Prenatal Diagnosis of Epidermolysis Bullosa: Current Aspects and Perspectives




© Springer-Verlag Berlin Heidelberg 2015
Dédée F. Murrell (ed.)Blistering Diseases10.1007/978-3-662-45698-9_20


20. Prenatal Diagnosis of Epidermolysis Bullosa: Current Aspects and Perspectives



Araksya Izmiryan  and Alain Hovnanian 


(1)
INSERM UMR 1163, Laboratory of Genetic Skin Diseases, Hôpital Necker Enfants Malades, Paris, France

(2)
INSERM UMR 1163, Laboratory of Genetic Skin Diseases, Department of Genetics, Imagine Institute, University Paris Descartes Sorbonne Cité, Necker Hospital for Sick Children, Paris, France

 



 

Araksya Izmiryan



 

Alain Hovnanian (Corresponding author)





20.1 Introduction


Inherited EB is a heterogeneous group of blistering diseases leading to variable but often severe and sometimes lethal phenotypes [12]. Despite progress in the treatment of some forms of EB, such as transplantation of autologous genetically corrected epithelia in nonlethal JEB [23], injection of allogenic fibroblasts [48] or allogenic bone marrow transplantation in RDEB [42], there is currently no cure for EB, and prenatal testing is most often appropriate considering the severity of many forms of EB. The delineation of the underlying disease genes and their defects in affected individuals with EB and their families has had a tremendous impact on early DNA-based prenatal testing and represents a major clinical implication of mutation analysis. This chapter is an overview on the current practice of early DNA-based prenatal diagnosis of EB and the first reports of preimplantation genetic diagnosis of EB. The future development of non-invasive prenatal diagnostic procedures is also discussed.


20.2 Principles


The principle of DNA-based prenatal diagnosis is to define the disease status of a fetus during a pregnancy at risk by genetic testing of its DNA, most often from the perspective of termination of the pregnancy should the fetus be predicted to be affected. Prenatal diagnosis regulations differ from country to country but are generally restricted to “diseases of a particular severity with no cure at the time of diagnosis”. The procedure is performed as early as possible (12 or 16 weeks of gestation) to be as ethically acceptable as possible. The period during which medical termination of pregnancy is allowed by the law varies between countries. In a majority of cases, DNA-based prenatal diagnosis relies on the identification of the familial mutation(s) causing the disease and rarely on the identification of the disease haplotype only.


20.3 The Role of the Geneticist


The prenatal diagnostic procedure is a specific aspect of genetic counselling, for which the geneticist plays a central role. Ideally, the geneticist and the genetic counsellor will have seen the couple at risk of a child being born with epidermolysis bullosa in the context of a genetic counselling clinic, prior to prenatal testing. Usually the type of EB in the family will already have been discerned by the EB dermatologist in advance, either in one of the parents or in a previous child born to parental carriers or one of their relatives. During this consultation, the geneticist confirms the diagnosis of EB on the basis of the family and personal history, the patient’s clinical presentation, on key biological investigations including antigen mapping and ultrastructural analysis of the dermal-epidermal junction and on the results of genetic testing. He assesses the severity and risk of recurrence of the disease in the family which is essential to justify a prenatal diagnostic procedure. This can be difficult in some forms of EB, in which disease expression can be highly variable between affected patients and with age. A precise information on the risk of recurrence of the disease is given to the couple, as well as on the predicted severity of the disease and the limitations of the treatment available if the fetus was affected. The couple is also informed of the risk of fetal loss after sampling, estimated to be approximately 1 % in trained centres. A written consent for genetic testing of the fetus must be obtained from the mother.


