Preface: Prenatal diagnosis of hereditary diseases usually requires genetic testing of the family’s preexisting patients (those with the genetic disease) and the identification of mutations in the causative gene. However, in some cases, it is difficult to provide a prenatal diagnosis for families when a family member with a preexisting condition dies soon after birth because the disease is too severe and early to be tested.
The Genetics Group of the Department of Dermatology at Peking University First Hospital has a long history of prenatal diagnosis of hereditary skin diseases and has accumulated a wealth of experience in genetic testing and prenatal diagnosis. In the absence of the results of genetic testing of the predisposing parents, it is also possible to infer the genetic defects of the predisposing parents by direct genetic testing of the parents, thus successfully providing accurate prenatal diagnosis for the family to conceive again. The following paper from the successful case of the genetic dermatology research team, including Lin Zhimiao from the Department of Dermatology, Peking University First Hospital, describes the testing process in detail.
Collodion baby (CB) is a newborn baby born with a tense gelatinous membrane covering the skin all over the body, which may be accompanied by flushing of the skin all over the body. As a result of the tense pulling of the skin, the child may develop deformities such as ectropion of the eyelids, ectropion of the lips, sparse hair, cartilage deformities of the ears and flexion of the fingers and toes [1]. CB is seen in a variety of genetic skin diseases, the most common cause being autosomal recessive congenital ichthyosis (ARCI) [2]. Prenatal diagnosis involves determining whether a fetus has a familially inherited disease by obtaining fetal tissue specimens for analysis of the corresponding gene mutation loci by chorionic villus puncture or amniocentesis during pregnancy after identifying the causative gene mutation loci in the patient or family carrier [3]. We successfully performed prenatal diagnosis by candidate gene exclusion in two couples who had delivered pyrogram-like babies.
Subjects and methods
I. Subjects
Family 1, a healthy couple who had two deliveries of CB and consulted at our hereditary dermatology clinic. 2 cases of CB were found to be covered with translucent gel-like membranous material after birth, with eyelid ectropion and flushing all over the body. Family 2, a healthy couple who had delivered a CB six months earlier, presented to the clinic. This infant also presented after birth with generalized flushing and tense membranous masses with ectropion of the eyelids and sparse hair. A few days after birth, the infant died within a week due to feeding difficulties, secondary infection, and water-electrolyte disturbances. The history of CB in both families was self-reported by the parents (neither could provide a photograph of the child), and CB was diagnosed by the dermatologist at the hospital of birth. Both couples had completely normal skin without any ichthyosis-like lesions and denied consanguineous marriage or else similar patients in the family. We were unable to obtain any fetal tissue specimens as both fetuses died shortly after birth.
II. Methods
1, DNA extraction: 5 ml of peripheral blood from each parent of the affected children in both families was taken, anticoagulated with 2% EDTA, and genomic DNA was extracted by hypotonic hemolysis as well as phenol-chloroform extraction.
2. PCR amplification of TGM1, NIPAL4 and ALOX12B genes and DNA sequencing: Specific primers were designed according to the gene sequences for amplifying the coding regions of the above genes and their flanking sequences. Based on the rate of occurrence of the causative genes in pyrogenic infants, the suspected causative genes were tested in the affected parents one by one until both parents were found to have causative mutation loci in the same causative gene. Pathogenicity of the mutated loci was predicted using Mutationtaste online software (http://www.mutationtaster.org/) and the identified mutated loci were ranked among 200 unrelated normal DNA.
3. Prenatal diagnosis: In family 1, the affected mother underwent chorionic villus aspiration sampling at 11 weeks of gestation, and PCR was performed to amplify exons of the TGM1 gene pathogenic locus after extracting fetal chorionic tissue DNA and sequencing. In Family 2, the mother of the child underwent amniocentesis at 18 weeks of gestation, and some of the amniotic fluid cells were used for direct DNA extraction, amplification of the ALOX12B pathogenicity locus exon and sequencing; some of the amniotic fluid cells were used for culture, and after the amniotic fluid cells were walled, the fluid was changed to remove the contamination of the suspended mother’s blood, and the DNA of the cultured amniotic fluid cells was extracted, and the ALOX12B pathogenicity locus exon was amplified and sequenced again. The DNA of the cultured amniotic fluid cells was amplified and sequenced again to exclude the mother’s blood contamination and to ensure the accuracy of the prenatal diagnosis.
Results
I. Pathogenic gene mutation loci
We detected a heterozygous mutation in c.C427T of the TGM1 gene in the genomic DNA of the father of the child in family 1, which resulted in a p.Arg143Cys amino acid substitution change in the encoded transglutaminase protein 1, a highly conserved mutation site previously reported in patients with lamellar ichthyosis; the mother of the child in family 1 detected a heterozygous mutation in c.1106 delG heterozygous mutation, resulting in a shift mutation in the encoded protein p.G369fsX13 and an early stop codon at the 13th amino acid downstream of the mutated amino acid, leading to the appearance of a truncated protein. No pathogenic mutations were found in the TGM1 and NIPAL4 genes of the parents of the affected children in family 2. The ALOX12B gene of the affected father had a c.1463G>A heterozygous mutation, resulting in a p.Arg488His amino acid substitution change in the encoded protein; the ALOX12B gene of the mother had a c.1642C>T heterozygous mutation, resulting in a p.Arg548Trp amino acid substitution change in the encoded protein, both mutant loci reported in pyrocyte-like infants These two mutation sites were reported in G. pyrifera-like infants, which are highly conserved amino acid sites. All four mutated loci were predicted to be disease-causing by Mutationtaste and were not seen in 200 normal individuals.
