diseasea Mutated geneb A¡ected protein/function Referencec

Ectrodactyly, ectodermal dysplasia, TP63 Tumor protein p63 Celli et al (1999) and cleft lip/palate syndrome 3 (EEC3) Hay^Wells syndrome (AEC) TP63 Tumor protein p63 McGrath et al (2001) Hyperammonemia with reduced P5CS Delta(1)-pyrroline-5-carboxylate synthase Baumgartner et al (2000) ornithine, citrulline, , and proline Hereditary angiodema C1NH C1 esterase inhibitor Bowen et al (2001) Hidrotic ectodermal dysplasia GJB6 Connexin 30 Lamartine et al (2000) Anhidrotic ectodermal dysplasia, ED1 Ectodysplacin A Kere et al (1996) X-linked autosomal recessive and dominant DL Ectodysplacin A receptor Monreal et al (1999) X-linked anhidrotic ectodermal IKBKG NEMO (modulator of NF-kappaB Do⁄nger et al (2001) dysplasia with immunode¢ciency signaling) (EDA-ID), osteopetrosis and (OL-EDA-ID) IKBKG NEMO (modulator of NF-kappaB Berlin et al (2002); Smahi et al (2000) signaling) Cartilage-hair hypoplasia RMRP Endoribonuclease RNase MRP RidanpÌÌ et al (2001) Bruton agammaglobulinemia BTK BPK, tyrosine kinase Jones and Gaspar (2000) Hyper^IgM syndrome TNSF5 CD40 ligand, a member of tumor necrosis Jones and Gaspar (2000) factors Chronic granulomatous disorder CYBA P22 phox, a-subunit of cytochrome b Jones and Gaspar (2000) CYBB Gp91 phox, b-subunit of cytochrome b NCF1 Neutrophil cytosol factor-1 NCF2 Neutrophil cytosol factor-2 Blau syndrome CARD15/NOD2 Member of the family of Miceli-Richard et al (2001) regulators Carney complex PRKAR1A Protein kinase A regulatory subunit 1a Kirschner et al (2000) Muckle^Wells syndrome CIAS1 Angiotensin/vasopressin receptor AII/Avp Dode et al (2002) Familial cold urticaria CIAS1 Angiotensin/vasopressin receptor AII/Avp Dode et al (2002) Acrodermatitis enteropathica SLC39A4 hZIP4, a zink transporter protein Kˇry et al (2002) Multiple-Lentigines/LEOPARD PTPN11 Protein-tyrosine , nonreceptor- Digilio et al (2002) syndrome type, 11 Homocystinuria CBS Cysiathione b-synthetase Kruger et al,1995 Hunter’s syndrome IDS Iduronate 2-sulfatase Wilson et al,1990 Hurler’s syndrome IDUA a-urodinase Scott et al,1995 Nail-Patella syndrome LMX1B Homeobox domain transcription factor Morello et al,2001 MEN 1 MEN1 Menin, Jun D binding protein Bassett et al,1998 CERP Cholesterol e¥ux regulatory protein Brooks-wilson et al,1999 Alkaptonuria HGD Homogentisic acid oxidase Fernandez-Canon et al,1996 Familial Mediterranean fever MEFV Marenstrin, PMN inhibitor International FMF Consortium, 1997 Farber’s disease ASAH Acid ceramidase Koch et al,1996 Gaucher’s disease GBA b-glucocerebrosidase Tsuji et al, 1987 Cancer disorders PTEN Phosphatase and tensin homolog Bonneau and Longy (2000) Bannayan^Zonan syndrome PTEN Phosphatase and tensin homolog Bonneau and Longy (2000) Basal cell syndrome PTC Patched (drosophila homolog) Bale et al (1998); Ingham et al (1998) Hereditary melanoma CDK4 Cyclin-dependent kinase 4 Platz et al (2000) CDKN2A Cyclin-dependent kinase inhibitor 2a Muir^Torre syndrome MSH2 Mismatch repair protein Dore et al (1999) Peutz^Jeghers syndrome STK11/LKB1 Protein kinase Rowan et al (1999); Dong et al (1998) XPA, XPB, XPC, Complementation groups A-G Berneburg and Lehmann (2001) DNA polymerase XPD, XPE, XPF, Z (eta) (di¡erent complementation groups) XPG, hRAD30 aEB, epidermolysis bullosa; PPK, . bFor details of the genes, see Online Mendelian Inheritance in Man (OMIM) database, www.ncbi-mim.nih.gov/Omim/, or the corresponding references. cIf a recent, comprehensive review on the molecular genetics of the corresponding disease exists, the reference is included. In other cases, pertinent original publications are referenced. dFor details on EB, see Tab l e I I and the text. (Modi¢ed from Pulkkinen et al, 2002a.) extracutaneous manifestations encountered in di¡erent variants junctional, and dystrophic types (Fine et al, 2000). More recently, of EB, can cause considerable morbidity and, in some cases, pre- we have proposed an additional subgroup, the hemidesmosomal mature demise of the a¡ected individuals. EB has been tradition- variants of EB, which include generalized atrophic benign EB, ally divided into three broad categories based on the level EB with pyloric atresia, and EB with muscular dystrophy (Pulk- of tissue separation within the cutaneous BMZ: the simplex, kinen and Uitto, 1998; Tab l e I I ). As of today, mutations have 10 UITTO ET AL JID SYMPOSIUM PROCEEDINGS

