Prenatal diagnosis of junctional epidermolysis bullosa associated with pyloric atresia.

Prenatal diagnosis of junctional epidermolysis bullosa associated with pyloric atresia was carried out in a couple at risk. Their two previous children had died during the first months of life of the same disorder despite surgery for the pyloric abnormality. Ultrastructural study of fetal skin biopsies obtained at 18 weeks' gestation showed dermal-epidermal separation at the lamina lucida level, while ultrasound showed marked stomach dilatation. Light microscopy of pyloric tissue obtained after termination showed the pyloric lumen to be replaced by loose connective tissue with no inflammatory reaction. Immunofluorescence studies on the skin specimens with the monoclonal antibody GB3, known to be absent in 'lethal' junctional epidermolysis bullosa skin, disclosed a marked positivity suggesting that the junctional epidermolysis bullosa in this case may be of the 'non-lethal' type.     

Aplasia cutis congenita in two sibs discordant for pyloric atresia

We report two sibs who were the products of a consanguineous mating, and who had an extensive form of aplasia cutis congenita (ACC). In one of them the generalized skin disorder was manifested by slipping off of the epidermis and mucous membranes with the slightest trauma. This sib also had pyloric atresia and other congenital malformations. Two hypotheses are presented to explain the discordance between the siblings for the abnormalities other than the ACC. One hypothesis assumes varying degrees of severity of the same autosomal recessive disease. The second suggests linkage between the gene for ACC and the gene for an epidermolysis bullosa (EB)-like disorder and pyloric atresia. a recombination event involving the EB-pyloric atresia gene in one carrier parent would then lead to an offspring with only ACC. Prenatal diagnosis is suggested by monitoring alpha-fetoprotein levels in aminotic fluid.     

Aplasia cutis congenita in two sibs discordant for pylori atresia

We report two sibs who were the products of a consanguineous mating, and who had an extensive form of aplasia cutis congenita (ACC). In one of them the generalized skin disorder was manifested by slipping off of the epidermis and mucous membranes with the slightest trauma. This sib also had pyloric atresia and other congenital malformations. Two hypotheses are presented to explain the discordance between the siblings for the abnormalities other than the ACC. One hypothesis assumes varying degrees of severity of the same autosomal recessive disease. The second suggests linkage between the gene for ACC and the gene for an epidermolysis bullosa (EB)-like disorder and pyloric atresia. A recombination event involving the EB-pyloric atresia gene in one carrier parent would then lead to an offspring with only ACC. Prenatal diagnosis is suggested by monitoring α-fetoprotein levels in amniotic fluid.     

Antigenic expression of integrin α6β4 in junctional epidermolysis bullosa

Integrin α6β4 is a major component of hemidesmosomes, considered to play a central role in the adhesion of basal epidermal cells to the underlying dermis. It is therefore of considerable interest in the study of the aetiology of inherited blistering disorders. We have examined the immunohistochemical characteristics of skin from 16 patients with epidermolysis bullosa using two antibodies directed against epitopes on the β4 subunit of αβ4 integrin (G71, 3E1), one antibody directed against an epitope on the α6 subunit (GoH3), GB3 an antibody for nicein, and LH7.2, an anti-collagen type VII antibody. All 10 patients with junctional epidermolysis bullosa showed markedly reduced or no immunoreactivity with G71. These patients included two with GB3-positive junctional epidermolysis bullosa associated with pyloric atresia, and four with other subtypes. By contrast, five patients with dystrophic epidermolysis bullosa and one patient with epidermolysis bullosa simplex showed normal immunoreactivity with G71. In this study, G71 is shown to have a high specificity and sensitivity for the diagnosis of junctional epidermolysis bullosa. Immunoreactivity with 3E1 and GoH3 was normal in most patients, consistent with published reports showing normal immunoreactivity with other β4 and α6 subunit antibodies. The data suggest a modification of the β4 subunit of integrin α6β4 at the dermo-epidermal junction in junctional epidermolysis bullosa.     