20.4 The Sources of Fetal DNA


For DNA-based prenatal analysis, fetal DNA is extracted preferably during the first trimester through chorionic villus sampling (CVS or trophoblast biopsy). Chorionic villi correspond to early placenta and are produced by the fetus, thus allowing access to fetal DNA without compromising fetal integrity. After verification of the term by ultrasonography, CVS is performed at 10–12 weeks of gestation by the obstetrician, most often transabdominally, under ultrasound guidance and after local anaesthesia. The CVS is subsequently carefully cleaned in the molecular genetic laboratory under a dissecting microscope to remove blood or maternal decidua which could contaminate the sample and lead to inaccuracy of the results. When the term of pregnancy does not allow CVS, amniotic fluid sampling (AFS) or amniocentesis is performed during the second trimester at 16 weeks of gestation. Fetal DNA is obtained transabdominally from amniotic cells isolated by pelleting amniotic fluid for direct DNA extraction or from cell culture which requires approximately two additional weeks. In both procedures, CV cells and AF cells are also grown in parallel for backup of the molecular analysis and for systemic karyotype analysis when appropriate, to rule out a major chromosomal abnormality independently of EB. The risk of fetal loss following CVS and AFS is around 1 %, depending on the expertise of the investigator.


20.5 Genetic Analysis Prior to Pregnancy


Ideally, the mutation search in the proband and his family is performed prior to the pregnancy in order to identify the disease mutation(s) in the family. Identification of the causative mutation(s) in the proband allows genetic testing of the parents to be performed, to confirm or deny genetic inheritance of the mutation(s) from the parent(s). This is of prime importance for accurate genetic counselling, in particular for recessive forms of EB, in which inheritance of each mutation needs to be verified to exclude a de novo mutation, non-paternity, hemizygosity (allele loss) or uniparental disomy (isodisomy) (both copies of the mutated gene result from the duplication of the gene from a parent), which obviously have a strong impact on genetic counselling and prenatal diagnostic testing.

When genetic analysis of the index case has not led to the identification of the causative mutation(s), indirect analysis can be used in familial dominant cases or in recessive cases to identify the parental allele(s) carrying the disease mutation(s). This type of analysis is not applicable to sporadic dominant cases of EB. Indirect analysis can be used only if the form of EB at risk shows no evidence for nonallelic genetic heterogeneity, which is the case for DEB and EBS with muscular dystrophy. This approach requires that the clinical presentation, the antigen mapping and the ultrastructural findings strongly support the implication of the gene in the affected member of the family. In that case, informative polymorphic markers (often microsatellite markers flanking the disease gene) are genotyped in the affected offspring and his parents in order to identify the maternal and/or paternal haplotypes inherited by the affected offspring and to identify the parental haplotypes corresponding to the mutated allele. In fact, this analysis is most often performed in parallel with direct mutation analysis (when the mutation has been identified), to secure the prenatal diagnosis.


20.6 Genetic Testing of the Fetus


Fetal DNA is tested by PCR amplification and sequencing for the presence or the absence of the mutation(s) previously identified in the index family case (direct analysis) and for genotyping using microsatellite markers linked to the mutated gene (indirect analysis). Usually, both approaches are used in parallel for safety. In rare cases, only the indirect analysis is possible since the causative mutation could not be identified in the proband because of time constraints. The parents’ DNA as well as the index case’s DNA are analysed in parallel, to serve as positive or negative controls and to exclude uniparental disomy. The assessment of the presence or absence of the mutation(s) in fetal DNA allows to predict the disease status of the fetus. This result is confirmed by genotype analysis using microsatellite markers closely linked with the disease locus. The genotype of the index case is then compared with the genotype of the fetus and his parents, allowing to conclude on the disease status of the fetus. In both approaches, polymorphic microsatellite markers on different chromosomal regions are also genotyped in parallel in the fetus and his parents, in order to exclude contamination by maternal DNA and non-paternity.

Prediction of the disease status of the child is based on the observation that penetrance of EB is complete and that individuals carrying a dominant mutation or two recessive mutations will always have a disease phenotype. However, the severity of EB is sometimes difficult to predict, depending on the form of EB and its underlying mutations, leading to variable disease expression. These aspects should have been previously discussed with the couple prior to prenatal diagnosis. It is essential that the geneticist and the genetic counsellor explain these results to the couple during a consultation of genetic counselling and that the couple fully understands this information in order to take the decision to continue or not the pregnancy in the light of this knowledge.


