Exome sequencing improves genetic diagnosis of structural fetal abnormalities revealed by ultrasound

The genetic etiology of non-aneuploid fetal structural abnormalities is typically investigated by karyotyping and array-based detection of microscopically detectable rearrangements, and submicroscopic copy-number variants (CNVs), which collectively yield a pathogenic finding in up to 10% of cases. We propose that exome sequencing may substantially increase the identification of underlying etiologies. We performed exome sequencing on a cohort of 30 non-aneuploid fetuses and neonates (along with their parents) with diverse structural abnormalities first identified by prenatal ultrasound. We identified candidate pathogenic variants with a range of inheritance models, and evaluated these in the context of detailed phenotypic information. We identified 35 de novo single-nucleotide variants (SNVs), small indels, deletions or duplications, of which three (accounting for 10% of the cohort) are highly likely to be causative. These are de novo missense variants in FGFR3 and COL2A1, and a de novo 16.8 kb deletion that includes most of OFD1. In five further cases (17%) we identified de novo or inherited recessive or X-linked variants in plausible candidate genes, which require additional validation to determine pathogenicity. Our diagnostic yield of 10% is comparable to, and supplementary to, the diagnostic yield of existing microarray testing for large chromosomal rearrangements and targeted CNV detection. The de novo nature of these events could enable couples to be counseled as to their low recurrence risk. This study outlines the way for a substantial improvement in the diagnostic yield of prenatal genetic abnormalities through the application of next-generation sequencing.


F3 and F16
A 28 year old woman presented to a tertiary referral centre at 17 weeks and six days gestation with monochorionic diamniotic twins (F3 and F16). F3 had an isolated cardiac VSD on USS. F16 had multiple anomalies on USS including possible sacral hypoplasia, abnormal left and right kidneys, and a dilated and tense bladder (probably due to a lower urethral tract obstruction). There were probable talipes and the head was lemon shaped with the presence of nuchal thickening. The couple decided to undergo a selective termination of pregnancy of F16 due to the USS features. F3 subsequently miscarried. No post-mortem examination was performed. Cytogenetics showed two normal male 46, XY karyotypes. Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome, Cambridge) and the higher resolution ISCA v2·0 60K array (Bluegnome). The source of DNA from F3 and F16 for sequencing was a tissue sample.

F5
A 31 year old woman presented to a tertiary referral centre at 21 weeks and five days gestation with a fetal cardiac anomaly on USS (probable aortic atresia, a VSD and absent pulmonary valve syndrome). She underwent an emergency Caesarean section at term, and a baby boy was born. Postnatally the cardiac defect was confirmed as a truncus arteriosus and type B interrupted aortic arch. Follow up at seven months of age reported that he had several cardiac operations and had a surgical repair of his pyloric stenosis. He was a healthy, alert and responsive child with development entirely appropriate to his age. Karyotyping had shown a normal male 46, XY karyotype. The 1Mb BAC array result showed a duplication Xp22·32p22·31 (RP11-60N3->RP11-769N24). This result was confirmed, and the breakpoints were refined, using an Affymetrix whole-genome 2·7M array. There were two separate duplications at Xp22·32p22·33 separated by ~412 kb. The first was 465 kb long and appears to be the more significant as it disrupts the NLGN4 gene. The second duplication was 201 kb with no HGNC mapped genes and most likely represents copy number polymorphism due to the amount of similar cases in the Database of Genomic Variation. Although not linked to cardiac anomalies NLGN4 is linked to autism. X inactivation studies were inconclusive. This was reported to the parents as a variant of unknown significance. This result has previously been described. (1) The source of DNA from F5 for sequencing was cord blood at delivery.

F6
A 34 year old woman presented to a tertiary referral centre at 22 weeks gestation with a complicated fetal cardiac defect on USS (levocardia with abdominal situs inversus (stomach on the right), a complete AVSD with malposed great arteries, multiple VSDs, and possible right atrial isomerism). The couple opted for a termination of pregnancy. A post-mortem examination showed a 23 week gestation fetus with appropriate measurements. A cardiac defect was confirmed showing malposition of the great arteries, transposition of the pulmonary veins, a double outlet right ventricle, an AVSD, and right atrial isomerism. There were bilateral trilobed lungs and a symmetrical liver. The gallbladder, stomach, duodenum, and pancreas were on the right side. There was no spleen present and the thymus was small. Some of these symptoms are consistent with Ivemark syndrome, the genetic cause of which is unknown.
Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome, Cambridge) and the higher resolution ISCA v2·0 60K array (Bluegnome). The