Prenatal testing in a fetus at risk for autosomal dominant polycystic kidney disese and autosomal recessive junctional epidermolysis bullosa with pyloric atresia

Amniocentesis and fetal skin biopsies were performed at 18 weeks of gestation in a fetus at risk autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive junctional epidermolysis bullosa (EBJ) with pyloric atresia. A previous son of the couple under investigation had disd at 3 months of EBJ. The mother of the propositus has ADPKD. Genetic linkage studies were carried out in 11 relatives (4 with ADPKD), and on fetal DNA obtained from cultured amniocytes, using 8 flanking DNA markers tightly linked to the PKD1 locus on chromosome 16p, and a DNA marker linked to another putative ADPKD locus on chromosome 2p. The linkage results indicated that the fetus had not inherited the ADPKD chromosome from the affected mother, with a diagnostic accuracy of >99%. Ultrastructural and immunohistochemical analyses of multiple fetal skin biopsies showed no EBJ-associated abnormalities. Thus, combining recent morphological and molecular diagnostic methods, we could show that the fetus was free from both diseases. After 40 weeks of gestation, a normal male infant was delivered. © 1993 Wiley-Liss, Inc.     

Fetal ultrasound abnormalities: Correlation with fetal karyotype,autopsy findings,and postnatal outcome—five-year prospective study

A 5-year prospective prenatal study in 151 pregnancies with 152 malformed fetuses detected by ultrasound was evaluated cytogenetically. Thirty-five fetuses (23%) had abnormal karyotypes. Specific anatomical fetal malformations identified by ultrasound increase the risk for fetal chromosome abnormalities. Risks of abnormal chromosomes in the fetus are present with both single and multiple anomalies including amniotic fluid volume although the risk is increased with specific anatomical systems and multiple malformations. An abnormal fetal karyotype was present in 17% with a single anatomical abnormality and 30% when two or more anatomical systems were involved. Fetal hydrops, duodenal atresia, and omphalocele were the most specific single ultrasound anomalies; fetal hydrops, IUGR, holoprosencephaly, congenital heart disease, diaphragmatic hernia, duodenal atresia, and omphalocele were the most specific multiple anomalies with abnormal amniotic fluid volume. Termination of pregnancy occurred in 32/58 patients diagnosed prior to the 20th week of pregnancy with most (31/32) having a chromosomal anomaly or severe fetal anomaly. Fetuses terminated after the 20th week had chromosomal (7/18) or lethal fetal anomalies (11/18). The most common aneuploidies were trisomy 21, trisomy 18, and 45,X. The decision to terminate the pregnancy was based in most cases on the fetal ultrasound findings. Correlation of ultrasound and clinical findings is important for accurate genetic counselling. © 1992 Wiley-Liss, Inc.     

"Blind" versus direct vision technique for fetal skin sampling in cases for prenatal diagnosis

Seven fetuses at risk of developing a serious inherited skin disorder (epidermolysis bullosa atrophicans generalisata gravis in 4, bullous ichthyosiform erythroderma in 2, and non-bullous ichthyosiform erythroderma in 1) were subjected to prenatal diagnosis by fetal skin sampling. The conventional "blind" biopsy procedure was used in the first 3 cases; a two-cannula technique (one cannula for the optic instrument and the other for the biopsy forceps) that permits biopsy of the skin under direct vision, was employed in the remaining 4 cases. With the "blind" technique, 8 to 10 biopsy specimens had to be taken to ensure that enough skin material would be available for the microscopic examination; only one specimen out of every two was found to consist of skin; the remainder comprised fetal membranes, myometrium, or tro-phoblast. In one case where the "blind" procedure had been used, leakage of amniotic fluid occurred and labor started in the 33rd week. With the two-cannula technique, the number of biopsy samples could be confined to two or three, and all proved to be of skin.     

Fetal ultrasound abnormalities: correlation with fetal karyotype, autopsy findings, and postnatal outcome--five-year prospective study.

