Fetus 1, family 1
A 20-year-old woman, gravida 2 para 0, was referred to the Guangxi Maternal and Child Health Hospital (Nanning, China) on March 14, 2017 at 24 weeks of gestation due to sonographic detection of fetal hydrocephaly. The family history was notable; a son with congenital hydrocephaly had been terminated at 28 weeks of gestation (Fig. 1A). A 3D ultrasound examination confirmed a fetus with bilateral dilatation of the cerebral posterior horns [2.54 cm (left), 2.16 cm (right)], consistent with hydrocephaly (Fig. 1B).
Fetus 2, family 2
A 27-year-old healthy woman, gravida 4 para 2, was referred to the Guangxi Maternal and Child Health Hospital (Nanning, China) on April 8, 2017 at 30 weeks of gestation due to sonographic detection of fetal hydrocephaly. The pregnancy progressed uneventfully, without exposure to any known teratogens. The family history was notable; a son had been terminated due to severe hydrocephalus. There were two healthy daughters (Fig. 1C). A standard second trimester ultrasound screening, performed at the 28th week of gestation, revealed that the width of the left lateral brain ventricle was 3.8 cm and the right 4.0 cm, consistent with severe hydrocephaly (Fig. 1D). No other fetal structural abnormalities were identified.
A total of 100 healthy individuals of Chinese ethnicity (50 males and 50 females, aged 1–60 years) were recruited at the Guangxi Maternal and Child Health Hospital between May 2017 to December 2017. With informed consent from all participants and approval from the Guangxi Maternal and Child Health Hospital Medical Ethics Committee [approval no. (2017) Lun Han Shen (3–10)], cordocentesis was performed for the two fetuses, and peripheral blood was obtained from the fetuses' family members and the healthy individuals.
Molecular analysis
Genomic DNA was extracted from the cord blood of the fetuses and the peripheral blood of both parents and family members according to the standard protocols of the QIAamp DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany). Fetal DNA was analyzed for L1CAM mutations by Sanger sequencing. The primers of the 28 exons with coding region and splicing junctions of L1CAM used for amplification and sequencing were designed by Primer3 web (http://primer3.ut.ee; version 4.0.0). The primer sequences are shown in Table I. Polymerase chain reaction was conducted in a 25 µl reaction volume using 12.5 µl Premix Taq™ (cat. no. RR901A; Takara Biotechnology Co., Ltd., Dalian, China), 9.5 µl distilled water, 200 nmol/l primer and 100 ng/µl DNA. The thermocycling conditions were as follows: Initial denaturation for 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 60°C, 60 or 90 sec at 72°C, and final extension for 7 min at 72°C. DNA sequencing was performed using an ABI3730XL Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to detect mutations in the entire coding region of L1CAM. Fetus sequence data was aligned with the reference sequence obtained from the University of California, Santa Cruz (Santa Cruz, CA, USA) Genome Browser (https://genome.ucsc.edu/; L1CAM, GRCh37/hg19, NM_000425.4) using Mutation Surveyor software (https://softgenetics.com/mutationSurveyor.php; version 4.0.4.0). Variants detected in the fetuses, but absent in the ClinVar (www.ncbi.nlm.nih.gov/clinvar/), Human Gene Mutation Database (www.hgmd.cf.ac.uk/ac/), L1CAM Mutation Database (http://www.l1cammutationdatabase.info/mutations.aspx), Single Nucleotide Polymorphism Database (www.ncbi.nlm.nih.gov/SNP), Ensembl (http://grch37.ensembl.org/index.html) and Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org/) were considered novel variants, and were further examined in the DNA samples of 100 healthy individuals, to exclude the possibility of ethnicity-specific polymorphisms.
In silico prediction and pathogenicity assessment
The amino acid sequences of human neural cell adhesion molecule L1CAM, obtained from the UniProt database (http://www.uniprot.org/), were aligned with orthologs from 6 different eukaryotic species by DNAMAN software (https://www.lynnon.com/; version 8.0), including Mus musculus, Bos taurus, Gallus gallus, Danio rerio, Xenopus tropicalis and Drosophila melanogaster. The pathogenicity of the identified variants was assessed using SIFT (http://sift.jcvi.org/), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/), and MutationTaster (http://www.mutationtaster.org/) prediction tools, and further classified following the guidelines of American College of Medical Genetics and Genomics (ACMG) /the Association for Molecular Pathology (AMP) (7).
Chromosome analysis revealed a normal male karyotype for the two fetuses, and molecular analysis identified two novel missense variants in L1CAM from two families: c.998C>T(p.Pro333Leu) in family 1, and c.2362G>T(p.Val788Phe) in family 2 (Fig. 2). The affected fetuses were hemizygous, and the mothers of the fetuses were heterozygous; fathers were wild type for the identified variants. The older sister of family 2 was heterozygous and the younger sister was wild type. The proline residue at position 333 and the valine residue at position 788 of L1CAM are highly conserved across 7 eukaryotic species. The two variants were predicted to be ‘disease causing’ based on SIFT, PolyPhen2 and MutationTaster prediction tools. The residues were absent in the dbSNP, Ensembl, ExAC and the 100 normal controls. Alterations in amino acids at position 333 (Fig. 3A) had been reported as pathogenic in patients with hydrocephaly, and so, according to the ACMG/AMP guidelines, PM1, PM2 and PM5, in addition to PP3 and PP4, apply to this variant and is classified as likely pathogenic. The variant at position 788 (Fig. 3B), PM1, PM2, PP3 and PP4 also apply, and can also be classified as likely pathogenic.