Table II. Molecular heterogeneity of epidermolysis bullosa Variant of EBa Cleavage plane Mutated genes/polypeptides Chromosomal A¡ected protein systems/interactions Locus

Simplex Basal keratinocytes KRT5/ (K5) 12q13 Keratins 5 and 14, the major components of KRT14/ (K14) 17q12-q21 intermediate ¢laments expressed in basal cells Hemidesmosomal Hemidesmosome^ lamina lucida interface EB-MD PLEC1/Plectin 8q24 Plectin, located in the inner plaque of hemidesmosomes, interacts with intermediate ¢laments providing integrity to the basal keratinocytes EB-PA ITGA6/a6 integrin 2q24-q31 The subunit components of a6b4 integrin, a ITGB4/b4 integrin 17q25 transmembrane complex extending from the outer plaque of hemidesmosomes to the lamina lucida GABEB COL17A1/a1 chain of type XVII 10q24.3 Type XVII collagen/the 180-kD bullous collagen (BPAG2/the 180-kD pemphigoid antigen, a transmembrane component bullous pemphigoid antigen of anchoring ¢laments, traverses lamina lucida providing integrity to the basement membrane zone Junctional Lamina lucida LAMA3/laminin a3-chain 18q11.2 The a3-, b3-,and g2-chains are subunit LAMB3/laminin b3 chain 1q32 polypeptides of laminin 5, located in the lamina LAMC2/laminin g2-chain 1q25-q31 densa/lamina lucida interface, interacts with a6b4 integrin and type VII collagen Dystrophic Papillary dermis COL7A1/a1 chain of type VII 3p21.1 TypeVII collagen is a major component of collagen anchoring ¢brils extending from lamina densa to the papillary dermis aEB, epidermolysis bullosa; EB-MD, EB with muscular dystrophy; EB-PA, EB with pyloric atresia; GABEB, generalized atrophic benign EB. been identi¢ed in 10 distinct genes expressed at the cutaneous inheritance, de novo dominant vs mitis recessive DEB (Hashimoto BMZ in di¡erent variants of EB (Pulkkinen and Uitto, 1999). et al, 1999). As discussed below, this dilemma can be resolved by Examination of the mutation database suggests that the types DNA analysis of the mutations in the type VII collagen gene and combinations of the mutations, their positions along the (COL7A1). a¡ected , and the dynamic interplay of the mutant Distinct mutations have been disclosed in COL7A1 both in alleles on the individuals’ genetic background will determine the DDEB and RDEB in a large number of families (Tab l e I I ). A continuum of severity in the spectrum of clinical manifestations characteristic genetic lesion in the most severe recessive DEB is of EB. premature termination codon-causing mutation, resulting either Particularly instructive regarding the progress made in under- from a nonsense mutation or from small out-of-frame insertions standing the molecular basis of di¡erent variants of EB and in or deletions or splicing mutations in both COL7A1 alleles. These explaining the clinical spectrum of the disease are the dystrophic mutations are distributed along the entire type VII collagen mo- forms of EB (DEB).There is considerable genetic and phenotypic lecule, and most of them are family speci¢c, only a few of them variability in DEB. First, the DEB can be inherited in either an being recurrent (Fig 1). A characteristic genetic lesion in domi- autosomal dominant or autosomal recessive fashion. Second, the nantly inherited forms of DEB is a glycine substitution mutation clinical severity is highly variable demonstrating a spectrum of in one allele of COL7A1 residing within the collagenous domain manifestations. For example, the most severe form of DEB, the of the proa1(VII) chain (Fig 1). The mutated polypeptides are Hallopeau^Siemens type, manifests with mutilating scarring full-length and capable of assembling into triple-helical collagen with joint contractures, corneal