F7
An 18 year old woman presented at 20 weeks and four days gestation to a tertiary referral centre with a complicated fetal cardiac defect on USS (mesocardia with a VSD and a possible over-riding aorta). A female infant was born and the cardiac defect was confirmed as congenitally corrected transposition of great arteries and VSD. She has since undergone two operative procedures. Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K (Bluegnome). The source of DNA from F7 for sequencing was cord blood at delivery. Mesocardia (HP:0011599); Ventricular septal defect (HP:0001629); Congenitally corrected transposition of the great arteries (HP:0011540); Overriding aorta (HP:0002623)

F8
A 21 year old woman presented at 28 weeks and two days gestation to a tertiary referral centre with a complicated fetal cardiac defect on USS (a congenitally corrected transposition of the great arteries, a VSD, mild sub-pulmonary obstruction, and a hypoplastic right valve). The couple opted for a termination of pregnancy. No post-mortem examination was performed. Cytogenetics showed a normal male 46, XY karyotype. Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the ISCA v2·0 higher resolution 60K (Bluegnome). The source of DNA from F8 for sequencing was a tissue sample.

F9
A 26 year old woman presented to a tertiary referral centre at 20 weeks and five days gestation with fetal anomalies on USS including severe ventriculomegaly bilaterally (>15mm) and a hypoplastic cerebellum. The fetus also appeared to have right-sided talipes. The couple opted for a termination of pregnancy. Post-mortem examination showed an appropriately sized male fetus. Confirmed anomalies included dysmorphic features (an enlarged head, wide sutures/fontanels and low set ears). Post-mortem examination of the CNS showed features consistent with rhombencephalosynapsis and ventriculomegaly. Cytogenetics showed a normal male 46, XY karyotype. Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher ISCA v2·0 resolution 60K (Bluegnome). The source of DNA from F9 for sequencing was a tissue sample.

F10
A 35 year old woman presented to a tertiary referral centre at 21 weeks and three days gestation. USS showed multiple fetal anomalies consistent with fetal akinesia syndrome (the legs were extended and both feet showed severe equinous deformity, the arms and hands were in a fixed flexed deformity, and the spine showed a lateral curvature).
There was also possible micrognathia present, a right-sided pleural effusion, the stomach was small, and there was evidence of a small cerebellum. The couple opted for a termination of pregnancy. A post-mortem examination showed a female fetus with measurements in keeping with the gestation. The face showed extremely low set ears, down-8 slanting palpebral fissures, a narrow nose, and severe micrognathia. Nuchal oedema and bilateral pleural effusions were identified. Musculoskeletal examination showed flexed arms at the elbows and wrists, internal rotation of the hips, hyperextended knees, and bilateral talipes with dislocation of the ankles and extreme loss of muscle bulk. There were pterygia at the shoulders, elbows, and groin. This phenotype resulted from early onset failure of fetal movement.
Histological examination of central nervous system samples showed that this akinesia was likely caused by congenital neuroaxonal dystrophy. Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome Cambridge). The source of DNA from F10 for sequencing was a tissue sample.

F11
A 24 year old woman presented to a tertiary referral centre at 25 weeks and two days gestation. USS showed multiple fetal anomalies including a cardiac defect (a dilated right heart ventricle with an anomaly of the crux consistent with an AVSD, the great vessels appeared dilated, and the crossing of the aortic arch could not be visualized). The cisterna magnum and the third ventricle of the brain were enlarged, and the long bones appeared shortened. The couple opted for a termination of pregnancy. Post-mortem examination showed a male fetus appropriately grown for 28 weeks gestation. There were dysmorphic facies with low-set ears, a long philtrum, and down-slanting palpebral fissures.
There was a midline cleft of the soft palate, a complete AVSD, and agenesis of the corpus callosum. Histologically there were abnormalities of the cerebellum (a dysplastic denate nucleus and glioneuronal heterotopia). The karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 array (Bluegnome). The source of DNA from F11 for sequencing was a tissue sample.

F12
A 36 year old woman presented to a tertiary referral centre at 20 weeks and six days gestation. USS showed severe fetal venticulomegaly (>15mm). The parents opted for a termination of pregnancy. A post-mortem examination confirmed ventriculomegaly and hydrocephalus. It additionally revealed right-sided talipes. Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (Bluegnome). The source of DNA from F12 for sequencing was a tissue sample.

