Preimplantation genetic diagnosis of skin fragility-ectodermal dysplasia syndrome.

Skin fragility-ectodermal dysplasia syndrome is an autosomal recessive disorder caused by loss-of-function mutations in the desmosomal protein, plakophilin 1. Clinically, there may be considerable morbidity from extensive skin erosions and painful fissures on the palms and soles. In the absence of any specific treatment, prenatal diagnosis is an option for couples at reproductive risk of recurrence. In 2000, we developed and applied a single cell nested polymerase chain reaction protocol to test one couple for compound heterozygous plakophilin 1 gene mutations by preimplantation genetic diagnosis (PGD). Although pregnancy was established, an unrelated trisomy 22 led to a spontaneous abortion. However, eight embryos of known genetic status were cryopreserved at that stage, and we planned to undertake subsequent frozen embryo replacement cycles that might lead to the birth of an unaffected child in this family. Embryo cryopreservation was carried out in June 2000 using standard protocols in a three-step freezing procedure. Four embryos were thawed in March 2003, one of which was viable and was used in a frozen embryo replacement cycle, but pregnancy did not occur. The remaining four embryos were thawed in February 2004, two of which were viable (both carriers of the paternal mutation) and these were used in a second frozen embryo replacement cycle, and a singleton pregnancy was established. The child's plakophilin 1 genotype was assessed by direct nucleotide sequencing across the site of both potential mutations. Following two frozen embryo replacement cycles, and almost 4 years after the initial embryo biopsy and mutation analysis, a pregnancy was achieved that progressed to term with the birth of a healthy baby girl. Nucleotide sequencing of cord blood DNA, taken immediately after delivery, showed that the child was a heterozygous carrier of the paternal mutation but not of the maternal mutation. This case demonstrates the value of embryo cryopreservation, which can increase the number of embryo replacement procedures and hence the cumulative pregnancy rate per retrieval cycle. Moreover, this is the first report of successful full-term pregnancy and birth of a healthy baby following exclusion of a severe genodermatosis by PGD. The successful outcome of PGD in this case illustrates what is technically possible for couples at risk of recurrence of a severe inherited skin disease.

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype.

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

Pseudodominant inheritance of spondylocostal dysostosis type 1 caused by two familial delta-like 3 mutations.

Spondylocostal dysostoses (SCD) are a heterogeneous group of disorders of axial skeletal malformation characterized by multiple vertebral segmentation defects and rib anomalies. Sporadic cases with diverse phenotypes, sometimes including multiple organ abnormalities, are relatively common, and monogenic forms demonstrating autosomal recessive (AR) and, more rarely, autosomal dominant (AD) inheritance have been reported. We previously showed that mutations in delta-like 3 (DLL3), a somitogenesis gene that encodes a ligand for the notch signaling pathway, cause AR SCD with a consistent pattern of abnormal segmentation. We studied an SCD family previously reported to show AD inheritance, in which the phenotype is similar to that in AR cases. Direct DLL3 sequencing of individuals in two generations identified the affected father as homozygous for a novel frameshift mutation, 1440delG. His two affected children were compound heterozygotes for this mutation and a novel missense mutation, G504D, the first putative missense mutation reported in the transmembrane domain of DLL3. Their two unaffected siblings were heterozygotes for the 1440delG mutation. Pseudodominant inheritance has been confirmed, and the findings raise potential consequences for genetic counseling in relation to the SCD disorders.

Molecular prenatal diagnosis in a case of an X-linked dominant chondrodysplasia punctata.

X-linked dominant chondrodysplasia punctata, (CDPX2-MIM302960) also known as Conradi-Hünermann-Happle syndrome, is a rare form of skeletal dysplasia that affects the skeleton, skin, hair, and eyes. The disorder is caused by mutations within the emopamil binding protein (Ebp) that functions as a delta(8), delta(7) sterol isomerase in the cholesterol biosynthesis pathway. To date, over 40 separate mutations have been reported in the Ebp gene, EBP, with no obvious correlation between the molecular defects and the severity of the clinical phenotype. We have studied a 30-year-old woman who presented in adulthood with skin, hair, and mild skeletal defects but no ocular abnormalities and have identified a heterozygous missense mutation within the third transmembrane domain of the protein. In addition, we have performed molecular prenatal testing on her unborn fetus. The results demonstrate inter-familial variability for missense mutations within the emopamil binding protein and add to the molecular data for CDPX2.

