Novel missense COL2A1 variant in a fetus with achondrogenesis type II

Achondrogenesis type II (ACG2) is a lethal skeletal disorder caused by pathogenic variants in COL2A1. We present a fetus with cystic hygroma and severe shortening of the limbs at 14 weeks of gestation. We performed postnatal genetic analysis of the parents and fetus to diagnose the disease. A novel missense variant of COL2A1 [NM_001844.5: c.2987G>A, (p. Gly996Asp)] was identified, which led to the ACG2 diagnosis.

10.5 cm long, and had marked limb shortening and a cystic hygroma (Fig. 2c, d).
Radiography taken after delivery showed marked shortening of the extremities. However, a detailed examination of ossification was difficult due to the fetus's immaturity. Chromosomal analysis (G-banding) of the products of conception revealed a normal female karyotype (46,XX).
Following genetic counseling of the couple and obtaining written consent, we performed whole-exome sequencing (WES) for a disease diagnosis. The study was approved by the Institutional Review Board (IRB) of the National Center for Child Health and Development and the Jikei University School of Medicine [IRB number: 234 and IRB number: 27-060 (7945)].
DNA was extracted from the peripheral blood samples of the couple (designated II-3 and II-6 as per pedigree chart: Fig. 1) and the umbilical cord of the infant (III-1) using a previously described method 4 . A whole-exome library was prepared from the DNA samples of III-1 using the Agilent SureSelect v6 Capture Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer's protocol. The libraries were sequenced on a HiSeq2500 (Illumina, San Diego, CA, USA) in the 101 bp pairedend mode. Sequence reads were mapped and aligned to the reference genome sequence hs37d5. Multisample calling of single nucleotide variations and short indels was performed with the RefSeq gene database in combination with 12 in-house control datasets. We extracted 1020 variants using our previously described method 4  To confirm the variant found in WES and whether the couple carried the same variant, we performed Sanger sequencing of the DNA from III-1, II-3, and II-6 using a previously reported method 4 . The missense variant was observed only in the affected fetus (III-1) and not in the couple (II-3 and II-6) (Fig. 2e). According to the American College of Medical Genetics and Genomics guidelines, the variant was considered "likely pathogenic (PS2+PM2+PP3+PP4)" 5 . The fetus was diagnosed with ACG2 resulting from a novel missense variant of COL2A1.
As mentioned above in the couple's inspection, this was a possible de novo variant. After informed consent was obtained, the female had a second spontaneous pregnancy and gave birth to a healthy child (III-2) with no symptoms of skeletal disorders.
ACG2 is an autosomal dominant fatal congenital skeletal disorder caused by defects in the COL2A1 gene 1 . COL2A1 is located on chromosome 12 and encodes a polypeptide chain of type 2 collagen. The triple-helical domain, which is the backbone of type 2 collagen, consists of a glycine-X-Y repeating motif. Glycine substitution in this domain causes ACG2, hypochondrogenesis, platyspondylic dysplasia (the Torrance type), SEDC, and SEMD (the Strudwick type) 1,6 . In this case, a substitution of glycine for asparagine acid was observed (p.Gly996Asp).
Prenatal diagnosis of skeletal disorders can be performed by fetal ultrasonography or computed tomography (CT) 3 . For postnatal diagnosis, radiographic examination and genetic analysis can be considered. In this case, fetal CT was not performed because of the early gestational period. The specific symptoms of ACG2 include fetal edema, marked limb shortening, a bell-shaped thorax, nonossification of the vertebral and pelvic bones, normal skull ossification, and internal rotation of the toes 1,3 . In our study, we observed a cystic hygroma and marked limb shortening in the fetus. There were some reports of cystic hygroma in ACG2, and the findings, in this case, were considered consistent with ACG2 7,8 . Since the fetus was immature, ossification could not be evaluated 3,9 . Therefore, we performed WES to differentiate ACG2 from other skeletal disorders and found a novel missense variant of COL2A1. Based on the clinical findings and genetic analysis, we diagnosed the fetus with ACG2.  Somatic and germline mosaicism is also found in some parents of ACG2 patients 7,10,11 , which when present at low levels, are difficult to detect by Sanger sequencing using peripheral blood DNA samples. Therefore, if parents are suspected of having somatic or germline mosaicism, based on the family history, a close examination should be considered 7,11 .

HGV DATABASE
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/ m9.figshare.hgv.3246.