A 5-year prospective prenatal study in 151 pregnancies with 152 malformed fetuses detected by ultrasound was evaluated cytogenetically. Thirty-five fetuses (23%) had abnormal karyotypes. Specific anatomical fetal malformations identified by ultrasound increase the risk for fetal chromosome abnormalities. Risks of abnormal chromosomes in the fetus are present with both single and multiple anomalies including amniotic fluid volume although the risk is increased with specific anatomical systems and multiple malformations. An abnormal fetal karyotype was present in 17% with a single anatomical abnormality and 30% when two or more anatomical systems were involved. Fetal hydrops, duodenal atresia, and omphalocele were the most specific single ultrasound anomalies; fetal hydrops, IUGR, holoprosencephaly, congenital heart disease, diaphragmatic hernia, duodenal atresia, and omphalocele were the most specific multiple anomalies with abnormal amniotic fluid volume. Termination of pregnancy occurred in 32/58 patients diagnosed prior to the 20th week of pregnancy with most (31/32) having a chromosomal anomaly or severe fetal anomaly. Fetuses terminated after the 20th week had chromosomal (7/18) or lethal fetal anomalies (11/18). The most common aneuploidies were trisomy 21, trisomy 18, and 45,X. The decision to terminate the pregnancy was based in most cases on the fetal ultrasound findings. Correlation of ultrasound and clinical findings is important for accurate genetic counselling.     

Plectin deficiency leads to both muscular dystrophy and pyloric atresia in epidermolysis bullosa simplex

Plectin is a cytoskeletal linker protein which has a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS‐MD), and EBS with pyloric atresia (EBS‐PA). Previous studies have demonstrated that loss of full‐length plectin with residual expression of the rodless isoform leads to EBS‐MD, whereas complete loss or marked attenuation of expression of full‐length and rodless plectin underlies the more severe EBS‐PA phenotype. However, muscular dystrophy has never been identified in EBS‐PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon‐causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient. © 2010 Wiley‐Liss, Inc.     

Use of type VII collagen gene (COL7A1) markers in prenatal diagnosis of recessive dystrophic epidermolysis bullosa.

Generalised recessive dystrophic epidermolysis bullosa (EB) is a severe inherited disease in which patients suffer from blistering and scarring of the skin and mucous membranes after minor mechanical trauma. Tight genetic linkage has been established to the type VII collagen gene (COL7A1) at 3p21, with no evidence of locus heterogeneity. Several COL7A1 mutations have now been identified in recessive dystrophic EB patients. Prenatal diagnosis has been performed by examination of a fetal skin biopsy taken at about 16 weeks' gestation, and relies on identification of characteristic ultrastructural and immunohistochemical changes. We have now achieved a first trimester prenatal diagnosis using intragenic and flanking COL7A1 markers in a pregnancy at risk for recessive dystrophic EB. Segregation of the informative markers predicted the baby would be an unaffected carrier. The pregnancy continued to term and a healthy baby was born, confirming this result.     

Partial deletion of 4p in fetal cells not present in chorionic villi

A case of a prenatal diagnosis at the second trimester is presented showing a normal karyotype in 12 metaphases from chorionic villi. In all cultured amniotic cells, however, and also in all fetal fibroblasts analyzed after abortion a structural anomaly (46,XY;del 4(pter----p15.2) was detected. Prenatal diagnosis was performed because of intrauterine growth retardation, cleft lip and esophagus atresia by ultrasound. The fetal stigmata are compatible with the Wolf Hirschhorn syndrome. We conclude that amniocentesis may be indicated notwithstanding a normal CV-diagnosis in those rare pregnancies with a characteristically abnormal ultrasound.     