Targetting of desmoglein 1 in inherited and acquired skin diseases.

Desmoglein 1 is a member of the desmosomal cadherin family that comprise the desmogleins and desmocollins. The desmoglein 1 gene (DSG1) is centromeric to the desmoglein gene cluster and spans approximately 45 kb of 18q12, comprising 15 exons. The transcript encodes a precursor protein of 1049 amino acids that is cleaved to yield a mature protein of 1000 residues. This mature protein is expressed in certain specialized epithelia, and in the epidermis is expressed within the superficial layers. Within the desmosome the extracellular domain of the protein is essential for calcium dependent heterophilic binding to the desmocollins, whereas the intracellular domain is essential for binding to the desmosomal plaque protein, plakoglobin. Desmoglein 1 has been implicated in several human diseases. Mutations within the extracellular domain lead to autosomal dominant striate palmoplantar keratoderma, whereas autoantibodies and strains of Staphylococcus aureus target the extracellular domain in the acquired bullous disorders pemphigus foliaceus and staphylococcal scalded skin syndrome, respectively. Therefore, intact and functionally active desmoglein 1 is essential to epidermal integrity. Here, we review the expression, protein structure, genetics, and molecular interactions of desmoglein 1 and outline the role it plays within the desmosome and how it becomes defective in human disease.

Genomic localization, organization and amplification of the human zinc transporter protein gene, ZNT4, and exclusion as a candidate gene in different clinical variants of acrodermatitis enteropathica.

Acrodermatitis enteropathica is an inherited disorder of zinc metabolism, the molecular basis of which is currently unknown. Recent transgenic mouse studies have highlighted the potential significance of certain zinc transport proteins, for example ZnT4, in providing clues to the pathogenesis of zinc-related disorders such as acrodermatitis enteropathica. Specifically, mice of any genotype suckled on ZnT4-deficient mice fail to absorb intestinal zinc and ZnT4-deficient mice also develop dermatitis, alopecia and stunted growth. Therefore, to assess human ZnT4 as a candidate gene/protein in acrodermatitis enteropathica or related disorders, we characterized the intron-exon organization of the human ZNT4 gene, which comprises seven distinct exons spanning approximately 38.7 kb. High-resolution radiation hybrid mapping placed ZNT4 on 15q21.1. We also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products. Using this approach, we sequenced DNA from five individuals with acrodermatitis enteropathica; no mutations were identified. Thus, ZNT4 is unlikely to be the correct candidate gene for this disorder. We also identified and characterized two common single nucleotide polymorphisms in exon 5 and in the 3' UTR of ZNT4, which will be useful for future genetic linkage studies in assessing ZNT4 as a candidate gene for other inherited disorders of zinc metabolism.

New mutations in keratin 1 that cause bullous congenital ichthyosiform erythroderma and keratin 2e that cause ichthyosis bullosa of Siemens.

The intermediate filaments of epithelial cells are formed by keratins, a family of structurally related proteins, which are expressed in pairs of acidic (type I) and basic (type II) polypeptides in a tissue- and differentiation-specific manner. Mutations in the genes encoding several keratins have been implicated in the pathogenesis of diseases of keratinization. We report molecular analysis of two patients with the rare autosomal dominant disorders bullous congenital ichthyosiform erythroderma (BCIE) and ichthyosis bullosa of Siemens (IBS). Previous studies have shown that these genodermatoses are due to mutations in the KRT1 and KRT2E genes, respectively. We report a new amino acid substitution mutation in codon 155 of KRT1 (valine to aspartic acid) in the conserved H1 domain of the protein in the patient with BCIE. We also report a novel amino acid substitution mutation in codon 192 of KRT2E (asparagine to lysine) in the conserved 1A helix initiation peptide of the protein in the patient with IBS. Our results demonstrate that these mutations are deleterious to keratin filament network stability and lead to specific clinical inherited disorders of keratinization.

Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.

The adhesive proteins of the desmosome type of cell junction consist of two types of cadherin found exclusively in that structure, the desmogleins and desmocollins, coded by two closely linked loci on human chromosome 18q12.1. Recently we have identified a mutation in the DSG1 gene coding for desmoglein 1 as the cause of the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK) in which affected individuals have marked hyperkeratotic bands on the palms and soles. In the present study we present the complete exon-intron structure of the DSG1 gene, which occupies approximately 43 kb, and intron primers sufficient to amplify all the exons. Using these we have analysed the mutational changes in this gene in five further cases of SPPK. All were heterozygotic mutations in the extracellular domain leading to a truncated protein, due either to an addition or deletion of a single base, or a base change resulting in a stop codon. Three mutations were in exon 9 and one in exon 11, both of which code for part of the third and fourth extracellular domains, and one was in exon 2 coding for part of the prosequence of this processed protein. This latter mutation thus results in the mutant allele synthesising only 25 amino acid residues of the prosequence of the protein so that this is effectively a null mutation implying that dominance in the case of this mutation was caused by haploinsufficiency. The most severe consequences of SPPK mutations are in regions of the body where pressure and abrasion are greatest and where desmosome function is most necessary. SPPK therefore provides a very sensitive measure of desmosomal function.

Genomic organization, amplification, fine mapping, and intragenic polymorphisms of the human hemidesmosomal tetraspanin CD151 gene.

CD151 is a member of the tetraspanin family that is involved in cellular processes including cell adhesion. The protein is expressed in a variety of tissues including vascular endothelium and epidermis, and has been shown to be a component of hemidesmosomes. Mutations in genes encoding other hemidesmosomal proteins give rise to a range of human disorders, characterized by fragility of the skin and/or mucous membranes. It is, therefore, plausible that inherited or acquired mutations in the gene encoding CD151 may be fundamental to the integrity and maturation of basal cell keratinocytes. To aid mutation analysis, we have characterized the intron-exon organization of the CD151 gene which comprises 8 exons spanning approximately 4.3 kb, and have developed a comprehensive PCR-based mutation detection strategy. In addition, to aid linkage analysis of CD151 in genetic disease we have fine-mapped the gene by radiation-hybrid methodology to 11p15.5, and detected a number of intragenic polymorphisms.

Genomic organization and amplification of the human desmosomal cadherin genes DSC1 and DSC3, encoding desmocollin types 1 and 3.

The desmosomal cadherins comprise the desmocollins and desmogleins and are involved in epithelial cell-cell adhesion. There are three desmocollins (DSC 1-3) and three desmogleins (DSG 1-3) that are expressed in a tissue- and development-specific manner. Desmosomal proteins have been implicated in a number of disorders characterized by loss of cell-cell adhesion and trauma-induced skin fragility. Therefore, the desmocollins are potential candidates for genodermatoses involving epithelial tissues. In order to screen the entire DSC1 and DSC3 genes, we have characterized their intron-exon organization. The DSC1 gene comprises 17 exons spanning approximately 33 kb on 18q12.1, and the DSC3 gene comprises 17 exons spanning approximately 49 kb on 18q12.1. We have also developed a comprehensive PCR-based mutation detection strategy for desmocollins 1, 2, and 3 using primers placed on flanking introns followed by direct sequencing of the PCR products.

Genomic organization and amplification of the human plakoglobin gene (JUP).

Plakoglobin is a globular protein common to the intracellular plaques of adhesive junctions, predominantly desmosomes and adherens junctions. Recently, a number of pathogenic mutations have been described in other components of desmosomes, specifically in plakophilin 1, desmoplakin and desmoglein 1. The phenotype of affected patients mainly involves thickening of palm and sole skin (keratoderma). Although no human mutations in plakoglobin have been described thus far, this protein represents an excellent candidate for other human genetic disorders, possibly involving skin and heart, sites of high plakoglobin expression. To facilitate future mutation detection analyses in such conditions, we have characterized the intron-exon organization of the human plakoglobin gene, which comprises 13 distinct exons spanning approximately 17 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.

The gene for Naegeli-Franceschetti-Jadassohn syndrome maps to 17q21.