II. Prenatal diagnosis
After PCR amplification and DNA sequencing, it was found that the fetus did not have any disease-causing mutations in either parent’s TGM1 gene after chorionic villus puncture sampling at 11 weeks of gestation in the mother of family 1, and was determined to be a healthy fetus. In family 2, PCR amplification and DNA sequencing of amniocentesis samples taken from mothers at 18 weeks of gestation revealed that the fetus had ALOX12B gene with paternal mutation locus c.1463G>A and without maternal mutation locus c.1642C>T. The results of repeat testing after amniotic fluid cell culture were consistent and the fetus was determined to be a healthy carrier. After follow-up, both fetuses were born as healthy newborns.
DISCUSSION
CB can be seen in a variety of genetic dermatoses, with ARCI being the most common [1]. CB can also be seen in a variety of syndromic and nonsyndromic ichthyoses, metabolic disorders such as Gaucher disease, and other conditions such as ectodermal dysplasia [4, 5].CB can have a completely different clinical course depending on the disease itself, ranging from complete self-resolution or only mild dry flaking of the skin (self-resolving flint-like infants) [6] to severe clinical transformation with lamellar ichthyosis [7]. severe clinical phenotype of lamellar ichthyosis [2]. However, in the early stages of life, CB can present with a significant increase in transcutaneous water loss due to severely impaired skin barrier function, resulting in thermoregulatory imbalances, water-electrolyte disturbances, and an increased chance of infection due to skin damage, making CB highly lethal during this period if poorly cared for [1]. All three children with CB in two families in this study died shortly after birth, which may be related to the high mortality rate in early CB and the lack of care experience in local hospitals. The final diagnosis of the disease was not obtained in the fire cotton gum-like infants of both families at that time because the infants died early, skin tissue specimens could not be obtained for pathological and histochemical testing, and further clinical manifestations of the disease were not available.
Prenatal diagnosis of hereditary diseases usually requires the identification of the causative gene mutation locus in the preexisting patient or patient, followed by testing of fetal amniotic fluid cells or chorionic villus tissue specimens at risk of recurrence for the appropriate locus to determine the fetal genotype and thus whether the fetus is affected [3, 7]. Since patient DNA could not be obtained from either family in this study, only the DNA of the parents who were established carriers could be searched for pathogenic mutation loci. Since CB is most commonly associated with ARCI, we first proposed to screen the parents of the children in both families for the six pathogenic genes causing ARCI. Due to the lack of characteristic clinical phenotypes to select from the 6 pathogenic genes, we screened each of the 6 genes from highest to lowest (TGM1>NIPAL4>ALOX12B>CYB4F22>ABCA12>ALOXE3) according to the literature search, ranked by the proportion of the 6 genes that have been reported to cause ARCI [8], until the pathogenic gene was identified. We considered the gene as the causative gene causing the fire cotton gum-like infant in the family only when both parents of the affected child were found to have pathogenic mutation loci in the same gene. Three of the mutant loci identified in both families (c.C427T for TGM1, c.G1463A and c. C1642T for ALOX12B) had been previously reported in ARCI cases. The unreported mutation site (c.1106delG mutation in TGM1) is a shift mutation that causes a truncated protein, which can cause significant shortening or alteration of the structure of the encoded protein, and is not seen in 200 normal people of the same race, making it highly likely to be a pathogenic mutation site. Since both couples are carriers of the disease-causing gene, there is a 25% chance of a second pregnancy with CB, and we recommend prenatal diagnosis during pregnancy.
Prenatal diagnosis can be performed by chorionic villus aspiration at 9-11 weeks of gestation or by amniocentesis at 16-20 weeks of gestation for chromosomal or DNA analysis. The advantage of chorionic villus aspiration is that it can be done earlier to know whether the fetus is sick or not, thus reducing unnecessary pain and burden on the pregnant woman, but it is slightly more difficult and risky than amniocentesis, and chorionic villus cell culture is difficult, making it difficult to verify later if maternal blood or maternal tissue contamination is suspected. In contrast, amniotic fluid cell culture is relatively easy, and later exclusion of maternal blood contamination is easier to achieve. Two families chose different ways of sampling fetal specimens depending on the situation. Our prenatal diagnostic results were accurate in both cases.
Although this study finally succeeded in providing prenatal diagnosis for two families, detection based on first-generation sequencing (Sanger sequencing) methods would be time-consuming, labor-intensive, and costly in the case of a large number of candidate causative genes. In recent years, the cost of second-generation sequencing based on massively parallel DNA sequencing has been decreasing [9, 10], and the concentration of a large number of candidate genes in one reaction system or microarray for capture and high-throughput sequencing will greatly reduce the human and material costs and accelerate the detection speed. It is highly likely to replace first-generation sequencing for clinical and laboratory testing in the future. In collaboration with the Huakang Institute of Genetics, our laboratory has developed a capture system containing the coding sequences of all reported disease-causing genes in hereditary ichthyosis for second-generation sequencing, and is currently undergoing sensitivity and specificity validation.
Figure 1 Sequencing map of the patient’s parents 1a shows heterozygous mutations in c.C427T and c.1106delG in the TGM1 gene of the parents of family 1, respectively; 1b shows heterozygous mutations in c.G1463A and c.C1642T in the ALOX12B gene of the parents of family 2, respectively.