Prenatal detection of intestinal obstructions, aneuploidy syndromes, and cystic fibrosis by microvillar enzyme assays (disaccharidases, alkaline phosphatase, and glutamyltransferase) in amniotic fluid

Microvillar enzymes (disaccharidases, alkaline phosphatase, and gamma-glutamyltransferase) were assayed in amniotic fluid from pregnancies with normal and abnormal fetuses to determine their specificity and reliability for the prenatal detection of intestinal obstructions and cystic fibrosis. All fetuses with imperforate anus, duodenal atresia, jejuno-ileal atresia, multiple intestinal atresia, or other forms of intestinal obstructions, with or without associated ventral wall defect or aneuploidy syndrome, showed diminished microvillar enzyme activities below the normal range of control amniotic fluid samples. The exclusively intestinal hydrolases maltase, sucrase, palatinase, and alkaline phosphatase were the most reliable and sensitive markers to detect intestinal obstructions whereas more widely distributed trehalase and gamma-glutamyltransferase activities were less sensitive. The combination of intestinal disaccharidase maltase, sucrase or palatinase and ALP assays is more accurate for prenatal diagnosis of CF than a combination of intestinal ALP and GGTF assays.     

Determination of cholinesterase and acetylcholinesterase in amniotic fluid

The determination of acetylcholinesterase (AChE) has been shown to be as specific as alphafetoprotein (AFP) for the prenatal detection of open neural tube defects although AFP remains the method of choice. This paper describes a semi-automated technique for the analysis of acetylcholinesterase in amniotic fluid that: A) reduces the cost of the procedure; B) allows for a larger number of samples to be run at a time; and C) provides for more accurate and reproducible procedures and results. Six fetuses with neural tube defects (2 with gastroschisis and 3 where one twin was dead) were detected and found to have elevated AChE, TChE and 2 bands by electrophoresis. Quality control procedures using both pure enzyme and amniotic fluid with low and high levels of the enzyme are described. The analysis of 340 amniotic fluids of normal pregnancies indicates that the normal value for AChE is 5.17 +/- 2.63 mU/ml (97% confidence interval for the mean 4.84-5.49 mU/ml. A group of 27 abnormal pregnancies provides evidence that fetal vomiting and regurgitation, fetal demise, multiple cysts syndrome, idiopathic IUGR, arthrogryposis multiplex, hydrocephaly (stenosis of aqueductus), trisomy 21, trisomy 18, hydronephrosis, pyloric stenosis, heart malformation, ectopia cordis and multiple gestation produce elevated levels of pseudocholinesterase (PChE) in amniotic fluid. The use of pseudocholinesterase levels in amniotic fluid for prenatal diagnosis is proposed and discussed in view of its elevated levels in abnormal pregnancies where AChE is normal. The normal values for PChE are 23.86 mU/ml (mean) and 5.83 for standard deviation. Electrophoretic analysis was performed on all samples with values higher than one standard deviation above the mean.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parental decision following prenatal diagnosis of fetal chromosome abnormality

In the event of prenatal diagnosis of fetal chromosome abnormality, parents must choose between continuation and termination of the pregnancy. To determine whether parents are capable of understanding differences in severity among aneuploidy syndromes, we examined the outcome chosen for all pregnancies in which a fetal chromosome disorder was diagnosed at Northwestern Memorial Hospital between January, 1977 and June, 1986. Among amniocentesis cases, 88% with autosomal aneuploidy were terminated, but only 41% with sex chromosome abnormalities and none with de novo structural rearrangements were terminated. Among a smaller group of chorionic villus sampling cases, all with abnormal results were terminated. Similar patterns of parental behavior were noted in other prenatal diagnosis units. We conclude that parents do distinguish among, and respond specifically to, fetal chromosome disorders of differing severity, at least in the second trimester of pregnancy. However, parents appear more inclined to terminate all pregnancies with chromosome abnormalities when the diagnosis has been made in the first trimester.     

Congenital myasthenic syndrome associated with epidermolysis bullosa caused by homozygous mutations in PLEC1 and CHRNE

Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.     

The dermo-epidermal junction and its acquired and hereditary pathology. A few recent advances]

The dermoepidermal junction (DEJ) is a key structure in the skin. Many acquired and inherited diseases affect the DEJ. New insight has been gained into the chemical composition of the DEJ of which at least 20 protein components have been identified to date. Some components of hemidesmosomes, which are critical DEJ structures, have also been identified. Hereditary epidermolysis bullosa is a heterogeneous group of DEJ disorders. Studies are beginning to shed light on the molecular mechanisms of these diseases. Dystrophic epidermolysis bullosa is due to alterations in the collagen IV molecule which is an essential component of anchoring fibrils. Several molecules, including BM-600 niceine, are apparently altered in the skin and other epithelia of patients with junctional epidermolysis bullosa. These molecules may be involved in the pathogenesis of inherited DEJ diseases with anomalies of hemidesmosomes. Lastly, DEJ antigens involved in a number of acquired bullous skin disorders (bullous pemphigoid, herpes gestationis, acquired epidermolysis bullosa...) have been identified recently.     