Naegeli-Franceschetti-Jadassohn syndrome is a rare autosomal dominant form of ectodermal dysplasia affecting sweat glands, nails, teeth, and skin. We have studied a multigeneration family of Anglo-Saxon British descent using microsatellite markers to screen candidate loci, including the epidermal differentiation complex on 1q, the keratin gene clusters on chromosomes 12q and 17q and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 17q was observed using marker D17S 1787, with a maximum two-point LOD score of 4.166 at a recombination fraction of theta = 0. Recombination events in the family place the gene in a 26.97 cM interval between markers D17S798 and D17S957, a region known to contain the type I keratin gene cluster and other genes expressed in epithelia. Keratins K15, K19, and K20, plakoglobin, and MEOX1 were excluded as candidates by direct sequencing of genomic polymerase chain reaction products.

Genomic amplification of the human plakophilin 1 gene and detection of a new mutation in ectodermal dysplasia/skin fragility syndrome.

Ectodermal dysplasia/skin fragility syndrome is a recently described autosomal recessive disease affecting skin, nails, and hair (MIM 604536), that results from mutations in plakophilin 1, a structural component of desmosomes. We report a new plakophilin 1 mutation in an affected patient as well as detailing the intron-exon organization of the gene to facilitate future polymerase chain reaction-based mutation screening. Using polymerase chain reaction amplification of genomic DNA, we identified 15 exons spanning approximately 50 kb. Direct sequencing disclosed several nonpathogenic intragenic polymorphisms, as well as a homozygous splice site mutation (1233-2 A-->T; GenBank Z73678) in a 17 y old affected male. The clinical features comprised skin erosions, dystrophic nails, sparse hair, and painful thickening and cracking of palms and soles. Skin biopsy showed negative immunolabeling with an anti-plakophilin 1 antibody and small desmosomes. These results expand the database of plakophilin 1 mutations and demonstrate the importance of this protein in the stabilization of desmosomal adhesion in terminally differentiating keratinocytes.

Genomic organization and amplification of the human epidermal type II keratin genes K1 and K5.

Keratins are a family of structurally related proteins that form the intermediate filament cytoskeleton in epithelial cells. Mutations in K1 and K5 result in the autosomal dominant disorders epidermolytic hyperkeratosis/bullous congenital ichthyosiform erythroderma and epidermolysis bullosa simplex, respectively. Most disease-associated mutations are within exons encoding protein domains involved in keratin filament assembly. However, some mutations occur outside the mutation hot-spots and may perturb intermolecular interactions between keratins and other proteins, usually with milder clinical consequences. To screen the entire keratin 1 and keratin 5 genes we have characterized their intron-exon organization. The keratin 1 gene comprises 9 exons spanning approximately 5.6 kb on 12q, and the keratin 5 gene comprises 9 exons spanning approximately 6.1 kb on 12q. We have also developed a comprehensive PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.

Genomic organization and amplification of the human keratin 15 and keratin 19 genes.

Keratin intermediate filaments are the major components of the cytoskeleton in epithelial cells. Mutations in keratin genes have been documented in many disorders of the skin, nails, hair, and mucous membranes. Although no mutations have been described in either keratin 15 or keratin 19, they are good candidates for other as yet uncharacterized genetic disorders of keratinization, particularly as the skin, nails, hair, and conjunctiva are sites of keratin 15 and 19 expression. To facilitate future mutation detection analyses, we have therefore characterized the intron-exon organization of the human keratin 15 and keratin 19 genes. The keratin 15 gene comprises 8 exons spanning approximately 5.1 kb on 17q21, and the keratin 19 gene consists of 6 exons covering approximately 4.7 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.

Preimplantation genetic diagnosis of compound heterozygous mutations leading to ablation of plakophilin-1 (PKP1) and resulting in skin fragility ectodermal dysplasia syndrome: a case report.

A new form of genodermatosis resulting from mutations in the gene plakophilin 1 (PKP1) has recently been identified. The clinical features of a functional knockout of PKP1 are a combination of skin fragility and a form of hypohydrotic ectodermal dysplasia. We have developed a single cell polymerase chain reaction (PCR) assay suitable for preimplantation genetic diagnosis (PGD) and here we report on the clinical application of this assay.