erosions, esophageal strictures, molecules in combination with wild-type polypeptides (Uitto and propensity to formation of cutaneous squamous cell carcino- and Pulkkinen, 2001); however, the presence of the glycine sub- mas that can lead to premature demise of the a¡ected individuals stitution destabilizes the collagenous triple-helix, and the pre- (Fine et al, 2000). A clinically milder form, the mitis variant of sence of the mutated polypeptides causes a dominantly inherited recessive DEB, is characterized by life-long blistering tendency, disease through dominant negative interference. Thus, analysis of with limited scarring and less frequent extracutaneous manifesta- the type of mutations and their combinations along the type VII tions. In fact, in some cases the clinical severity of mitis recessive collagen gene provides an explanation for the di¡erent modes of DEB can manifest only with subtle nail changes and occasional inheritance (dominant versus recessive) and the severity of the dis- blistering. ease, even though the mutations reside in the same gene In general, patients with dominantly inherited forms of DEB (COL7A1) (Pulkkinen and Uitto, 1999). tend to have a relatively mild phenotype, and they often show blistering only on the hands and feet, and occasionally there is only minimal cutaneous involvement with nail dystrophy. Thus, THE GENETIC BASIS OF PXE the dominantly inherited DEB can be indistinguishable from the mitis recessive DEB on clinical, immunohistochemical, and ul- Another example of a condition in which recent progress has trastructural grounds. Speci¢cally, besides the mild clinical fea- provided understanding of the molecular basis of a tures, immunohistochemistry is positive, although occasionally is PXE, a systemic heritable disorder character- attenuated, for type VII collagen epitopes, and diagnostic trans- ized by progressive calci¢cation of elastic structures in the skin, mission electron microscopy usually reveals the presence of some the eyes, and the cardiovascular system. Certain clinical features, anchoring ¢brils which, however, can be either reduced in num- including delayed onset of the clinical manifestations and consid- ber or morphologically altered in both forms of DEB. This situa- erable intra- and interfamilial phenotypic variability, have been tion then poses a diagnostic dilemma concerning the mode of puzzling (Neldner, 1988). Adding to the complexity of PXE is VOL. 7, NO. 1 DECEMBER 2002 HERITABLE SKIN DISEASES 11

Figure1. Illustration of the molecular heterogeneity in the dystrophic forms of EB. The structural organization of the type VII collagen a1(VII) polypeptide deduced from the primary nucleotide sequence of full-length cDNA. The polypeptide consists of a collagenous domain that is £anked by N- and C-terminal noncollagenous domains with modular structures, as indicated on the lower left corner. The arrows indicate positions of distinct mutations disclosed in families with dystrophic forms of EB. The mutations depicted above the a1(VII) collagen polypeptide are recessive, and most of them cause premature termination codons, either as a result of nonsense mutations, insertions, or deletions, or as out-of-frame exon skipping mutations, predicting synthesis of a truncated polypeptide or downregulation of the corresponding mRNA transcript through nonsense-mediated mRNA decay.The mutations depicted below the polypeptide are glycine substitution mutations within the collagenous domain. The majority of these missense mutations cause dom- inantly inherited dystrophic EB through dominant negative interference. (Modi¢ed from Pulkkinen and Uitto, 1999.) the fact that both autosomal recessive and autosomal dominant some 16, and the critical interval was further narrowed to B500 inheritance patterns have been reported, and in many cases PXE kb within the chromosomal region 16p13.1 (Le Saux et al, 1999; is sporadic without family history, the precise mode of inheri- Cai et al, 2000). Examination of the genome database revealed