F13
A 38 year old woman presented to a tertiary referral centre at 21 weeks gestation. USS showed multiple fetal anomalies including bilateral multicystic dysplastic kidneys, microcephaly, a banana-shaped cerebellum, hemivertebrae, an Arnold Chiari malformation, nuchal thickening, and possible talipes. The couple opted for a termination of pregnancy. Post-mortem examination showed a male fetus whose measurements were less than expected for 22 weeks gestation. Congenital anomalies included a cystic-dysplastic horseshoe kidney, a high cardiac VSD and a vertebral segmentation defect with distorted ribs and scoliosis of the thoracic/upper lumbar spine. There was a thoraco-lumbar myelomeningocele present and confirmation of the Arnold-Chiari malformation and bilateral talipes. Cytogenetics showed a normal male 46, XY karyotype. Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome Cambridge). The source of DNA from F13 for sequencing was a tissue sample.

F14
A 34 year old woman presented to a tertiary referral centre at 35 weeks gestation. USS showed severe left sided fetal ventriculomegaly (>15mm) and agenesis of the corpus callosum. The couple opted for a termination of pregnancy.
Post-mortem examination showed a female fetus whose weight was on the 18 th centile. Post-mortem examination confirmed hydrocephalus, agenesis of the corpus callosum, focal cerebral cortical dysplasia, and possible dysplasia of the denate nucleus and cerebellum. The face showed mild micrognathia and hypertelorism, and the left uterine horn and fallopian tube were absent. Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome, Cambridge). The higher resolution ISCA v2·0 60K array (Bluegnome) identified a rare, de novo deletion in Xp22.2 (g.13770535_13787331del), which overlaps the protein coding genes OFD1 and GPM6B. This result was reported to the parents. This deletion (with slightly different estimated breakpoints) was also identified from the exome sequencing data using CoNVex. The source of DNA from F14 for sequencing was a fetal blood sample.

F15
A 31 year old woman presented to a tertiary referral centre at 12 weeks and 3 days gestation. Fetal USS showed megacystis, hydronephrosis of the right kidney and a multicystic left kidney. There was a one-segment thoracic hemivertebrae, and an urachal cyst. A female infant was born. Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome Cambridge). The source of DNA from F15 for sequencing was cord blood at delivery.

F17
A 24 year old woman presented to a tertiary referral centre at 23 weeks gestation. USS revealed fetal renal agenesis and anhydramnios. The couple opted for a termination of pregnancy. Post-mortem examination showed a female fetus appropriately grown for 23 weeks gestation. It confirmed unilateral renal agenesis accompanied by contralateral simple renal hypoplasia. Also seen were "Potter-type" facial features (low-set ears, flat nose, and forehead), bilateral talipes, discoid adrenals, and a small bladder. Cytogenetics showed a normal female 46, XX karyotype. Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (Bluegnome). The source of DNA from F17 for sequencing was a tissue sample.

F18
A 26 year old woman presented to a tertiary referral centre at 21 weeks and three days gestation. Fetal USS showed an exomphalos and mild kyphosis. A live baby boy was delivered at 37 weeks and four days gestation. A small exomphalos was confirmed and cloacal exstrophy was diagnosed. In addition he had sacral dysgenesis, spina bifida, bilateral talipes, an imperforate anus, shortening of his bowel, and left-sided renal ectopia. Some of these symptoms are consistent with OEIS complex. He underwent five corrective operations within the first two years of life. His motor developmental milestones were delayed: he crawled at over one year of age and walked at 21 months. At 23 months of age he can speak over 50 words and has good understanding. Cytogenetics showed a normal male 46, XY karyotype. Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (BlueGnome). The source of DNA from F18 for sequencing was cultured

F19
A 26 year old woman of Indian ancestry presented to a tertiary referral centre at 20 weeks and three days gestation. On USS a fetal bilateral cleft lip and palate were visualized. A live baby boy was born. Postnatally he was also shown to have a small atrial septal defect (ASD), patent ductus arteriosis, and oesophageal atresia. He has since undergone surgery to repair the oesophagus. Karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 array (BlueGnome). The source of DNA from F19 for sequencing was a postnatal blood sample.

F20
A 24 year old woman presented to a tertiary referral centre at 12 weeks and two days gestation. Fetal USS showed an increased nuchal translucency of 5·6mm, tricuspid regurgitation, choroid plexus cysts and an echogenic cardiac focus.
The legs and feet appeared very abnormal with an extended attitude and talipes. The pregnancy subsequently miscarried and there was no post-mortem examination. Karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (Bluegnome). The source of DNA for F20 was a chorionic villus sample.