Striate palmoplantar keratoderma resulting from desmoplakin haploinsufficiency.

Recently, the first example of a human mutation in the gene encoding the desmosomal plaque protein, desmoplakin, has been described in a patient with autosomal dominant striate palmoplantar kerato-derma. We now report a further case of a desmoplakin mutation in a proband with striate palmoplantar keratoderma that also results in a null allele and haploinsufficiency. The mutation was a heterozygous G > A transition at the donor + 1 site of intron 7 of the desmoplakin gene (939 + 1 G > A; Genbank M77830). The aberrant splicing leads to retention of the entire intron 7, which contains a premature termination codon within the N-terminal domain of the peptide. Because the mutant null allele could not be identified on cDNA sequencing, we determined by polymerase chain reaction the exon-intron organization of the desmoplakin gene to facilitate analysis of genomic DNA. The gene spans approximately 45 kb of chromosome 6 and comprises 24 exons ranging in size from 51 bp to 3922 bp. We have also characterized fully the 3'UTR of the desmoplakin cDNA. This study demonstrates the relevance of haploinsufficiency for desmoplakin in the pathogenesis of this genodermatosis. Assessment of family members bearing the mutant allele also emphasizes the significance of an individual's age and exposure to skin trauma in manifesting full phenotypic expression of the disorder.

Comparative mutation detection screening of the type VII collagen gene (COL7A1) using the protein truncation test, fluorescent chemical cleavage of mismatch, and conformation sensitive gel electrophoresis.

Mutations in the type VII collagen gene, COL7A1, give rise to the blistering skin disease, dystrophic epidermolysis bullosa. We have developed two new mutation detection strategies for the screening of COL7A1 mutations in patients with dystrophic epidermolysis bullosa and compared them with an established protocol using conformational sensitive gel electrophoresis. The first strategy consisted of an RNA based protein truncation test that amplified the entire coding region in only four overlapping nested reverse transcriptase-polymerase chain reaction assays. These fragments were transcribed and translated in vitro and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have used the protein truncation test procedure to characterize 15 truncating mutations in 13 patients with severe recessive dystrophic epidermolysis bullosa yielding a detection sensitivity of 58%. The second strategy was a DNA-based fluorescent chemical cleavage of mismatch (fl-CCM) procedure that amplified the COL7A1 gene in 21 polymerase chain reaction assays. Mismatches, formed between patient and control DNA, were identified using chemical modification and cleavage of the DNA. We have compared fl-CCM with conformational sensitive gel electrophoresis by screening a total of 50 dominant and recessive dystrophic epidermolysis bullosa patients. The detection sensitivity for fl-CCM was 81% compared with 75% for conformational sensitive gel electrophoresis (p = 0.37 chi2-test). Using a combination of the three techniques we have screened 93 dystrophic epidermolysis bullosa patients yielding an overall sensitivity of 87%, detecting 79 different mutations, 57 of which have not been reported previously. Comparing all three approaches, we believe that no single method is consistently better than the others, but that the fl-CCM procedure is a sensitive, semiautomated, high throughput system that can be recommended for COL7A1 mutation detection.

Dystrophin point mutation screening using a multiplexed protein truncation test.

We report here the first use of a multiplexed protein truncation test for the high throughput screening of dystrophin point mutations. We have developed a substantially more robust and efficient procedure incorporating large savings in cost which uses muscle biopsy or lymphocyte total RNA as the template. The entire dystrophin open reading frame is screened in only five overlapping fragments using a long RT-PCR strategy to amplify dystrophin cDNA in excess of 3.7 kb. These five fragments are uniquely transcribed and translated in vitro in a single multiplexed reaction containing magnesium ions to reduce nonspecific internal initiation of translation. We have used this system to analyze mutations in 11 Duchenne muscular dystrophy patients (10 unrelated) with previously uncharacterized mutations. A single truncating mutation was identified in all patients, which was confirmed at the genomic level. Multiplex PTT provides the most efficient method for point mutation screening in this large gene and has potential applications to several disease genes with a significant proportion of truncating mutations.