tance thus being unclear (Pope, 1975). Furthermore, the genetic that this region contained four distinct genes with no apparent complexity has been compounded by suggestions that obligate relation to the extracellular matrix of connective tissue in general heterozygous carriers in families with autosomal recessive forms or the elastic ¢ber system in particular. Two of the genes, ABCC1 of PXE may show minimal manifestations of the disease and ABCC6, encode proteins, MRP1 and MRP6, respectively, (Bacchelli et al, 1999; Sherer et al, 2001). Nevertheless, the involve- that are members of the multiple drug resistance-associated pro- ment of the elastic structures in the skin, the eyes, and the cardi- tein (MRP) family. Another gene, NPIP, was found to encode ovascular system, and the accumulation of a number of the nuclear pore interacting protein, whereas the fourth gene, extracellular matrix components, including ¢bronectin and pM5, encodes a protein with unknown function but shares some vitronectin as well as various proteoglycan/glycosaminoglycan similarity with the conserved domain within the collagenase complexes, in the lesional areas of skin in association with elastic family of proteins. ¢bers, have suggested that PXE is a primary heritable connective Sequencing of the four candidate genes within the critical tissue disease. In fact, it has been considered as a prototype of PXE interval resulted in identi¢cation of pathogenetic mutations such conditions with the primary involvement of elastic ¢bers in the ABCC6 gene, which encodes MRP6, a member of the (McKusick, 1972). ATP-dependent membrane transporter family of proteins (Le The underlying molecular defect in PXE has remained un- Saux et al, 2000; Ringpfeil et al, 2000). Sequence information known until recently. Initial observations indicating pathology derived from full-length ABCC6 cDNA predicts that MRP6 in the elastic ¢bers suggested that elastin and elastin-associated has three transmembrane spanning domains and two intracellular micro¢brillar proteins could serve as candidate gene/protein sys- nucleotide-binding folds (NBF1 and NBF2) (Fig 2). The NBF tems in PXE. Subsequently, however, the genes encoding elastin domains contain conserved Walker motifs, critical for ATP bind- as well as elastin-associated proteins, such as ¢brillins 1 and 2 and ing and for the function of the protein as a transmembrane trans- lysyl oxidase on 15 and 5, were excluded by genetic porter. Mutation detection strategies have identi¢ed a number of linkage analysis. Subsequent positional cloning approaches al- single- substitutions, resulting in missense or nonsense lowed mapping of the PXE gene to the short arm of chromo- mutations, as well as several small insertions and deletions or 12 UITTO ET AL JID SYMPOSIUM PROCEEDINGS

Figure 2. Schematic representation of MRP6, a putative transporter protein, altered by mutations in PXE. As shown, MRP6 consists of three transmembrane domains with ¢ve, six, and six transmembrane spanning segments, respectively.The protein is predicted to have two intracellular nucleotide binding folds (NBF1 and NBF2). The repertoire of mutations within MRP6, as recently published (Cai et al, 2001; Le Saux et al, 2001; Meloni et al,2001; Ringpfeil et al, 2001; Pulkkinen et al, 2001) is illustrated by arrows pointing to the regions a¡ected by mutations. The majority of the mutations replace critical residues within the intracellular domains of MRP6 or cause premature termination of . Note the presence of large deletion mutations depicted on the top of the ¢gure. Two recurrent mutations, R1141X and del exons 23^29, are in bold. EC, extracellular; IC, intracellular. large deletions in the ABCC6 gene (Fig 2). The majority of the strong with other members of the MRP fa- mutations reside in the carboxyl-terminal half of the MRP6 mily, particularly with MRP1, the prototypic protein within the polypeptide, a¡ecting critical arginine residues or other con- family, which functions as an e¥ux pump for amphipathic and served