Restrictive dermopathy (RD, MIM# 275210) is a rare neonatal lethal condition, with a prevalence <1/1,000,000 newborns [1]. Neonatal features are represented by thin and tight skin, typical facial dysmorphism (sparse eyelashes and eyebrows, small mouth fixed in the “O” position, micrognathia, dysplastic ears), and arthrogryposis multiplex. Bone mineralization defects and dysplastic clavicles are also frequent. Most affected newborns die in the first week due to respiratory insufficiency [2].

      Most cases are caused by autosomal recessive mutations in the Zinc Metallopeptidase STE24 gene ( ZMPSTE24 ); less frequently by de novo dominant mutations in the Lamin A/C gene ( LMNA ) [3]. Pregnancies with fetal RD shows intrauterine demise, polyhydramnios, prematurity, premature rupture of membranes, and fetal akinesia deformation sequence (decreased fetal movements, intrauterine growth restriction (IUGR), arthrogryposis and pulmonary hypoplasia) [4]. Prenatal diagnosis is very challenging without a genetic test, due to non-specific late ultrasonography findings, often appearing only in the third trimester [4].

      Here we describe a case report of a couple of apparently healthy, non-consanguineous parents from Caucasian origin that requested a genetic counselling after two consecutive complicated pregnancies that ended with the premature delivery of two male infants with multiple congenital anomalies; the first was a stillborn, while the second died at 8 days old (Figure 1A). Molecular analyses were performed in the second child, from whom biological material was available, and both parents.

Patient 1

      The first pregnancy presented normal ultrasonography in the first trimester. At 21+0 week gestational age (wGA) appropriate fetal growth was observed, but several fetal anomalies were suspected: low set ears, retromicrognathia, pinched nose, mouth often fixed in an open position, upper lip with downslanting corners, thick philtrum, hyperechogenic bowel, ascites, and tendency to fixed positions of some joints (Figures 1B and 1C). Maternal perception of reduced fetal movements and repeated episodes of vaginal leaks (suspected preterm premature rupture of membranes [PPROM]) were referred starting from ≈24 wGA. Oligohydramnios, hydrothorax with mediastinal shift, hyperechogenic small lungs and bowel, bilateral hydrocele testis, intrauterine growth restriction (IUGR), and abnormal cardiotocography (CTG) were observed. A male stillborn was delivered by cesarean section at 31 wGA. Major anomalies included very tight skin with several lacerations, diffuse rigidity with proximal and distal arthrogryposis, very small nose, protruding eyes, and micrognathia. Autopsy was limited by maceration. A karyotype was performed from skin and showed a normal male 46, XY karyotype.

Patient 2

      The second pregnancy presented normal ultrasonography in the first trimester. At 21+4 wGA fetal growth was appropriate; left ventricular hyperechogenic focus, dilated left renal pelvis and poorly filled stomach were observed; micrognathia was observed. PPROM was suspected at 22 wGA. At 24 wGA, amniotic fluid index was at the 20^th percentile. Amniocentesis for fetal karyotyping was performed at 24+5 wGA and proved to be normal [46,XY]. FISH test for 22q11.2 deletion was negative. At 28 wGA PPROM, abnormal CTG, and IUGR leaded to cesarean section. The newborn was intubated; APGAR score was 4 at 1 minute and 5 at 5 and 10 minutes. The baby presented with progeroid appearance with tight translucent skin with underlying blood vessels clearly visible, severe microretrognathia, small nose, long philtrum, small “O”-shaped mouth, low set and small ears, stiff joints with severe restriction of movements, choanal stenosis, atrial septal defect, mild hepatomegaly, and dilated renal pelvis (Figures 1D and 1E). The baby died at 8 days old due to cardiocirculatory arrest and impossibility of intubation because of severe micrognathia and neck rigidity.

Figure 1: Clinical and genetic data of reported family. (A) Family pedigree. Squares and circles indicate male and female family members, respectively. Arrow indicates proband. Solid symbols are affected individuals. DOD, date of death; SB, stillbirth; wk, week. (B) Ultrasound ecography of the first fetus presenting with microretrognathia. (C) Ultrasound ecography of the first fetus presenting with low set ears and dysmorphic mouth with down turned upper lip at the 21st week of gestation; (D) Picture of the second child, presenting with the typical RD facial dysmorphisms, thin and translucent skin, and arthrogryposis. (E) Total body Rx revealing hypoplasia of the distal third of clavicles, a defect of ossification at C2, and abnormal positioning of the scapula.

      A blood sample was obtained from patient 2 and both parents; cytogenomic microarray was normal (data not shown). A clinical exome sequencing (CES) analysis was then requested for the trio. A TruSightOne sequencing panel kit (Illumina, San Diego, CA, USA) was used to analyze 4813 genes associated to human genetic diseases. The enriched libraries were sequenced by a NextSeq500 instrument (Illumina). CES data were processed and analyzed using an in-house implemented pipeline as previously described [5]. Among a total of 8,000 variants we identified 10 variants compatible with autosomal recessive mode of inheritance. We considered as the best candidate a homozygous variant in the ZMPSTE24 gene (NM_005857.4: c.954+1G>A; rs781706477) (Supplementary Figure 1A) located in a canonical splicing site with a minor allele frequency (MAF) of 0.0012% in the gnomAD public database of genetic variants (http://gnomad.broadinstitute.org/). This variant, confirmed by Sanger sequencing, was present in heterozygous state in both parents (Supplementary Figure 1B), and was deemed to completely abolish splicing function by in silico prediction tools (Supplementary Figure 1C).

      The majority of RD patients are homozygous or compound heterozygous for null mutations in the ZMPSTE24 gene [3] resulting in lack of protein activity, consequent accumulation of prelamin-A isoforms and absence of mature lamin A. This leads to a fatal clinical course of the disease and a prenatal or neonatal death [3]. Generally, the severity of phenotype varies according to the type of ZMPSTE24 mutations and to the degree of enzyme activity reduction. The c.954+1G>A mutation is a plausible deleterious loss-of-function (null) mutation, as expected by the phenotype of the two male infants compatible with classical RD clinical features [3].

      In routine ultrasonography, prenatal diagnosis of RD is a challenge since most fetal ultrasonography RD findings are nonspecific and appear only late in the second/third trimester. Ultrasound examinations in the two pregnancies reported here revealed that some fetal anomalies associated with RD could be recognized at ultrasound scans already in the second trimester. In particular, a comparison of prenatal data of the present patients with those from a review of the literature [6-8] strongly suggest that some fetal abnormalities, such as microretrognathia, small/pinched nose, reduction of fetal movements, and intrauterine join contractures are recurrent in RD and their recognizable combination could help the prenatal and/or postnatal diagnosis of this rare condition (Table 1).

Table 1

Table 1: Comparison of selected clinical features from pregnancies complicated by RD of the fetus.

      In conclusion, we have successfully used NGS to diagnose RD in two male infants with multiple congenital anomalies and identified a novel homozygous ZMPSTE24 mutation. Present data expand the mutation spectrum of ZMPSTE24 gene and provide further support to the use of CES in the early diagnosis of fetuses with multiple congenital anomalies. Moreover, they provide clear evidence that some specific RD-associated prenatal sonographic findings can be recognized from the second trimester and can be used to drive molecular diagnosis.

      We would like to express our gratitude to the family, which made this study possible. This work was supported by grants from the Italian Ministry of Health RC2017.