14
A 23 year old woman presented to a tertiary referral centre at 21 weeks and six days gestation. USS showed short fetal long bones and ambiguous genitalia. A baby boy was born prematurely at 26 weeks gestation. Postnatally, he was confirmed to have ambiguous genitalia and also a cardiac ASD. He was transferred to the intensive care area of the neonatal unit and was intubated. He developed thrombocytopenia and a subsequent intraventricular haemorrhage. He died at 17 days of age secondary to lung hypoplasia and respiratory infection. The karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (BlueGnome). The source of DNA from F21 for sequencing was cord blood at delivery.

F22
A 24 year old woman presented at 30 weeks and five days gestation to a tertiary referral centre. USS showed an isolated fetal cleft lip. A baby boy was born at 36 weeks gestation by Caesarean section for breech presentation. After birth a cleft lip and palate were confirmed and a cardiac cor triatriatum was diagnosed. He has since undergone operative procedures for both structural anomalies. The child was walking at 14 months old and had normal speech at 24 months. Karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (BlueGnome). The source of DNA from F22 for sequencing was a tissue sample.

F23
A 31 year old woman presented at 22 weeks and three days gestation to a tertiary referral centre. USS showed findings consistent with fetal skeletal dysplasia including shortened long bones, and a "telephone receiver" appearance of the femur and humerus. The hands and feet were difficult to visualize, the chest was small, and the ribs were shortened.
An abnormality such as thanatophoric dysplasia was suggested. The couple opted for a termination of pregnancy. A subsequent post-mortem examination showed a male fetus whose measurements reflected severe osteochondrodyplasia. Skeletal examination showed disproportionate dwarfism with short limbs, a large head, and dysmorphic facial features. In addition, the chest was narrow and bell-shaped, and there was megaloencephaly.
Collectively X-ray features, brain abnormalities, and histology of bones and joints were consistent with thanatophoric dysplasia type 1. The karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (BlueGnome).
The source of DNA from F23 for sequencing was a tissue sample.

F25
A 35 old woman presented at 28 weeks and one day gestation to a tertiary referral centre. USS showed that the fetus had a right-sided hydrothorax with a mediastinal shift. A baby boy was born at term by Caesarean section weighing 3·77 kg and his length and head circumference were on the 50 th centile. At 34 months of age he was meeting developmental milestones normally and was not under paediatric follow up, as he was healthy. The karyotype was that of a normal male (46, XY). Microarray testing was normal on the 1Mb BAC targeted array platform (BlueGnome Cambridge). The source of DNA from F25 for sequencing was cord blood at delivery.

F26
A 35 year old woman presented at 12 weeks and three days gestation to a tertiary referral centre. USS showed that the fetus had megacystis with possible lower urethral tract obstruction. The pregnancy miscarried and no post-mortem examination was performed. The karyotype was that of a normal male (46, XY). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (BlueGnome). The source of DNA for F26 was a chorionic villus sample.

F27 and F33
A 33 year old woman with neurofibromatosis type 2 presented at 15 weeks and six days gestation to a tertiary referral centre. USS of the fetus (F27) showed features consistent with a lower urethral tract obstruction (enlarged bladder, oligohydramnios, and echogenic parenchyma of the right kidney). The couple opted for a termination of pregnancy.
Post-mortem examination showed a female fetus with appropriate growth for 16 weeks gestation. It confirmed a distended bladder secondary to urethral atresia, and also showed a recto-vesical fistula, slightly dilated ureters and kidneys, and low-set ears. The karyotype of the fetus was that of a normal female (46, XX). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge) and the higher resolution ISCA v2·0 60K array (BlueGnome). The source of DNA from F27 for sequencing was a tissue sample.
The couple went on to have a further pregnancy. An USS at 13 weeks gestation showed that the fetus (F33) had ascites and a massively dilated bladder. The pregnancy was terminated at 13 weeks gestation. A post-mortem examination showed anal and urethral atresia. The uterus was incorporated into the posterior wall of the bladder (probably pressure related), and a dilated bladder, ureters, and slightly dilated renal pelvises were visualized. F33 also had micrognathia, ambiguous genitalia, and possible hypoplasia of the cerebellar vermis. Quantitative fluorescent PCR showed no evidence of trisomy 13,18, 21, or sex chromosome aneuploidy. Multiplex ligation-dependent probe amplification showed no evidence of deletions or duplications in the subtelomeric regions. Microarray testing was not preformed. The source of DNA from F33 for sequencing was a tissue sample.