amino acids. Furthermore, premature termination codon glutathione conjugates. These observations suggest that MRP6 is mutations or large deletions frequently lead to elimination of a metabolic pump expressed in the liver and the kidneys, and the third transmembrane domain and the second nucleotide- PXE could potentially be considered as a metabolic disorder, binding fold. Finally, gene conversion between ABCC6 and one rather than being a primary connective tissue disease (Uitto et al, of its pseudogenes has been suggested as a pathogenetic event in 2001). One could speculate that when MRP6 as a cellular trans- some families with PXE (Cai et al, 2001). The mutations in porter is genetically altered to be nonfunctional, accumulation of ABCC6 are present either in a homozygous or in a compound yet to be identi¢ed compounds in the circulation could take heterozygous state in the a¡ected individuals, whereas heterozy- place. Such compounds could have a⁄nity for elastic ¢bers and gous carriers are clinically una¡ected, consistent with autosomal calcium and potentially lead to progressive calci¢cation of elastic recessive inheritance pattern. In fact, no molecular evidence for structures in the a¡ected organs, resulting in progressive degen- autosomal dominant mode of inheritance has been demonstrated eration and pathology in the target tissues. as yet, although some of the heterozygous carriers may demonstrate minimal manifestations of the disease (Sherer et al,2001).Also, in some families, the proposed autosomal dominant mode THE IMPACT OF MOLECULAR GENETICS ON PATIENT of inheritance could be explained by a pseudodominant family CARE pedigree due to consanguinity in the family (Ringpfeil et al, 2000). The progress made in understanding EB, PXE, and other geno- A surprising ¢nding in the context of clinical manifestations dermatoses raises a number of critical questions: What are the of PXE is the fact that ABCC6/MRP6 is expressed predomi- bene¢ts of this progress in basic research to the patients and nantly, if not exclusively, in the liver and the kidneys. The biolo- their families? How can we translate this basic information to gical function of MRP6 is currently unknown, but there is improved patient care? Is there anything that the practicing VOL. 7, NO. 1 DECEMBER 2002 HERITABLE SKIN DISEASES 13 dermatologist can convey to the bene¢t of the patients concern- disease. Such testing can be performed from a chorionic villus ing their disease and its molecular basis? In other words, what is sample as early as the tenth week of gestation or from early am- the impact of molecular genetics on the patient care? Collectively, niocentesis performed at the twelfth week. In fact, prenatal test- signi¢cant bene¢ts are already accruing from the basic research ing has already been established for EB and is readily available for on heritable blistering skin diseases, and expansion of the re- the severe forms of recessive DEB and for the Herlitz type of JEB search database will provide additional insights into the clinical during the ¢rst trimester of pregnancy (Christiano et al,1996, perspective of these conditions. 1997). Prenatal predictions have also been made in cases with EB For example, the immediate bene¢ts for EB have already ma- with pyloric atresia, a frequently lethal variant of EB (Nakano terialized through improved, molecularly based diagnosis with et al, 2001b). DNA-based analysis has essentially replaced the pre- re¢ned classi¢cation, which allows better prognostication regard- viously employed fetal skin biopsy, which is performed late dur- ing the severity and the natural progress of the disease. An exam- ing the second trimester, as a prenatal diagnostic tool for EB. ple is provided by the junctional form of EB (JEB), which has An extension of DNA-based prenatal testing is the develop- been traditionally divided, on the basis of the clinical outcome, ment of preimplantation genetic diagnosis (PGD), which is per- to two broad categories: (i) the Herlitz variant of JEB that