20.7 Which EB Should Benefit from DNA-Based Prenatal Diagnosis?


Inherited EB represent the most frequent genetic skin diseases for which prenatal diagnosis is requested. This is due to their prevalence in the general population and the severity of several forms of EB. There is currently no cure for these diseases, and EB were among the first severe genetic skin diseases to benefit from the translational application of the discovery of the defective genes for early DNA-based prenatal diagnosis. Major progresses in prenatal diagnosis of EB came from the identification of the genes underlying the majority of inherited EB. Prenatal diagnosis of EB is now routinely performed in several specialised centres.

Epidermolysis bullosa (EB) comprise a heterogeneous group of skin fragility disorders in which mild physical trauma leads to skin and/or mucosal blistering since birth, leading to a wide range of disease severity. Because several forms are devastating disorders with dramatic functional and life-threatening complications leading to demise in infancy or in childhood, early prenatal diagnosis of EB is most often highly recommended. EB can be caused by mutations in more than 18 genes expressed at the dermal-epidermal junction and can be inherited in an autosomal dominant or recessive manner. Several recent reviews have provided useful information on genotype-phenotype correlations in the major forms of EB, namely, EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler syndrome (KS), which has recently been included in the EB classification. They are useful to clinicians to predict the disease severity but have also their limitations due to possible inter- and intrafamilial variability.

New forms of EB due to mutations in exophilin 5 [26] and in integrin alpha 3 [15] have also added to the complexity of EB [33, 45]. Although they are indistinguishable at birth, EB differ from each other by their clinical course, their complications, their mode of inheritance and their genetic bases.

The phenotype, mode of inheritance and mutated genes underlying epidermolysis bullosa simplex (EBS) are highly variable, and several clinical forms of EBS are recognised. A majority of EBS is due to dominant mutations in the genes for keratin 5 (KRT5) or keratin 14 (KRT14), with three forms recognised: EBS generalised severe (previously referred as EBS, Dowling-Meara), EBS localised and EBS generalised intermediate (previous generalised other). Thus, skin and mucosal blistering varies from relatively mild involvement affecting only the hands and/or feet (localised EBS) to severe forms with widespread cutaneous and mucous blistering, palmoplantar keratoderma and severe itching in the generalised severe forms. Generalised severe EBS often improves during childhood or adulthood, although palmoplantar keratoderma can be severe and remain painful. Prenatal diagnosis of EBS mainly applies to generalised severe forms and has already been used in several families [33, 35]. Some KRT5 and KRT14 missense mutations cause generalised EBS with a particular severity which fully justify prenatal diagnosis [40]. Generalised intermediate and localised forms are significantly milder, and a request for prenatal diagnosis does not seem to be justified. In contrast, rare autosomal recessive forms of EBS due to KRT14 loss of function mutations have been reported, and their severity warrants prenatal diagnosis [14, 39, 50]. Other unfrequent forms of recessive localised EBS have been described, caused by rare missense mutations in KRT14 [17]. Recently, very rare entities have been classified as recessive EBS with specific features and very different severity, caused by six distinct new genes, TGM5, DSP, JUP, PKP1, EXPH5 and DST [12, 13].

However, the most severe and potentially life-threatening forms of EBS are associated with muscular dystrophy (EBS-MD) and with pyloric atresia (EBS-PA), which are caused by recessive plectin mutations [34]. EBS-MD represents a unique group of EBS in which skin blistering can be severe at birth but tends to improve with age, while a severe muscular weakness develops progressively to become very disabling or life-threatening. The muscular defect in EBS-MD can lead to major loss of autonomy and respiratory distress and to death. For these reasons, prenatal diagnosis of EBS-MS is fully justified. Recessive plectin mutations can also cause EBS with pyloric atresia, which has a very poor prognosis with demise soon after birth, and for which prenatal diagnosis is fully warranted also [28]. In contrast to these severe plectin mutations, a specific, dominant plectin mutation has been shown to underlie the EBS-Ogna type, which is responsible for a mild phenotype [20].