F28
A 28 year old woman presented at 11 weeks and six days gestation to a tertiary referral centre. USS showed that the fetus had an increased nuchal translucency of 3·5 mm, and evidence of tricuspid regurgitation. The pregnancy resulted in a live birth of a female baby. We were not able to contact the parents for follow up information. Karyotype of the fetus was that of a normal female (46, XX). Microarray testing was normal both on the 1Mb BAC targeted array platform (BlueGnome Cambridge and the higher resolution ISCA v2·0 60K array (BlueGnome). The source of DNA for F28 was a chorionic villus sample.

F29
A 31 year old woman presented at 12 weeks and four days gestation to a tertiary referral centre. USS showed that the fetus had an increased nuchal translucency of 5·9 mm, tricuspid regurgitation, and an intra-cardiac focus. The pregnancy resulted in a live birth of a female baby.

Exome sequencing
All samples were whole exome sequenced at the Wellcome Trust Sanger Institute, Cambridge, UK. Genomic DNA was extracted from the samples using standard protocols, and fragmented using an ultrasonicator. 100-400 bp fragments were prepared using Illumina paired-end DNA library preparation, enriched for exonic sequences using a SureSelect_All_Exon_50Mb_GRCh37_hs37d5 and then sequenced using the HiSeq TM platform (Illumina) as pairedend 75-bp reads according to the manufacturer's protocol. All samples were indexed and pooled four to a lane for sequencing, except P5, which was not pooled. In all other respects this sample was treated the same as the others, but it does have higher coverage (see Figure S1). we filtered out variants with minor allele frequency >0·01, in non-coding regions, depth <10x (in any of trio), in a tandem repeat or segmental duplication, we removed variants which occur in >10% of either parental read, and those where the calls in the VCF files were not consistent with a de novo mode of inheritance. Finally we visually inspected plots of the reads using the Integrative Genomics Viewer (IGV) and removed variants that appeared incorrectly mapped. (11) For genes in DDG2P we used a slightly less stringent filtering process. We removed variants with minor allele frequency >0·01, in non-coding regions, and those that appeared incorrectly mapped on IGV plots.
To calculate whether our final list of de novo mutations is enriched for functional mutations over what would be expected by chance, we calculated that the proportion of de novo variants in exons expected to be functional, by chance is 71·4%. (12) We compared this to the proportion of de novo variants that are functional in our cohort using a binomial test. To calculate the probability that a given number of functional de novo variants will occur in the same gene in this cohort by chance, we calculated the number that are expected to occur using the known exome mutation rate, and the proportion of variants that are expected to be functional, taking into account the length of the coding sequence of the gene of interest. (12,13) We compared this to the observed number of such variants.

Identification of inherited recessive and X-linked SNPs and indels
For each of the samples, we merged the VCF files from the different variant callers using VCFtools. (14) We identified inherited SNPs and indels under different Mendelian models using in-house Python scripts. We only considered variants that passed the callers' quality filters, were functional (predicted protein consequences were essential splice site, stop gained, frameshift coding, non synonymous, or stop lost), and had an allele frequency of <0·01 in both the 1000 Genomes project, and an internal control cohort of 2172 individuals exome sequenced at the same laboratory, using the same pipelines and analysis methods. We also only considered variants in which the genotypes of the three members of the trio were consistent with inherited recessive (homozygous or compound heterozygous) or X-linked model of inheritance, with unaffected parents.

Validation of SNVs and indels using Sanger sequencing
We whole genome amplified ~50 ng genomic DNA from each sample using Illustra Genomiphi V3 ready-to-go kit (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer's instructions. We used this as a template to amplify a fragment containing each the variant of interest in the relevant trios using REDTaq ® DNA

22
Polymerase (Sigma-Aldrich, Dorset, UK) and capillary sequenced using BigDye v31 kit and ABI 3730 sequencer according to the manufacturers' instructions.

Identification of CNVs from exome data
To call CNVs from the exome data we used CoNVex (manuscript in preparation, ftp://ftp.sanger.ac.uk/pub/users/pv1/CoNVex/). Not to be confused with ADTEx, previously known also as CoNVex.
(15) CoNVex detects copy number variation from exome data using comparative read depth. It corrects for technical variation between samples and detects copy number variable segments using a heuristic error-weighted score and the Smith-Waterman algorithm. It detects deletions and duplications of targeted sequences from few hundred base pairs in size to a few Mb or more.