is formed in conjunction with in vitro fertilization (McGrath and usually lethal during the ¢rst few months of life; and (ii) the Handyside, 1998). In this procedure, the fertilized embryos are al- non-Herlitz variant that demonstrates persistent blistering ten- lowed to grow in vitro to the eight-cell stage level, at which time dency throughout the life, without signi¢cantly compromising one cell is removed for mutation analysis. Subsequently, embryos the overall lifespan. Molecular analysis of the JEB patients’ DNA lacking the mutation are implanted into the uterus to establish has revealed that those with the Herlitz variant harbor, as a gen- pregnancy, as routinely done as part of in vitro fertilization proto- eral rule, a premature termination codon mutation, i.e., a gene col, therefore excluding the recurrence of the disease in the fa- defect that predicts synthesis of a truncated and nonfunctional mily. Thus, couples with a child previously a¡ected with EB, or polypeptide, in both alleles of any of the three genes encoding any other severe genodermatosis in which the molecular basis is laminin 5 subunits (LAMA3, LAMB3, and LAMC2) (Nakano known, can now initiate the next pregnancy by knowing that et al, 2000). In contrast, patients with the non-Herlitz variants there are ways to ¢nd out the genotype of the fetus at the early harbor frequently a missense mutation, i.e., an amino acid substi- stages of pregnancy through DNA-based prenatal testing or even tution-causing mutation, in one or both alleles of the corre- before the pregnancy is established by applying PGD. sponding genes. Thus, analysis of DNA from a newborn with Finally, it is evident that identi¢cation of the underlying mo- clinical, histopathologic, and ultrastructural evidence of JEB al- lecular defects in genodermatoses is a prerequisite for develop- lows general predictions as to whether the disease is the severe ment of successful therapies in the future. In particular, Herlitz (lethal) variant, or whether the individual is expected to development of approaches requires precise knowl- have relatively mild non-Herlitz JEB with a normal lifespan (Na- edge of the mutations in the a¡ected genes and their conse- kano et al,2001a). quences at the mRNA and protein levels (Uitto and Pulkkinen, Another example of the impact of molecular genetics relates 2000). Although successful application of gene therapy for treat- to genetic counseling of families at risk for recurrence of the ment of EB may still be several years away, rapid development of disease in the same and subsequent generations. An example new technologies or promising breakthroughs could lead to is provided by a ‘‘sporadic’’ patient with relatively mild DEB durable gene therapy for these devastating skin diseases in with no family history of the disease, and speci¢cally, both the future. parents being clinically normal. The disease in the a¡ected individual could result either from a new (de novo) dominant mutation in one allele of the type VII collagen gene, or the FUTURE PROSPECTS: COMPLEX DISORDERS AND disease could be a mild (mitis) recessive DEB due to mutations GENETIC SUSCEPTIBILITY in both type VII collagen alleles, inherited separately from each parent. These two possibilities are indistinguishable by As illustrated above, signi¢cant progress has been made towards clinical examination, as well as by histopathologic, immuno- understanding the molecular basis of a large number of genoder- histochemical, and ultrastructural analysis (Hashimoto et al, matoses, and in fact, the majority of the single gene disorders 1999). This diagnostic dilemma can be solved, however, by analy- may have been mapped to distinct genetic loci and the underly- sis of mutations in the DNA from the a¡ected individual and his/ ing mutated genes have been identi¢ed. Nevertheless, the mole- her parents. Speci¢cally, presence of a single mutation in the cular basis of a number of Mendelian disorders still awaits proband, in the absence of the corresponding mutation in