Junctional EB (JEB) form a heterogeneous group of blistering diseases due to mutations in six different genes. The generalised severe form of JEB, which is due to mutations in one of the three laminin 332 genes (LAMA3, LAMB3 and LAMC2), was among the first EB to benefit from DNA-based prenatal diagnosis [3, 25, 37, 38, 44]. The first reported case of DNA analysis in a twin pregnancy at risk of generalised severe JEB with successful diagnosis and selective termination of one affected twin (nonidentical twins) was subsequently reported [8]. Prenatal diagnosis of JEB with pyloric atresia (JEB-PA), which often shares a very poor prognosis with generalised severe JEB and is due to mutations in alpha 6-beta 4 integrins (ITGA6, ITGB4), has also been successfully performed [21]. Of note, late ultrasound examination can reveal enlarged stomach bubble and gastric outlet obstruction in pregnancy with no known familial disease suggestive of JEB [22]. Recently, a new method using immunofluorescence analysis of chorionic villus biopsies has been developed for prenatal diagnosis of JEB and EBS with pyloric atresia [6]. The authors concluded that chorionic villus immunofluorescence examination with anti-integrin alpha 6-beta 4 and plectin antibodies is a reliable method for prenatal diagnosis of JEB-PA and EBS-PA, respectively, and suggest that this procedure could be devised for EB with muscular dystrophy, which is also caused by plectin mutations. These results provide a new alternative method for first trimester prenatal diagnosis of JEB with PA and would be particularly useful in affected kindred carrying as yet unidentified genetic mutations.

Dystrophic EB (DEB) were the first forms of EB to benefit from DNA-based prenatal diagnostic procedures [2, 16]. These were initially performed for the most severe forms of RDEB, the generalised severe forms which lead to major complications and early demise [2, 7, 16, 19, 24, 47]. Prenatal diagnosis of other RDEB subtypes is also often requested. These include generalised nonsevere forms, inversa RDEB, RDEB pruriginosa and localised RDEB forms. Although these forms do not display the dramatic features of generalised RDEB, they can lead to severe skin and/or mucosal involvement with major complications, suffering and distress. Genotype-phenotype correlations have established that mutations leading to PTCs on both alleles cause severe generalised forms, whereas missense mutations or other inframe mutations are most often associated with less severe forms. The combination of certain missense mutations with null alleles can lead to inversed forms, which often develop severe oesophageal stenosis, or to generalised forms with fusion of fingers but no dramatic extension of skin lesions as seen in the generalised severe forms. Therefore, the precise history and clinical presentation of the patient, together with the nature and position of COL7A1 mutations, should be carefully considered for prenatal diagnosis of RDEB subtypes distinct from the generalised severe forms. Genetic counselling in RDEB is also complicated by the variability of the disease severity between and within affected families. The identification of MMP1 as the first modifier gene in RDEB has brought to light the role of matrix remodelling in the modulation of the disease severity [41]. However, MMP1 genotyping cannot be used at present to predict the extent to which the phenotype could be modified for a given mutation, as other unknown genetic and environmental factors are also likely to be involved. An illustration of the wide variability in the disease phenotype for identical mutations is pretibial DEB pruriginosa, which can be recessively or dominantly inherited. Finally, dominant forms of DEB usually do not share the severity of generalised RDEB. However, their phenotype is highly variable, and they can cause severe and extended blistering with considerable distress for which prenatal diagnosis can be requested. Of note, in the absence of family history, dominant DEB can be indistinguishable from generalized nonsevere RDEB. In these cases, the identification of the causative COL7A1 mutation in the proband and the verification of its absence in the parents are essential to establish the mode of inheritance of DEB [8, 10, 32]. Of note, in rare cases, dominant and recessive COL7A1 mutations can segregate in the same family.

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Jun 3, 2017 | Posted by in Dermatology | Comments Off on Prenatal Diagnosis of Epidermolysis Bullosa: Current Aspects and Perspectives

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