Materials and analysis
The proband (Child A) is the second child (female) of a non-consanguineous couple and was 7 months old at time of death. Blood samples from Child A were collected post-mortem. The family’s first child (male) (Child B) was 4 years old by the time of analysis.
Peripheral blood for RNA extraction was collected into Tempus Blood RNA Tubes (Ambion, Life Technologies) and RNA was isolated using Tempus Spin RNA Isolation Kit (Ambion). The whole transcriptome RNA-seq libraries were constructed with SOLiD Total RNA-seq Kit (Ambion). 75 bp in forward direction was sequenced using SOLiD 5500 W platform (Life Technologies). RNA-seq raw data were mapped using LifeScope software (Applied Biosystems, Thermo Fisher Scientific) to the human reference genome (hg19) and raw counts were analysed with R statistical software using edgeR package .
Quantitative real-time (qRT)-PCR was used to validate the RNA-seq results of all four family members. In addition, ten control family trios with appropriate for gestational age (AGA) newborn (average ± SD: birth weight 3684 ± 467 g and gestational age 39 weeks 6 days ± 1 week 3 days), and five trios and four mother-child duos with small for gestational age (SGA) newborns (birth weight 1920 ± 738 g and gestational age 38 weeks 3 days ± 2 weeks 1 day) were also analysed. Samples added for validation were extracted from the maternal and paternal peripheral and newborns’ umbilical cord blood.
SNP genotyping experiment was performed on genomic DNA extracted from peripheral blood of both siblings. Samples were genotyped for 542,585 markers using the Infinium HumanCoreExome BeadChip (Illumina Inc). Genotypes were called by GenomeStudio software Genotyping Module v.3.1 (Illumina Inc.). Log R Ratio (LRR) and B Allele Frequency (BAF) values generated by the GenomeStudio software were used for CNV calling using Hidden Markov Model-based software PennCNV .
Child A was mothers’ second child from her second complicated pregnancy. The mother suffered from a respiratory viral infection (not treated) on the 17th week of the pregnancy. During weeks 19–21 foetal growth retardation was diagnosed and at the 22nd week of pregnancy decrease in amniotic fluid was seen in ultrasound (US). TORCH test was performed on maternal peripheral blood and it was negative for infections. The amniotic fluid chromosomal testing revealed a normal female-foetus with karyotype of 46, XX. Maternal laparoscopic cholecystectomy was performed on the 27th week of pregnancy due to a gall bladder infection.
As a result of reduced uteroplacental blood flow (UBF) of 3rd class and maternal infection, emergency caesarean section was performed at gestational age of 27 weeks and 4 days. Child A Apgar scores were 6, 7 and 8 at 1, 5 and 10 min, respectively. The child was SGA with following measurements: weight 470 g, length 27 cm and head circumference 23 cm. Placenta was very small - 108 g (10th percentile is 192 g), with the umbilical cord positioned distally. Placental histological study results of child “A” and “B” were highly similar demonstrating areas of ischemic infarction, decidual vasculopathy, calcification of infarction zone, subcortical fibrinoid deposits and non-inflammatory pattern. Moreover, even weight of placenta was identical.
The newborn was intubated immediately after birth due to respiratory failure. Subsequent problems were liver failure (persistent hyperbilirubinemia, elevated alkaline phosphatase levels and hepatosplenomegaly) and coagulopathy (thrombocytopenia, low antithrombin II activity, low protein C and S and lower than normal prothrombin).
Medical geneticists’ consultation was obtained at postnatal age of 1 month and 16 days (corrected age of 35 weeks) due to peculiar phenotype – extremities and a small trunk compared to the head and micro-anomalies (mongoloid eye shape, long inner canthal distance, low glabella, large tongue, high forehead, and small mandible). Due to the unexpected course of the disease, metabolic diseases and chromosomal abnormalities were suspected. However, genetic testing, including the disease-associated mutations in the cystic fibrosis transmembrane conductance regulator gene, alpha-1-antithrypsin deficiency (SERPINA1 S and Z alleles), galactosemia (GALT gene mutations) and imprinting disorders within Silver–Russell syndrome region at 11p15.5 were found to be normal.
Despite of intensive neonatal care the proband died from pulmocardiac insufficiency at age 7 months and 7 days.
Family history revealed that the father of Child A was healthy and the proband was his first child. Mother (height 160 cm and weight approximately 110 kg) had no previous miscarriages. She was tested for antinuclear antibodies (1:10, neg.), gastric parietal cell antibodies (1:100, pos.), anticardiolipin antibodies (<12 RU/ml, neg.), beta-2 glycoprotein 1 antibodies (neg.), protein C (70–130 %) and free protein S (60–140 %). Thrombophilia was not confirmed for the mother.
Child B, who was also born via emergency caesarean section at 30 weeks of gestation with birth weight of 660 g and Apgar scores of 4, 7 and 7 at 1, 5 and 10 min, respectively. Similarly before delivery, reduced UBF of 3rd class was diagnosed, with almost no detection of amniotic fluid with US and a small placenta (109 g) at delivery. Child B was intubated shortly after birth due to respiratory failure caused by congenital sepsis and quickly followed by deterioration into a septic shock. Subsequent problems were perinatal bronchopulmonary dysplasia, feeding disorders and other pathologies. The child left the hospital at 2.5 months of age and weighed 2560 g at the time with constant mild respiratory disorders and cerebral palsy. At age four, when present study was conducted, Child B had non-allergic asthma and behavioural problems. Father of Child B died in an accident 3 weeks before the birth. Timeline summarizing the clinical course of the children is presented in Additional file 1.