clari¢cation, and it is becoming increasingly evident that a num- the parents’ DNA isolated from peripheral blood, indicates a ber of disorders can be caused by defects in di¡erent genes with de novo dominant mutation. In contrast, identi¢cation of two resulting phenocopies or closely resembling phenotypes (Tab l e I ). mutant alleles in the proband’s DNA and demonstration of their An example of such molecular diversity is Vohwinkel’s syndrome, presence in the respective parents signi¢es mitis recessive DEB. characterized by mutilating keratoderma with pseudoainhum, The implications are, of course, that the risk of an a¡ected indivi- ichthyosiform erythroderma, and deafness. Our original attempts dual in the case of de novo dominant DEB of having an a¡ected to clarify the genetic basis of Vohwinkel’s syndrome in a family child is one in two, or 50%, whereas the risk of the individual with keratoderma and erythroderma resulted in identi¢cation of with recessive DEB having an a¡ected o¡spring is very low, re- mutations in the gene encoding loricrin, a corni¢ed envelope £ecting the relatively low carrier frequency of EB (Pfendner et al, protein expressed in the epidermis (Maestrini et al, 1996). Subse- 2001). At the same time, the risk for the parents of the patient quently, however, examination of families with classic, more with a de novo dominant mutation of having another a¡ected complete forms of Vohwinkel’s disease manifesting with deafness, child is relatively low, whereas the risk for the parents of the pa- led to identi¢cation of mutations in the GJB2 gene encoding tient with a recessive DEB having another a¡ected o¡spring is connexin 26, a cell^cell communication protein (Maestrini et al, one in four, or 25%. 1999). As another example, di¡erent variants of X-linked anhi- drotic ectodermal dysplasia have been shown to result from mu- tations, in addition to ED1, in the IKBKG gene that encodes PRENATALTESTING, PREIMPLANTATION GENETIC NEMO, a subunit of the IkB kinase complex (Tab l e I ). At the DIAGNOSIS, AND GENE THERAPY same time, incontinentia pigmenti, a complex X-linked domi- nant disorder, is also due to mutations in the IKBKG gene (Berlin A consequence of the identi¢cation of speci¢c mutations in gen- et al, 2002). The dramatic phenotypic di¡erences in X-linked ec- odermatoses has been the development of DNA-based prenatal todermal dysplasias, either with immunode¢ciency (EDA-ID) or testing for families at risk for recurrence of severe forms of the together with osteopetrosis and lymphedema (OL-EDA-ID), and 14 UITTO ET AL JID SYMPOSIUM PROCEEDINGS

Table III. Examples of putative susceptibility genes in complex skin diseases Disease Chromosomal region Candidate gene Corresponding protein References

Systemic lupus erythematosus (SLE) 16p13.3 DNASE1 Deoxynuclease 1 Yasutomo et al (2001) 6p22.2-p21.3 PRL Prolactin Stevens et al (2001) -related SLE 17p13 SLEV1 Unknown Nath et al (2001) Atopic dermatitis 5q32 SPINK LEKT1 (see Tab l e I ) Walley et al (2001) Psoriasis 6p21 HCR Putative transcription factor Asumalahti et al (2002) 6p21 HLA-C Major histocompatibility Mallon et al (1999) complex, class I, C; HLA-C Systemic sclerosis 15q21.1 FBN1 Fibrillin1 Tan et al (2001) Systemic sclerosis with ¢brosing alveolitis 2q34 FN Fibronectin Avila et al (1999) in incontinentia pigmenti can be explained by the consequences REFERENCES of the mutations within the same gene. Speci¢cally, loss of func- tion mutations in IKBKG, which abolish NFkB signaling, result Ahmad W,PanteleyevAA, Christiano AM:The molecular basis of congenital atrichia in humans and mice: mutations in the hairless gene. J Invest Dermatol Symp Proc in incontinentia pigmenti (Smahi et al, 2000). In contrast, muta- 4:240^243, 1999 tions which impair but do not abolish the NFkB signaling are Anikster Y, Huizing M,White J, et al: Mutation of a new gene causes a unique form associated with EDAvariants with immune de¢ciency (Do⁄nger of Hermansky^Pudlak syndrome in a genetic isolate of central Puerto Rico. et al, 