Homozygous alpha6 integrin mutation in junctional epidermolysis bullosa with congenital duodenal atresia

Junctional epidermolysis bullosa with congenital pyloric or duodenal atresia is a distinct variant within this group of autosomal recessive blistering skin diseases. In this study we demonstrate, for the first time, a homozygous mutation in the alpha6 integrin gene (ITGA6) in a family with three affected individuals. For this purpose, we first determined the genomic organization of ITGA6, and placed the gene on chromosome 2q by high resolution radiation hybrid mapping. Heteroduplex analysis of PCR products containing the individual exons of ITGA6, followed by direct nucleotide sequencing, revealed that the proband was homozygous for a G-to-T transversion in the +1 position of intron 12. This mutation, 1856+1G-->T, affects an invariant base of the 5' donor splice site predicting aberrant splicing involving exon 12. The mutation was verified in the proband's DNA by restriction enzyme digestion which also confirmed that the parents were heterozygous carriers of this mutation. Altered expression of alpha6 integrin, which forms a heterodimer with the beta4 subunit at the dermal-epidermal junction, would explain fragility and blistering as a result of minor trauma to the skin.      

Epidermolysis bullosa simplex (Dowling-Meara type) associated with pyloric atresia and congenital urologic abnormalities

We report a case of epidermolysis bullosa simplex, Dowling-Meara type (EBS-DM), which was associated with congenital pyloric atresia (PA) and various urologic abnormalities, a diagnosis confirmed by immunofluorescence mapping and electron microscopic findings. Immunofluorescent mapping showed the serum from a patient with bullous pemphigoid faintly binding to the floor of the blister, and monoclonal antibodies against type IV and VII collagens were also stained on the floor of the blister. Electron microscopy showed epidermolytic cleavage and prominent clumping of tonofilaments in the basal and suprabasal keratinocytes. An abdominal radiograph and barium swallow showed a complete obstruction at the pyloric channel level. The widespread bullae healed without any scar formation and the bullae formation was localized on the extremities after 3 months of age without any specific treatment. Multiple urologic abnormalities such as bilateral hydronephrosis, hydroureter and a distended bladder with trabeculation were observed at 12 months of age. Currently, with the patient at 4 years of age, bullae still appear on the hands and feet and nail shedding can be observed. The patient's father, a paternal uncle and a paternal aunt had had similar bullous eruptions in infancy, all of which had improved spontaneously by the age of one.     

Monoclonal antibody GB3 defines a widespread defect of several basement membranes and a keratinocyte dysfunction in patients with lethal junctional epidermolysis bullosa

An antigen expressed at the dermal-epidermal junction as well as in some other human basement membranes (BM) has been detected by the use of a monoclonal antibody termed GB3. This antigen, synthesized by cultured normal human keratinocytes, has been identified as a 600-kilodalton glycoprotein different from other known components of BM. Using indirect immunofluorescence, GB3 was found to be not reactive with the epidermal BM in patients with lethal junctional epidermolysis bullosa. The present study demonstrates (by indirect immunofluorescence) that GB3 defines a widespread defect of several BM in these patients. Furthermore, it gives evidences for an intrinsic biologic defect of lethal junctional epidermolysis bullosa epidermal keratinocytes using in vitro culture of these cells. Whether the lack of GB3 reactivity is the consequence of a true absence of the antigen or an alteration of its molecular structure is not yet known. Nevertheless, GB3 is a useful probe for both rapid and prenatal diagnosis of lethal junctional epidermolysis bullosa, which will give new insights into the molecular comprehension of this disorder.