2001). Furthermore, EDA variants without immune de¢- Nat Genet 28:376^380, 2001 Armstrong DK, McKenna KE, Purkis PE, Green KJ, Eady RA, Leigh IM, Hughes ciency, can be caused by mutations in two di¡erent genes. The AE: Haploinsu⁄ciency of desmoplakin causes a striate subtype of palmoplan- X-linked variants are due to mutations in the ED1 gene encoding tar keratoderma. Hum Mol Genet 8:143^148, 1999 ectodysplacin A, whereas the autosomal variants with essentially Asumalahti K, Veal C, Latinen T, et al: Coding haplotype analysis supports HCR as identical phenotypes are due to mutations in the ectodysplacin A the putative susceptibility gene at the MHC PSORS1 locus. Hum Mol Genet 11:589^597, 2002 receptor gene (DL) (Kere et al, 1996; Monreal et al, 1999). Both of Attie T,Till M, Pelet A, et al: Mutation of the endothelin-receptor B gene in Waar- these gene products participate in NFkB signaling. Thus, further denburg-Hirschsprung disease. Hum Mol Genet 4:2407^2409, 1995 expansion of the molecular basis of single gene disorders, with Avi la JJŁ, Lympany PA, Pantelidis P,Welsh KI, Black CM, duBois RM: Fibronectin extended mutation databases towards re¢nement of genotype/ gene polymorphisms associated with ¢brosing alveolitis in systemic sclerosis. Am J Respir Cell Mol Biol 20:106^112, 1999 phenotype correlations, is still required for understanding of a Azuma H: Genetic and molecular pathogenesis of hereditary hemorrhagic telangiec- number of single gene disorders. tasia. J Med Invest 47:81^90, 2000 At the same time, the molecular diagnostics of genodermatoses Bacchelli B, Ouaglino D, Gheduzzi D, et al: Identi¢cation of heterozygote carriers in is expanding to include complex genetic disorders towards iden- families with a recessive form of pseudoxanthoma elasticum (PXE). Mod Pathol 12:1112^1123, 1999 ti¢cation of susceptibility genes (Tab l e I I I ). A prime example of Bale SJ, Falk RT, Rogers GR: Patching together the genetics of Gorlin syndrome. such conditions is psoriasis, which is clearly known to have a ge- J Cutan Med Surg 3:31^34, 1998 netic contribution with a major susceptibility locus residing on Bassett JHD, Forbes SA, Pannett AAJ, et al: Characterization of mutations in patients 6 in association with the HLA locus. In fact, a num- with multiple endocrine neoplasia type 1. Am J Hum Genet 62:232^244, 1998 Baumgartner MR, Hu CA, Almashanu S, et al: Hyperammonemia with reduced or- ber of candidate genes have been recently identi¢ed, and evidence nithine, citrulline, arginine and proline: a new inborn error caused by a muta- in support of certain genes or in favor of exclusion of the corre- tion in the gene encoding delta(1)-pyrroline-5-carboxylate synthase. Hum Mol sponding genes is rapidly appearing in the literature (see, for ex- Genet 9:2853^2858, 2000 ample, Mallon et al, 1999; Chia et al, 2000; O’Brien et al,2001; Bergen AA, Plomp AS, Schuurman EJ, et al: Mutations in ABCC6 cause pseudox- anthoma elasticum. Nat Genet 25:228^231, 2000 Asumalahti et al, 2002). Further examples of such conditions are Berlin AL, Paller AS, Chan LS: Incontinentia pigmenti. A review and update autoimmune disorders, such as systemic lupus erythematosus and on the molecular basis of pathophysiology. JAmAcadDermatol47:169^187, 2002 vitiligo, for which a shared genetic susceptibility locus has been Berneburg M, Lehmann AR: Xeroderma pigmentosum and related disorders: recently linked to chromosome 17p13 (Nath et al,2001). Defects in DNA repair and transcription. Adv Genet 43:71^102, 2001 Bignell GR, Warren W, Seal S, et al: Identi¢cation of the familial cylindromatosis Another example of a relatively common, complex disorder is tumour-suppressor gene. Nat Genet 25:160^165, 2000 atopic dermatitis, and the loci in£uencing atopy have been loca- Boissy RE, Nordlund JJ: Molecular basis of congenital hypopigmentary disorders in lized to a number of chromosomal regions, including chromo- humans: a review. Pigment Cell Res 10:12^24, 1997 some 5q31. 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